Protein Surface Recognition by Synthetic Molecules

4.09

Protein Surface Recognition by Synthetic Molecules

K Samanta, P Jana, C Hirschha¨user, and C Schmuck, University of Duisburg-Essen, Essen, Germany Ó 2017 Elsevier Ltd. All rights reserved.

4.09.1 4.09.2 4.09.2.1 4.09.2.2 4.09.2.3 4.09.2.4 4.09.2.5 4.09.2.6 4.09.2.7 4.09.2.8 4.09.2.9 4.09.2.10 4.09.3 4.09.3.1 4.09.3.1.1 4.09.3.1.2 4.09.3.2 4.09.3.2.1 4.09.3.2.2 4.09.3.3 4.09.3.3.1 4.09.3.3.2 4.09.3.3.3 4.09.4 4.09.4.1 4.09.4.1.1 4.09.4.1.2 4.09.4.1.3 4.09.4.1.4 4.09.4.2 4.09.4.2.1 4.09.4.3 4.09.4.3.1 4.09.4.4 4.09.4.4.1 4.09.4.4.2 4.09.4.5 4.09.4.5.1 4.09.4.5.2 4.09.4.6 4.09.4.6.1 4.09.5 4.09.5.1 4.09.6 4.09.6.1 4.09.6.2 4.09.6.3 4.09.6.4 4.09.7 4.09.7.1 4.09.7.2 4.09.8 4.09.8.1 4.09.9

Introduction Protein Surface Recognition Using a-Helix Mimetics NK3 Protein Surface Bak/Bcl-xL Protein–Protein Interaction p53-hDM2 Protein–Protein Interaction (HIF-1a)/p300 Protein–Protein Interaction Amyloid Protein Surface Nuclear Receptor Box Cdc42/Dbs Protein–Protein Interaction CaM-smMLCK Protein Surface AKAP-PKA Protein–Protein Interaction (KSHV Pr) Dimerization Surface Protein Surface Recognition Using Peptide Foldamers Mixed a/b-Peptides as a-Helix Mimetics Bak/Bcl-xL protein–protein interaction VEGF protein surface b-Peptides as a-Helix Mimetics gp41 protein surface p53-hDM2 protein–protein interaction a-Peptides as a-Helix Mimetics CBP KIX protein surface Bak/Bcl-2 or Bcl-XL protein–protein interaction Protein kinase protein surface Constrained Peptides as a-Helix Mimetics for Protein Surface Recognition Hydrocarbon-Stapled Peptides as a-Helix Mimetics Bak/Bcl-2 protein–protein interaction Epidermal growth factor protein surface ExoS-14-3-3 protein–protein interaction Rab-GTPase protein–protein interaction Hydrogen Bond Surrogate (HBS) Peptides as a-Helix Mimetics (HIF1a)/p300 protein–protein interaction Light-Regulated Stapled Peptides as a-Helix Mimetics Clathrin protein surface Lactam Bridges Containing Peptides as a-Helix Mimetics gp41 protein surface CSP-1, HIV-1 Rev protein, and nociception protein surface Disulfide Bridges Containing Peptides as a-Helix Mimetics Nuclear receptor-coactivator protein surface Androgen receptor-cofactor protein surface b-Hairpin Scaffold Peptides as a-Helical Mimetics p53/hDM2 protein surface Flexible Peptides Protein Phosphatase-1 (PP1)–Interacting Proteins (PIP) Protein Surface Protein Surface Recognition Using Small Molecules MDM2-p53 Protein–Protein Interaction Bcl-xL/Bcl-2 Protein–Protein Interaction Interleukin-2 (IL-2)/IL-2Ra Protein Surface The von Hippel–Lindau Protein (VHL)/HIF-1a Protein Surface Protein Surface Recognition With Dendrimers b-Tryptase Protein Surface Chymotrypsin (ChT) Protein Surface Polymers for Protein Surface Recognition Arginine- and Lysine-Specific Protein Surface Molecular Tweezers as Receptors for Protein Surface Recognition

Comprehensive Supramolecular Chemistry II, Volume 4

http://dx.doi.org/10.1016/B978-0-12-409547-2.12533-8

296 296 296 297 301 304 305 306 306 307 308 308 309 309 309 310 312 312 312 312 312 314 314 315 315 315 316 316 317 317 317 319 319 320 320 321 322 322 322 323 323 323 324 324 324 326 328 329 330 330 331 332 332 332

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4.09.9.1 4.09.10 4.09.10.1 4.09.10.2 4.09.11 4.09.11.1 4.09.11.2 4.09.11.3 4.09.11.4 4.09.12 4.09.12.1 4.09.12.2 4.09.12.3 4.09.12.4 4.09.12.5 4.09.13 4.09.13.1 4.09.13.2 4.09.14 References

4.09.1

Lysine-Specific 14-3-3 Protein Surface Stabilization of Protein–Protein Interactions 14-3-3/PMA2 Protein Surface 14-3-3/Tau Protein Surface Protein Surface Recognition Using Metal Complexes Cytochrome c (Cyt-c) Protein Surface a-Chymotrypsin Protein Surface Carbonic Anhydrase (Bovine Erythrocyte) Protein Surface Protein Kinase Protein Surface Calixarenes Derived Receptors for Protein Surface Recognition Histone Deacetylase Protein Surface a-Chymotrypsin Protein Surface PDGF Protein Surface p53 Protein Surface Potassium Channels (Kv1.x) Protein Surface Protein Surface Recognition Using Porphyrins Cytochrome c Protein Surface Potassium Channels Protein Surface Conclusion

332 334 335 335 336 336 337 337 338 340 341 341 342 343 343 345 345 346 346 347

Introduction

Synthetic molecules that can effectively bind to protein surfaces are a fascinating field in modern biomedicine. Such small molecules provide many new principal routes for the discovery of treatments for a multitude of fatal diseases.1 In principle, there are two types of interaction sites available in proteins: (a) inside or interior interaction protein sites and (b) surface interaction protein sites. In most cases, enzyme active sites are found in the interior part of proteins. The success of functionalized small molecules that inhibit enzyme activity is due to the convergent nature of the enzyme’s active site. However, protein surface functional groups are divergent in nature. It is known that each protein surface has a unique composition of charged, hydrophobic and hydrophilic domains. Synthetic molecules that match the electrostatic features and topology of such protein targets might be expected to bind the protein surface and prevent protein–protein interactions (PPIs).2 Therefore, tight binding requires the involvement of large surface areas and multiple functional group interactions. The understanding and manipulation of PPIs is of growing interest due to their critical role in the cellular function/organelle structure, immune response, protein enzyme inhibition, signal transduction, and apoptosis. Rational design of synthetic molecules for recognition of protein surfaces may provide better insights into exactly how proteins interact with one another and open up an alternative approach to disease therapy. Identification of “hot spots” on protein surfaces is an important development in the protein structure field.3 A “hot spot” is defined as locale of ca.600 Å2 on the surface of a protein at or near the geometric center of the protein–protein interface. The functional groups or residues that constitute the hot spot contribute significantly to the thermodynamic stability of the protein–protein complex. This chapter will present current strategies for binding protein surfaces with an emphasis on the use of designed molecules. The principles of molecular recognition learned from these model systems constitute the foundation for progress in the field of protein surface recognition.

4.09.2

Protein Surface Recognition Using a-Helix Mimetics

The design of low-molecular-weight ligands with the desired helical propensity that disrupts PPIs is a challenging area in medicinal chemistry, due to the involvement of large interfacial surface areas.4 Over 30% of protein secondary structure is helical in nature, making it the most abundant secondary structural motif. Therefore, it stands to reason that a significant part of PPIs should involve a-helices, making them a generic template for the design of inhibitors.5 Two outstanding reviews have been published by the Hamilton6 and the Wilson group7 on this topic.

4.09.2.1

NK3 Protein Surface

The pioneering work of Horwell et al. showed that conformationally constrained, nonpeptide templates (1,1,6-trisubstituted indanes) (Fig. 1) allow for the incorporation of two adjacent amino acid side chains, plus a third binding group in an orientation similar to that found in a-helices. Six racemic and two homochiral Phe-Phe and Trp-Phe mimetics were synthesized, and their affinity in tachykinin receptor binding assays was evaluated. Many of them were found to bind with micromolar affinity to the

Protein Surface Recognition by Synthetic Molecules

Figure 1

297

The indane template (right) mimicking an a-helix.

NK, and/or NK3 receptor. The X-ray crystallography of the homochiral indanes, (1R)-N-((S)-L-hydroxymethylbenzyl)-l,6dibenzylindan-L-carboxamide was analyzed and found to be in an a-helix conformation.8

4.09.2.2

Bak/Bcl-xL Protein–Protein Interaction

Kutzki et al. designed a potent a-helix mimetic based on the crystal and solution structures of the Bak/Bcl-xL complex (Fig. 2). Many types of cancer are linked to overexpression of Bcl-2 family members (of which Bcl-xL is representative), which protect mutated cells from undergoing apoptosis, i.e., cell death, thus leading to uncontrolled cell growth. A designed Bak/Badmimetic could inhibit the Bak/Bcl-xL interaction and thus enable the apoptotic cascade.9 A series of terphenyl molecules have been designed, containing alkyl or aryl substituents on the three ortho positions to mimic the key hydrophobic substituents (i, i þ 3, and i þ 7) on the helical exterior of Bak or Bad and carboxylic acid substituents on either end to mimic further additional ion pairing interactions. A key synthetic route to terphenyl derivatives 1–5 was developed involving sequential Suzuki coupling of the corresponding methoxyphenylboronate and phenyltriflate derivatives. A fluorescence polarization (FP) assay demonstrated that the terphenyl molecule 4 shows the strongest binding to Bcl-xL with a Kd value of 114 nM. The less hydrophobic terphenyls 1 and 3 showed lower affinity (Kd 2.09 and 1.89 mM, respectively), emphasizing the importance of hydrophobic interactions for binding to the recognition cleft in Bcl-xL. When the position of the naphthyl substituent was changed in 5, this leads to a significant drop in binding affinity (Kd 2.70 mM), suggesting an effective shape complementarity between 4 and the natural peptide (Fig. 3). Finally, the partial removal of the carboxylate groups, as in 6 (Kd 6.8 mM), or conversion to positively charged groups, as in 2, (Kd 13.7 mM), lead in both cases to a significant loss of activity.10 Yin et al. reported a series of terephthalamide-based helical scaffolds that mimic the a-helical region of the Bak peptide. Among the library of terephthalamide derivatives, compounds 7 (Ki ¼ 0.78  0.07) and 8 (Ki ¼ 1.85  0.32 mM) showed favorable in vitro activities in disrupting the Bcl-xL/Bak BH3 domain complex (Fig. 4). NMR experiments and computational docking simulations indicated that the synthetic inhibitors bind to the cleft of the BH3 domain of the Bak peptide on the surface of Bcl-xL. When terephthalamide derivative 8 was treated with human HEK293 cells, the result was disruption of the Bax/Bcl-xL interaction with

Figure 2

List of compounds tested in the fluorescence polarization assay.

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Protein Surface Recognition by Synthetic Molecules

Figure 3 Overlay of peptide and postulated binding location for 4. Reprinted with permission from Kutzki, O.; Park, H. S.; Ernst, J. T.; Orner, B. P.; Yin, H.; Hamilton, A. D. J. Am. Chem. Soc. 2002, 124, 11838–11839. Copyright 2002, American Chemical Society.

Figure 4

Generic structure of terephthalamide helical mimetics.

an IC50 of 35.0 mM. The intramolecular hydrogen bond between the amide eNH and the alkoxy oxygen atom was proposed to play an important role in the conformational constraint in the molecule, resulting in the preferred orientation of the 2-isopropoxy group, as well as the upper isobutyl side chain, towards the same side of the terephthalamide.11 The Hamilton group designed trispyridylamide-based a-helix mimetics to disrupt the Bak BH3/Bcl-xL complex (Fig. 5). An X-ray crystal structure showed that the polyamide backbone is planar, and all the side chain alkyl groups are projected on one side in the expected conformation. FP assays showed that three trimer analogues 9b, 9d, and 9e were found to inhibit the Bak BH3/Bcl-xL complex with low micromolar affinity (Ki values of 2.3, 9.8, and 1.6 mM, respectively). The trismethoxy derivative 9a as well as the isopropoxy dimer 10 showed no binding below a concentration of 25 mM. Tetramer compound 11 mimics all four key hydrophobic side chains of Bak, but it showed a slightly lower affinity (Ki ¼ 4.17 mM) for Bcl-xL than 9b.12 Biros et al. reported a pyridazine-based library of a-helix peptidomimetics prepared by reverse electron demand Diels–Alder reactions (Fig. 6).13 The potency of the resulting mimetics to disrupt the Bak/Bcl-xL interaction was tested using an in vitro FP assay. The molecules containing b-hydroxyamides (compounds 12, 13, and 14) appeared to have little to no affinity for Bcl-xL. The free rotation of amide bonds is frozen upon receptor binding, which is entropically unfavorable. The use of the more rigid oxazole moiety in place of the b-hydroxy amide (as present in the oxazole–pyridazine–piperazine and bisoxazole scaffolds 15, 16, 17a, and 17b) helped avoid this entropy “penalty” and led to the desired binding results with mP values. Three of these compounds (15, 17a, and 17b) are neutral, while the fourth (16) contains a hydrophobic phenyl ring. These are the strongest binders for Bcl-xL of the small libraries. However, they did not reach the affinity demonstrated by terpheny-based a-helix mimetics.14 Rodriguez et al. reported a biphenyl 4,40 -dicarboxamide scaffold-based structure designed to mimic the i, i þ 4, i þ 7, and i þ 11 residues of an Bcl-xL/Bak protein–helix interface (Fig. 7). This scaffold combines the hydrophobic core of the oligophenyl series and the synthetically accessible carboxamide groups of the terephthalamides. An energy-minimized structure of the biphenyl (in which R1 ¼ R2 ¼ R3 ¼ R4 ¼ Me) showed good overlap with the i, i þ 4, i þ 7, and i þ 11 residue side chains of an a-helix with

Protein Surface Recognition by Synthetic Molecules

Figure 5

299

Trispyridylamide scaffold-based a-helix mimetics.

an RMSD value of 1.368 Å. The results shown in Table 1 demonstrate that the biphenyl derivatives inhibit the Bcl-xL/Bak interaction. The best biphenyl, 27, was found to have a Ki value of 1.8 mM by FP and a Kd of 7.1 mM, as determined by isothermal titration microcalorimetry (ITC). Furthermore, 15N HSQC experiments confirmed that the biphenyls bind to the Bak binding region of Bcl-xL.15

Figure 6

Pyridazine-based libraries of a-helix peptidomimetics.

300

Figure 7

Protein Surface Recognition by Synthetic Molecules

List of biphenyl-based a-helical mimetics (18–28) and their FP assay results.

Table 1

Competition fluorescence polarization assay results for biphenyls 18–28

Compound

R1

R2

R3

R4

Ki (mM)

18 19 20 21 22 23 24 25 26 27 28

Me iBu Me iBu Me iBu Me iBu Bn iBu iBu

iPr iPr 2-Naphthyl 2-Naphthyl iPr iPr 2-Naphthyl 2-Naphthyl iPr iPr iPr

Ph Ph iPr iPr 2-Naphthyl 2-Naphthyl Ph Ph 2-Naphthyl 1-Naphthyl 1-Naphthyl

iPr iPr iPr iPr iPr iPr iPr iPr iPr iBr iPr

180  55 36  6.3 37  7.0 17  6.1 29  6.2 2.1  0.57 8.6  0.67 9.6  0.57 >500 1.8  0.63 2.3  0.57

The same group reported a new foldamer family based on benzoylurea oligomers in which intramolecular hydrogen bonding favors a linear conformation that mimics the position of residues as in an a-helix under aqueous conditions, thus disrupting the Bcl-xL/Bak interaction (Fig. 8). To test the validity of these acylureas as a-helix mimetics, a benzoylurea derivative 30 was synthesized as an exact isostere of best terphenyl inhibitor 29 (Ki of 114 nM) that was developed by the same group for Bcl-xL/Bak interaction. A FP competition assay showed that the compound 30 displaced a fluorescently labeled Bak peptide from Bcl-xL with a Ki value of 2.4 mM. This value is comparable to those of various reported terphenyl derivatives (0.114–13.6 mM) which are known to be effective mimics of the helical Bak peptide binding to Bcl-xL.16

Figure 8

Benzoylurea oligomers-based a-helix mimetics (29–30).

