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Peptide-based inhibitors of protein–protein interactions
Accepted Manuscript Peptide-based inhibitors of protein-protein interactions Paulina Wójcik, Łukasz Berlicki PII: DOI: Reference:
S0960-894X(15)30394-2 http://dx.doi.org/10.1016/j.bmcl.2015.12.084 BMCL 23445
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
18 October 2015 22 December 2015 23 December 2015
Please cite this article as: Wójcik, P., Berlicki, L., Peptide-based inhibitors of protein-protein interactions, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.084
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Peptide-based inhibitors of protein-protein interactions
Paulina Wójcik, Łukasz Berlicki*
Department of Bioorganic Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
* Corresponding author, e-mail: [email protected]
Keywords: stapled peptide; hydrogen bond surrogate; miniproteins; hairpins; beta-peptides; D-peptides; peptoids
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Abstract Protein-protein interactions (PPIs) are key elements of several important biological processes and have emerged as valuable targets in medicinal chemistry. Importantly, numerous specific protein-protein interactions (e.g., p53 - hDM2 and Bcl-2 - BH3 domains) were found to be involved in the development of several diseases, including various types of cancer. In general, the discovery of new synthetic PPI inhibitors is a challenging task because protein surfaces have not evolved in a manner that allows for specific binding of low molecular weight compounds. Here, we review the discovery strategies for peptide-based PPI inhibitors. Although peptide-based drug candidates exhibit significant drawbacks (in particular, low proteolytic stability), modifications of either the side chains or backbone could provide molecules of interest. Moreover, due to the large molecular size of peptide-based compounds, the discovery of molecules that specifically interact with extended protein surfaces is possible. Two major strategies for constructing peptide-based PPI inhibitors are as follows: (a) cyclization (e.g., stapled peptides) and (b) modification of the backbone structure (e.g., -peptides and peptoids). These approaches for constructing PPI inhibitors enhance both the inhibitory activity and pharmacokinetic properties compared with non-modified -peptides. Graphical abstract
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Introduction. Interactions between proteins are involved in the control of nearly all cellular functions. The network of binary protein-protein interactions (PPIs), the so-called interactome, is extremely expanded, and over 14,000 PPIs have been characterized in humans to date [1]. The highly important role of PPIs in living organisms contributes to various pathological states, which has been demonstrated for numerous PPIs associated with the development of human diseases, especially cancer [2-4]. As valuable medicinal chemistry molecular targets, PPIs have gained tremendous attention and substantial efforts have been undertaken to identify effective PPI inhibitors [5-7]. Unfortunately, this task is far from being trivial. Design strategies that have been developed over decades for ligands of enzymes and receptors are not effective to the desired extent. Proteins typically interact via large surfaces, although it is possible to indicate ‘hot spots’ that are crucial for these processes in many cases [8]. Because of the type of interactions between proteins, it is often problematic to develop low-molecular-weight compounds that can reach small area of the proteins (300-1000 Å2); as a result, the affinity of these low-molecular-weight compounds is often low. Therefore, in the case of protein-protein interactions, medium-sized inhibitors (MW 1000-2000 Da) could be much more effective. Among the various groups of compounds with such characteristics, peptides are the most widely studied [9,10]. They have numerous advantages, including direct similarity to protein fragments, affordable synthesis and the possibility to incorporate a wide variety of functional groups. Obviously, peptides are not preferred drug candidates because of their low proteolytic stability. Moreover, short linear peptides also have low conformational stability, which could decrease binding to the target. Several approaches to develop peptidebased PPI inhibitors have been studied and it was proven that the drawbacks of -peptides could be effectively reduced.
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Figure 1. Major modification types of peptide-based protein-protein interaction inhibitors. Increasing stability of the active conformation and decreasing susceptibility toward proteolysis are the most important goals of the introduced peptide modifications. There are two major groups of structural changes, (a) cyclizations and (b) modifications of the backbone (Figure 1). The key effect of cyclization of a peptide is rigidification of its structure in the active conformation. Various strategies (e.g., hydrogen bond surrogate, stapling, and hairpins) were developed to stabilize turns, helices and extended conformations [11,12]. The second approach, which is based on changes in the backbone, usually alternates the properties of the compound more profoundly, and the obtained sequences and three-dimensional structures differ significantly from the original protein fragment. The major possibilities are as follows: (a) variation in the stereochemistry (the use of D-amino acids), (b) extension of the backbone (incorporation of -amino acids), and (c) shift in the side chains to nitrogen atoms (peptoids). Cyclizations that result in peptidic macrocycles is one of the most widely chosen methods for developing PPI inhibitors [11]. Typically, the first design step is based on the crystal structure of the molecular target with the fragment of its natural counterpart. The 4
overall strategy must be elucidated based on the conformation of the native ligand. Often, it is based on stabilization of the secondary structure (turn/loop/helix/strand). A recent, very elegant example of this approach was delivered by Wang and co-workers, who discovered inhibitors of menin-MLL interaction [13], a PPI that is related to the development of acute leukemia [14]. A short loop conformation of menin binding peptide was stabilized by cyclization using ring-closing metathesis (Figure 2). This approach for cyclization was deduced from the hydrogen bonding distance between the arginine guanidine function and proline carbonyl. The inhibition constant of cyclized peptide 1 (Ki = 23.8 nM) was significantly lower than that measured for the analogous fragment of the natural ligand (Ki = 71.1 nM). A
B
5
Figure 2. Cyclized peptide interacting with menin (A) and the crystal structure of its complex with menin (B) (PDB id: 4I80) [13]. Menin is shown in ribbon representation with transparent surface colored according to interpolated charges. The ligand is presented in tube representation. N-Terminal amino acids and hydrocarbon bridge are shown as sticks. The same method of presentation is used in all following figures.
The hydrogen bond surrogate (HBS) approach is also based on the construction of a peptide macrocycle that is indicated by hydrogen bonds. The interaction of the N-terminal i to i + 4 amino acid residues is replaced by a covalent bond. This modification induces an α-helical conformation in short peptide sequences [15]. Importantly, the use of the HBS approach makes all amino acid side chains available for molecular recognition [9]. Typically, the hydrocarbon linker obtained by a ring closing metathesis reaction is used to build the HBS ligand [16]. A hydrazone bridge can also be effectively applied to mimic the hydrogen bond, as shown by Cabezas and Satterthwait [17]. Synthesis of hydrocarbon HBS can be performed using standard SPPS, where the N-terminal i and i+4 amino acid residues are replaced by pentenoic acid and N-allyl-glycine, respectively, which is followed by an RCM reaction [18, 19]. The conformational stability of the HBS helix significantly improves its resistance to proteolysis and increases the cell penetration properties [16, 20]. The construction of a peptide inhibitor of the myoA- MTIP (myoA tail interacting protein) is an example of the effective use of the HBS approach (Figure 3) [20]. This PPI is considered to be a promising target for developing drugs against Plasmodium falciparum, the parasite responsible for malaria [21]. This case is particularly demanding because the interacting peptide is fully buried inside the host protein. It was demonstrated that the HBS strategy is effective because the N-terminal motif of the HBS does not interfere with the native MTIP-myoA contacts and it increases the affinity from IC50 = 4.4 M for the native peptide to IC50 = 2.4 M for HBS (Figure 3A). The 6
crystal structure of the complex of the HBS peptide and MTIP showed that mode of binding for the HBS ligand is almost exactly the same as that observed for the wide type peptide (Figure 3B) [20]. A
B
Figure 3. Hydrogen bond surrogate peptide analogous to the helical tail of the malaria parasite invasion motor myosin (myoA) (A) and the X-ray crystal structure of its complex with myoA tail interacting protein (MTIP) (B), PDB id. 4MZL [20]. Stapled peptides are among the most common peptide inhibitors of protein-protein interactions that restore and enhance the natural α-helical structure of a peptide [22-24]. The principle of the design of such modifications is based on joining side chains that are in spatial 7
proximity to the -helical conformation by covalent bonds. Usually, i, i+4 or i, i+7 hydrocarbon staples that contain 8 or 11 carbon atoms, respectively, are formed. Alternatively,
disulfide
bridges,
lactamisation,
cysteine
crosslinking,
azide-alkyne
cycloadditions, biaryl linkages formation using borylated phenylalanine derivatives can be used [25]. Recently, it was shown that tryptophan-phenylalanie/tyrosine linkages could be obtained using C-H activation with Pd catalyst [26]. Moreover, light-regulated staples based on azobenzene derived crosslinkers were also described [27]. Typically, stapled peptides are synthesized using the SPPS approach with selected amino acids replaced by those containing a terminal double bond in the side chain of a chosen length (all necessary derivatives are commercially available). The RCM reaction leads to the formation of a peptide macrocycle that shows a higher propensity for the helical conformation. The appropriate stereochemistry and additional C methyl substituent at introduced residues have an additional structuring effect. Recently, the possibility of introduction of multiple hydrocarbon staples was explored [28]. The most important advantage of this approach is the possibility to introduce constraints at any chosen part of the helix, allowing for proper modulation of the conformational stability and the discovery of staples that do not interfere with ligand-protein interactions [29]. The resulting stapled peptides could have a biological function that is analogous to their natural αhelical counterparts; however, success depends on the introduction of a staple that does not exchange or cover interacting residues [30]. Preferably, the staple should enhance binding through hydrophobic interactions with the target molecule. Importantly, properly designed stapled peptides exhibit other important features, including an increased resistance to proteolysis and high cellular uptake [29-31]. Numerous examples of successful applications of stapled peptides for inhibiting proteinprotein interactions that are mediated by helical fragments are present in the literature, including inhibitors of: the p53-MDM2 [32,33], BCL-2 family-BH3 domains [34], -catenin-
8
TCF [35], Rab-GTPase–Effector [36], ER-coactivator protein [37], Cullin3 – BTB [38], VDR-coactivator protein [39] and β-arrestin/β-Adaptin [27]. Stapled peptides that bind to the estrogen receptor (ER) are typical representatives of this class of compounds for which all advantages of this approach could be shown [37]. ER protein is the molecular target in the treatment of breast and endometrial cancer and osteoporosis [40]. The staple was introduced to a short linear peptide (11 residues) at positions 3 and 7 (Figure 4A), resulting in a welldefined helical conformation that was observed in both the free and bound states. The binding constant was significantly lower for the stapled peptide (e.g., peptide 3, KD = 0.352 M) than for the linear ligand (KD = 2.5 M). The crystal structure of the complex of peptide 3 and ER revealed that the assumed mode of binding, in particular, the docking of the staple on the surface of the protein, caused additional hydrophobic interactions. It was also shown that the position of the staple in the peptide considerably influences the binding affinity, which ranges from 0.075 to >15 M.