Protein Surface Recognition by Synthetic Molecules

4.09.2.3

301

p53-hDM2 Protein–Protein Interaction

The Wilson group described oligobenzamide scaffold-based a-helix proteomimetic inhibitors of the p53-hDM2 PPI (Fig. 9). P53 is the major tumor suppressor, and its activity is regulated by binding to hDM2.17 Overexpression of hDM2 prevents p53 from its normal activity, and so this PPI is a major target for cancer chemotherapy. Three key hydrophobic residues Phe19, Trp23, and Leu26 from p53 are involved in the interaction between p53 and hDM2 by binding in a helical conformation to a hydrophobic cleft on hDM2 (Fig. 10).18 A series of oligobenzamide compounds were synthesized, considering the mimicry of key residues at i, i þ 4, and i þ 7 of the p53 helix. The inhibitory potency was tested in a fluorescence anisotropy (FA) assay. All compounds (31a–f) were found to inhibit the interaction with low mM activity. The best inhibitor was 31e for which an IC50 of 1.0 mM was determined. Compounds 32a–b were used as negative controls.19 The same group introduced “hybrid” a-helix mimetics in which the backbone was varied by substitution of the middle aryl unit with an a-amino acid to mimic the i, i þ 4, and i þ 7 side chains of the p53 a-helix, which forms a PPI with hDM2 (Fig. 11). The Phe19, Trp23, and Leu26 of p53 are known hot-spot residues, which were imitated. The foldamer recognizes its target protein hDM2 and inhibits its PPI with p53. All mimetics were tested in a FA competition assay. Hybrid 33 displayed low micromolar affinity (IC50 of 11.9  0.6 mM), and all controls 34–37 were found to be inactive. The inactivity of all controls (34–37) indicated that the “top” and “bottom” units as well as hydrophobic side chains play important roles in binding.

Figure 9

Oligobenzamide scaffold-based a-helix proteomimetics.

Figure 10 (A) Crystal structure of hDM2 in complex with a p53 peptide (PDB ID: 1YCR) and (B) the p53 peptide showing side chains key for binding. Reprinted with permission from Plante, J. P.; Burnley, T.; Malkova, B.; Webb, M. E.; Warriner, S. L.; Edwards, T. A.; Wilson, A. J. Chem. Commun. 2009, 5091–5093. Copyright 2007, Royal Society of Chemistry.

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Protein Surface Recognition by Synthetic Molecules

Figure 11

List of “hybrid” a-helix mimetics.

Most significantly, mimetic 33 acted as a selective inhibitor for p53/hDM2 against four other targets including Bcl-xL/BAK, HIF-1a/p300, eIF4E/eIF4G, and Mcl-1/NOXA-B.20 Barnard et al. described the design and synthesis of a library of N-alkylated helix mimetics (Fig. 12). They studied the correlation between biophysical and cellular selectivity to highlight the potential for off-target effects, which had not previously been explored for proteomimetics. These mimetics were screened for inhibition of the p53/hDM2 interaction, in a FA competition assay. Six of the proteomimetics (38, 41, 42, 44, 46, and 47) displaced p53 from hDM2 with low mM IC50 values. All six mimetics have a large aromatic group on the central residue, which effectively mimic the central tryptophan residue on the p53 helix. The mimetics 46 and 47 are the most effective inhibitors, with IC50 values of around 4 mM in both U2OS and SJSA-1 cells. They (46 and 47) promoted cell death through p21 expression (apoptotic pathway). Biotinylated congeners of 46 and 47 confirmed hDM2 binding in a streptavidin pull-down experiment in both U2OS and SJSA-1 cells. Moreover 46 and 47 were shown to act in a selective manner on Mcl-1/NOXA-B over Bcl-xL/BH3 in both biophysical assays and a cellular context. Four compounds (39, 40, 43, and 45) showed no inhibition of the p53/hDM2 interaction up to a concentration of 100 mm. At the central position, these compounds have a small or polar side chain, which is likely to abrogate inhibition. The mimetic 45 inhibited the p53/hDM2 interaction in the FA screen reasonably well but did not score highly for induction of apoptosis or cell viability. Compound 41 acted as an inhibitor of p53/hDM2 but only reduced cell number in the HCS to

Figure 12

A library of N-alkylated helix mimetics.

Protein Surface Recognition by Synthetic Molecules

Figure 13

303

Pyrrolopyrimidine-based a-helix mimetics.

a limited extent. The results suggest that these helix mimetics can reproduce their binding selectivity in cells. Interestingly, dual inhibition of hDM2 and Mcl-1 may also open a new way to further develop anticancer chemotherapeutics.21 The Lim group reported of a novel class of pyrrolopyrimidine scaffold-based a-helix mimetics with increased conformational rigidity. They identified the most potent, dual inhibitors of MDMX- and MDM2-p53 interactions by high-throughput screening (Fig. 13). They constructed a 900-member library containing compounds of type 48, following a facile and divergent solid-phase synthesis. The binding affinities of the selected compounds (48a and 48b) were tested using a FP-based assay. A 15-mer peptide derived from p53 and a known MDM2 inhibitor, MI-63, were used as controls. Compounds 48a and 48b inhibited the p53-MDMX binding (Ki ¼ 0.62 and 0.45 mM, respectively). The Ki-values were comparable to that of a 15-mer p53 peptide (Ki ¼ 0.8 mM). These compounds were found to inhibit the p53-MDM2 interaction as well, with similar binding affinities as for MDMX (Ki ¼ 0.62 and 0.84 mM, respectively).22 Computational modeling showed that the scaffold 48 could mimic MDMX-bound p53 peptide (Fig. 14).

Figure 14 Overlay of compound 48 (R1, R2, R3 ¼ CH3) and an MDMX-bound p53 peptide. Reprinted with permission from Lee, J. H.; Zhang, Q.; Jo, S.; Chai, S. C.; Oh, M.; Im, W.; Lu, H.; Lim, H. -S. J. Am. Chem. Soc. 2011, 133, 676–679. Copyright 2011, American Chemical Society.

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4.09.2.4

(HIF-1a)/p300 Protein–Protein Interaction

Arora and coworkers described the design and synthesis as well as biochemical, and in vivo evaluation of oxopiperazine helix mimics (OHM) as potential inhibitors of hypoxia-inducible signaling. These compounds capture the topography of the a-helical domain at the interface of hypoxia-inducible factor (HIF-1a) and p300 (Fig. 15). HIF regulates the transcription of key genes, whose expression contributes to metastasis, angiogenesis, and altered energy metabolism in cancer.23 The designed OHM (OHM 49, Fig. 16) bound to the hot spot of the target protein with high affinity, thereby inhibiting the interactions of HIF-1a with coactivator p300/CBP. This significantly downregulated the expression of a specific set of genes and reduced the tumor burden in mouse xenograft models. OHM 49 targets CH1 with an affinity of Kd ¼ (5.3  1.4)  10 7 M and OHM 50, which comprises the two critical leucine residues, but lacks Gln824, bound with a slightly reduced affinity Kd ¼ (6.2  1.1)  10 7 M. The two negative controls OHMs 51 and 52 showed very weak affinities for p300-CH1, with Kd values of [1.0  10 5 M in each case.24 Burslem et al. described the first examples of biophysically characterized aromatic oligoamide helix mimetics as inhibitors (Fig. 17) of the HIF-1a/p300 PPI. These compounds were designed to mimic the key functionalities at the i, i þ 4, and i þ 7 positions and spatial orientation of the C-terminal helix (helix 3) of HIF-1a. IC50 values of the mimetics are summarized in Table 2. The compounds 53 and 54 were designed upon the basis of helix 3, which was shown to be inactive in the FA assay. Compound 56 was the most potent inhibitor.25

Figure 15

Synthesis of topographical HIF-1a mimetics as modulators of hypoxia-inducible gene expression.

Figure 16 (A) Model depicting complex of HIF-1a and the CH1 domain of p300/CBP were retrieved from the Protein Data Bank (PDB), ID code 1L8C. The key residues Leu818, Leu822, and Gln824 of HIF-1a CTAD (shown in blue) are positioned in the binding pocket of the p300/CBP CH1 domain (illustrated in orange). Magnified is an overlay of the HIF1a helix spanning residues 816–824 (blue) and OHM 49 (green). Reprinted with permission from Lao, B. B.; Grishaginb, I.; Mesallati, H.; Brewera, T. F.; Olenyukb, B. Z.; Arora, P. S. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 7531–7536. Copyright 2014, HighWire.

Protein Surface Recognition by Synthetic Molecules

Figure 17

305

List of aromatic oligoamide helix mimetic inhibitors and their IC50 values.

Table 2

Structures and IC50 values for compound library

Compound

R1

R2

R3

IC50 (mM)

Helix 3 peptide 53 54 55 56 57 58 59 60 61 62 63b

Ac-GTEELLRALDQVNAAG-NH2 iBu iPr Me iBu iBu Benzyl iPr iBu Benzyl 2-Hydroxyethyl iPr

iBu iBu iPr iBu iPr Benzyl iPr iPr iPr iPr iPr

iPr iBu iBu iBu iBu Benzyl iPr iPr iPr iPr iPr

Inactivea 9.2  0.9 24  1.6 216  16 9.8  1.3 13  1.5 56  6.0 39  4.0 17  0.7 20  0.8 416  64 Inactive

Up to >250 mM. N-Alkylated scaffold.

a

b

4.09.2.5

Amyloid Protein Surface

Saraogi et al. proposed an alternative mechanism of amyloid inhibition in which small-molecule helix mimetics interfere with the helix assembly process in IAPP aggregation.26 They designed oligopyridylamide-based a-helix mimetics (64a–e and 65a–e) (Fig. 18) containing four or five carboxy-terminated side chains. These compounds exhibit their functionalities on one side in direct analogy to an a-helix. In compounds 64a–e, intramolecular hydrogen bonding of the amide to the alkoxy as well as to the pyridine moiety rigidifies the backbone. In contrast the oligobenzamide series 65a–e was synthesized to increase the conformational

Figure 18

Oligopyridylamide scaffold-based a-helix mimetics (64 and 65).

306

Protein Surface Recognition by Synthetic Molecules

flexibility of the aryl-C (]O) bonds, as the amide-pyridine H-bond falls away. The rate of fibrillogenesis of IAPP under lipid-catalyzed conditions significantly diminished in the presence of the helix mimetics. Compound 64e showed a dosedependent inhibition, and an IC50 of 8 mM was obtained. The lipid-catalyzed aggregation showed a gradual increase in antagonistic activity for both series of compounds from the dimer to the longer oligomers, whereas the monomer had virtually no effect on the kinetics. The molecules acted as agonists of amyloid formation in the absence of the lipid, and compound 64e showed twoto three-fold acceleration in the aggregation kinetics. Presumably, the compounds do not effectively partition into the membrane, while IAPP is bound at the membrane surface.27

4.09.2.6

Nuclear Receptor Box

Becerril et al. employed a pyrdylpyridone scaffold to mimic the surface functionality of an a-helical LXXLL motif, also known as a nuclear receptor box (where L is leucine and X is any amino acid including leucine). This short LXXLL sequence is necessary and sufficient for binding to the Estrogen receptor (ER)28 and is therefore essential for the expression of ER-regulated genes. A series of substituted pyridylpyridone derivatives (66–72) was synthesized as shown in Fig. 19. Most compounds bound with Ki values in the low micromolar range in a FP assay (Table 3). Compounds 68 and 71 containing one and two benzyl groups at the i and i þ 3 mimicking positions gave Ki values of 9.4 and 6.5 mM, respectively. Introduction of a more hydrophobic naphthyl group in 72 led to a further improvement and a Ki value of 4.2 mM.29

4.09.2.7

Cdc42/Dbs Protein–Protein Interaction

Cummings et al. developed a more water-soluble 5-6-5 imidazole–phenyl-thiazole-based a-helix mimetic 73 (Fig. 20), which mimics the Q770, K774, and L777 residues of Dbs, corresponding to the i, i þ 4, and i þ 7 of a key Dbs a-helix. Cdc42 is a GTPase

Figure 19

Pyridylpyridone scaffold-based a-helical mimetics and their FP assay results.

Table 3

Results of the fluorescence polarization assay

Compound

Ki (mM)

Compound

Ki (mM)

SRC-1 NR II 66 67 68

1.0 (0.3) 34 (3) 16 (3) 9.4 (2.0)

69 70 71 72

>50 >50 6.5 (0.5) 4.2 (0.5)

The values in brackets are the corresponding standard deviations.

Figure 20

Imidazole-phenyl-thiazole core-based novel a-helix mimetic.

Protein Surface Recognition by Synthetic Molecules

307

(guanine nucleotide triphosphatase) shown to mediate cancer cell resistance30 and has also been linked to diabetes and cardiovascular and neurodegenerative diseases.31 Cdc42 is activated by interaction with the GEF (guanine nucleotide exchange factor) Dbs. A mant-GDP fluorescence-based activity assay showed the disruption of Cdc42/Dbs PPI at micromolar affinity (67 mM). A nonplanar conformation of diacid 73 could match the positions of the i, i þ 4, and i þ 7 side chains in the key Dbs a-helix.32

4.09.2.8

CaM-smMLCK Protein Surface

The DeGrado group synthesized arylamide-based synthetic inhibitors of CaM33 that are intended to mimic a CaM-binding helical peptide smMLCK (Fig. 21). CaM regulates the intracellular Ca2 þ level and activates a large number of regulatory proteins, including kinases, phosphatases, and ion channels. It also plays important roles in many critical biological processes, such as inflammation, metabolism, apoptosis, muscle contraction, intracellular movement, and short- and long-term memory.34 Compound 74, which has two D-Phe residues, showed a Ki value of 7.10  1.48 nM in a FP assay (Table 4). (1H,15N)-HSQC NMR spectroscopy experiments indicate that compound 74 binds to CaM in a similar way as smMLCK does (Fig. 22).35

Figure 21

Arylamide scaffold-based helix mimetics inhibitors.

Table 4

Results of the fluorescence polarization assay

Entry

R1

R2

Ki (nM)

74 75 76 77 78

D-Phe D-3-PyA D-2-Nal L-2-Nal D-3-PyA

D-Phe D-3-PyA D-2-Nal L-2-Nal H

7.10  1.48 25.7  3.42 83.4  6.2 >120 >1200

PyA, pyridylalanine; Nal, naphthylalanine.

Figure 22 Overlay of arylamide 74 (stick) and smMLCK (red ribbon) complexed with CaM (purple cartoon). Reprinted with permission from Yin, H.; Frederick, K. K.; Liu, D.; Wand, A. J.; DeGrado, W. F. Org. Lett. 2006, 8, 223–225. Copyright 2006, American Chemical Society.

308

Protein Surface Recognition by Synthetic Molecules

4.09.2.9

AKAP-PKA Protein–Protein Interaction

The Klussmann group developed polypyridines as helix mimetics for inhibition of AKAP/PKA interactions (Fig. 23). Protein kinase A (PKA) phosphorylates a broad variety of substrates. Dysregulation of cellular processes that depend on AKAP/PKA interactions are associated with several diseases.36 Their sophisticated design takes into account solubility, conformational orientation of the side chain residues, as well as hydrophobic and hydrophilic interactions with the binding cavity. The meta-methyl group of the second pyridine ring in molecule 79, the condensed cyclopentyl ring of the third pyridine moiety, and the following two benzyl rings correspond to the hydrophobic face of the AKAP18d-L314E helix. The carboxy group of the external pyridine ring mimics the hydrophilic side chain of E300 and is likely to interact with Q4 of the D/D domain. The mimetics 79b and 79f bound to the D/D domain with Kd values of 148 and 31 mM, respectively, as determined by isothermal titration calorimetry. However, the inhibitory potency of 79a and 79f was very low. SAR and NMR data suggested that 79b bound with the D/D domain of RIIa subunits in which 79b adopts a conformation that mimics the a-helical AKAP18d fragment (Fig. 24).37

4.09.2.10 (KSHV Pr) Dimerization Surface Shahian and coworkers identified a small-molecule inhibitor of human Kaposi’s sarcoma-associated herpesvirus (KSHV Pr) dimerization by screening a helix-mimetic library (Fig. 25). The herpesvirus is one of the most prevalent viral families, including eight human types which can cause a variety of devastating illnesses such as mononucleosis (Epstein–Barr virus, EBV), genital herpes (herpes simplex virus, HSV), shingles (varicella zoster virus, VZV), retinitis (cytomegalovirus, CMV), and cancer (Kaposi’s sarcoma-associated herpes-virus, KSHV).38 The inhibitor 80 (IC50 ¼ 8.8  0.3 mM) was identified after screening a library of 182 small-molecule helix-mimetics. Furthermore, seven structural analogs of 80 were synthesized, and all compounds were screened

Figure 23

Polypyridines as helix mimetics inhibitors.

Figure 24 Model of the interaction between terpyridine 79b (cyan) and the D/D domain of human regulatory RIIa subunits of PKA. The possible hydrogen-bond donors Q4 and Q14 at the rim (blue) are predicted to interact with the carboxy groups of the terpyridine moiety of 79b. Reprinted with permission from Schäfer, G.; Milic, J.; Eldahshan, A.; Götz, F.; Zühlke, K.; Schillinger, C.; Kreuchwig, A.; Elkins, J. M.; Azeez, K. R. A.; Oder, A.; Moutty, M. C.; Masada, N.; Beerbaum, M.; Schlegel, B.; Niquet, S.; Schmieder, P.; Krause, G.; Kries, J. P. v.; Cooper, D. M. F.; Knapp, S.; Rademann, J.; Rosenthal, W.; Klussmann, E. Angew. Chem. Int. Ed. 2013, 52, 12187–12191. Copyright 2011, Wiley.

Protein Surface Recognition by Synthetic Molecules

Figure 25

309

Compounds (81–87) are structural analogs of initial hit compound 80.

using a fluorogenic activity assay. The most potent compounds were the pyridine derivative 81, which had an IC50 of 3.1  0.2 mM, and its anisole congener 82, IC50 ¼ 16.4  0.7 mM. The improved IC50 of 81 may be due to the added pyridine nitrogen, which permits for either intermolecular hydrogen bonding to residues on the protease or intramolecular hydrogen bonding that rigidifies its structure. The latter could also lead to a lower entropy “penalty” upon binding, as rotation around the amide bond becomes less favorable and its loss upon binding thus less problematic. Compound 81 inhibited protease dimerization by associating with the interface residues, including Trp109, Met197, and Ile201, probably in the i and i þ 4 positions on a-helix 5. Heteronuclear single quantum coherence (HSQC) spectra of selectively labeled 13C-Met KSHV Pr revealed that increasing concentrations of 81 induced a chemical shift perturbation and an increase in the peak volume of the Met197-monomer resonance. At the same time, they observed a reduction in the peak volume of the Met197-dimer peak.39

4.09.3

Protein Surface Recognition Using Peptide Foldamers

Nowadays unnatural peptide oligomers with discrete folding propensities can be created that adopt distinct secondary structures. These foldamers can be used to mimic protein secondary structural elements, such as the a-helixes discussed before, and thereby block PPIs. Given the straight forward synthesis and the number of medicinally relevant PPIs, these compounds appear as interesting candidates for the treatment of several biological disorders.

4.09.3.1 4.09.3.1.1

Mixed a/b-Peptides as a-Helix Mimetics Bak/Bcl-xL protein–protein interaction

The Gellman group designed peptide-based helical foldamers that bind to a specific cleft of an a-helical BH3 domain and disrupt protein–protein Bak/Bcl-xL interaction. The peptides’ foldamer backbone contained a and b amino acids and was designed to adopt a 14/15-helical secondary structure40 (Fig. 26). Chimeric oligomer 89, in which an N-terminal a/b-peptide segment is fused to a C-terminal a-peptide part ((a/b þ a)-peptides), tightly bound to Bcl-xL ligands in a FP competition assay (Ki 0.0019 mM). Compound 89 is 10-fold more potent than the Bak 16-mer. Oligomer 91 in which b3-hNle-9 was replaced by b3-hLeu showed a slightly improved affinity for Bcl-xL (IC50 0.029 mM, Ki 0.0007 mM). The results suggested that oligomers 89 and 91 are well suited to occupy a portion of the BH3-recognition cleft on Bcl-xL. In cell lysates the most potent foldamer induced cytochrome c (cyt c) release from mitochondria, which is an early step in the apoptosis cascade. In (a þ a/b) oligomer 90, which was found to be

310

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Figure 26

Sequences of Bak 16-mer peptide foldamers.

less potent (IC50 > 700 mM), the first nine residues in the N-terminal part are replaced by an a-amino acid segment, while the seven amino acids towards the C-terminal part contain a- and b-peptides. Binding selectivity studies using surface plasmon resonance (SPR) measurements were also performed with the most potent oligomer 89. Oligomer 89 binds tightly to Bcl-w as well as to Bcl-xL, while somewhat weaker to Bcl-2. However, no binding was detected with Mcl-1.41

4.09.3.1.2

VEGF protein surface

Haase et al. developed peptidic foldamers (Fig. 27) that bind to an irregular receptor-recognition surface of vascular endothelial growth factor (VEGF) and inhibit VEGF-induced HUVEC proliferation with modest potency. Binding of VEGF at the domain 2 of the extracellular portion of tyrosine kinase receptors VEGFR1 and VEGFR2 (Fig. 28) stimulates vasculogenesis and angiogenesis.42 Agents that disrupt these interactions are thus used to treat cancer, as tumor tissue requires the formation of new blood vessels for nutrition. In this study the effect of a-to-b-amino acid building block substitution on the VEGF affinity of a peptide ligand was explored. The overall goal was to maintain affinity for VEGF and increase resistance to proteolysis, which is one of the key strengths of b-amino acids. A competition FP assay showed that the 19-mer peptides v107 and v114 bind to the receptor-recognition surface of VEGF with Ki ¼ 0.60 mM and Ki ¼ 0.070 mM, respectively. When M10 is replaced with norleucine (designated v114*), this caused no loss of affinity. After this encouraging result, they prepared v114* derivatives containing multiple b amino acid building blocks. Their affinity and the proteolytic stability data are listed in Table 5. The results showed that peptide 95 has a nearly 190-fold higher resistance to proteinase K than v114* and a 10-fold higher resistance compared to peptide 94. However, peptide 95 has a c.3-fold lower affinity than 94. a/b-Peptides 94 and 95 inhibit VEGF-induced HUVEC proliferation with modest potency. This clearly suggests that these a/b-peptides bind to the receptor recognition surface and block VEGF-mediated signal transduction via cell-surface receptors.43

Protein Surface Recognition by Synthetic Molecules

Figure 27 Sequences of a- and a/b-peptides. Blue circles indicate b3-residues, and Z indicates the cyclic b-residue. nL denotes norleucine. All peptides have a disulfide bridge between the two Cys residues (underlined).

Figure 28 The VEGF9–108 homodimer (gray) bound to (A) domain 2 of VEGFR1 (VEGFR1D2 is green; PDB: 1QTY) or (B) peptide v107 (v107 is green; PDB: 1KAT). Reprinted with permission from Haase, H. S.; Peterson-Kaufman, K. J.; Levengood, S. K. L.; Checco, J. W.; Murphy, W. L.; Gellman, S. H. J. Am. Chem. Soc. 2012, 134, 76527655. Copyright 2012, American Chemical Society.

Table 5

Binding and proteolysis data of various peptides

Peptide foldamer

Ki (mM) a

t1/2 (min) b

v107 v114 v114* 92 93 94 95

0.60 0.070 0.060 0.26 0.74 1.6 4.6

  1.6   24 300

a

Ki values determined by using competitive FP assay. Half-life of a- and a/b-peptides at 50 mM in the presence of proteinase K (10 mg mL 1) in TBS, pH 7.5, with 5% DMSO.

b

311

312

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Figure 29 Sequences of bWWI-1-4 and bWAI-1. b3-homoamino acids are recognized by the single letter code used for the corresponding a-amino acid. O indicates ornithine.

4.09.3.2 4.09.3.2.1

b-Peptides as a-Helix Mimetics gp41 protein surface

Stephens et al. synthesized a short 14-helix-shaped b-peptide foldamer derived from b3-decapeptides bWWI-1-4 (Fig. 29). These compounds bind to the N-peptide region of gp41, thereby inhibiting the gp41-mediated PPI that powers fusion of viral and host cell membranes.44 All four b-peptides were fluorescently labeled at the N-terminus, and their affinity were tested on the gp41 model IZN17. All peptides bound the IZN17 model well, with equilibrium affinities of 0.75  0.1, 1.0  0.3, 2.4  0.7, and 1.5  0.4 mM, respectively. Competition FP experiments were conducted to assess whether bWWI-1-4 competed with a fluorescent analogue of the gp41 ligand C14wtFlu (suc-MTWMEWDREINNYTCFlu), which binds the IZN17 model with an affinity of 4.1 mM. All peptides competed well with IC50 values of 4.0  0.7, 4.6  0.4, 13  4.1, and 3.3  1.4 mM, respectively. Furthermore, bWWI-1-4 inhibited cell–cell fusion with EC50 values of 27  2.5, 15  1.6, 13  1.9, and 5.3  0.5 mM, respectively, whereas the negative control bWAI-1 was inactive.45

4.09.3.2.2

p53-hDM2 protein–protein interaction

Kritzer et al. reported a set of b3-peptides with significant 14-helix stability in water. This was achieved by exploiting electrostatic macrodipole and side chain–side chain salt bridge interactions stabilizing the helical conformation (Fig. 30).46 The presence of the 14-helix structure was confirmed by circular dichroism (CD) spectra and two-dimensional NMR spectroscopy. The peptides were designed to mimic a short a-helical domain of p53 and disrupt the p53/hDM2 interaction. The Kd (233  32 nM) was measured by direct FP analysis of p53ADFlu/hDM2. b53-1 inhibited p53ADFluo/hDM2 complexation with an IC50 value of 94.5  4.4 mM. Next, a series of b3-decapeptides were prepared to investigate whether the affinity of b53-1 for hDM2 required all, or just part of the functional epitope composed of p53AD side chains F19, W23, and L26. b53-Tryp6 was the most potent inhibitor, with an IC50 value that was twofold higher than that of b53-1, whereas b53-Phe9 was only moderately potent. Three peptides (b53-Leu3, b53-Leu6, and b53-Leu9) with a single b3-leucine residue showed no inhibition up to a concentration of 20 mM. Similarly, b53-iso-Leu3, b53-iso-Leu6, and b53-iso-Leu9 containing only a single b3-isoleucine residue showed no inhibition up to 1 mM. Other peptides (b53-Phe3 and b53-Phe6) with a single b3-phenylalanine residue showed no inhibition at 1 mM. These results suggest that b53-1 interacts with hDM2 with specific influences from two of three residues involving the p53AD functional epitope, b3W and b3F.47

4.09.3.3 4.09.3.3.1

a-Peptides as a-Helix Mimetics CBP KIX protein surface

Rutledge et al. developed a technique, called protein grafting, (Fig. 31) for the identification of functional miniature proteins by stabilization of a-helical-binding epitopes on a protein scaffold. A functional CBP KIX-binding epitope of CREB KID was grafted

Figure 30 Helical net diagrams of b3-peptides. b3X denotes a b3-homoamino acid where X is the corresponding a-amino acid. Red and blue highlight electrostatic features; residues that involve the p53AD epitope are in yellow.

Protein Surface Recognition by Synthetic Molecules

313

Figure 31 Protein grafting applied to the KIDP$KIX interaction. Schematic representation of the protein grafting process. Reprinted with permission from Rutledge, S. E.; Volkman, H. M.; Schepartz, A. J. Am. Chem. Soc. 2003, 125, 14336–14347. Copyright 2005, American Chemical Society Adopted from Rutledge, S. E.; Volkman, H. M.; Schepartz, A. J. Am. Chem. Soc. 2003, 125, 14336–14347.

onto the solvent-exposed a-helical face of the small, yet stable protein aPP (avian pancreatic polypeptide).48 Even in the absence of phosphorylation the aPP scaffold is capable of binding with high affinity and specificity to CBP KIX. The CBP KIX-binding miniature protein (PPKID) library was derived from the alignment of an a-helix of aPP and helix B of the CREB KID domain as shown in Fig. 32. A large library (5  107-members) of miniature proteins (PPKID Library1) was produced for use in phage display selection experiments, and the affinity results are summarized in Table 6. The results revealed that PPKID101U, PPKID102U, and PPKID103U exhibit exceptionally high affinity for HisKIX, with Kds ranging from 1.5 to 3.1 mM (“U” indicates an unphosphorylated peptide). In comparison with a KID-ABU control peptide, HisKIX-binding affinity was improved by at least 37- to 77-fold for these peptides and at least 96- to 198-fold relative to KID-BU. Furthermore, peptides PPKIDU 101–103 bind HisKIX with 7- to 14-fold higher binding affinity than peptide CU. The selected polyproline helix and turn regions of the PPKIDU 101–103 peptides compensate  1.2 to  1.6 kcal mol 1 compared to the free energy of complex formation of KID with CBP KIX. Also the binding modes of PPKID99P (“P” indicates a phosphopeptide) and PPKID101U were investigated. The results suggest that the interaction of

Figure 32 Library design (PPKID). The sequence of helix B of CREB KID is grafted with the sequence of the a-helix of aPP. The blue residues are important for aPP folding. The PKA recognition site is in green, and the red hydrophobic residues of helix B are important for CBP KIX binding. X denotes the randomized residues.

Table 6

HisKIX-binding affinity of PPKID and control peptidesa

Selection 1 PPKID96 PPKID97 PPKID98 Selections 2 & 4 PPKID99 PPKID100 Selections 3 & 4 PPKID101 PPKID101 S18E PPKID102 PPKID103 Controls peptides KID-AB KID-B KID-C

GASDMTYWGDDAPVRRLSFFYILLDLYLDAPGVC GMSRVTPGGDDAPVRRLSFFYILRDLYLDAPGVC GASPHTSSGDDAPVRRLSFFDILLDLYLDAPGVC GPSQPTYPGDDAPVRRLSFFYILLDLYLDAPGVC GLSWPTYHGDDAPVRRLSFFYILLDLYLDAPGVC GISWPTFEGDDAPVRRLSFFYILLDLYLDAPGVC GISWPTFEGDDAPVRRLEFFYILLDLYLDAPGVC GLSPYTEWGDDAPVRRLSFFYILLDLYLDAPGVC GLSWKTDPGDDAPVRRLSFFYILLDLYLDAPGVC TDSQKRREILSRRPSYRKILNDLSSDAPGVC RRPSYRKILNDLSSDAPGVC RRLSFFYILLDLYLDAPGVC

Kd PPKIDP (nM)

Kd PPKIDU (mM)

591  59 729  36 1200  100 Kd PPKIDP (nM) 515  44 534  31 Kd PPKIDP (nM) 624  49

24.1  4.0 12.6  1.4 6.7  0.2 Kd PPKIDU (mM) 12.1  2.4 6.6  2.0 Kd PPKIDU (mM) 1.5  0.1 10.9  2.0 2.3  0.2 3.1  0.5 U: Kd (mM) >116 >297 21.5  2.6

P: Kd 562  41 nM 51.6  4.0 mM 2.4  0.2 mM

a Each peptide was labeled with acetamidofluorescein on the C-terminal Cys residue for use in fluorescence polarization experiments. “P” indicates a phosphopeptide, whereas “U” indicates an unphosphorylated peptide. The phosphoserine residue in phosphopeptides is marked in bold.

314

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both PPKID99P and PPKID101U takes place within the CREB KIDP-binding cleft of CBP KIX. The peptides PPKID99P and PPKID101U bind to HisKIX with a high specificity over carbonic anhydrase (CA) and calmodulin.49

4.09.3.3.2

Bak/Bcl-2 or Bcl-XL protein–protein interaction

Gemperli et al. synthesized paralog-selective inhibitors for Bcl-2 proteins. PPBH3-104 is a miniature protein that binds the antiapoptotic protein paralogs Bcl-2 and Bcl-xL with nanomolar affinity. Two new miniature proteins PPBH3-105 and PPBH3106 were identified from the modification of PPBH3-104 whose specificity is reversed (Fig. 33). These two paralog proteins bind Bcl-2 with nanomolar affinity over Bcl-xL. The affinity of PPBH3-5Flu and PPBH3-106Flu for Bcl-2 and Bcl-xL was measured using a FP assay. The two molecules bound Bcl-2 well, with equilibrium dissociation constants (Kd) of 505 and 543 nM, respectively. These dissociation constants are 10-fold higher than that of the PPBH3-104Flu Bcl-2 complex (Kd ¼ 52  5 nM) and 10-fold lower than that of the Bak72-87/Bcl-2 complex (Kd ¼ 6.1  1.5 mM). However, unlike Bak72-87 or PPBH3-104, both PPBH3-105 and PPBH3-106 prefer Bcl-2 over Bcl-xL with equilibrium dissociation constants (Kd) of the Bcl-xL complexes of 2.7  0.5 and 5.4  0.7 mM, respectively.50

4.09.3.3.3

Protein kinase protein surface

Schneider et al. described a miniature protein (107) that selectively recognizes the cAMP-dependent PKA specificity epitope and inhibits its function. Covalent conjugation of 107 to the nonselective kinase inhibitor K252a leads to a dramatic increase in its inhibitory potency and kinase specificity (Fig. 34). PKAs play important roles in maintaining several signal transduction pathways, and its aberrant activity is linked to a plethora of human diseases. The active side N-terminal a-helix of protein kinase inhibitor

Figure 33 Bcl-2-selective miniature protein evolution. (A) Alignment of Bak72-87, PPBH3-104, the two paralogs protein PPBH3-105, and PPBH3-106. Residues in red indicate the binding of Bcl-xL by Bak72-87; residues in blue and yellow contribute to aPP folding. Positions varied in the sequence are indicated by X. (B) Binding affinities.

Figure 34 Synthesis of miniature protein 107 and 108 using protein grafting technique and a miniature protein conjugate 107-K252a. Blue residues within aPP contribute to a-helical folding or light yellow (PPII helix). The pseudosubstrate residues of PKI5–24 are depicted in red; residues that comprise the PKI5–24 a-helical specificity element are shown in green.