A
B
9
Figure 4. Stapled peptide (A) and the crystal structure of its complex with ER 2YJA [37].
PDB id.
Hairpins are particularly interesting tools for the molecular recognition of PPI interfaces. The approach to the design of the β-hairpin involves the use of a protein epitope [9]. The starting point for designing this synthetic molecule is the identification of key epitopes that are involved in protein-protein interactions. The discovered epitopes are transferred on semi-rigid macrocyclic templates of hairpin loop sequences. The β-hairpin template is a universal scaffold that can be used to mimic epitopes based on various types of secondary structures [41]. To obtain the stable β-hairpin conformation, a cyclic peptide that contains a turn inducing unit must be designed. D-Pro-L-Pro is one of the most commonly used fragments for this purpose [42], but other rigid units could also be applied, e.g. squaramide-based modules have been recently proposed [43]. Typically, cyclization of -haipins is done by coupling Nand C-termini, although other methods using reactivity of side chains were also applied, including, disulfide formation [44], azide-alkyne cycloaddition [45], Trp-Trp crosslinking [46]. The use of -hairpins seems to be universal and offers many possible changes, such as the size of the hairpin loop, template, and sequences, particularly, incorporation of nonnatural amino acids. β-Hairpins are easily accessible through synthesis, and they take on a 10
conformation that is very similar to the natural protein. They can exhibit high affinity and selectivity as well as several drug-like ADMET properties [41]. Robinson and co-workers designed a β-hairpin that mimics the α-helix of p53, which inhibits the p53-HDM2 interaction [47]. This PPI is one of the most widely studied because it is related to several cancer types [48]. The cyclic antiparallel template uses a dimer of D-Pro-LPro as a turn inducer. The three key residues (Phe19, Trp23 and Leu26) were added along one face of the -hairpin, which interacted with the hydrophobic pocket on the surface of HDM2 (Figure 5) [47]. Many peptides with variation in the amino acid residues have been tested, and most of them exhibited IC50 values in the micromolar range. The most active compound (peptide 4) had an IC50 = 0.14 M, and the crystal structure of its complex with HDM2 confirmed the mode of binding is mediated by three key residues (Figure 5). This example clearly shows that -hairpins can effectively mimic other secondary structures, especially helices.
A
B
11
Figure 5. -Hairpin peptide (A) and its mode of binding to HDM2 (B, PDB id. 2AXI) [47].
Miniproteins are naturally occurring oligopeptides (usually with a length not exceeding 80 residues) with a well-defined three-dimensional structure that is mainly stabilized by disulfide bonds [49,50]. High conformational stability in solution allows for the use of these structures as scaffolds for constructing biologically active compounds, especially PPI inhibitors. The general strategy of miniprotein usage, so called protein grafting, relies on the identification of an epitope of the native ligand and then placement, i.e., grafting, of this epitope onto the scaffold [51]. In the majority of cases, small changes that are introduced to a miniprotein do not affect the overall three-dimensional structure if only the network of disulfide bridges is preserved. The major concern during the design of this class of inhibitors is the proper choice of the scaffold that should fit to the protein interacting surface as well as the appropriate placing of the epitope [51]. A wide range of various scaffolds of this type is known, including the following: animal toxins, cyclotides, defensins and others [52]. The compounds obtained using this approach exhibit usually high biological compatibility, which is expressed by good
12
plasma stability profiles; however, in some cases, inefficient cellular uptake could be problematic [53,54]. Therefore, miniproteins are considered to be promising candidates for medicines because they are synthetically available molecules, which have extended structural complexity, can have high specificity and affinity for proteins with large interacting sites. The p53-MDM2/MDMX interaction was the most widely studied target using the miniprotein approach [6]. In this case, a stable -helical fragment that incorporates Phe, Trp and Leu residues is necessary for effective binding to the target. Schepartz and co-workers obtained miniproteins that can inhibit the p53-MDM2 interaction, grafting the aforementioned epitope on the α-helix of the avian pancreatic polypeptide (aPP) [55]. With the same goal, Lu and coworkers used a disulfide-rich miniprotein — the potassium ion channel toxin BmBKTx1. The introduction of various modifications to the scaffold allowed for the development of miniprotein-based binders of MDM2 and MDMX with submicromolar affinity [56]. Subsequent studies by the same research group included the use of apamin — a twentyresidue miniprotein containing 2 disulfide bridges [57]. Five analogues containing Phe, Tyr, Trp and Leu residues incorporated into the helical fragment of the scaffold showed nanomolar binding affinities toward both MDM2 and MDMX proteins (e.g., stygmin-5, Figure 6A). Structural studies have demonstrated the mode of binding for the obtained miniprotein inhibitors, which reproduced the native network of interactions (Figure 6B) [57]. Recently, cyclotide MCoTI-I was used to construct p53-MDM2 inhibitor with low nanomolar activity in vitro [51]. Importantly, the developed compound was tested also in vivo and caused significant reduction of tumor growth .
A
13
B
Figure 6. Miniprotein (so-called stygmin-5, panel A) and the crystal structure of its complex of with MDM2 (panel B, PDB id. 3IUX) [57]. Residues marked in bold are involved in protein binding.
The second group of peptide modifications concerns changes in the main chain and comprises variation in the stereochemistry (D-peptides), chain length (-peptides) and location of substitution (peptoids) [58]. D-Peptides usage in medicinal chemistry is one of the longest-known strategies for developing peptide-based bioactive compounds. The major advantage of this approach is avoidance of the fast proteolytic degradation that is typical for the L-peptides in blood plasma. Unfortunately, D-peptides exhibit problematic features, namely low conformational stability
14
and mirror three-dimensional arrangements in relation to L-peptides that make structure-based design quite challenging. In particular, helices formed by D-peptides have the opposite handness of the natural ones, and the binding mode for such fragments usually differs from that observed in the native ligand-protein complexes. In some cases, the retro-inverso strategy could be applied [59], whereas the mirror-image phage display technique is of more general utility [60]. D-peptide inhibitors was found effective for the following protein-protein interactions: p53/MDM2 [60], VEGF/VEGF-receptor [61], PD-1/PD-L1 [62]. Lu and coworkers applied mirror-image phage display to obtain D-peptide inhibitors of MDM2-p53 interactions [60,63]. Dodecapeptides exhibiting binding constants in the nanomolar range were found (e.g., peptide 6, Figure 7). The crystal structure of the 6-MDM2 complex provided insight into the mode of interaction, and the trp, leu and leu residues of peptide 6 were identified to occupy the clefts of Phe, Trp, and Leu in the native ligand. Further optimization of this D-peptide led to the discovery of subnanomolar inhibitors of the MDM2-p53 interaction [64]. A
B
15
Figure 7. D-peptide DPMI- (A) and the crystal structure of its complex with MDM2 (B, PDB id 3LNJ) [60]. Key residues involved in protein binding are marked in bold. D-amino acid residues are denoted by lowercase.
β-Peptides, i.e., oligomers containing beta-amino acids, have several key advantages in the construction of effective PPI inhibitors [65, 58]. This group of compounds is often classified as foldamers — oligomers that have a high tendency to form stable conformations in solution. The possibility to predict the relationship between the sequence and three-dimensional structure makes the -peptides very useful for discovering new bioactive compounds [66, 67]. There are two general approaches for designing β-peptides, (a) exclusive usage of β-residues (typically non-constrained) or (b) combination usage of constrained β-residues and natural αresidues. The β-peptides may adopt similar conformations to α-peptides in spite of the presence of an additional backbone carbon atom in each -amino acid residue [68, 69]. However, the replacement of -amino acid residues with -analogues in the peptide sequence can cause mismatch, careful design by appropriate placement of interacting residues give possibility to construct tightly binding compounds. Due to high conformational stability, 16
peptides containing -residues can be significantly shorter than -peptides but achieve a similar degree of folding. Moreover, a number of -amino acids are commercially available or can be obtained by reliable synthetic methods; therefore, the synthesis of this class of compounds based on standard SPPS protocols can be considered feasible [70]. An additional advantage of this class of compounds (observed both for - and -peptides) is that they have an unusual resistance to degradation by proteases [71]. -Amino acid containing peptides were elaborated as inhibitors of protein-protein interactions for: Bcl-2/BH3 domain [72,73], p53/hDM2 [74], VEGF/VEGFR1 [75]. Gellman and co-workers developed peptides containing -amino acid residues that inhibit BCL-xL/BH3 domain interactions [76], which is considered an attractive target for anticancer therapy [77]. Initially, chimeric α/β-peptides with an N-terminal region containing -residues that are responsible for folding and a C-terminal region containing only α-amino acid residues that possess side chain groups relevant for recognizing the target protein were discovered. Further optimizations of (+) peptides led to finding of tightly binding peptides that have high proteolytic stability (e.g., peptide 7 exhibits a Ki = 2 nM, Figure 8A) [78-80]. Although the three-dimensional structure of the helix at the N-terminus of peptide 7 differs from the helix, the crystal structure of the 7-Bcl-xL protein indicates that the peptide is well docked in the protein cleft (Figure 8B). The aforementioned peptides contained fragments with a 1:1 amino acid residue alternating pattern, but further studies indicated that and patterns could be effectively used to design highly active BCL-xL inhibitors [81]. Moreover, the αααβ peptide has sufficient stability against proteolytic degradation [82]. These successful examples confirm that the proper design of -amino acid containing peptides could lead to the discovery of compounds with excellent inhibitory and metabolic properties. A 17
B
Figure 8. Crystal structure of an -peptide bound to Bcl-xL (PD id: 3FDM) [80]. Peptoids consist of repeating N-substituted glycine units [83]. They are able to fold into helices that mimic the peptide structure and function [84, 58]. Peptoid monomers differ from natural α-amino acids in that the side chain is attached to the backbone nitrogen instead of the α-carbon. Due to the lack of the H-bonding capacity of the backbone, the induction of folding into well-defined helical structures must be performed through the careful choice of the Nsubstituent groups. As for -peptides, peptoids exhibit several advantages, including a particularly high resistance to proteolytic degradation [85]. The major problem in the design of peptoids is the appropriate matching to the surface of the protein, and it often requires many attempts to position the key interacting residues in the right place. The synthesis of 18
peptoids is achieved by the iterative coupling of bromoacetic acid and substitution by a chosen amine, which can be effectively performed on a solid support, allowing for the incorporation of a wide variety of substituents. Peptoid-based inhibitors were found for a number of PPIs, including p53-MDM2 [85], VEGF−VEGFR2 [86], Apaf [87], and skp2/p300 [88]. Appella and co-workers attempted to design peptoid inhibitors of p53-hDM2 interactions [85]. A rigid helical peptoid scaffold was decorated with substituents, mimicking Phe, Trp and Leu residues placed on one side of the helix. Surprisingly, this rigid construction did not interact with the target, but its analogues that lack helix-promoting groups were found active in the micromolar range. The most active peptoid was achiral and exhibited an IC50 = 6.6 M (structure 8, Figure 9).