Protein Surface Recognition by Synthetic Molecules

315

protein (PKI5–24) selectively recognizes PKA and inhibits its function. The sequence of the N-terminal active side is grafted to the sequence of the a-helix of aPP (avian pancreatic polypeptide). The relative affinities of 107Flu (Kd ¼ 99  39 nM), 108Flu (Kd ¼ 570  123 nM), and PKI5–24 Flu (Kd ¼ 31  8 nM) for PKA were measured by FP analysis in the presence of ATP. Surprisingly, 107Flu retained significant affinity for PKA in the absence of ATP (Kd ¼ 230  34 nM), whereas PKI5–24 Flu bound with a 50-fold decrease in affinity (Kd ¼ 1.6  0.4 mM). Next, the miniature protein conjugates 107-K252a and PKI-K252a were synthesized, and the relative abilities of 107, 107-K252a, PKI-K252a, and K252a itself to inhibit the catalytic activity of PKA were measured. The conjugated protein 1-K252a was 30-fold more active as an inhibitor than 107 (IC50 ¼ 3.65  0.13 and 117  14 nM, respectively) and only 26-times less active than K252a (IC50 ¼ 0.140  0.003 nM). Interestingly, PKI-K252a was 60-times less active as an inhibitor of PKA (IC50 ¼ 221  2 nM) compared to 107-K252a. The natural inhibitor K252a inhibited all four distinct protein kinases including Akt kinase (PKB), protein kinase CR, Ca2 þ/calmodulin kinase II, and cGMP-dependent protein kinase (PKG), whereas both 107 and 107-K252a showed remarkable specificity for PKA, inhibiting no other kinase at concentrations as high as 100 nM.51

4.09.4

Constrained Peptides as a-Helix Mimetics for Protein Surface Recognition

Short synthetic peptide sequences derived from small folded protein epitopes do not have a specific conformation in water. However, as a specific secondary structure, such as an a-helix is necessary for a binding event, it becomes entropically less favorable. In a protein this problem does not occur, as the desired three-dimensional structure is fixed by the protein architecture. To circumvent this problem, scientists have cleverly introduced constraints into peptides by cyclization. This stabilizes a desired secondary structure and thereby enhances protein-like biological activities and thus potencies.52 In the following paragraphs we will discuss different techniques that help induce such constraints:

4.09.4.1 4.09.4.1.1

Hydrocarbon-Stapled Peptides as a-Helix Mimetics Bak/Bcl-2 protein–protein interaction

Walensky et al. used a chemical strategy, termed hydrocarbon stapling, to generate BH3 peptides with improved pharmacologic properties. The strategy relies on the stabilization of an a-helix by tethering suitable amino acid positions in a macrocycle and thereby shifting the equilibrium to the a-helix form. The hydrocarbon-stapled peptides (Fig. 35), with a helical content ranging from 35% to 87%, were designed to mimic the BH3 domain of the BID peptide. a-Helixes (SAHBs) were stabilized as macrocycles by ring closing metathesis of suitable olefin residues. The hydrocarbon chains were attached to the a-carbon of the constituting amino acids. The resulting macrocycles were protease-resistant (both in vitro and in vivo) and cell-permeable. By targeting the

Figure 35

List of staple peptides (substitution positions at i, i þ 4, or i, i þ 7) and ring cyclization was carried out using metathesis reaction.

316

Protein Surface Recognition by Synthetic Molecules

natural Bcl-2 binding pockets with increased affinity, they specifically activated cyt c release from mitochondria, which is an early step in the apoptosis cascade. A FP-binding assay revealed a more than sixfold enhancement in binding affinity of SAHBA (Kd, 38.8 nM) compared to that of the untethered BID BH3 peptide (Kd, 269 nM). A Gly-to-Glu mutation [SAHBA (G / E)] reduced binding affinity (Kd, 483 nM), and HSQC experiments showed similar spectral changes in 15N-Bcl-xL upon binding SAHBA or BID BH3 peptide. SAHBA caused a dose-dependent increase in cyt c release, thus effectively inhibiting the growth of human leukemia xenografts in vivo.53

4.09.4.1.2

Epidermal growth factor protein surface

Sinclair et al. described hydrocarbon-stapled peptides that inhibit the epidermal growth factor (EGFR) in a new way. These compounds block the formation of a coiled-coil dimer in the juxtamembrane (JM) segment that is critical for assembly of the active, asymmetric kinase dimer.54 Generally, the EGFR inhibitors bind the extracellular or intracellular ATP-domain as well as the growth factor domain.55 The EGFR tyrosine kinase is involved in a large number of human cancers.56 Four new peptides (E1S, E2S, E4S, and T4S), derived from the wild-type sequence JMWT (Fig. 36), were synthesized. One peptide (T1S) was prepared as a negative control. All EGFR-expressing cell lines (A431, H2030, H3255, H1975, and SK-NMC) were sensitive to the hydrocarbon-stapled peptides, with potency following the order E1S > E2S [ T4S  E4S. The control (T1S) was inactive in all cell lines, whereas JMWT is not sensitive to any cell lines. E1S was 10 times more potent than E1DL S, in which the two leucines were mutated to alanine. It was also 2–10 times more potent than the previously reported TE-64562 peptide. Peptide E1S was incubated with A431 cells at concentrations of 1–50 mM leading to a dose-dependent decrease in EGFR phosphorylation at positions Y845, Y1045, Y1086, and Y1173. This inhibits downstream factors such as Erk and Akt. Taking into account the cell viability, pull-down, and immunoblotting experiments, the authors proposed that E1S allosterically inhibits EGFR, by blocking the intradimer coiled-coil formation within the JM segment.57

4.09.4.1.3

ExoS-14-3-3 protein–protein interaction

Glas et al. reported the structure-based design of constrained peptides that are capable of inhibiting the ExoS-14-3-3 interaction (Fig. 37).58 ExoS is a virulence factor of the pathogenic bacterium Pseudomonas aeruginosa, a major reason for infections.59 They used the ESp wild-type sequence as a starting point for the design of macrocyclic PPI inhibitors. Three cross-linked peptides aSS8, bRS8, and gRS8 were synthesized, and an FP assay was used to determine the binding affinity toward 14-3-3z protein. Two

Figure 36 Sequences of hydrocarbon-stapled peptides; Z, X, and B represent (R)-2-(70 -octenyl) alanine, (S)-2-(40 -pentenyl)-alanine, and (R)-2-(40 pentenyl)alanine, respectively and positioned for macrocyclic bridges. The superscript S indicates peptides with a hydrocarbon staple.

Figure 37 Modified peptides with three different cross-link architectures (a, b, g). Absolute configuration of a-methylated nonnatural amino acids is designated by subscript.

Protein Surface Recognition by Synthetic Molecules

317

Figure 38 Side view of superimposed structures of ESp (semitransparent (gray) and bSS12 (orange) in ribbon representation bound to 14-3-3z (gray surface). Reprinted with permission from Rossolini, G. M.; Mantengoli, E. Clin. Microbiol. Infect. 2005, 11, 17–32. Copyright 2014, Wiley.

peptides f-aSS8 and f-bRS8 (“f” means fluorescein-labeled) exhibited around 20-fold weaker binding to 14-3-3z (Kd z 20 mM), while a 4.6-fold increased affinity was observed for f-gRS8 (Kd ¼ 0.25 mM) compared to the starting sequence f-ESp (Kd ¼ 1.14 mM). The 14-33z and bRS8 cocrystal structures revealed that the N-terminal part of the backbone in bRS8 is rearranged compared to ESp, resulting in the loss of direct as well as water-mediated polar interactions. Furthermore, a disposition of the crucial leucine residue L423 is detected and leads to a complete absence of hydrophobic interactions between L423 of bRS8 and 14-3-3z. Next, they considered a variation of the linker length and the absolute configuration of both nonnatural amino acids. Peptide f-bSS12 (C12-linker) is the strongest 14-3-3z binder with (Kd ¼ 41 nM), exhibiting a sixfold higher affinity than f-bRS8 and a 28-fold higher affinity than f-ESp. They again produced a cocrystal structure of f-bSS12 with 14-3-3z. It revealed that the arrangement of the backbone in bSS12 is parallel to the unmodified peptide ESp resulted in an almost indistinguishable interaction of 14-3-3z, with leucine L423 in bSS12 (Fig. 38).60

4.09.4.1.4

Rab-GTPase protein–protein interaction

Spiegel et al. reported the inhibition of Rab-PPI, a subfamily of GTPases using a-helical peptides that were stabilized by means of hydrocarbon-peptide stapling. The peptides were derived from relevant a-helical motifs of Rab-interacting proteins (Fig. 39), which were identified by the analysis of known crystal structures of Rab-protein binder cocomplexes. Small GTPases are involved in a variety of key cellular processes including cell growth, survival, and membrane trafficking.61 Abnormal activation of small GTPases can result in numerous human diseases such as neurodegenerative diseases and various forms of cancer.62 After an initial screening, the four wild-type peptides (wtR6IP, wtLidA2, wtRabin8, and wtREP) were chosen for stapling. Peptides with good binding affinity are shown in Fig. 40. For Rab8a (NF) and Rab11a (NF) several ligands showed dissociation constants in the submicromolar range, with StRIP110/Rab11 [Kd ¼ (0.40  0.02) mM] and StREP109/Rab8a [Kd ¼ (0.42  0.03) mM] exhibiting the highest affinity. Competition FP experiments were performed with Rab8 effector-binding protein oculocerebrorenal syndrome of Lowe (Lowe syndrome) (OCRL1, Kd 1.7 mM) to investigate the ability of StRIP110 to disrupt this interaction. In this assay, concentration-dependent dislocation of labeled OCRL1539–901 by acetylated StRIP110 was observed (IC50 490  65 mM). Control peptide wtR6IP did not interfere with OCRL1 binding.63

4.09.4.2 4.09.4.2.1

Hydrogen Bond Surrogate (HBS) Peptides as a-Helix Mimetics (HIF1a)/p300 protein–protein interaction

Kushal et al. employed a hydrogen bond surrogate (HBS) approach64 to stabilize a-peptides. This relied on the replacement of intramolecular hydrogen bonds stabilizing the a-helices by carbon–carbon bonds (Fig. 41). The constrained peptides HBS 119 and HBS 120 showed characteristic a-helical CD spectra in aqueous buffers. In contrast, their unconstrained congener 121 showed no discernible helicity, as one would expect for such a very short peptide. HBS 119 mimics the aB domain of HIF-1a, and four residues significantly contribute to binding (L818, L822, D823, and Q824). HBS 120 is identical to 119 with the exception of L822, which

Figure 39

Design of stapled peptides derived from Rab6-interacting protein 1.

318

Protein Surface Recognition by Synthetic Molecules

Figure 40

Selected stapled peptides (* indicates the amino acid residue positions, choose for the stapling) and dissociation constants values.

was mutated to an alanine group. The HBS helices mimic protein subdomains of the p300/CBP coactivator and disrupt the HIF-1a C-TAD-p300/CBP PPI with high affinity and specificity. A tryptophan fluorescence spectroscopy assay was used to evaluate the binding affinity of all peptides for the 15N-labeled p300 CH1 domain. HBS 119 bound to p300-CH1 with a Kd of 690  25 nM, while HIF-1aC-TAD786-826 bound p300-CH1 with a Kd of 38  0.14 nM. HBS 120, which was synthesized as a specificity control, had a fourfold weaker binding affinity (Kd ¼ 2820  140 nM). Peptide 121, the unconstrained analog of HBS 119, bound to the CH1 domain with a Kd of 6060  320 nM. Thus the stabilization of the peptide conformation offers a ninefold increase in binding affinity. In a FP assay, HBS 119 showed a concentration-dependent inhibitory constant with Ki ¼ 3.5  1.2 mM. HBS 120 or peptide 121 did not lead to reproducible inhibition of the complex. The compound HBS 119 showed a downregulation of hypoxia-induced transcription of target genes in a luciferase-based reporter assay and significantly suppressed tumor growth in the murine xenograft models of renal cell carcinoma of the clear cell type.65 Henchey et al. reported a structure-based design mimicking the a-helical conformation of the 799DCEVNA804 sequence, which is known to be the critical component for the interaction between the CBP/p300 CH1 domain and HIF-1a. Mutagenesis data revealed that Cys-800 and Asn-803 play pivotal roles in HIF-1a and p300/CBP complex formation and thus signal transduction in hypoxia.66 Three constrained peptides HBS 122, HBS 123, HBS 124 and an unconstrained analogue 125 were synthesized. Key residues of HBS 122 were placed outside of the HBS macrocycle. An extra arginine at the C-terminus of HBS 123 was incorporated to enhance uptake in certain cell lines. HBS helix 124 was prepared as a negative control in which the key Cys-800 residue is mutated to an arginine. The N-terminal residues Ser-797 and Tyr-798 are replaced with alanine residues as they are not directly involved in the interaction with the coactivator surface. HBS 122 did not show measurable inhibition of transcription in the cell-based assay, which indicates poor cellular uptake of 122.67 The binding affinity of the helix mimetics toward GST-p300 and the ability of these compounds to downregulate HIF-1a-induced transcription of the VEGF gene in HeLa cells (under hypoxic conditions) are shown in Table 7.

Figure 41

Structures of stabilized helices (HBS 119 and HBS 120) and linear peptide 121. Peptide 121 is an unconstrained negative control.

Protein Surface Recognition by Synthetic Molecules

Table 7

Summary of key biophysical and in vitro data for peptides designed to target HIF1a-p300 interactions Helicity (%) b

Kd (nM) c

HBS 122

40

950  90

03

HBS 123

53

420  35

45  8

HBS 124

51

>2200

15 –

825  50 120  25

Compound

HBS 125 Chetomin

319

Sequence a

–

Transcription inhibition d

27 83 50  5

a

X denotes pentenoic acid residue in the HBS macrocycle. Helicity determined from circular dichroism spectroscopy studies. From ITC analysis. d % Inhibition of VEGF gene measured by real-time qRT-PCR assays in HeLa cells with 1 mM peptide or 200 nM chetomin. b c

4.09.4.3 4.09.4.3.1

Light-Regulated Stapled Peptides as a-Helix Mimetics Clathrin protein surface

Nevola et al. took the constraining concept a step further and generated photoswitchable inhibitors (Fig. 42) that target the AP2 complex based on a helical interaction motif, thereby inhibiting clathrin-mediated endocytosis (CME) in living cells. By introducing a photoswitchable azobenzene moiety into the tether they were able to regulate the secondary structure of the peptide and thus its affinity for AP2 (Table 8). CME is a key process in all eukaryotic cells, which controls surface expression of proteins, the uptake of nutrients, cell signaling, and the turnover of membrane components.68 The compounds were derived from the b-arrestin C-terminal peptide sequence that binds to the b-appendage of AP2 (b-adaptin).69 In its a-helical structure four highly conserved residues

Figure 42 (A) Schematic representation of the photoswitchable peptide inhibitors when the azobenzene cross-linker is conjugated at positions i and i þ 11, the binding would be favored in trans configuration; (i þ 4), (i þ 7) positions would be favored for cis configuration. Yellow cysteine residues ( ) were selected for cross-linking. (B) Representation of the AP2 b-appendage (gray) and key interacting residues in red (modified from,13 PDB ID 2V8). Reprinted with permission from Burns, D. C.; Zhang, F.; Woolley, G. A. Nat. Protoc. 2007, 2, 251–258. Copyright 2013, Wiley.

320

Protein Surface Recognition by Synthetic Molecules

Table 8 Sequences and binding constants of unmodified (*) peptides and designed traffic light (TL) peptides.The binding affinity (Ki) values of TL peptides were obtained from a FP competition assay. @ denotes a-aminoisobutyric acid Peptide

Sequence

BAP long* BAP short* ARH* TL-126 TL-127 TL-128 TL-129

Design

Kd (mM)

Ki (mM) (

– – –

2.1 NB 4.7 – – – –

– – – 81 240  150 NB NB

)

Ki (mM) (

)

– – – 31  3 19  0.3 NB NB

(D, F, F, and R) are aligned along one side of the helix that interacts with the binding pocket. To achieve photosensitivity a photoisomerizable crosslinker 3,30 -bis(sulfonato)-4,40 -bis (chloroacetamido)azobenzene (BSBCA)70 was incorporated in-between the pairs of cysteines in the BAP-long sequence (yellow residues). By switching between the cis and trans isomers, using wavelengths of 380 and 500 nm, respectively, a change in the stability of the helix conformation was achieved. A FP competition assay determined the binding affinity of wild-type peptides and photoswitchable inhibitors in each state (i.e., after a 3 min exposure to 380 and 500 nm light). TL-126 bound satisfactorily in the trans state and lost binding strength in response to irradiation at 380 nm (cis state). In contrast TL-127 bound weakly in the trans and more tightly in the cis state. To assess the capacity of the TL peptides to inhibit CME intracellularly, transferrin receptor uptake was studied in living cells using confocal microscopy. In the absence of CME inhibitors, cells internalized fluorescent transferrin, whereas preincubation of cells with TL-126 accumulated transferrin on the membrane, indicating CME inhibition.71

4.09.4.4 4.09.4.4.1

Lactam Bridges Containing Peptides as a-Helix Mimetics gp41 protein surface

Sia et al. synthesized constrained peptides (Fig. 43) that contained 14-residues derived from the C-terminal heptad repeat of HIV-1 gp41 (C-peptides). These compounds target the hydrophobic pocket region of HIV-1 gp41 and inhibit HIV fusion. As the C-peptides bind to the gp41 N-region only in a-helical conformation, the short unmodified sequences exhibited only low binding affinity for gp41 and thus poor inhibitory activity. To increase helicity chemical cross-linking as well as substitution with unnatural helix-favoring amino acids was employed. The inhibitory activities of the constrained peptides are given in Table 9. A constrained peptide (C14linkmid) inhibited cell–cell fusion at micromolar concentration, while the short linear

Figure 43 List of peptides used in the study: C14wt (wild-type); C14Aib (Asn-636 and Met-629 mutated to Aib); C14linkmid (the middle of the peptide at positions 629 and 636 are cross-linked); C14unlinkmid (N-3-propylglutamine at positions 629 and 636); C14linkN (cross-linked at the N terminus at positions 626 and 633); and C14linkNAib (cross-linked at the N terminus and Aib substitutions in the middle of the peptide). “B” denotes Aib residue, “am” and “suc” denotes amide, succinimide, respectively.