Figure 9. The structure of the peptoid-based inhibitor of p53-HDM2 interactions [85].
In summary, the targeting of protein-protein interactions is a very rapidly growing field of medicinal chemistry. The number of PPI targets of interest is increasing significantly, and considering the current knowledge of the human interactome, several disease-related PPIs are waiting to be discovered. Substantial effort has been made to develop rational strategies in designing PPI inhibitors for target proteins that have no well-defined binding site (the so19
called ‘hot spot’) and, thus, have been considered undruggable. Peptide-based compounds could be considered as one of the most interesting options for targeting PPIs, especially the most problematic ones. Early achievement in the filed were mainly based on application of helix mimetics (e.g. for inhibition of p53-hDM2), while now the scope of possible threedimensional structures of PPI inhibitors has grown and more complicated patterns can be mimicked (e.g. -peptide inhibitors of VEGF/VEGFR1 [75]). The impact of new synthetic strategies for construction of more extended compounds has also to be mentioned. New reactions allowing orthogonal coupling of side chains (e.g. selective Pd-catalyzed Phe-Trp coupling [26]) as well as new modifications of backbone (e.g. formation of oligooxopiperazines [89]) are enhancing possibilities of construction of functional peptidomimetics that exhibit the properties desired in drug candidates.
Acknowledgements The work was financed by the National Science Centre, Poland (grant no. DEC2013/10/E/ST5/00625; Ł.B.), and Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018. Pictures were prepared using the Discovery Studio package (BIOVIA) used under a Polish country-wide license. The use of software resources (BIOVIA Discovery Studio program package) of the Wrocław Centre for Networking and Supercomputing is also kindly acknowledged.
References 1. Rolland, T.; Taşan, M.; Charloteaux, B.; Pevzner, S. J.; Zhong, Q.; Sahni, N.; Yi, S.; Lemmens, I.; Fontanillo, C.; Mosca, R.; Kamburov, A.; Ghiassian, S. D.; Yang, X.; Ghamsari, L.; Balcha, D.; Begg, B. E.; Braun, P.; Brehme, M.; Broly, M. P.; Carvunis, A. R.; Convery-Zupan, D.; Corominas, R.; Coulombe-Huntington, J.; Dann, E.; Dreze, M.; Dricot, A.; Fan, C.; Franzosa, E.; Gebreab, F.; Gutierrez, B. J.; Hardy, M. F.; Jin, M.; Kang, S.; Kiros, R.; Lin, G. N.; Luck, K.; MacWilliams, A.; Menche, J.; Murray, 20
R. R.; Palagi, A.; Poulin, M. M.; Rambout, X.; Rasla, J.; Reichert, P.; Romero, V.; Ruyssinck, E.; Sahalie, J. M.; Scholz, A.; Shah, A. A.; Sharma, A.; Shen, Y.; Spirohn, K.; Tam, S.; Tejeda, A. O.; Trigg, S. A.; Twizere, J. C.; Vega, K.; Walsh, J.; Cusick, M. E.; Xia, Y.; Barabási, A. L.; Iakoucheva, L. M.; Aloy, P.; De Las Rivas, J.; Tavernier, J.; Calderwood, M. A.; Hill, D. E.; Hao, T.; Roth, F. P.; Vidal, M. Cell, 2014, 159, 1212. 2. Jaeger, S.; Aloy, P. IUBMB Life, 2012, 64, 529. 3. Taylor, I. W.; Wrana, J. L. Proteomics, 2012, 12, 1706. 4. Ivanov, A. A.; Khuri, F. R.; Fu, H. Trends Pharmacol. Sci., 2013, 34, 393. 5. Arkin, M. R.; Tang, Y.; Wells, J. A. Chem. Biol., 2014, 21, 1102. 6. Milroy, L. G.; Grossmann, T. N.; Hennig, S.; Brunsveld, L.; Ottmann, C. Chem. Rev., 2014, 114, 4695. 7. Higueruelo, A. P.; Jubb, H.; Blundell, T. L. Curr Opin Pharmacol, 2013, 13, 791. 8. London, N.; Raveh, B.; Schueler-Furman, O. Curr Opin Chem Biol, 2013, 17, 952. 9. Tsomaia, N. Eur J Med Chem, 2015, 94, 459 - 470. 10. Nevola, L.; Giralt, E. Chem. Commun. (Camb.), 2015, 51, 3302. 11. Bhat, A.; Roberts, L. R.; Dwyer, J. J. Eur J Med Chem, 2015, 94, 471. 12. Gao, M.; Cheng, K.; Yin, H. Biopolymers, 2015, 104, 310. 13. Zhou, H.; Liu, L.; Huang, J.; Bernard, D.; Karatas, H.; Navarro, A.; Lei, M.; Wang, S. J. Med. Chem., 2013, 56, 1113. 14. Cierpicki, T.; Grembecka, J. Future Med Chem, 2014, 6, 447. 15. Mahon, A. B.; Arora, P. S. Chem. Commun. (Camb.), 2012, 48, 1416. 16. Henchey, L. K.; Jochim, A. L.; Arora, P. S. Curr. Opin. Chem. Biol., 2008, 12, 692. 17. Cabezas, E.; Satterthwait, A. C. J. Am. Chem. Soc., 1999, 121, 3862. 18. Patgiri, A.; Jochim, A. L.; Arora, P. S. Acc. Chem. Res., 2008, 41, 1289. 19. Miller, S. E.; Thomson, P. F.; Arora, P. S. Curr. Protoc. Chem. Biol., 2014, 6, 101. 20. Douse, C. H.; Maas, S. J.; Thomas, J. C.; Garnett, J. A.; Sun, Y.; Cota, E.; Tate, E. W. ACS Chem. Biol., 2014, 9, 2204. 21. Bosch, J.; Turley, S.; Daly, T. M.; Bogh, S. M.; Villasmil, M. L.; Roach, C.; Zhou, N.; Morrisey, J. M.; Vaidya, A. B.; Bergman, L. W.; Hol, W. G. Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 4852. 22. Verdine, G. L.; Hilinski, G. J. Meth. Enzymol., 2012, 503, 3. 23. Guerlavais, V.; Sawyer, T. K. Ann. Rep. Med. Chem., 2014, 49, 331. 24. Walensky, L. D.; Bird, G. H. J. Med. Chem., 2014, 57, 6275. 25. Lau, Y. H.; de Andrade, P.; Wu, Y.; Spring, D. R. Chem. Soc. Rev., 2015, 44, 91. 26. Mendive-Tapia, L.; Preciado, S.; García, J.; Ramón, R.; Kielland, N.; Albericio, F.; Lavilla, R. Nat. Commun, 2015, 6, 7160. 27. Nevola, L.; Martín-Quirós, A.; Eckelt, K.; Camarero, N.; Tosi, S.; Llobet, A.; Giralt, E.; Gorostiza, P. Angew. Chem. Int. Ed. Engl., 2013, 52, 7704. 28. Hilinski, G. J.; Kim, Y. W.; Hong, J.; Kutchukian, P. S.; Crenshaw, C. M.; Berkovitch, S. S.; Chang, A.; Ham, S.; Verdine, G. L. J. Am. Chem. Soc., 2014, 136, 12314. 29. Bird, G. H.; Gavathiotis, E.; LaBelle, J. L.; Katz, S. G.; Walensky, L. D. ACS Chem. Biol., 2014, 9, 831. 30. Bernal, F.; Wade, M.; Godes, M.; Davis, T. N.; Whitehead, D. G.; Kung, A. L.; Wahl, G. M.; Walensky, L. D. Cancer Cell, 2010, 18, 411. 31. Chu, Q.; Moellering, R. E.; Hilinski, G. J.; Kim, Y.-W.; Grossmann, T. N.; Yehab, J. T.-H.; Verdine, G.L. Med. Chem. Commun., 2015, 6, 111. 32. Bernal, F.; Tyler, A. F.; Korsmeyer, S. J.; Walensky, L. D.; Verdine, G. L. J. Am. Chem. Soc., 2007, 129, 2456. 21
33. Baek, S.; Kutchukian, P. S.; Verdine, G. L.; Huber, R.; Holak, T. A.; Lee, K. W.; Popowicz, G. M. J. Am. Chem. Soc., 2012, 134, 103. 34. Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science, 2004, 305, 1466. 35. Grossmann, T. N.; Yeh, J. T.; Bowman, B. R.; Chu, Q.; Moellering, R. E.; Verdine, G. L. Proc. Natl. Acad. Sci. U.S.A., 2012, 109, 17942. 36. Spiegel, J.; Cromm, P. M.; Itzen, A.; Goody, R. S.; Grossmann, T. N.; Waldmann, H. Angew. Chem. Int. Ed. Engl., 2014, 53, 2498. 37. Phillips, C.; Roberts, L. R.; Schade, M.; Bazin, R.; Bent, A.; Davies, N. L.; Moore, R.; Pannifer, A. D.; Pickford, A. R.; Prior, S. H.; Read, C. M.; Scott, A.; Brown, D. G.; Xu, B.; Irving, S. L. J. Am. Chem. Soc., 2011, 133, 9696. 38. de Paola, I.; Pirone, L.; Palmieri, M.; Balasco, N.; Esposito, L.; Russo, L.; MazzĂ , D.; Di Marcotullio, L.; Di Gaetano, S.; Malgieri, G.; Vitagliano, L.; Pedone, E.; Zaccaro, L. PLoS ONE, 2015, 10, e0121149. 39. Misawa, T.; Demizu, Y.; Kawamura, M.; Yamagata, N.; Kurihara, M. Bioorg. Med. Chem., 2015, 23, 1055. 40. Heldring, N.; Pike, A.; Andersson, S.; Matthews, J.; Cheng, G.; Hartman, J.; Tujague, M.; Ström, A.; Treuter, E.; Warner, M.; Gustafsson, J. A. Physiol. Rev., 2007, 87, 905. 41. Robinson, J. A.; Demarco, S.; Gombert, F.; Moehle, K.; Obrecht, D. Drug Discov. Today, 2008, 13, 944. 42. Robinson, J. A. Acc. Chem. Res., 2008, 41, 1278. 43. Martínez, L.; Martorell, G.; Sampedro, Á.; Ballester, P.; Costa, A.; Rotger, C. Org. Lett., 2015, 17, 2980. 44. Ganesh Kumar, M.; Mali, S. M.; Raja, K. M.; Gopi, H. N. Org. Lett., 2015, 17, 230. 45. Park, J. H.; Waters, M. L. Org. Biomol. Chem., 2013, 11, 69. 46. Makwana, K.M.; Mahalakshmi, R. Org. Lett., 2015, 17, 2498. 47. Fasan, R.; Dias, R. L.; Moehle, K.; Zerbe, O.; Obrecht, D.; Mittl, P. R.; Grütter, M. G.; Robinson, J. A. ChemBioChem, 2006, 7, 515. 48. Popowicz, G. M.; DĂśmling, A.; Holak, T. A. Angew. Chem. Int. Ed. Engl., 2011, 50, 2680. 49. Zoller, F.; Markert, A.; Barthe, P.; Zhao, W.; Weichert, W.; Askoxylakis, V.; Altmann, A.; Mier, W.; Haberkorn, U. Angew. Chem. Int. Ed. Engl., 2012, 51, 13136. 50. Stricher, F.; Martin, L.; Vita, C. Methods Mol. Biol., 2006, 340, 113. 51. Ji, Y.; Majumder, S.; Millard, M.; Borra, R.; Bi, T.; Elnagar, A. Y.; Neamati, N.; Shekhtman, A.; Camarero, J. A. J. Am. Chem. Soc., 2013, 135, 11623. 52. Craik, D. J.; Swedberg, J. E.; Mylne, J. S.; Cemazar, M. Expert Opin. Drug. Discov., 2012, 7, 179. 53. Daniels, D. S.; Schepartz, A. J. Am. Chem. Soc., 2007, 129, 14578. 54. Smith, B. A.; Daniels, D. S.; Coplin, A. E.; Jordan, G. E.; McGregor, L. M.; Schepartz, A. J. Am. Chem. Soc., 2008, 130, 2948. 55. Kritzer, J. A.; Zutshi, R.; Cheah, M.; Ran, F. A.; Webman, R.; Wongjirad, T. M.; Schepartz, A. ChemBioChem, 2006, 7, 29. 56. Li, C.; Liu, M.; Monbo, J.; Zou, G.; Li, C.; Yuan, W.; Zella, D.; Lu, W. Y.; Lu, W. J. Am. Chem. Soc., 2008, 130, 13546. 57. Li, C.; Pazgier, M.; Liu, M.; Lu, W. Y.; Lu, W. Angew. Chem. Int. Ed. Engl., 2009, 48, 8712. 58. Mándity, I. M.; Fülöp, F. Expert Opin Drug Discov, 2015, 10, 1163. 59. Li, C.; Zhan, C.; Zhao, L.; Chen, X.; Lu, W. Y.; Lu, W. Bioorg. Med. Chem., 2013, 21, 4045.
22
60. Liu, M.; Pazgier, M.; Li, C.; Yuan, W.; Li, C.; Lu, W. Angew. Chem. Int. Ed. Engl., 2010, 49, 3649. 61. Mandal, K.; Uppalapati, M.; Ault-RichĂŠ, D.; Kenney, J.; Lowitz, J.; Sidhu, S.S.; Kent, S.B. Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 14779. 62. Chang, H.N.; Liu, B.Y.; Qi, Y.K.; Zhou, Y.; Chen, Y.P.; Pan, K.M.; Li, W.W.; Zhou, X.M.; Ma, W.W.; Fu, C.Y.; Qi, Y.M.; Liu, L.; Gao, Y.F. Angew. Chem. Int. Ed., 2015, 54, 11760. 63. Liu, M.; Li, C.; Pazgier, M.; Li, C.; Mao, Y.; Lv, Y.; Gu, B.; Wei, G.; Yuan, W.; Zhan, C.; Lu, W. Y.; Lu, W. Proc. Natl. Acad. Sci. U.S.A., 2010, 107, 14321. 64. Zhan, C.; Zhao, L.; Wei, X.; Wu, X.; Chen, X.; Yuan, W.; Lu, W. Y.; Pazgier, M.; Lu, W. J. Med. Chem., 2012, 55, 6237. 65. Cabrele, C.; Martinek, T. A.; Reiser, O.; Berlicki, Ł. J. Med. Chem., 2014, 57, 9718. 66. Horne, W. S. Expert Opin. Drug Discov., 2011, 6, 1247. 67. Koyack, M. J.; Cheng, R. P. Methods Mol. Biol., 2006, 340, 95. 68. Martinek, T. A.; Fülöp, F. Chem. Soc. Rev., 2012, 41, 687. 69. Pilsl, L. K.; Reiser, O. Amino Acids, 2011, 41, 709. 70. Fülöp, F.; Martinek, T. A.; Toth, G. K. Chem Soc Rev, 2006, 35, 323. 71. Weiss, H. M.; Wirz, B.; Schweitzer, A.; Amstutz, R.; Rodriguez Perez, M. I.; Andres, H.; Metz, Y.; Gardiner, J.; Seebach, D. Chem. Biodivers., 2007, 4, 1413. 72. Peterson-Kaufman, K. J.; Haase, H. S.; Boersma, M. D.; Lee, E. F.; Fairlie, W. D.; Gellman, S. H. ACS Chem. Biol., 2015, 10, 1667. 73. Smith, B. J.; Lee, E. F.; Checco, J. W.; Evangelista, M.; Gellman, S. H.; Fairlie, W. D. ChemBioChem, 2013, 14, 1564 - 1572. 74. Bautista, A. D.; Appelbaum, J. S.; Craig, C. J.; Michel, J.; Schepartz, A. J. Am. Chem. Soc., 2010, 132, 2904. 75. Haase, H. S.; Peterson-Kaufman, K. J.; Lan Levengood, S. K.; Checco, J. W.; Murphy, W. L.; Gellman, S. H. J. Am. Chem. Soc., 2012, 134, 7652. 76. Sadowsky, J.D.; Schmitt, M.A.; Lee, H.S.; Umezawa, N.; Wang, S.; Tomita, Y.; Gellman, S.H. J. Am. Chem. Soc., 2005, 127, 11966. 77. Besbes, S.; Mirshahi, M.; Pocard, M.; Billard, C. Oncotarget, 2015, 6, 12862. 78. Sadowsky, Jack D.; Fairlie, W. Douglas; Hadley, Erik B.; Lee, Hee-Seung; Umezawa, Naoki; Nikolovska-Coleska, Zaneta; Wang, Shaomeng; Huang, David C. S.; Tomita, York; Gellman, Samuel H. J. Am. Chem. Soc., 2007, 129, 139. 79. Sadowsky, J.D.; Murray, J.K.; Tomita, Y.; Gellman, S.H. ChemBioChem, 2007, 8, 903. 80. Lee, E.F.; Sadowsky, J.D.; Smith, B.J.; Czabotar, P.E.; Peterson-Kaufman, K.J.; Colman, P.M.; Gellman, S.H.; Fairlie, W.D. Angew. Chem. Int. Ed., 2009, 48, 4318 4322. 81. Boersma, M. D.; Haase, H. S.; Peterson-Kaufman, K. J.; Lee, E. F.; Clarke, O. B.; Colman, P. M.; Smith, B. J.; Horne, W. S.; Fairlie, W. D.; Gellman, S. H. J. Am. Chem. Soc., 2012, 134, 315. 82. Lee, E. F.; Smith, B. J.; Horne, W. S.; Mayer, K. N.; Evangelista, M.; Colman, P. M.; Gellman, S. H.; Fairlie, W. D. ChemBioChem, 2011, 12, 2025. 83. Laursen, J. S.; Engel-Andreasen, J.; Olsen, C. A. Acc. Chem. Res., 2015, 48, 2696. 84. Zuckermann, R. N.; Kodadek, T. Curr. Opin. Mol. Ther., 2009, 11, 299. 85. Hara, T.; Durell, S. R.; Myers, M. C.; Appella, D. H. J. Am. Chem. Soc., 2006, 128, 1995. 86. Udugamasooriya, D. G.; Dineen, S. P.; Brekken, R. A.; Kodadek, T. J. Am. Chem. Soc., 2008, 130, 5744.
23
87. Mondragón, L.; Orzáez, M.; Sanclimens, G.; Moure, A.; Armiñán, A.; Sepúlveda, P.; Messeguer, A.; Vicent, M. J.; Pérez-Payá, E. J. Med. Chem., 2008, 51, 521. 88. Oh, M.; Lee, J. H.; Moon, H.; Hyun, Y. J.; Lim, H. S. Angew. Chem. Int. Ed. Engl., 2015, DOI: 10.1002/anie.201508716. 89. Lao, B. B.; Drew, K.; Guarracino, D. A.; Brewer, T. F.; Heindel, D. W.; Bonneau, R.; Arora, P. S. J. Am. Chem. Soc., 2014, 136, 7877.