Protein Surface Recognition by Synthetic Molecules

Table 9

321

Inhibition of cell–cell fusion by C14 peptides

Peptide

IC50 for cell–cell fusion (mM) a

C14linkmid C14Aib C14wt C14unlinkmid C14linkN C14linkNAib

35 144 >500 >500 No activity No activity

a

C14wt and C14unlinkmid display very weak inhibition.

peptide showed no significant inhibitory activity. X-Ray crystallography studies confirmed that the constrained peptides bound to the gp41 hydrophobic pocket.72

4.09.4.4.2

CSP-1, HIV-1 Rev protein, and nociception protein surface

Harrison et al. demonstrated that even short pentapeptides, Ac-(1,5-cyclo)-[KXRXD]-NH2 featuring an amide linkage between the side chains of lysine and aspartate, were a-helical73 in water (as shown by CD and NMR spectroscopy). This general strategy of stabilizing helicity by connecting lysine and aspartate residues, which are separated by three amino acids, should thus be more widely applicable. To assess the general utility of this approach they chose four a-helices with diverse functions and quite different receptors: (i) viral (RSV Fusion protein),74 (ii) bacterial competence stimulating peptide (CSP-1),75 (iii) RNA binding human protein (HIV-1) Rev protein,76 and (iv) ORL-1-binding nociceptin protein. They evaluated the biological activity and compared short peptide sequences in both unconstrained (130, 132, 134, and 136) and a-helix-constrained (131, 133, 135, and 137) forms (Fig. 44). The CD and NMR results revealed that the constrained analogues (131, 133, 135, and 137) had a significantly higher helicity compared to the unconstrained peptides (130, 132, 134, and 136). As expected these helix-constrained compounds showed improved biological potencies compared to their linear counterparts. The helix-constrained peptide 131 inhibited recombinant RSV F-mediated fusion in a cell-to-cell assay at picomolar concentrations (IC50190 pM, pIC50  9.72  0.03), whereas unconstrained 130 showed no inhibition up to a concentration < 1 mM. The analogue 133 had a > 10-fold enhanced antibacterial activity compared to its unconstrained congener 132. Constrained peptide-mimetic 135 showed submicromolar binding affinity (pIC50  6.65  0.04), to the stem IIB region of the Rev responsive element of RNA, whereas unconstrained analogue (134) had submicromolar-affinity (pIC50  6.05  0.07), an advantage in free energy of 3.4 kJ mol 1 K 1. The nociceptin analogue 137 was a ninefold more active ORL-1 agonist (EC50 40 pM, pEC50  10.39  0.14) than the unconstrained variant 136 (360 pM, pEC50  9.43  0.17) as evaluated by ERK phosphorylation in mouse neuroblastoma cells Neuro-2a. This work represents a blueprint for the creation of a-helical protein segments as short as five amino acids by constraining lysine and aspartate residues, which are separated by three amino acids.77

Figure 44

List of peptides both in native and constrained forms. Lactam bridges were used to induce constrained in the peptide backbone.

322

Protein Surface Recognition by Synthetic Molecules

4.09.4.5 4.09.4.5.1

Disulfide Bridges Containing Peptides as a-Helix Mimetics Nuclear receptor-coactivator protein surface

Leduc et al. synthesized short helix-stabilized cyclic peptides, containing a copy of the LXXLL nuclear receptor box pentapeptide, introducing cysteine disulfide-bridges at i, (i þ 3) positions (Fig. 45). These compounds bind tightly to the receptor and selectively inhibit the steroid receptor–coactivator interactions. The steroid receptors (ERa & ERb), which are members of the nuclear receptor (NR) superfamily, are ligand-activated transcription factors that regulate a wide variety of physiological and developmental processes.78 Agents that inhibit the interactions between the receptor and coactivators have recently been proposed as attractive targets for new anticancer drugs. The disulfide-bridged compound 138 (PERM-1) showed the greatest helical character in CD experiments and also displayed a higher binding affinity for ERa (Ki ¼ 0.025 nM) than for ERb (Ki ¼ 0.390 nM), showing a selectivity z15.6 times higher for ERa over ERb. To investigate the mode of peptide binding to the receptor, X-ray crystal structures of the LBD of ERa, together with both, the native ligand, estradiol, as well as PERM-1 were produced. The cyclic peptide 138 bound to the expected hydrophobic groove created by helices 3, 4, 5, and 12. The three hydrophobic leucine residues as well as the additional isoleucine residue at position  1 made close contact with the receptor. The two XX residues (here, Cys-Arg) are pointing away from the groove as expected for an amphiphilic helix.79

4.09.4.5.2

Androgen receptor-cofactor protein surface

Vaz et al. synthesized a diverse set of natural miniprotein mimetics based on in silico modeling. These compounds mimic an a-helical domain of the androgen receptor (AR) cofactor and were found to be potent AR80 binders. Until now, the AR has attracted only little attention, although it is an attractive target for potentially treating prostate cancer and other human diseases. A structural analysis of the AR–cofactor interaction shows that a short helix segment (FXXLF motif) binds to the AR at the activation function 2 (AF-2). The helical structures of mini-protein scaffolds Apamin,81 k-hefutoxin1,82 CD4M3 (a scyllatoxin analogue),83 and Om-toxin384 were stabilized by means of 2 or 3 disulfide bridges. The AR-specific FXXLF motif was introduced in the miniprotein helix at positions that did not affect the conformationally crucial disulfide bridges. The eight new miniproteins were synthesized based on in silico calculations. AR-binding affinities were determined by a FP assay and are summarized in Table 10. All the natural toxins (Apa-141, Scy-143, Het-145, Omt-149) showed no binding affinity to the AR-LBD, indicating that the intrinsic helical character is not enough for sufficient binding. The aspartic (D) to arginine (R) point mutation of the first helix of the miniprotein

Figure 45 List of cyclic peptides: inhibition of estrogen receptor/coactivator recognition was determined by a time-resolved fluorescence-based coactivator interaction assay.

Table 10

Miniproteins, sequences, and AR binding affinities

Name

Sequences a

Ki (mM)

Apa-141 Apa-142 Scy-143 Scy-144 Het-145 Het-146 Het-147 Het-148 Omt-149 Omt-150 Omt-151 Omt-152

CNCKAPETALCARRCQQH CNCKAPETAFCALFCQQH CNLARCQLSCKSLGLKGGCQGSFCTCG CNLAFCQLFCKSLGLKGGCQGSFCTCG GHACYRNCWREGNDEETCKERC GHACYFNCLFEGNDEETCKERC ACYFNCLFEGNDEETCKERC GHACYRNCWREGNDRFTCLFRC NDPCEEVCIQHTGDVKACEEACQ NRFCELFCIQGTGDVKACEEACQ CRFVCLFHTGDVKACEEACQ NDPCEEVCIQHTGDVFACLFACQ

n.a. 4.9  1.0 n.a. 160 n.a. 17.9  2.7 1.6  0.3 10.2  1.4 n.a. 1.3  0.2 0.5  0.2 40.3  7.4

a The italics residues represent helical segments, disulfide bridged cysteines are underlined, and inserted mutations are shown in bold. n.a. ¼ not active.

Protein Surface Recognition by Synthetic Molecules

323

(Omt-150) resulted in a remarkable binding affinity of 1.3 mM. In miniprotein Omt-151, mutation at position 2 (E to R) delivered the best AR binder with a Ki of 0.5 mM.85

4.09.4.6 4.09.4.6.1

b-Hairpin Scaffold Peptides as a-Helical Mimetics p53/hDM2 protein surface

Fasan et al. designed a b-hairpin peptide 153 (Fig. 46) to mimic the side chains of Phe19 and Trp23 (and possibly also Leu26) of an a-helical segment of the tumor-suppressor p53. This protein can interact simultaneously with the p53 binding site on HDM2 and inhibit the PPI. In cyclic mimetics, D-Pro-L-Pro can function as a template for stabilizing b-hairpin loop conformations (Fig. 47).86 The affinity of 153 for HDM2 was weak (IC50 ¼ 125 mM) as determined by a BIAcore (Biacore AB) solution-phase competition assay. Ala-scan revealed that the expected side chains of not only Phe1, Trp3, and Leu4 but also Lys6 make important energetic contributions to HDM2 binding. Taking into account these two effects, a series of cyclic peptides were synthesized. Point mutation of tryptophan at position 3 to 6-chlorotryptophan increased the affinity for HDM2 by a factor of almost 900, compared to the initial lead 153 and an improvement over the affinity of the linear p53 analogue by almost eightfold. The mimetics interact with HDM2 at the p53 binding site, which was evidenced by NMR spectroscopy.87

4.09.5

Flexible Peptides

While the previous examples show that the introduction of constraining tethers is a good method for the fixation of active conformations and thus a valuable tool for enhancing binding capacities, this approach does not always lead to increased affinities. Sometimes a certain flexibility is necessary for binding as the following example illustrates.

Figure 46

Structure of b-hairpin peptide mimetic 153.

Figure 47 A model b-hairpin (yellow) superimposed on the p53 helical peptide (red). Reprinted with permission from (a) Favre, M.; Moehle, K.; Jiang, L.; Pfeiffer, B.; Robinson, J. A. J. Am. Chem. Soc. 1999, 121, 2679–2685; (b) Shankaramma, S. C.; Moehle, K.; James, S.; Vrijbloed, J. W.; Obrecht, D.; Robinson, J. A. Chem. Commun. 2003, 1842–1843. Copyright 2004, Wiley.

324

Protein Surface Recognition by Synthetic Molecules

4.09.5.1

Protein Phosphatase-1 (PP1)–Interacting Proteins (PIP) Protein Surface

Chatterjee et al. developed proteolytically stable, cell-permeable peptides that specifically disrupt the interaction between protein phosphatase-1 (PP1) and interacting proteins (PIP), thus modulating PP1 signaling in living cells. PP1 is a ubiquitous enzyme that catalyzes the majority of Ser/Thr dephosphorylations in eukaryotic cells.88 Malfunction of these processes is associated with the development and progress of several human diseases. About 90% of all validated PIPs employ the RVxF type PP1-binding motif (single-letter amino acid code, x ¼ any amino acid). RVxF binds to a site on PP1 that is remote from the active site.89 Only, the RVxF motif containing synthetic peptides disrupt a subset of PIP–PP1 complexes. The efficacy (EC50) of the peptides was determined by an in vitro phosphatase assay, measuring the activity of PP1 toward its 32P-labeled substrate glycogen phosphorylase a (Table 11). Cell-permeability of these peptides was enhanced by the sequential addition of arginine/lysine residues to the N-terminus, without significantly compromising its efficacy as a PP1–PIP-disrupting peptide. The crystal structure of the PP1:PDP156 complex showed that PDP156 bound in the same groove as PP1, similar to the other structurally characterized PIPs containing the RVxF motif.90 PDP157 enhanced histone H3 dephosphorylation during mitosis, indicating that it activates PP1 in living cells.91 However, introducing a constraint in the peptide backbone by cyclization resulted in the loss of inhibition potency, which suggests that a certain conformational flexibility of the RVxF motif and its flanking sequences is required for binding to PP1.

4.09.6

Protein Surface Recognition Using Small Molecules

Design and synthesis of small-molecule inhibitors to disrupt PPIs remains an attractive target from a drug-development perspective. Small molecules that do not necessarily mimic the original helix structure still manage to bind to the protein surface and inhibit PPIs. Furthermore, applying classical medicinal chemistry principles, modifications of existing bioactive compounds lead to enhanced drug-like properties and well-established medicinal chemistry principles can be applied here.

4.09.6.1

MDM2-p53 Protein–Protein Interaction

Vassilev et al. reported the development of potent and selective small-molecule inhibitors of the MDM2/p53 interaction with in vitro and in vivo antitumor activity. A series of cis-imidazoline analogs named Nutlins were identified and optimized for potency and selectivity (Fig. 48). These compounds displaced the p53 protein from its MDM2/p53 complex with inhibitory concentration (IC50) values in the 100–300 nM range. Nutlin-160, arbitrarily called enantiomer-a, showed a strong binding activity (IC50 ¼ 90 nM), whereas its enantiomer was 150 times less active. The cocrystal structure of the human MDM2-Nutlin-159 complex revealed that the inhibitor binds to the p53-binding site on MDM2. With its arrangement of functional groups, this cis-imidazoline inhibitor mimics the helical portion of the p53 peptide, with one bromophenyl moiety sitting deeply in the Trp pocket, whereas the

Table 11

Efficacy of the peptides in disrupting the PP1:I2 complex

Peptide

Sequence

EC50 (nM)

PDP154 PDP155 PDP156 PDP156m PDP157 PDP157m

RPKRKRKNSRVTFSEDDEII RPKRKRKNARVTFAEAAEII RRKRPKRKRKNARVTFAEAAEII RRKRPKRKRKNARATAAEAAEII RRKRPKRKRKNARVTFBpaEAAEII RRKRPKRKRKNARATABpaEAAEII

87  10 21  2 53  8 Inactive 176  13 Inactive

The RVxF motif is underlined for each peptide sequence. m stands for RATA mutant of PDP156.

Figure 48

Structure of MDM2 inhibitors.

Protein Surface Recognition by Synthetic Molecules

Figure 49

325

Structure of antagonists for the HDM2-p53 interaction.

other bromophenyl group inhabits the Leu pocket. The ethyl side chain is fixed in the Phe pocket. The Nutlin-158 upregulates p53 in a dose-dependent manner in all cancer cell lines, leading to cell cycle arrest and apoptosis. Treatment of mice bearing tumors of 100–300 mm3 with Nutlin-160 resulted in 90% inhibition of tumor growth relative to vehicle controls.92 Grasberger et al. screened more than 338,000 compounds for HDM2 binding using a ThermoFluor affinity-based assay. This resulted in the discovery of a novel series of benzodiazepinedione antagonists for the HDM2-p53 interaction (Fig. 49). A 2.7 Å crystal structure of 161 bound to HDM293 revealed that the antagonist 161 buries roughly 25% less exposed surface area in the p53-binding pocket (804 Å2). This is the first example of a benzodiazepinedione acting as an a-helix mimetic. The optimized molecule showed high binding affinity (IC50 ¼ 420 nM) and upregulated the transcription of p53 target genes and decreased proliferation of tumor cells expressing wild-type p53.94 Rew et al. described the discovery of novel inhibitors of the MDM2/p53 PPI. A structure-based design approach led to the identification of the most potent inhibitor 163 (Fig. 50). Its dissociation constant (Kd ¼ 0.4 nM) was determined in a SPR spectroscopy binding assay. The binding affinity of this compound for MDM2 was achieved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. The ability of compound 163 to inhibit tumor growth was also evaluated in the SJSA-1 mouse xenograft model. Tumor growth inhibition in a dose-dependent way was observed for 163. It significantly reduced tumor growth at 150 and 200 mg kg 1 q.d. compared to the control vehicle (ED50 of 78 mg kg 1). Importantly, 200 mg kg 1 q.d. of 163 caused tumor regression (R ¼ 27%). No body weight loss was observed in any of the treatment groups. Compound 163 had a relatively high clearance (CL ¼ 3.5 L h 1 kg 1) and short half-life time (t1/2 ¼ 2.6 h) in mice.95 Shangary et al. reported a new spiro-oxindole based class of inhibitors for the MDM2/p53 interaction. Their structure-based approach was based on the crystal structure of the MDM2/p53 complex. MI-63 bound to MDM2 with high affinity Ki ¼ (3  1) nM. It activates the p53 pathway and selectively prevents cell growth in cancer cell lines with wild-type p53. However, MI-63 had a poor pharmacokinetic (PK) profile and is unsuitable for in vivo evaluation. Extensive modifications of MI-63 generated MI-219 as a potent and selective MDM2 inhibitor with a desirable PK profile (Fig. 51). MI-219 bound to human MDM2 with a Ki value of 5 nM and is thus > 10,000-fold more potent than p53. Computational modeling projected that MI-219 mimics all the key binding residues in p53 (Phe-19, Leu-22, Trp-23, and Leu-26). It is also 10,000 times less potent in binding to MDMX than to MDM2. It disrupts the MDM2/p53 interaction and dose-dependently induced p53 accumulation. It also upregulates the p53 pathway in cells with wild-type p53, which leads to cell cycle arrest and potently reduced cell growth in the SJSA-1, LNCaP, and 22Rv1 cancer cells, but importantly not in normal cells. IC50 values range from 0.4 to 0.8 mM. MI-219 stimulates rapid but transient in vivo activation of p53, which was proven in mouse xenograft models of human cancer. A single oral dose of MI-219 inhibited cell proliferation and induced apoptosis in tumor tissues in a time-dependent manner and achieved maximum effects at 24 h. MI-219 is not toxic to animals and activates p53 in normal tissues with only minimal p53 accumulation.96

Figure 50

Structure of the most potent inhibitor.

326

Protein Surface Recognition by Synthetic Molecules

Figure 51

4.09.6.2

Chemical structures of MDM2 inhibitors.

Bcl-xL/Bcl-2 Protein–Protein Interaction

Wendt et al. described a potent class of biarylacylsulfonamide antagonists (164a) for the antiapoptotic protein Bcl-xL, binding with a Ki of 0.8 nM. In a cellular assay, the inhibitor 164a effectively negated the survival advantage afforded by Bcl-xL overexpression against cytokine withdrawal in FL5.12 cells with an EC50 of 0.47 mM. It thus enhanced the cytotoxic activity of multiple cytotoxic agents and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model. However, 164a showed considerably lower affinity for Bcl-2 (Fig. 52).97 A dual subnanomolar inhibitor for both Bcl-xL and Bcl-2 was discovered by Bruncko et al. who used a structure-guided design that exploited a deep hydrophobic binding pocket on the surface of these proteins. This study led to the identification of 165 (Fig. 52), which exhibited EC50 values of 8 and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Here replacement of the piperidine ring of 164a with a piperazine unit, connected to a chloro-diphenyl moiety (165), enhanced Bcl-2 affinity significantly while maintaining Bcl-xL affinity. Compound 165 showed a > 20-fold (Bcl-xL) and > 250-fold (Bcl-2) improvement in cellular efficacy compared to starting compound 164a. This strongly improved cellular activity, however, may be due to decreased interaction with serum components rather than increased affinity for Bcl-2 proteins. Compound 165 showed great efficacy (EC50 values < 1 mM) against three human tumor cell lines, DoHH2, SUDHL-4, and RS11380, that express high levels of Bcl-2, whereas 164a shows no cellular activity up to 30 mM. Compound 165 reduced tumor growth by 60–65% to an average size of 225 mm3 during therapy prior to tumor rebound and even reduced tumor growth by 90% in combination with etoposide.98 Zhou et al. described another class of potent small-molecule inhibitors of the antiapoptotic proteins Bcl-2 and Bcl-xL. The crystal structure revealed that an initial lead compound (166) containing a 3,4-diphenyl-1H-pyrrole-2-carboxamide scaffold and the p-chlorobiphenyl fragment of Abbott’s drug ABT-737 both occupy site 1 in the Bcl-xL protein. The fragment 167 occupies site 2 in the Bcl-xL protein. Therefore, they reasoned that connecting compounds 166 and 167 with a suitable linker could afford new compounds with high affinity for Bcl-xL. This strategys lead to conjugate 168, in which the linker length is 10.6 Å. It was found to have a Ki of 2.0 nM for Bcl-2 and Ki < 1 nM for Bcl-xL and is thus > 10,000 times more potent than either 166 or 167. The best inhibitor (169) which is an analogue of 168 (Fig. 53) bound to Bcl-xL and Bcl-2 with Ki < 1 nM. It improved cell-growth inhibition in H146 and H1417 cancer cell lines, with IC50 values of 61 and 90 nM, respectively. Compound 169 induced substantial cell death in a dose-dependent manner in H146 cells at 30–100 nM and led to more than 70% cell death after 24 h at 300 nM.99

Figure 52

Structures of Bcl-2 family protein inhibitors.

Protein Surface Recognition by Synthetic Molecules

Figure 53

327

Structure-based design of inhibitor 168. Compound 169 is the analogue of 168.

Gräber et al. presupposed that some small molecule drugs already approved for human use against other targets can inhibit Bcl-xL/Bak PPIs in their therapeutic concentration range. They identified oral disinfectants chlorhexidine (170)100 and alexidine (171)101 (Fig. 54) as potentially selective inhibitors of the Bcl-xL/Bad interaction, after screening a library of 4088 compounds containing a high proportion of clinically used small molecules. Based on FP-binding assays, the affinity of 170 and 171 against the Bcl-xL/Bak interaction was determined to be in the low micromolar range with IC50 ¼ (21.0  1.0) mM and (18.2  1.6) mM, respectively. These activities are well in the used concentration range of the oral disinfectant chlorhexidine (170), alexidine (171), respectively. Amino acids with strong chemical shift perturbations included Phe146, Val126, Glu129, Leu108, Asn136, and Gly94, suggesting that the small molecules bound to the hydrophobic BH3 binding pocket of Bcl-xL, which was confirmed by 1H,15 N-HSQC NMR experiments. As visualized by a TUNEL assay, both 170 and 171 induced DNA-strand breaks, characteristic of apoptosis

Figure 54

Chemical structure of chlorhexidine and alexidine that inhibit protein–protein interactions mediated by Bcl-xL.

328

Protein Surface Recognition by Synthetic Molecules

Figure 55

Chemical structure of the inhibitor 172 and negative control 173.

in three tongue squamous cell carcinoma lines (SCC-4, SCC-9, and SCC-15) and two pharynx carcinoma cell lines (FaDu and Detroit 562). Both compounds led to a strong reduction of the cationic dye tetramethylrhodamine ethyl ester (TMRE) uptake, consistent with depolarization of the mitochondrial membrane leading to the subsequent induction of apoptosis by these agents102.

4.09.6.3

Interleukin-2 (IL-2)/IL-2Ra Protein Surface

Based on structural information obtained by X-ray crystallographic103 and NMR studies104, Tilley et al. developed a small molecule inhibitor (172) of the IL-2/IL-2Ra receptor interaction, which mimics the R38-F42 region of Interleukin-2 (IL-2). Compound 172 was found to be a competitive inhibitor of IL-2/IL-2Ra binding with a mid-micromolar IC50. IL-2 is a cytokine that stimulates T-cell proliferation by binding on the T-cell surface with picomolar affinity.105 Antibodies that recognize the a receptor subunit (IL-2Ra) and prevent IL-2 binding have proven clinically effective as immunosuppressive agents. 15N-labeled NMR studies of IL-2 revealed that the resonances of residues in the vicinity of the binding epitope were altered by 172 in a concentration-dependent manner, but not by its inactive enantiomer 173 (Fig. 55). The results indicate that 172 disrupts the IL-2/IL-2Ra interaction by binding IL-2 with an IC50 of 3 mM at pH 7.4. This is the first well-characterized example of a small molecule, nonpeptide inhibitor of a cytokine/cytokine receptor interaction.106 Erlanson et al. developed a tethering strategy,107 which allows for the rapid identification of small, low-affinity fragments. By connecting these fragments to a disulfide moiety, small libraries can be swiftly generated. These libraries are then allowed to interact with peptides containing native or artificially introduced cysteine residues near a proposed binding site. In the presence of a reducing agent like 2-mercaptoethanol, the disulfide moieties are reversibly cleaved. This leads to an equilibrium between the cysteine containing target protein and the library members in which even weak interactions lead to the accumulation of cysteinecaptured ligands, which can be easily identified by MS (Fig. 56). This way, small target affine fragments can be identified. These can then be added to a lead compound to enhance its binding affinity. Braisted et al. demonstrated the potential of this approach by improving the affinity of the lead compound (174, black scaffold) which had hit a low mM affinity plateau in a traditional medicinal chemistry screening. The aim was binding to specific sites on the cytokine interleukin-2, in order to inhibit the IL-2/IL-2Ra receptor interaction. Molecular modeling suggested that the covalent attachment of fragments could occupy a deep hydrophobic cavity within the adaptive region. Table 12 shows conjugates of 174 with hits from a tethering screening. Eight of the 20 compounds containing a carboxylic acid inhibited IL-2/IL-2RR binding at submicromolar concentrations, demonstrating a 5- to 50-fold improvement in potency over the lead compound. Thus, the acidic functionality recognized by tethering was clearly essential for the improved binding. For example, the benzoic acid derivative 177 displayed an IC50 of 0.20 mM, representing a 15-fold improvement over 174, whereas control compounds 179 and 181 were at least 10-fold less potent than 175 and 200-fold less potent than 177. Compound 183 was the most potent compound with an IC50 of only 60 nM.108

Figure 56

Schematic representation of the cysteine-fragment tethering strategy.

329

Protein Surface Recognition by Synthetic Molecules

Table 12

Selected examples from the 20 member library prepared to merge tethering hits onto compound 174

No.

R

175

H

176 177

IC50 (mM)

R

IC50 (mM)

3.0

180

0.60

35.0

181

0.30

182

0.40

183

0.060

0.20

178

40.0

179

1.8

4.09.6.4

No.

The von Hippel–Lindau Protein (VHL)/HIF-1a Protein Surface

Buckley et al. described the rational design and synthesis of the first small molecule targeting the von Hippel–Lindau protein (VHL), the substrate recognition subunit of an E3 ligase disrupting the VHL/HIF-1a interaction.109 The primary substrate of VHL is HIF-1a. This key transcription factor upregulates numerous genes under hypoxia (not enough O2) such as the proangiogenic growth factor, VEGF, glucose transporter, and the red blood cell inducing cytokine, erythropoietin.110 In contrast, under normoxia (enough O2) HIF-1a undergoes VHL-mediated ubiquitination.111 Small molecules that disrupt the VHL/HIF-1a interaction under normoxia would lead to increased endogenous erythropoietin production. According to their hypothesis, rationally designed hydroxyproline (Hyp) bearing small molecules could inhibit the VHL/HIF-1a interaction, since residue Hyp564 on HIF-1a makes key interactions with VHL19, which is also crucial for HIF-1a binding. Initially, the de novo design software BOMB was used to guide the selection of plausible hydroxyproline analogues.112 Compounds 184 and 185 were synthesized (Fig. 57), and their ability to bind to VHL was measured: IC50 ¼ 117 and 120.1 mM, respectively (the assay monitored competition of a fluorescent HIF-1a peptide and FAM-DEALAHyp-YIPD whose Kd value is 560 nM).113 Having these promising results, a series of compounds was synthesized in order to enhance the affinity to VHL. The benzylamine moiety of 184 was modified while maintaining the methyl-isoxazole fragment. The library of compounds and their respective binding affinities are shown in Table 13.

Various benzylamines halogenated at the para position showed improved affinity, and there was only a slight difference of affinity between chloride and bromide, while the fluoride congener was significantly less potent. Substitution with electron

Figure 57

Synthesis of initial ligands for VHL, with IC50 values.

330

Protein Surface Recognition by Synthetic Molecules

Table 13

Binding of different ligands to VHL

No.

R

IC50 (mM)

SEM (mM)

184 186 187 188 189 190 191 192 193 194 195 196 197

3-Cl 4H 2-Cl 4-Cl 4-F 4-Br 4-tBu 4-OMe 4-CO2Me 4-NO2 4-CN 4-COMe 4-(5-oxazoyl)

117 130 149 20.5 149 32.0 >250 106 39.4 16.0 60.3 22.6 4.1

10 10 13 1.9 13 3.6 N/A 13 2.2 0.6 5.3 2.0 0.4

withdrawing groups ( CO2Me,  NO2,  CN, and ketones) delivered more potent ligands than substitution with electrondonating groups ( OMe and tBu). The cocrystal structure of VHL bound to 197 established that it bound to VHL on the same site as HIF-1a did.114

4.09.7

Protein Surface Recognition With Dendrimers

As discussed in the previous chapter, small molecules can be tailored to bind specifically to certain parts of the protein surface e.g., by holding on to clefts and ridges. But sometimes bigger areas, with a less versatile topography, need to be addressed. Therefore larger scaffolds are often necessary that can present multiple functional group interactions or cover large areas of the protein surface. That way they can cover active sites and thus prevent the protein’s function. Dendrimers can adopt a wide variety of defined three-dimensional shapes. Their size depends on the generation and can thus be easily controlled by synthesis. As a dendrimer’s surface contains multiple copies of same functional group, they are capable of engaging in multivalent interactions. The following two examples illustrate dendrimers that interact with charged hot spots on protein surfaces.

4.09.7.1

b-Tryptase Protein Surface

Wich et al. developed a multivalent cationic peptide that is able to interact with anionic b-tryptase surface hot spots, which are formed by clusters of acidic amino acids (Glu, Asp) which are deprotonated under physiological pH (Fig. 58). The

Figure 58 The surface of the entrance to the central cavity b-tryptase features clusters of negatively charged amino acids (red) in which the active sites are located. Heparin on top of the enzyme stabilizes the tetrameric structure of b-tryptase. Reprinted with permission from: (a) Wich, P.; Schmuck, C.; Angew. Chem. Int. Ed. 2010, 49, 4113–4116. Copyright 2010, Wiley.

Protein Surface Recognition by Synthetic Molecules

331

Figure 59 Combinatorial library of 216 tetravalent inhibitors with four identical arms, in which the last three amino acids are combinatorially varied with six amino acids in each variable position.

ligands inhibit the enzyme by binding tightly to the enzyme’s surface, and thereby block entrance to the active sites, which lie in a tunnel-like pore. The serine protease b-tryptase plays an important role in the pathogenesis of asthma and other allergic and inflammatory disorders.115 For the design of inhibitors, a second generation lysine dendrimer116 was selected to which four identical tetrapeptide arms were attached. Each arm contained three variable positions AA1–AA3. A combinatorial split-mix library with 63 ¼ 216 different tetravalent peptide ligands was synthesized on biocompatible PEGA resin, with the following six proteinogenic L-amino acids at each variable position: lysine, arginine, tryptophan, glutamic acid, phenylalanine, and alanine (Fig. 59). The library was screened directly on-bead for inhibition of b-tryptase in a high-throughput fluorescence assay, measuring the highly fluorescent 7-amino-4-methylcoumarin, which is released from a small substrate by tryptase. The 10 best inhibitors, which showed an inhibition above 90%, are listed in Table 14; the best ones carrying (RWKG)4 and (KWKG)4 sequences exhibited approximately 95% inhibition each. The two basic amino acids (lysine and arginine), in the library, are the most important amino acids for obtaining strong inhibition, and a cationic amino acid at AA3 position is crucial. The solution-phase inhibition studies showed that the peptide ligand (RWKG)4 inhibited b-tryptase strongly with a Ki value of 170 nM. The kinetic data suggested that the tetravalent peptide ligands are reversible, noncompetitive inhibitors that do not bind to the active site of the enzyme, but to the surface of the protein like a “molecular plug.”117

4.09.7.2

Chymotrypsin (ChT) Protein Surface

Klaikherd et al. synthesized amphiphilic biaryl dendrimers and compared them to benzyl ether amphiphilic dendrimers for molecular recognition toward chymotrypsin (ChT). The idea was actually to test their structural hypothesis, which presupposed that these biaryl dendrimers adopt a conformation in which all hydrophilic functionalities (eCO2H) throughout the molecule’s backbone are directed on the solvent-exposed exterior in water, and all the hydrophobic functionalities (eCH2Ph) are presented in the interior of the assembly. This battery of negatively charged carboxylates was expected to interact with a cationic patch that

Table 14 bold)

The ten best inhibitors of the on-bead screening (inhibition in %; cationic amino acids are marked in

AA1

AA2

AA3

Inhibition

Arg Lys Trp Phe Lys Phe Lys Lys Lys Phe

Trp Trp Lys Arg Arg Lys Trp Phe Lys Trp

Lys Lys Phe Lys Arg Arg Arg Arg Trp Lys

95 94 93 92 91 91 90 90 90 90

332

Protein Surface Recognition by Synthetic Molecules

Table 15

Comparison of number of carboxylic group, biding ratio, and binding constant of biaryl dendrimers and classical dendrimers

Compound

No. of carboxylic acid groups

Binding ratio (no. of ChT per dendron)

No. of carboxylic groups per ChT

Dissociation constant Kd (M)

198 199 200 201 202 203 207

1 3 7 15 2 4 8

n/a 0.7 2.0 5.7 0.4 1.2 3.3

n/a 4.4 3.5 2.6 5.0 3.3 2.4

n/a 5.78  10 6 1.47  10 6 6.02  10 7 2.03  10 2 1.81  10 5 3.61  10 6

surrounds the ChT active sited. The binding affinity of all dendrimers and the binding ratio of dendrons versus ChT were determined using a well-established enzymatic assay.118 The results are given in Table 15. The biaryl dendrimers 200 and 201 (Fig. 60) bind more ChT molecules compared to the dendrimer 203 and 204. That could be explained due to the higher number of carboxylic units presented in the biaryl dendrons 200 and 201 compared to dendrons 203 and 204. This result is consistent with their hypothesis that the internal layers of the facially amphiphilic biaryl dendrons are solvent-exposed and accessible for recognition. Furthermore, 94% of the enzyme activity of ChT was inhibited in the presence of 199 at 48 mM and then saturated at higher concentrations. Dendrimers 200 and 201 inhibited 96% of enzyme activity at 9.6 mM and 1.9 mM concentrations, respectively. Dendrimers 202–204 showed a generation dependent inhibition activity. Compounds 199–201 exhibited substrate-specific inhibition. This is due to a micelle-type assembly approximately 10–40 nm in size for all these three dendrons, which is enough to exert the maximum effect on substrate selectivity, whereas the size of the Frechet-type dendrons (202–204) does not show the substrate selectivity of the enzyme. These dendrons do not exhibit any evidence of aggregation in DLS studies.119

4.09.8

Polymers for Protein Surface Recognition

Polymeric macromolecules are even larger scaffolds than dendrimers. However, they are also less well defined and contain heterogeneous mixtures. The three-dimensional structures of polymers are variable and difficult to predict due to their flexible nature.