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S0960-894X(15)30394-2 http://dx.doi.org/10.1016/j.bmcl.2015.12.084 BMCL 23445
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
18 October 2015 22 December 2015 23 December 2015
Please cite this article as: Wójcik, P., Berlicki, L., Peptide-based inhibitors of protein-protein interactions, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.084
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Peptide-based inhibitors of protein-protein interactions
Paulina Wójcik, Łukasz Berlicki*
Department of Bioorganic Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
* Corresponding author, e-mail: [email protected]
Keywords: stapled peptide; hydrogen bond surrogate; miniproteins; hairpins; beta-peptides; D-peptides; peptoids
1
Abstract Protein-protein interactions (PPIs) are key elements of several important biological processes and have emerged as valuable targets in medicinal chemistry. Importantly, numerous specific protein-protein interactions (e.g., p53 - hDM2 and Bcl-2 - BH3 domains) were found to be involved in the development of several diseases, including various types of cancer. In general, the discovery of new synthetic PPI inhibitors is a challenging task because protein surfaces have not evolved in a manner that allows for specific binding of low molecular weight compounds. Here, we review the discovery strategies for peptide-based PPI inhibitors. Although peptide-based drug candidates exhibit significant drawbacks (in particular, low proteolytic stability), modifications of either the side chains or backbone could provide molecules of interest. Moreover, due to the large molecular size of peptide-based compounds, the discovery of molecules that specifically interact with extended protein surfaces is possible. Two major strategies for constructing peptide-based PPI inhibitors are as follows: (a) cyclization (e.g., stapled peptides) and (b) modification of the backbone structure (e.g., -peptides and peptoids). These approaches for constructing PPI inhibitors enhance both the inhibitory activity and pharmacokinetic properties compared with non-modified -peptides. Graphical abstract
2
Introduction. Interactions between proteins are involved in the control of nearly all cellular functions. The network of binary protein-protein interactions (PPIs), the so-called interactome, is extremely expanded, and over 14,000 PPIs have been characterized in humans to date [1]. The highly important role of PPIs in living organisms contributes to various pathological states, which has been demonstrated for numerous PPIs associated with the development of human diseases, especially cancer [2-4]. As valuable medicinal chemistry molecular targets, PPIs have gained tremendous attention and substantial efforts have been undertaken to identify effective PPI inhibitors [5-7]. Unfortunately, this task is far from being trivial. Design strategies that have been developed over decades for ligands of enzymes and receptors are not effective to the desired extent. Proteins typically interact via large surfaces, although it is possible to indicate ‘hot spots’ that are crucial for these processes in many cases [8]. Because of the type of interactions between proteins, it is often problematic to develop low-molecular-weight compounds that can reach small area of the proteins (300-1000 Å2); as a result, the affinity of these low-molecular-weight compounds is often low. Therefore, in the case of protein-protein interactions, medium-sized inhibitors (MW 1000-2000 Da) could be much more effective. Among the various groups of compounds with such characteristics, peptides are the most widely studied [9,10]. They have numerous advantages, including direct similarity to protein fragments, affordable synthesis and the possibility to incorporate a wide variety of functional groups. Obviously, peptides are not preferred drug candidates because of their low proteolytic stability. Moreover, short linear peptides also have low conformational stability, which could decrease binding to the target. Several approaches to develop peptidebased PPI inhibitors have been studied and it was proven that the drawbacks of -peptides could be effectively reduced.
3
Figure 1. Major modification types of peptide-based protein-protein interaction inhibitors. Increasing stability of the active conformation and decreasing susceptibility toward proteolysis are the most important goals of the introduced peptide modifications. There are two major groups of structural changes, (a) cyclizations and (b) modifications of the backbone (Figure 1). The key effect of cyclization of a peptide is rigidification of its structure in the active conformation. Various strategies (e.g., hydrogen bond surrogate, stapling, and hairpins) were developed to stabilize turns, helices and extended conformations [11,12]. The second approach, which is based on changes in the backbone, usually alternates the properties of the compound more profoundly, and the obtained sequences and three-dimensional structures differ significantly from the original protein fragment. The major possibilities are as follows: (a) variation in the stereochemistry (the use of D-amino acids), (b) extension of the backbone (incorporation of -amino acids), and (c) shift in the side chains to nitrogen atoms (peptoids). Cyclizations that result in peptidic macrocycles is one of the most widely chosen methods for developing PPI inhibitors [11]. Typically, the first design step is based on the crystal structure of the molecular target with the fragment of its natural counterpart. The 4
overall strategy must be elucidated based on the conformation of the native ligand. Often, it is based on stabilization of the secondary structure (turn/loop/helix/strand). A recent, very elegant example of this approach was delivered by Wang and co-workers, who discovered inhibitors of menin-MLL interaction [13], a PPI that is related to the development of acute leukemia [14]. A short loop conformation of menin binding peptide was stabilized by cyclization using ring-closing metathesis (Figure 2). This approach for cyclization was deduced from the hydrogen bonding distance between the arginine guanidine function and proline carbonyl. The inhibition constant of cyclized peptide 1 (Ki = 23.8 nM) was significantly lower than that measured for the analogous fragment of the natural ligand (Ki = 71.1 nM). A
B
5
Figure 2. Cyclized peptide interacting with menin (A) and the crystal structure of its complex with menin (B) (PDB id: 4I80) [13]. Menin is shown in ribbon representation with transparent surface colored according to interpolated charges. The ligand is presented in tube representation. N-Terminal amino acids and hydrocarbon bridge are shown as sticks. The same method of presentation is used in all following figures.
The hydrogen bond surrogate (HBS) approach is also based on the construction of a peptide macrocycle that is indicated by hydrogen bonds. The interaction of the N-terminal i to i + 4 amino acid residues is replaced by a covalent bond. This modification induces an α-helical conformation in short peptide sequences [15]. Importantly, the use of the HBS approach makes all amino acid side chains available for molecular recognition [9]. Typically, the hydrocarbon linker obtained by a ring closing metathesis reaction is used to build the HBS ligand [16]. A hydrazone bridge can also be effectively applied to mimic the hydrogen bond, as shown by Cabezas and Satterthwait [17]. Synthesis of hydrocarbon HBS can be performed using standard SPPS, where the N-terminal i and i+4 amino acid residues are replaced by pentenoic acid and N-allyl-glycine, respectively, which is followed by an RCM reaction [18, 19]. The conformational stability of the HBS helix significantly improves its resistance to proteolysis and increases the cell penetration properties [16, 20]. The construction of a peptide inhibitor of the myoA- MTIP (myoA tail interacting protein) is an example of the effective use of the HBS approach (Figure 3) [20]. This PPI is considered to be a promising target for developing drugs against Plasmodium falciparum, the parasite responsible for malaria [21]. This case is particularly demanding because the interacting peptide is fully buried inside the host protein. It was demonstrated that the HBS strategy is effective because the N-terminal motif of the HBS does not interfere with the native MTIP-myoA contacts and it increases the affinity from IC50 = 4.4 M for the native peptide to IC50 = 2.4 M for HBS (Figure 3A). The 6
crystal structure of the complex of the HBS peptide and MTIP showed that mode of binding for the HBS ligand is almost exactly the same as that observed for the wide type peptide (Figure 3B) [20]. A
B
Figure 3. Hydrogen bond surrogate peptide analogous to the helical tail of the malaria parasite invasion motor myosin (myoA) (A) and the X-ray crystal structure of its complex with myoA tail interacting protein (MTIP) (B), PDB id. 4MZL [20]. Stapled peptides are among the most common peptide inhibitors of protein-protein interactions that restore and enhance the natural α-helical structure of a peptide [22-24]. The principle of the design of such modifications is based on joining side chains that are in spatial 7
proximity to the -helical conformation by covalent bonds. Usually, i, i+4 or i, i+7 hydrocarbon staples that contain 8 or 11 carbon atoms, respectively, are formed. Alternatively,
disulfide
bridges,
lactamisation,
cysteine
crosslinking,
azide-alkyne
cycloadditions, biaryl linkages formation using borylated phenylalanine derivatives can be used [25]. Recently, it was shown that tryptophan-phenylalanie/tyrosine linkages could be obtained using C-H activation with Pd catalyst [26]. Moreover, light-regulated staples based on azobenzene derived crosslinkers were also described [27]. Typically, stapled peptides are synthesized using the SPPS approach with selected amino acids replaced by those containing a terminal double bond in the side chain of a chosen length (all necessary derivatives are commercially available). The RCM reaction leads to the formation of a peptide macrocycle that shows a higher propensity for the helical conformation. The appropriate stereochemistry and additional C methyl substituent at introduced residues have an additional structuring effect. Recently, the possibility of introduction of multiple hydrocarbon staples was explored [28]. The most important advantage of this approach is the possibility to introduce constraints at any chosen part of the helix, allowing for proper modulation of the conformational stability and the discovery of staples that do not interfere with ligand-protein interactions [29]. The resulting stapled peptides could have a biological function that is analogous to their natural αhelical counterparts; however, success depends on the introduction of a staple that does not exchange or cover interacting residues [30]. Preferably, the staple should enhance binding through hydrophobic interactions with the target molecule. Importantly, properly designed stapled peptides exhibit other important features, including an increased resistance to proteolysis and high cellular uptake [29-31]. Numerous examples of successful applications of stapled peptides for inhibiting proteinprotein interactions that are mediated by helical fragments are present in the literature, including inhibitors of: the p53-MDM2 [32,33], BCL-2 family-BH3 domains [34], -catenin-
8
TCF [35], Rab-GTPase–Effector [36], ER-coactivator protein [37], Cullin3 – BTB [38], VDR-coactivator protein [39] and β-arrestin/β-Adaptin [27]. Stapled peptides that bind to the estrogen receptor (ER) are typical representatives of this class of compounds for which all advantages of this approach could be shown [37]. ER protein is the molecular target in the treatment of breast and endometrial cancer and osteoporosis [40]. The staple was introduced to a short linear peptide (11 residues) at positions 3 and 7 (Figure 4A), resulting in a welldefined helical conformation that was observed in both the free and bound states. The binding constant was significantly lower for the stapled peptide (e.g., peptide 3, KD = 0.352 M) than for the linear ligand (KD = 2.5 M). The crystal structure of the complex of peptide 3 and ER revealed that the assumed mode of binding, in particular, the docking of the staple on the surface of the protein, caused additional hydrophobic interactions. It was also shown that the position of the staple in the peptide considerably influences the binding affinity, which ranges from 0.075 to >15 M.