4.09.8.1

Arginine- and Lysine-Specific Protein Surface

Renner et al. synthesized arginine- and lysine-specific homo and copolymers for protein surface recognition and immobilization (Fig. 61).120 Aryl bisphosphonates on their own bind arginine and lysine well in DMSO, but affinity drops drastically in water. Nevertheless, polymerization of corresponding methacrylamides transformed a multitude of weak binders into an efficient receptor site for binding arginine-rich proteins surfaces. A broad spectrum of biological proteins with pI values from slightly acidic ( 5) to strongly basic ( 10) was chosen for recognition. The strong binding affinities are reflected by association constants (Ka values) in the range of 105–106 M 1. Stoichiometries between 3:2 and 4:1 indicate the efficient complexation of multiple protein molecules by one single polymer strand (Table 16). The least basic protein Ferritin, with a pI of 5.5, bound very tightly to both polymers with a capacity to hold nine copies at a time. The polymeric hosts are more sensitive to arginine-rich protein surfaces compared to lysine surfaces. The polymers bind Arg-rich proteins one order of magnitude tighter than their lysine-rich congeners of comparable pI, and size ratio. Another important factor for the efficiency of protein binding is the sheer size of the target molecule. The immobilized bisphosphonate polymer with (Arg)n and protein solutions was observed, in most cases, and a marked increase in optical thickness was monitored in a time-resolved manner by reflectometric interference spectroscopy in a flow-through system.121

4.09.9

Molecular Tweezers as Receptors for Protein Surface Recognition

While the last example showed how polymerization can be used to turn a structurally simple but only moderately affine guanidinium and ammonium binder into an excellent receptor, a more elaborately tailored motiff is able to tightly bind ammonium residues on its own. Klaerner and Schrader developed a horseshoe-arene equipped with phosphate moieties that is able to tightly bind small ammonium residues, as present in the lysine side chain.

4.09.9.1

Lysine-Specific 14-3-3 Protein Surface

Bier et al. employed the lysine-specific molecular tweezers (207) (Fig. 62) to bind the 14-3-3 adapter protein and modulate its interaction with protein-binding partners. The 14-3-3s are small (25–30 kDa) eukaryotic adapter proteins that influence a plethora of

Protein Surface Recognition by Synthetic Molecules

Figure 60

Chemical structures of amphiphilic biaryl dendrimers 198–201 and amphiphilic dendrimers 202–204.

333

334

Protein Surface Recognition by Synthetic Molecules

Figure 61 (A) Structural key element: bisphosphonate dianion interacts with arginine residue. Schematic representation of oligoarginine recognition by bisphosphonate units. (B) Structure of homo- and copolymer 205 and 206. A dansyl label was attached for the purpose of analysis.

physiological processes, for example, the kinase C-Raf,122 the transcriptional modulator YAP,123 and the tumor suppressor p53.124 In the presence of the tweezers molecule 207, specific binding of the FAM-labeled peptides C-RafpS259 and ExoS to the 14-3-3 protein was inhibited in a dose-dependent manner with IC50 values of 480 and 520 mM, respectively. Protein crystallography showed that the tweezers molecule bound to a surface-exposed lysine (Lys214) of the 14-3-3 protein. Interestingly no other surface-exposed lysine residues were bound, which normally interact with the partner proteins. A combination of structural analysis and computer simulations afforded the following empirical rules for efficient surface lysine complexation by molecular tweezers: (i) steric accessibility at the edges of secondary structures; (ii) additional hydrogen bonding to neighboring residues; (iii) lack of multiple lysine or arginine residues in close proximity, which lead to binding by external ion pairs. In the 14-3-3 adapter protein only Lys214 met all three requirements.125

4.09.10 Stabilization of Protein–Protein Interactions As can be seen in this chapter, inhibition of PPIs with synthetic molecules gained substantial interest in pharmaceutical research and provides novel prospects for the treatment of many diseases. However, the development of small molecules that stabilize PPIs has been realized in only very few cases, two of which, realized for 14-3-3 interactions, are discussed below. Table 16

The binding experiments involved homo and copolymers with proteins in free solution Homopolymer (205)

Protein

pItheor

Hist H1 Cyt c Lysozyme ADH Trypsin Prot K BSA Ferritin

10.4 9.2 9.1 8.0 8.3 7.7 5.8 5.4

pIexptl

11.0 5.4 10.5 5.8 6.0

MW (kDa) 22.0 12.3 14.3 141 22.0 28.9 66.0 455.3

Copolymer (206)

Stoich

Ka

2:1

>e þ 6 6e þ 5 >e þ 5

No shift

5:2 1:9

>e þ 5 >e þ 4 2e þ 5 1e þ 6

Stoich

3:1 4:1 No saturation 4:1 4:1 3:2 1:9

Ka a >e þ 5 2e þ 5 4e þ 6 4e þ 4 2e þ 4 2e þ 5 1e þ 6

a Averaged virtual 1:1 binding constant, calculated for each complexation step is determined from fluorescence titration. Dansyl-labeled homo- and copolymers were used for the titration.

Protein Surface Recognition by Synthetic Molecules

335

Figure 62 Chemical structure of supramolecular ligand (molecular tweezers) 207 and 3D representation of the specific interaction with the ammonium moiety of lysine.

4.09.10.1 14-3-3/PMA2 Protein Surface After screening a 37,000-member compound library Rose et al. identified two small molecules that selectively stabilize the 14-3-3/ PMA2 PPI by binding to two adjacent sites in the interface (Fig. 63). The hit compound, pyrrolidone 208, exhibited association kinetics similar to fusicoccin a known stabilizer but led to more rapid dissociation, with a Kd of 80 mM. The dipeptide epibestatin stabilized the 14-3-3/PMA2 complex, with a slower association than fusicoccin and pyrrolidone 208. However, once the complex is formed, binding of 14-3-3 to immobilized PMA2 was measured. The cocrystal structures revealed different binding modes of pyrrolidone 208 and epibestatin, resulting in different association/dissociation kinetics of pyrrolidone 208 and epibestatin. Pyrrolidone 208 shared most of its protein contact surface with 14-3-3 (288.2 Å2 from a total of 349.7 Å2; 83%), whereas epibestatin shared a roughly equal contact surface with 14-3-3 (164.4 Å2, 55%) and PMA2 (135.5 Å2, 45%). It was also buried more deeply in the protein complex.126

4.09.10.2 14-3-3/Tau Protein Surface Milroy et al. reported the first rational design of 14-3-3/Tau PPI inhibitors that was guided by the X-ray cocrystal structure of a protein-stabilizer (14-3-3/fusicoccin A) complex. The Tau protein stabilizes the microtubules. Tau hyperphosphorylation (pTau) leads to enhanced formation of neurofibrillary tangles (NFTs), by disrupting Tau binding to microtubules,127 which causes Alzheimer’s disease (AD).128 The adaptor protein 14-3-3 binds to pTau at two epitope sites, pS214 and pS324. It also regulates Tau phosphorylation.129 Thus inhibitors that disrupt the 14-3-3/Tau interactions carry the potential for downstream applications in AD treatment. A series of chimeric inhibitors of 14-3-3/Tau based on the Tau pS214 epitope (Tau epitope) were synthesized (Table 17). A FA assay showed that the Tau epitope (AcNH-RTPpSLPTP-OH) exhibited only weak activity (IC50 ¼ 1330.0), whereas compounds 209a and 209b were stronger inhibitors with IC50 values of 83 and 90 mM, respectively. The cocrystal structure of analogue 209b with 14-3-3 protein revealed that the peptidic region of 209b bound to the amphiphilic groove of 14-3-3. Phe, Met, and Lys form a highly conserved hydrophobic (FC) pocket within the amphiphilic groove of the protein. Importantly the methoxy substituent ( OMe) of 209b bound here, similar to the fusicoccin A (FC)-derived methoxy group. Introducing bulky aromatic groups to the Tau epitope enhanced the binding affinity through a specific interaction at the FC pocket. The sterically bulky and conformationally rigid benzhydryl pyrrolidine moiety, in peptide 211b, gives the most potent inhibitor with an IC50 value of 5.9 mM and a near 1000-fold increase in 14-3-3 binding affinity compared to the

Figure 63

Chemical structure of the hit compounds.

336

Protein Surface Recognition by Synthetic Molecules

Table 17

Structure and activity of chimeric inhibitors

R1R2NH

Analogue

IC50 (mM)

209a

83.1

209b

90.1

210a

301.3

210b

178.5

210c

55.4

211aa

60.1

211b

5.9

a

Data based on the major diastereomer of 211a.

unmodified Tau epitope. The presence of 211b notably inhibited the binding of phosphorylated full-length Tau to 14-3-3 protein, according to NMR spectroscopy studies.130

4.09.11 Protein Surface Recognition Using Metal Complexes The incorporation of metals into surface binders in the form of complexes opens up several new possibilities for these binders to interact with proteins. Metal centers can either interact directly with a protein by participating in enzymatic redox processes or provide binding sites for ligands like the imidazolium moiety of a histidine residue. Besides that metal complexes allow access to new three-dimensional structures that can allow for addressing binding sites that could not be easily reached by strictly organic scaffolds.

4.09.11.1 Cytochrome c (Cyt-c) Protein Surface Takashima et al. described series of Ru(bpy)3 complexes functionalized with branched carboxylates (Fig. 64) that bind to the positively charged protein surface of Cyt-c. Cyt c is a particularly attractive target since it plays a key role in apoptosis,131 mediated by complex formation with other proteins such as cyt c oxidase and Apaf1. The affinity of 212a–c for various proteins (myoglobin, horseradish peroxidase, Cyt-c, cytochrome b562) was assessed by ultrafiltration binding assays. The results revealed that the fraction of proteins bound by Ru(bpy)3 increased with an increasing number of carboxylate groups. Poly-anionic 212c, bearing 18 carboxylates, bound to the positive-charged surface of Cyt-c at neutral pH, 10 times more tightly than the original Ru(bpy)3 complex. Based on the redox potentials of Cyt-c [0.26 V (Fe3 þ/Fe2 þ) vs. NHE] and Ru(bpy)3 [ 0.86 V (Ru3 þ/*Ru2 þ) vs. NHE],132 they studied the photoreduction of Cyt-c by visible light. The most effective electron transfer (ET) relay was accomplished by 212b, having 12-carboxylates, instead of 212c, having 18-carboxylates. The results suggested that not only the number of carboxylate groups but also the asymmetric spatial orientation around the Ru center is critical in the net photoreduction efficiency. No photoreduction was observed for Mb, HRP, and Cyt-b562.133 Muldoon et al. synthesized RuII tris-(5,50 -biscarboxamidobipyridine) derivative 213 (Fig. 65), which bound selectively to cyt c with a 1:1 stoichiometry (Kd ¼ 2 nM). It was tested against a range of other proteins (acetylated cyt c, myoglobin, ferredoxin,

Protein Surface Recognition by Synthetic Molecules

Figure 64

337

Chemical structures of Ru(bpy)3-based poly-anionic complexes, 212a–c.

horseradish peroxidase, and lysozyme), but no response was observed. The dissociation constant (Kd) for lysozyme is 273  43 nM, whereas for other proteins, it was more than 30,000 nM.134 Filby et al. described the influence of the geometric arrangement of ligands around the ruthenium(II) core of compound 214. The diastereomers were then tested on their recognition ability toward the positively charged protein-surface of cyt c, at physiological pH (pI  10). Receptor 214 was functionalized with an optically pure recognition motif incorporating two L-aspartyl groups at the 5 position of bipyridine. All the protected fac/mer and D/L isomers were separated by chromatography (Fig. 66). All isomers of 214 were tested in a functional ascorbate reduction assay in which the reduction of cyt c by ascorbate occured at a much slower rate, in the presence of the receptor 214, indicating binding of the receptors to the heme region. The binding affinity for all isomers to cyt c is listed in Table 18. Ruthenium complex 215 was synthesized as a negative control.135

4.09.11.2 a-Chymotrypsin Protein Surface Ohkanda et al. reported the protein surface binding of dendritic ruthenium(II) tris(bipyridine) complexes (Fig. 67) to a-chymotrypsin, as well as inhibition of the enzyme activity. The binding affinity of the metal complexes to ChT was evaluated by titration, monitoring the change in luminescence at 620 nm at pH 7.4. The association of 216a with ChT involved 1:1 and 1:2 (216a: ChT) complex formation. The dissociation constants for each equilibrium step were 1.3  10 7 and 4.3  10 7 M, respectively. They further evaluated the ChT inhibition activity of the dendritic [Ru(bpy)3] complexes 216a, which showed significant inhibitory activity (74  17%). The amino functionalized 216b showed no inhibition activity at (pH 7.4), which is most likely due to the electrostatic repulsion of the protonated lysine 3-amino groups (pKa  10.5) with the ChT surface. The above results support that 216a recognized the protein surface through complementary ion–pair interaction between the negatively charged carboxyl groups and the positively charged amino residues.136

4.09.11.3 Carbonic Anhydrase (Bovine Erythrocyte) Protein Surface Fazal et al. described transition metal complexes (Fig. 68) that target the surface histidine pattern of CA (bovine erythrocyte). CA has six histidines on the surface, at positions 1, 7, 12, 14, 61, and 93, as determined from the X-ray structure.137 It was deduced that either His-14 or His-12 should be able to participate in metal binding. Strong and selective binding only occurs when the pattern of cupric ions on a complex matches the surface pattern of histidines of the protein. The binding affinity for the metal complexes was

Figure 65

Chemical structures of Ru(bpy)3-based poly-anionic complexes, 213.

338

Protein Surface Recognition by Synthetic Molecules

Figure 66

Structure of compound 214 and negative control 215.

measured by ITC, and the data were fitted to a multiple independent binding site model. The tris-Cu2 þcomplex 217 in which the distances between the metal centers matched those of the histidine residues bound strongly to the target protein (CA) with (K) ¼ (299,000  30,000 M 1). The other metal complexes showed lower affinities. To check selectivity, three other proteins were used (chicken egg albumin (CEA), horse skeletal muscle myoglobin, and chicken egg lysozyme) which are known to interact with transition metal ions and form complexes with the surface-exposed histidines. However, complex 217 proved very selective for CA over CEA (300:1) although with myoglobin, the selectivity was more moderate (20:1), whereas lysozyme was precipitated in the presence of complex 217. The flexible complex 219 showed a moderate affinity for the (CA) protein (K ¼ 70,600 M 1). Protein– ligand interactions were very much dependent on the pH of the buffer as this regulates the protonation–deprotonation equilibria of the imidazole nitrogens (pKa ¼ 6.8) and thus can prohibit complex formation with copper. Also the binding constant decreased with increasing salt concentrations, but it was not dependent on buffer structure and concentration.138

4.09.11.4 Protein Kinase Protein Surface Feng et al. presented six carefully tailored, stable octahedral pyridocarbazole metal complexes (Fig. 69) in which unique and defined molecular geometries with Staurosporine-like structural complexity are constructed around octahedral ruthenium(II) or iridium(III) metal centers. Each of the six metal compounds displayed high selectivity and high binding affinity for an individual PKA, namely, GSK3R, PAK1, PIM1, DAPK1, MLCK, and FLT4 although all six complexes contain ligands around the same canonical octasporine scaffold. To elucidate the exact binding mode of L-OS221 to GSK3, a cocrystal structure of GSK3 bound to L-OS221 was produced (Fig. 70). A typical two-lobe PKA architecture139 was found connected by the “hinge region,” with the catalytic site positioned in a deep intervening cleft. The metal of L-OS221 is located at a “hot spot” within the Table 18 Dissociation constant for all isomers to cyt c were determined by fluorescence titrationa Compound

Kd (nM)

D-mer 214 L-mer 214 D-fac 214 L-fac 214

25  0.8 29  0.8 172  0.2 130  0.3

1 mM 214, 5 mM sodium phosphate buffer, pH 7.4, ex 467 nm.

a

Protein Surface Recognition by Synthetic Molecules

Figure 67

339

Structure of [Ru(bpy)3] complexes 216.

ATP binding pocket, similar to the binding mode of staurosporine.140 The maleimide moiety of L-OS221 made two canonical hydrogen bonds with the hinge region by contributing a hydrogen bond from the maleimide NH to the backbone amide carbonyl group, whereas the ruthenium-coordinated CO ligand interacts extensively with the flexible glycine-rich loop. Unique structural features of the octahedral coordination geometry contribute significantly to binding potencies and selectivities.141

Figure 68

Carbonic anhydrase targeting metal complexes.

340

Protein Surface Recognition by Synthetic Molecules

Figure 69 Molecular structures of highly selective octasporine protein kinase inhibitors. The compounds L-OS221 and L-OS222 are single enantiomers, whereas OS223-OS226 are racemic mixtures (only one enantiomer shown). IC50 values were determined in the presence of 100 mM ATP.

4.09.12 Calixarenes Derived Receptors for Protein Surface Recognition The calixarene core structure offers a very versatile template for noncovalent interactions involved in the molecular recognition of protein surfaces.142 Calix[4]arene can adopt, or be forced to adopt a chalice conformation and thus provide a unique shape that can be easily functionalized on its lower (endo) and/or upper (exo) rim, representing the closed and open side of the chalice, respectively. Also, the inside of the chalice provides a versatile pocket that can bind a multitude of guests.