A
B
9
Figure 4. Stapled peptide (A) and the crystal structure of its complex with ER 2YJA [37].
PDB id.
Hairpins are particularly interesting tools for the molecular recognition of PPI interfaces. The approach to the design of the β-hairpin involves the use of a protein epitope [9]. The starting point for designing this synthetic molecule is the identification of key epitopes that are involved in protein-protein interactions. The discovered epitopes are transferred on semi-rigid macrocyclic templates of hairpin loop sequences. The β-hairpin template is a universal scaffold that can be used to mimic epitopes based on various types of secondary structures [41]. To obtain the stable β-hairpin conformation, a cyclic peptide that contains a turn inducing unit must be designed. D-Pro-L-Pro is one of the most commonly used fragments for this purpose [42], but other rigid units could also be applied, e.g. squaramide-based modules have been recently proposed [43]. Typically, cyclization of -haipins is done by coupling Nand C-termini, although other methods using reactivity of side chains were also applied, including, disulfide formation [44], azide-alkyne cycloaddition [45], Trp-Trp crosslinking [46]. The use of -hairpins seems to be universal and offers many possible changes, such as the size of the hairpin loop, template, and sequences, particularly, incorporation of nonnatural amino acids. β-Hairpins are easily accessible through synthesis, and they take on a 10
conformation that is very similar to the natural protein. They can exhibit high affinity and selectivity as well as several drug-like ADMET properties [41]. Robinson and co-workers designed a β-hairpin that mimics the α-helix of p53, which inhibits the p53-HDM2 interaction [47]. This PPI is one of the most widely studied because it is related to several cancer types [48]. The cyclic antiparallel template uses a dimer of D-Pro-LPro as a turn inducer. The three key residues (Phe19, Trp23 and Leu26) were added along one face of the -hairpin, which interacted with the hydrophobic pocket on the surface of HDM2 (Figure 5) [47]. Many peptides with variation in the amino acid residues have been tested, and most of them exhibited IC50 values in the micromolar range. The most active compound (peptide 4) had an IC50 = 0.14 M, and the crystal structure of its complex with HDM2 confirmed the mode of binding is mediated by three key residues (Figure 5). This example clearly shows that -hairpins can effectively mimic other secondary structures, especially helices.
A
B
11
Figure 5. -Hairpin peptide (A) and its mode of binding to HDM2 (B, PDB id. 2AXI) [47].
Miniproteins are naturally occurring oligopeptides (usually with a length not exceeding 80 residues) with a well-defined three-dimensional structure that is mainly stabilized by disulfide bonds [49,50]. High conformational stability in solution allows for the use of these structures as scaffolds for constructing biologically active compounds, especially PPI inhibitors. The general strategy of miniprotein usage, so called protein grafting, relies on the identification of an epitope of the native ligand and then placement, i.e., grafting, of this epitope onto the scaffold [51]. In the majority of cases, small changes that are introduced to a miniprotein do not affect the overall three-dimensional structure if only the network of disulfide bridges is preserved. The major concern during the design of this class of inhibitors is the proper choice of the scaffold that should fit to the protein interacting surface as well as the appropriate placing of the epitope [51]. A wide range of various scaffolds of this type is known, including the following: animal toxins, cyclotides, defensins and others [52]. The compounds obtained using this approach exhibit usually high biological compatibility, which is expressed by good
12
plasma stability profiles; however, in some cases, inefficient cellular uptake could be problematic [53,54]. Therefore, miniproteins are considered to be promising candidates for medicines because they are synthetically available molecules, which have extended structural complexity, can have high specificity and affinity for proteins with large interacting sites. The p53-MDM2/MDMX interaction was the most widely studied target using the miniprotein approach [6]. In this case, a stable -helical fragment that incorporates Phe, Trp and Leu residues is necessary for effective binding to the target. Schepartz and co-workers obtained miniproteins that can inhibit the p53-MDM2 interaction, grafting the aforementioned epitope on the α-helix of the avian pancreatic polypeptide (aPP) [55]. With the same goal, Lu and coworkers used a disulfide-rich miniprotein — the potassium ion channel toxin BmBKTx1. The introduction of various modifications to the scaffold allowed for the development of miniprotein-based binders of MDM2 and MDMX with submicromolar affinity [56]. Subsequent studies by the same research group included the use of apamin — a twentyresidue miniprotein containing 2 disulfide bridges [57]. Five analogues containing Phe, Tyr, Trp and Leu residues incorporated into the helical fragment of the scaffold showed nanomolar binding affinities toward both MDM2 and MDMX proteins (e.g., stygmin-5, Figure 6A). Structural studies have demonstrated the mode of binding for the obtained miniprotein inhibitors, which reproduced the native network of interactions (Figure 6B) [57]. Recently, cyclotide MCoTI-I was used to construct p53-MDM2 inhibitor with low nanomolar activity in vitro [51]. Importantly, the developed compound was tested also in vivo and caused significant reduction of tumor growth .
A
13
B
Figure 6. Miniprotein (so-called stygmin-5, panel A) and the crystal structure of its complex of with MDM2 (panel B, PDB id. 3IUX) [57]. Residues marked in bold are involved in protein binding.
The second group of peptide modifications concerns changes in the main chain and comprises variation in the stereochemistry (D-peptides), chain length (-peptides) and location of substitution (peptoids) [58]. D-Peptides usage in medicinal chemistry is one of the longest-known strategies for developing peptide-based bioactive compounds. The major advantage of this approach is avoidance of the fast proteolytic degradation that is typical for the L-peptides in blood plasma. Unfortunately, D-peptides exhibit problematic features, namely low conformational stability
14
and mirror three-dimensional arrangements in relation to L-peptides that make structure-based design quite challenging. In particular, helices formed by D-peptides have the opposite handness of the natural ones, and the binding mode for such fragments usually differs from that observed in the native ligand-protein complexes. In some cases, the retro-inverso strategy could be applied [59], whereas the mirror-image phage display technique is of more general utility [60]. D-peptide inhibitors was found effective for the following protein-protein interactions: p53/MDM2 [60], VEGF/VEGF-receptor [61], PD-1/PD-L1 [62]. Lu and coworkers applied mirror-image phage display to obtain D-peptide inhibitors of MDM2-p53 interactions [60,63]. Dodecapeptides exhibiting binding constants in the nanomolar range were found (e.g., peptide 6, Figure 7). The crystal structure of the 6-MDM2 complex provided insight into the mode of interaction, and the trp, leu and leu residues of peptide 6 were identified to occupy the clefts of Phe, Trp, and Leu in the native ligand. Further optimization of this D-peptide led to the discovery of subnanomolar inhibitors of the MDM2-p53 interaction [64]. A
B
15
Figure 7. D-peptide DPMI- (A) and the crystal structure of its complex with MDM2 (B, PDB id 3LNJ) [60]. Key residues involved in protein binding are marked in bold. D-amino acid residues are denoted by lowercase.
β-Peptides, i.e., oligomers containing beta-amino acids, have several key advantages in the construction of effective PPI inhibitors [65, 58]. This group of compounds is often classified as foldamers — oligomers that have a high tendency to form stable conformations in solution. The possibility to predict the relationship between the sequence and three-dimensional structure makes the -peptides very useful for discovering new bioactive compounds [66, 67]. There are two general approaches for designing β-peptides, (a) exclusive usage of β-residues (typically non-constrained) or (b) combination usage of constrained β-residues and natural αresidues. The β-peptides may adopt similar conformations to α-peptides in spite of the presence of an additional backbone carbon atom in each -amino acid residue [68, 69]. However, the replacement of -amino acid residues with -analogues in the peptide sequence can cause mismatch, careful design by appropriate placement of interacting residues give possibility to construct tightly binding compounds. Due to high conformational stability, 16
peptides containing -residues can be significantly shorter than -peptides but achieve a similar degree of folding. Moreover, a number of -amino acids are commercially available or can be obtained by reliable synthetic methods; therefore, the synthesis of this class of compounds based on standard SPPS protocols can be considered feasible [70]. An additional advantage of this class of compounds (observed both for - and -peptides) is that they have an unusual resistance to degradation by proteases [71]. -Amino acid containing peptides were elaborated as inhibitors of protein-protein interactions for: Bcl-2/BH3 domain [72,73], p53/hDM2 [74], VEGF/VEGFR1 [75]. Gellman and co-workers developed peptides containing -amino acid residues that inhibit BCL-xL/BH3 domain interactions [76], which is considered an attractive target for anticancer therapy [77]. Initially, chimeric α/β-peptides with an N-terminal region containing -residues that are responsible for folding and a C-terminal region containing only α-amino acid residues that possess side chain groups relevant for recognizing the target protein were discovered. Further optimizations of (+) peptides led to finding of tightly binding peptides that have high proteolytic stability (e.g., peptide 7 exhibits a Ki = 2 nM, Figure 8A) [78-80]. Although the three-dimensional structure of the helix at the N-terminus of peptide 7 differs from the helix, the crystal structure of the 7-Bcl-xL protein indicates that the peptide is well docked in the protein cleft (Figure 8B). The aforementioned peptides contained fragments with a 1:1 amino acid residue alternating pattern, but further studies indicated that and patterns could be effectively used to design highly active BCL-xL inhibitors [81]. Moreover, the αααβ peptide has sufficient stability against proteolytic degradation [82]. These successful examples confirm that the proper design of -amino acid containing peptides could lead to the discovery of compounds with excellent inhibitory and metabolic properties. A 17
B
Figure 8. Crystal structure of an -peptide bound to Bcl-xL (PD id: 3FDM) [80]. Peptoids consist of repeating N-substituted glycine units [83]. They are able to fold into helices that mimic the peptide structure and function [84, 58]. Peptoid monomers differ from natural α-amino acids in that the side chain is attached to the backbone nitrogen instead of the α-carbon. Due to the lack of the H-bonding capacity of the backbone, the induction of folding into well-defined helical structures must be performed through the careful choice of the Nsubstituent groups. As for -peptides, peptoids exhibit several advantages, including a particularly high resistance to proteolytic degradation [85]. The major problem in the design of peptoids is the appropriate matching to the surface of the protein, and it often requires many attempts to position the key interacting residues in the right place. The synthesis of 18
peptoids is achieved by the iterative coupling of bromoacetic acid and substitution by a chosen amine, which can be effectively performed on a solid support, allowing for the incorporation of a wide variety of substituents. Peptoid-based inhibitors were found for a number of PPIs, including p53-MDM2 [85], VEGF−VEGFR2 [86], Apaf [87], and skp2/p300 [88]. Appella and co-workers attempted to design peptoid inhibitors of p53-hDM2 interactions [85]. A rigid helical peptoid scaffold was decorated with substituents, mimicking Phe, Trp and Leu residues placed on one side of the helix. Surprisingly, this rigid construction did not interact with the target, but its analogues that lack helix-promoting groups were found active in the micromolar range. The most active peptoid was achiral and exhibited an IC50 = 6.6 M (structure 8, Figure 9).
Figure 9. The structure of the peptoid-based inhibitor of p53-HDM2 interactions [85].
In summary, the targeting of protein-protein interactions is a very rapidly growing field of medicinal chemistry. The number of PPI targets of interest is increasing significantly, and considering the current knowledge of the human interactome, several disease-related PPIs are waiting to be discovered. Substantial effort has been made to develop rational strategies in designing PPI inhibitors for target proteins that have no well-defined binding site (the so19
called ‘hot spot’) and, thus, have been considered undruggable. Peptide-based compounds could be considered as one of the most interesting options for targeting PPIs, especially the most problematic ones. Early achievement in the filed were mainly based on application of helix mimetics (e.g. for inhibition of p53-hDM2), while now the scope of possible threedimensional structures of PPI inhibitors has grown and more complicated patterns can be mimicked (e.g. -peptide inhibitors of VEGF/VEGFR1 [75]). The impact of new synthetic strategies for construction of more extended compounds has also to be mentioned. New reactions allowing orthogonal coupling of side chains (e.g. selective Pd-catalyzed Phe-Trp coupling [26]) as well as new modifications of backbone (e.g. formation of oligooxopiperazines [89]) are enhancing possibilities of construction of functional peptidomimetics that exhibit the properties desired in drug candidates.
Acknowledgements The work was financed by the National Science Centre, Poland (grant no. DEC2013/10/E/ST5/00625; Ł.B.), and Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018. Pictures were prepared using the Discovery Studio package (BIOVIA) used under a Polish country-wide license. The use of software resources (BIOVIA Discovery Studio program package) of the Wrocław Centre for Networking and Supercomputing is also kindly acknowledged.
References 1. Rolland, T.; Taşan, M.; Charloteaux, B.; Pevzner, S. J.; Zhong, Q.; Sahni, N.; Yi, S.; Lemmens, I.; Fontanillo, C.; Mosca, R.; Kamburov, A.; Ghiassian, S. D.; Yang, X.; Ghamsari, L.; Balcha, D.; Begg, B. E.; Braun, P.; Brehme, M.; Broly, M. P.; Carvunis, A. R.; Convery-Zupan, D.; Corominas, R.; Coulombe-Huntington, J.; Dann, E.; Dreze, M.; Dricot, A.; Fan, C.; Franzosa, E.; Gebreab, F.; Gutierrez, B. J.; Hardy, M. F.; Jin, M.; Kang, S.; Kiros, R.; Lin, G. N.; Luck, K.; MacWilliams, A.; Menche, J.; Murray, 20
R. R.; Palagi, A.; Poulin, M. M.; Rambout, X.; Rasla, J.; Reichert, P.; Romero, V.; Ruyssinck, E.; Sahalie, J. M.; Scholz, A.; Shah, A. A.; Sharma, A.; Shen, Y.; Spirohn, K.; Tam, S.; Tejeda, A. O.; Trigg, S. A.; Twizere, J. C.; Vega, K.; Walsh, J.; Cusick, M. E.; Xia, Y.; Barabási, A. L.; Iakoucheva, L. M.; Aloy, P.; De Las Rivas, J.; Tavernier, J.; Calderwood, M. A.; Hill, D. E.; Hao, T.; Roth, F. P.; Vidal, M. Cell, 2014, 159, 1212. 2. Jaeger, S.; Aloy, P. IUBMB Life, 2012, 64, 529. 3. Taylor, I. W.; Wrana, J. L. Proteomics, 2012, 12, 1706. 4. Ivanov, A. A.; Khuri, F. R.; Fu, H. Trends Pharmacol. Sci., 2013, 34, 393. 5. Arkin, M. R.; Tang, Y.; Wells, J. A. Chem. Biol., 2014, 21, 1102. 6. Milroy, L. G.; Grossmann, T. N.; Hennig, S.; Brunsveld, L.; Ottmann, C. Chem. Rev., 2014, 114, 4695. 7. Higueruelo, A. P.; Jubb, H.; Blundell, T. L. Curr Opin Pharmacol, 2013, 13, 791. 8. London, N.; Raveh, B.; Schueler-Furman, O. Curr Opin Chem Biol, 2013, 17, 952. 9. Tsomaia, N. Eur J Med Chem, 2015, 94, 459 - 470. 10. Nevola, L.; Giralt, E. Chem. Commun. (Camb.), 2015, 51, 3302. 11. Bhat, A.; Roberts, L. R.; Dwyer, J. J. Eur J Med Chem, 2015, 94, 471. 12. Gao, M.; Cheng, K.; Yin, H. Biopolymers, 2015, 104, 310. 13. Zhou, H.; Liu, L.; Huang, J.; Bernard, D.; Karatas, H.; Navarro, A.; Lei, M.; Wang, S. J. Med. Chem., 2013, 56, 1113. 14. Cierpicki, T.; Grembecka, J. Future Med Chem, 2014, 6, 447. 15. Mahon, A. B.; Arora, P. S. Chem. Commun. (Camb.), 2012, 48, 1416. 16. Henchey, L. K.; Jochim, A. L.; Arora, P. S. Curr. Opin. Chem. Biol., 2008, 12, 692. 17. Cabezas, E.; Satterthwait, A. C. J. Am. Chem. Soc., 1999, 121, 3862. 18. Patgiri, A.; Jochim, A. L.; Arora, P. S. Acc. Chem. Res., 2008, 41, 1289. 19. Miller, S. E.; Thomson, P. F.; Arora, P. S. Curr. Protoc. Chem. Biol., 2014, 6, 101. 20. Douse, C. H.; Maas, S. J.; Thomas, J. C.; Garnett, J. A.; Sun, Y.; Cota, E.; Tate, E. W. ACS Chem. Biol., 2014, 9, 2204. 21. Bosch, J.; Turley, S.; Daly, T. M.; Bogh, S. M.; Villasmil, M. L.; Roach, C.; Zhou, N.; Morrisey, J. M.; Vaidya, A. B.; Bergman, L. W.; Hol, W. G. Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 4852. 22. Verdine, G. L.; Hilinski, G. J. Meth. Enzymol., 2012, 503, 3. 23. Guerlavais, V.; Sawyer, T. K. Ann. Rep. Med. Chem., 2014, 49, 331. 24. Walensky, L. D.; Bird, G. H. J. Med. Chem., 2014, 57, 6275. 25. Lau, Y. H.; de Andrade, P.; Wu, Y.; Spring, D. R. Chem. Soc. Rev., 2015, 44, 91. 26. Mendive-Tapia, L.; Preciado, S.; García, J.; Ramón, R.; Kielland, N.; Albericio, F.; Lavilla, R. Nat. Commun, 2015, 6, 7160. 27. Nevola, L.; Martín-Quirós, A.; Eckelt, K.; Camarero, N.; Tosi, S.; Llobet, A.; Giralt, E.; Gorostiza, P. Angew. Chem. Int. Ed. Engl., 2013, 52, 7704. 28. Hilinski, G. J.; Kim, Y. W.; Hong, J.; Kutchukian, P. S.; Crenshaw, C. M.; Berkovitch, S. S.; Chang, A.; Ham, S.; Verdine, G. L. J. Am. Chem. Soc., 2014, 136, 12314. 29. Bird, G. H.; Gavathiotis, E.; LaBelle, J. L.; Katz, S. G.; Walensky, L. D. ACS Chem. Biol., 2014, 9, 831. 30. Bernal, F.; Wade, M.; Godes, M.; Davis, T. N.; Whitehead, D. G.; Kung, A. L.; Wahl, G. M.; Walensky, L. D. Cancer Cell, 2010, 18, 411. 31. Chu, Q.; Moellering, R. E.; Hilinski, G. J.; Kim, Y.-W.; Grossmann, T. N.; Yehab, J. T.-H.; Verdine, G.L. Med. Chem. Commun., 2015, 6, 111. 32. Bernal, F.; Tyler, A. F.; Korsmeyer, S. J.; Walensky, L. D.; Verdine, G. L. J. Am. Chem. Soc., 2007, 129, 2456. 21