Figure 70 Interactions of L-OS221 within the active site of GSK3b. Labeled amino acids are involved in hydrogen bonds to the ruthenium complex. Reprinted with permission from Bertrand, J. A.; Thieffine, S.; Vulpetti, A.; Cristiani, C.; Valsasina, B.; Knapp, S.; Kalisz, H. M.; Flocco, M. J. Mol. Biol. 2003, 333, 393–407. Copyright 2011, American Chemical Society.

Protein Surface Recognition by Synthetic Molecules

Figure 71

341

Structural features of calix[4]arene derivatives 227–237 candidate for HDAC inhibition.

4.09.12.1 Histone Deacetylase Protein Surface Chini et al. described conformationally locked calixarenes that inhibit histone deacetylase (HDACs) (Fig. 71). The compounds were locked in the cone conformation, by propoxy groups at the endo rim, and the appropriate functional groups are directed at the exo rim (the open side of the chalice) to bind to the receptor surface.143 HDACs are a family of metalloenzymes that play an important role in the epigenetic regulation of gene expression.144 They are associated with carcinogenesis and tumor progression by removing acetyl groups from lysine or arginine residues145 in the tails of histones. They are also involved in the remodeling of chromatin. Thus, HDAC inhibitors (HDACi) are considered to be promising candidates for anticancer agents. Molecular docking studies guided the selection of (i) the metal binders, (ii) aromatic cap groups, (iii) length of the linker chain, and (iv) length of an amide aliphatic chain. The inhibition of HDAC activity in HeLa cells was monitored by a fluorescence-based assay. The IC50 values of selected compounds and Trichostatin A (TSA), a well-known HDAC inhibitor, are listed in Table 19. The larger aromatic ring bearing compounds 234, 235, and 237 showed higher inhibitory activities (IC50 ¼ 0.14–0.86 mM), whereas derivative 229 equipped with a larger alkyl amide (IC50 > 10 mM) and the small aromatic phenyl derivative 233 (IC50 ¼ 5.10 mM) were the least active compounds.146

4.09.12.2 a-Chymotrypsin Protein Surface The Hamilton group described a receptor based on a calix[4]arene core around which four peptide loops are arranged on the open side of the chalice, which is also conformationally locked by butyloxy groups at the endo-rim (Fig. 72). The compound was designed to target the predominantly positively charged surface region of a-chymotrypsin. Each peptide loop of the receptor 238 (GDGD) contained two anionic residues, whereas receptor 239 contained one anionic and one hydrophobic residue (GDGY). It is likely that the residues are flexible enough to contact multiple basic moieties across the surface of the protein. They also prepared four biscationic (GKGK, 240) peptide loop derivatives, as a negative control. The binding of synthetic receptors to specific regions of ChT’s protein surface and inhibition of the enzyme’s activity by disrupting key PPIs are visualized in Fig. 73. Initial screening for inhibition activity showed that receptor 238 (GDGD) was found to decrease hydrolysis to 10% of that of the untreated enzyme compared to little or no inhibition by 239 and 240. Importantly, the activity of another serine protease, elastase, was not inhibited by 238, 239, or 240, indicating selectivity of 238 for the surface of a-chymotrypsin. This was further confirmed by nondenaturing gel electrophoresis on agarose gel. The most potent receptor (238) showed slow binding inhibition kinetics in an analogous manner to several of the natural protein proteinase inhibitors. Detailed kinetic analysis showed that 238 is a competitive inhibitor and bound Table 19

In vitro HDAC inhibitory activity (IC50  sd)

Compound

IC50 (mM)

229 233 234 235 237 TSA

> 10 5.10 (1.00) 0.14 (0.02) 0.14 (0.02) 0.86 (0.10) 0.02 (0.009)

342

Protein Surface Recognition by Synthetic Molecules

Figure 72

Designs of artificial receptors for chymotrypsin surface recognition.

tightly to ChT in both its initial (Ki ¼ 0.81 mM) and final (Ki* ¼ 0.11 mM) enzyme–inhibitor complexes. In contrast, receptors 239 and 240 showed significantly lower inhibition potency with Ki-values of 15 and 36 mM, respectively.147

4.09.12.3 PDGF Protein Surface The same group also employed their calix[4]arenedpeptide loop design (v.s.) to target the platelet-derived growth factor (PDGF) protein surface. The PDGF induces growth and motility in several cell types such as fibroblasts, endothelial cells, and smooth muscle cells. It causes cell proliferation, angiogenesis, wound healing, and chemotaxis and inhibits apoptosis.148 Overexpression of PDGF is thus common in malignant diseases where uncontrolled cell proliferation occurs. Consequently, synthetic derivatives that antagonize PDGF binding are potential anticancer agents. By varying the amino acids that build up the individual loops they were able to

Figure 73

Schematic representation of enzyme inhibition by protein surface recognition.

Protein Surface Recognition by Synthetic Molecules

Table 20

343

Inhibition of PDGF-dependent receptor tyrosine phosphorylation by growth factor binders (GFBs)

Compounds

Loop sequence

IC50 (mM)

GFB-241 GFB-242 GFB-243 GFB-244 GFB-245 GFB-246 GFB-247 GFB-248 GFB-249 GFB-250 GFB-251 GFB-252 GFB-253 GFB-254 GFB-255

GDFD GDDD GDGD d-ADGD GDLD GDAD GDGY ADGD GDSD GKGF GKGK GDND PDGD GDDG GDDY

2.4 8 2.5 9 7.5 1.7 0.25 29 2.8 50 40 5.8 20 1.3 1.7

target the regions of PDGF (loops I and III) involved in binding to the platelet-derived growth factor receptor (PDGFR). A series of derivatives with varying amino acids were synthesized, in which four peptide loop domains are attached. First a screening assay with NIH 3T3 cells was used to detect molecules capable of blocking PDGF-BB-induced tyrosine autophosphorylation of the PDGFR. GFB-247 was identified as the most potent (IC50 ¼ 250 nM) inhibitor (Table 20). It was also selective for PDGF over EGF, IGF1, aFGF, bFGF, and HRGb (IC50 values > 100 mM) but inhibits VEGF-induced Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation with an IC50 of 10 mM. To determine the in vivo antitumor efficacy of GFB-247 in the nude mouse human xenograft model, daily feeding of GFB-247 (50, 100, and 200 mg kg 1) to nude mice implanted subcutaneously with the human glioblastoma U87MG resulted on day 32 in a dose-dependent inhibition of tumor growth of 56%, 81%, and 88%, respectively. It was also effective at inhibiting the tumor growth in nude mice of the human lung adenocarcinoma A-549 and the rat glioma C6. In vivo GFB-247 inhibited angiogenesis strongly in U87MG and A-549 tumors.149 The power of this design certainly lies in its flexibility, as variation of the amino acids in the loops allows for tailoring the scaffold to a potential protein surface.

4.09.12.4 p53 Protein Surface Gordo et al. synthesized a conical calix[4]arene scaffold with four guanidinomethyl residues at the upper (open) rim and a hydrophobic surface at the lower-rim loops of the calixarene (Fig. 74). This compound stabilized the four monomers of the mutated p53R337H protein. The tetrameric protein p53, often known as the “genome guardian,” is a key transcription factor. It induces both cell arrest when DNA is damaged and triggers the expression of DNA repair machinery. When the damage is irreversible, it induces apoptosis.150 The inherited mutation of p53-R337H, in which argenine 337 is replaced by histidine, suffers from destabilization of the tetramer, probably because the histidine is not fully protonated at physiological conditions.151 This leads to an increased tendency for tumor formation in organisms.152 Hence, molecules that are able to stabilize the tetrameric structure of the mutated protein could be valuable therapeutic tools. The tetraguanidiniomethylcalix[4]arene ligand 256 stabilized the tetrameric structure of p53TD-R337H by interacting with glutamates E336 and E339 via ion–pairing and hydrogen bonding as well as interactions with the hydrophobic pocket of the calixarene. The decrease in the rmsd value and the attenuation of the fluctuations in molecular dynamics simulation suggested that it strongly stabilized mutant p53-R337H, at thermal denaturing conditions (400 K). The presence of ligand 256, in vitro, at 400 mM induced thermal stabilization from 62 C to 82 C in mutant p53TD-R337H and a changed from 85.5 C to 86.9 C in highly stable wild-type p53TD. This indicates that ligand 256 stabilizes the tetramerization of the mutated protein. The interaction of ligand 256 with p53TD-R337H was also shown by 15N-1H-HSQC NMR spectroscopy.153

4.09.12.5 Potassium Channels (Kv1.x) Protein Surface Martos et al. reported a series of multivalent calix[4]arene ligands (Fig. 75) that bind to the surface of voltage-dependent potassium channels (Kv1.x) and a reversible inhibition was observed. Many diseases, mostly nervous disturbances, are linked to the aberrant function of potassium channels, which makes them major targets in biomedical and pharmacological research.154 More than 90 genes for different Kþ channel subunits have been identified so far in the human genome. The voltage-gated potassium channel Kv1.3 plays a crucial role in human T-lymphocyte activation.155 The groups hypothesized that the cone-shaped calixarenes would fit into the outer vestibule of the potassium channel. Structures 257–263 thus carry no substituents, but only free phenol groups at the endo-rim of the calix[4] arene core, whereas 264 contains two short loops, which have difficulties entering the channel simultaneously but could allow the molecule to bind in a slightly distorted way. This, however, is not possible with longer or less-

344

Protein Surface Recognition by Synthetic Molecules

Figure 74 Structure of the tetraguanidiniomethylcalix[4]arene ligand 256. Two molecules of ligand 256 stabilize the tetramerization domain of p53TD-R337H (A) with interactions of the ligand guanidinium groups and glutamates E336 and E339, as well as the fitting of the lower rim of the ligand into the hydrophobic pocket of the protein. Reprinted with permission from Achatz, M. I. W.; Olivier, M.; Calvez, Florence, L.; Martel-Planche, G.; Lopes, A.; Rossi, B. M.; Ashton-Prolla, P.; Giugliani, R.; Palmero, E. I.; Vargas, F. R. Rocha, J. C. C. D.; Vettore, A. Luiz.; Hainaut, P. Cancer Lett. 2007, 245, 96–102. Copyright 2008, HighWire.

structured side chains, such as in 265 and 266. The upper rim of the calix[4]arenes were fitted with cationic substituents (guanidines) that specifically interact with the negatively charged extracellular Asp-379 residue of the channel at the “turret loop,” in Kv1.2 by both electrostatic and hydrogen bonding interactions. Inhibition of Kv1 channels by the calixarenes was investigated by using electrophysiological methods on Xenopus laevis oocytes expressing the Shaker potassium channel. Compounds 257–266 caused different reduction levels of Shaker ionic current, at 50 mM which in most cases was reversible. Among them, ligand 260

Figure 75

Ligand structures of calix[4]arenes 257–266.

Protein Surface Recognition by Synthetic Molecules

345

was the most potent and showed a concentration-dependent inhibition of both Shaker and Kv1.3, a subfamily of ion channels. The Hill coefficients for Shaker and Kv1.3 were 1.0  0.3 and 1.1  0.7, respectively. However, shaker ionic current was not completely inhibited, suggesting an incomplete pore block. Docking studies employing the coordinates of the crystal structure of the Kv1.2 potassium channel,156 suggesting that the conical platform of OH-free calix[4]arenes 260 was best suited to deeply penetrate the channel and bind the protein with high affinity by establishing 4 hydrogen bonds with the Asp-379 residue.157

4.09.13 Protein Surface Recognition Using Porphyrins Owing to their comparatively large size, rigidity, and photophysical properties, porphyrins have been used in numerous artificial receptors and model systems in bioorganic and bioinorganic chemistry.158 In nature, hemoproteins have a number of important functions, and these are regulated by interactions with the apoprotein. Porphyrin is an ideal scaffold for preorganized binding pockets. It is also possible to introduce functional groups at the peripheral positions for recognition. Porphyrins are more flat and rigid compared to calixarene making them a highly complementary scaffold.

4.09.13.1 Cytochrome c Protein Surface Jain et al. synthesized tetraphenylporphyrin-based receptors (Fig. 76), bearing different charged and hydrophobic groups and investigated binding to the surface of cyt c. The tetraarylporphyrin scaffold closely matches the arrangement of hydrophobic and charged domains (lysine and arginine residues) surrounding the exposed heme edge surface.159 The interactions of compounds 267–270, with horse heart cyt c, were studied by fluorescence spectroscopy. Dissociation constants (Kd) values are given in Table 21. Receptor 270 was identified to be the strongest synthetic receptor (Kd 20 nM) for cyt c. The differences in affinity among the receptors revealed a strong dependence on the number of anionic and hydrophobic groups.160 The same group further used ligand 270 as cyt c denaturant. The ligand binds tightly to the heme edge region, thus dramatically lowering the melting temperature (Tm) of cyt c, a protein commonly used in unfolding studies. Upon addition of 1.2 equiv. of 270, Tm dropped to 64 C. Increasing the concentration of 270 to 60 mM (6 equiv.) leads to a further decrease in Tm to 53 C, corresponding to a 32 K reduction relative to the unbound cyt c (c ¼ 10 mM, Tm ¼ 85 C) at pH 7.4 as monitored by the loss of the CD signal at 222 nm.161

Figure 76

Structure of synthetic receptors 267–270.

Table 21

Dissociation constantsa and structural properties of synthetic receptors and other water-soluble porphyrins

Compound

Kd (nM)

Charge

Aryl groups

267 268 269 270 Coproporphyrin I

950  250 860  90 160  20 20  5 7700  270

4 4 8 8 4

4 4 8 8 0

a

Determined at 5 mM sodium phosphate, pH 7.4, 298 K.

346

Protein Surface Recognition by Synthetic Molecules

Figure 77

Table 22

Structure of all ligands.

Inhibition constant (Ki) for ligands based on displacement assays

Ligand

Ki (mM)

Ligand

Ki (mM)

Ligand

Ki (mM)

273 275 276

0.020 0.138 0.157

277 278 279

0.144 0.026 0.013

280 281 282

0.038 1.918 0.156

4.09.13.2 Potassium Channels Protein Surface Gradl et al. developed tetraphenylporphyrin-based ligands (Fig. 77) for potassium channels. These fourfold symmetrical molecules are designed to mimic peptide toxins that interact with all four channel subunits simultaneously, resulting in a strong synergy effect.162 The binding affinity of all ligands was determined in a competitive binding assays with 125I-hongotoxin1-A19Y/Y37F (125I-HgTX1A19Y/Y37F) (Kd ¼ 0.046 pM), and in electrophysiological assays using the Xenopus oocyte system. The Ki values for inhibition of 125I-HgTX1A19Y/Y37F binding are listed in Table 22. Most of these ligands bind to voltage-gated potassium channels of the Kv1x class, such as Shaker and Kv1.3, with nanomolar affinities. Ligands 274 and 283 are an exception and have no effect at concentrations up to 10 mM. This is probably due to lack of positive-charged side chains. Tetracationic porphyrin 273 has the highest binding affinity (Kd ¼ 0.020 mM) and significantly inhibited the Shaker current, in a reversible manner. Ligand 273 does not completely block ionic current through Shaker channels even at high concentrations, suggesting it binds in the outer vestibule of potassium channels.163

4.09.14 Conclusion The examples of protein surface binders discussed in here are based on a multitude of chemical scaffolds such as a-helices and smallmolecule a-helix mimetics, peptide foldamers, dendrimers, molecular tweezers, metal complexes, calixarenes, and porphyrins. An important requirement of suitable molecular scaffolds is that they allow for the presentation of selected functional groups in a specific orientation. In general terms synthetic molecules with a large surface area are well suited for that task and thus for protein surface recognition. The large number of weak interactions available in this way can overcome the highly solvated protein surface character which is a key challenge in protein surface recognition. Thus the task synthetic protein surface binders must face is to strike the critical balance of hydrophobic and charged residues that will lead to complementarity and high affine interactions with a surface target, without resulting in insolubility or aggregation of these agents. Although the examples discussed above have been shown to interact strongly with the desired protein surfaces, most of the studies currently were performed on isolated proteins in vitro. A key question with regards to downstream applications in medicinal-/pharmaceutical chemistry is thus selectivity (for a specific target) and specificity (for a certain organ or tissue). Thus the next big challenges concern applications under physiological conditions and ultimately in vivo. Therefore questions of toxicity, membrane permeability, metabolic distribution, and stability have to be addressed. While several of the systems described in this chapter already show good activity in cell assays and some are even effective in vivo, there is still a need for the development of general strategies to overcome these challenges. A pivotal key is certainly the ability to specifically address a particular protein function. Here, one approach could be to generate highly target selective binders that only address the desired surface region of the target protein. These binders should have extremely high and specific binding

Protein Surface Recognition by Synthetic Molecules

347

constants, to avoid off-target effects. However, an alternative approach could be to design molecules that bind less tightly and can thus reversibly interact with a multitude of protein surfaces. This strategy might proof effective, when the on-target binding generates the desired effect, e.g., on a specific PPI, while off-target interactions produce no or only a negligible effect. This could be the case, as binding to the protein surface occurs far away from the active site, which would leave a majority of proteins unencumbered. The latter approach illustrates the conceivably game changing potential of protein surface binders, which will certainly continue to be a highly interesting field of investigation.

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