33. Baek, S.; Kutchukian, P. S.; Verdine, G. L.; Huber, R.; Holak, T. A.; Lee, K. W.; Popowicz, G. M. J. Am. Chem. Soc., 2012, 134, 103. 34. Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science, 2004, 305, 1466. 35. Grossmann, T. N.; Yeh, J. T.; Bowman, B. R.; Chu, Q.; Moellering, R. E.; Verdine, G. L. Proc. Natl. Acad. Sci. U.S.A., 2012, 109, 17942. 36. Spiegel, J.; Cromm, P. M.; Itzen, A.; Goody, R. S.; Grossmann, T. N.; Waldmann, H. Angew. Chem. Int. Ed. Engl., 2014, 53, 2498. 37. Phillips, C.; Roberts, L. R.; Schade, M.; Bazin, R.; Bent, A.; Davies, N. L.; Moore, R.; Pannifer, A. D.; Pickford, A. R.; Prior, S. H.; Read, C. M.; Scott, A.; Brown, D. G.; Xu, B.; Irving, S. L. J. Am. Chem. Soc., 2011, 133, 9696. 38. de Paola, I.; Pirone, L.; Palmieri, M.; Balasco, N.; Esposito, L.; Russo, L.; MazzĂ , D.; Di Marcotullio, L.; Di Gaetano, S.; Malgieri, G.; Vitagliano, L.; Pedone, E.; Zaccaro, L. PLoS ONE, 2015, 10, e0121149. 39. Misawa, T.; Demizu, Y.; Kawamura, M.; Yamagata, N.; Kurihara, M. Bioorg. Med. Chem., 2015, 23, 1055. 40. Heldring, N.; Pike, A.; Andersson, S.; Matthews, J.; Cheng, G.; Hartman, J.; Tujague, M.; Ström, A.; Treuter, E.; Warner, M.; Gustafsson, J. A. Physiol. Rev., 2007, 87, 905. 41. Robinson, J. A.; Demarco, S.; Gombert, F.; Moehle, K.; Obrecht, D. Drug Discov. Today, 2008, 13, 944. 42. Robinson, J. A. Acc. Chem. Res., 2008, 41, 1278. 43. Martínez, L.; Martorell, G.; Sampedro, Á.; Ballester, P.; Costa, A.; Rotger, C. Org. Lett., 2015, 17, 2980. 44. Ganesh Kumar, M.; Mali, S. M.; Raja, K. M.; Gopi, H. N. Org. Lett., 2015, 17, 230. 45. Park, J. H.; Waters, M. L. Org. Biomol. Chem., 2013, 11, 69. 46. Makwana, K.M.; Mahalakshmi, R. Org. Lett., 2015, 17, 2498. 47. Fasan, R.; Dias, R. L.; Moehle, K.; Zerbe, O.; Obrecht, D.; Mittl, P. R.; Grütter, M. G.; Robinson, J. A. ChemBioChem, 2006, 7, 515. 48. Popowicz, G. M.; DĂśmling, A.; Holak, T. A. Angew. Chem. Int. Ed. Engl., 2011, 50, 2680. 49. Zoller, F.; Markert, A.; Barthe, P.; Zhao, W.; Weichert, W.; Askoxylakis, V.; Altmann, A.; Mier, W.; Haberkorn, U. Angew. Chem. Int. Ed. Engl., 2012, 51, 13136. 50. Stricher, F.; Martin, L.; Vita, C. Methods Mol. Biol., 2006, 340, 113. 51. Ji, Y.; Majumder, S.; Millard, M.; Borra, R.; Bi, T.; Elnagar, A. Y.; Neamati, N.; Shekhtman, A.; Camarero, J. A. J. Am. Chem. Soc., 2013, 135, 11623. 52. Craik, D. J.; Swedberg, J. E.; Mylne, J. S.; Cemazar, M. Expert Opin. Drug. Discov., 2012, 7, 179. 53. Daniels, D. S.; Schepartz, A. J. Am. Chem. Soc., 2007, 129, 14578. 54. Smith, B. A.; Daniels, D. S.; Coplin, A. E.; Jordan, G. E.; McGregor, L. M.; Schepartz, A. J. Am. Chem. Soc., 2008, 130, 2948. 55. Kritzer, J. A.; Zutshi, R.; Cheah, M.; Ran, F. A.; Webman, R.; Wongjirad, T. M.; Schepartz, A. ChemBioChem, 2006, 7, 29. 56. Li, C.; Liu, M.; Monbo, J.; Zou, G.; Li, C.; Yuan, W.; Zella, D.; Lu, W. Y.; Lu, W. J. Am. Chem. Soc., 2008, 130, 13546. 57. Li, C.; Pazgier, M.; Liu, M.; Lu, W. Y.; Lu, W. Angew. Chem. Int. Ed. Engl., 2009, 48, 8712. 58. Mándity, I. M.; Fülöp, F. Expert Opin Drug Discov, 2015, 10, 1163. 59. Li, C.; Zhan, C.; Zhao, L.; Chen, X.; Lu, W. Y.; Lu, W. Bioorg. Med. Chem., 2013, 21, 4045.
22
60. Liu, M.; Pazgier, M.; Li, C.; Yuan, W.; Li, C.; Lu, W. Angew. Chem. Int. Ed. Engl., 2010, 49, 3649. 61. Mandal, K.; Uppalapati, M.; Ault-RichĂŠ, D.; Kenney, J.; Lowitz, J.; Sidhu, S.S.; Kent, S.B. Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 14779. 62. Chang, H.N.; Liu, B.Y.; Qi, Y.K.; Zhou, Y.; Chen, Y.P.; Pan, K.M.; Li, W.W.; Zhou, X.M.; Ma, W.W.; Fu, C.Y.; Qi, Y.M.; Liu, L.; Gao, Y.F. Angew. Chem. Int. Ed., 2015, 54, 11760. 63. Liu, M.; Li, C.; Pazgier, M.; Li, C.; Mao, Y.; Lv, Y.; Gu, B.; Wei, G.; Yuan, W.; Zhan, C.; Lu, W. Y.; Lu, W. Proc. Natl. Acad. Sci. U.S.A., 2010, 107, 14321. 64. Zhan, C.; Zhao, L.; Wei, X.; Wu, X.; Chen, X.; Yuan, W.; Lu, W. Y.; Pazgier, M.; Lu, W. J. Med. Chem., 2012, 55, 6237. 65. Cabrele, C.; Martinek, T. A.; Reiser, O.; Berlicki, Ł. J. Med. Chem., 2014, 57, 9718. 66. Horne, W. S. Expert Opin. Drug Discov., 2011, 6, 1247. 67. Koyack, M. J.; Cheng, R. P. Methods Mol. Biol., 2006, 340, 95. 68. Martinek, T. A.; Fülöp, F. Chem. Soc. Rev., 2012, 41, 687. 69. Pilsl, L. K.; Reiser, O. Amino Acids, 2011, 41, 709. 70. Fülöp, F.; Martinek, T. A.; Toth, G. K. Chem Soc Rev, 2006, 35, 323. 71. Weiss, H. M.; Wirz, B.; Schweitzer, A.; Amstutz, R.; Rodriguez Perez, M. I.; Andres, H.; Metz, Y.; Gardiner, J.; Seebach, D. Chem. Biodivers., 2007, 4, 1413. 72. Peterson-Kaufman, K. J.; Haase, H. S.; Boersma, M. D.; Lee, E. F.; Fairlie, W. D.; Gellman, S. H. ACS Chem. Biol., 2015, 10, 1667. 73. Smith, B. J.; Lee, E. F.; Checco, J. W.; Evangelista, M.; Gellman, S. H.; Fairlie, W. D. ChemBioChem, 2013, 14, 1564 - 1572. 74. Bautista, A. D.; Appelbaum, J. S.; Craig, C. J.; Michel, J.; Schepartz, A. J. Am. Chem. Soc., 2010, 132, 2904. 75. Haase, H. S.; Peterson-Kaufman, K. J.; Lan Levengood, S. K.; Checco, J. W.; Murphy, W. L.; Gellman, S. H. J. Am. Chem. Soc., 2012, 134, 7652. 76. Sadowsky, J.D.; Schmitt, M.A.; Lee, H.S.; Umezawa, N.; Wang, S.; Tomita, Y.; Gellman, S.H. J. Am. Chem. Soc., 2005, 127, 11966. 77. Besbes, S.; Mirshahi, M.; Pocard, M.; Billard, C. Oncotarget, 2015, 6, 12862. 78. Sadowsky, Jack D.; Fairlie, W. Douglas; Hadley, Erik B.; Lee, Hee-Seung; Umezawa, Naoki; Nikolovska-Coleska, Zaneta; Wang, Shaomeng; Huang, David C. S.; Tomita, York; Gellman, Samuel H. J. Am. Chem. Soc., 2007, 129, 139. 79. Sadowsky, J.D.; Murray, J.K.; Tomita, Y.; Gellman, S.H. ChemBioChem, 2007, 8, 903. 80. Lee, E.F.; Sadowsky, J.D.; Smith, B.J.; Czabotar, P.E.; Peterson-Kaufman, K.J.; Colman, P.M.; Gellman, S.H.; Fairlie, W.D. Angew. Chem. Int. Ed., 2009, 48, 4318 4322. 81. Boersma, M. D.; Haase, H. S.; Peterson-Kaufman, K. J.; Lee, E. F.; Clarke, O. B.; Colman, P. M.; Smith, B. J.; Horne, W. S.; Fairlie, W. D.; Gellman, S. H. J. Am. Chem. Soc., 2012, 134, 315. 82. Lee, E. F.; Smith, B. J.; Horne, W. S.; Mayer, K. N.; Evangelista, M.; Colman, P. M.; Gellman, S. H.; Fairlie, W. D. ChemBioChem, 2011, 12, 2025. 83. Laursen, J. S.; Engel-Andreasen, J.; Olsen, C. A. Acc. Chem. Res., 2015, 48, 2696. 84. Zuckermann, R. N.; Kodadek, T. Curr. Opin. Mol. Ther., 2009, 11, 299. 85. Hara, T.; Durell, S. R.; Myers, M. C.; Appella, D. H. J. Am. Chem. Soc., 2006, 128, 1995. 86. Udugamasooriya, D. G.; Dineen, S. P.; Brekken, R. A.; Kodadek, T. J. Am. Chem. Soc., 2008, 130, 5744.
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87. Mondragón, L.; Orzáez, M.; Sanclimens, G.; Moure, A.; Armiñán, A.; Sepúlveda, P.; Messeguer, A.; Vicent, M. J.; Pérez-Payá, E. J. Med. Chem., 2008, 51, 521. 88. Oh, M.; Lee, J. H.; Moon, H.; Hyun, Y. J.; Lim, H. S. Angew. Chem. Int. Ed. Engl., 2015, DOI: 10.1002/anie.201508716. 89. Lao, B. B.; Drew, K.; Guarracino, D. A.; Brewer, T. F.; Heindel, D. W.; Bonneau, R.; Arora, P. S. J. Am. Chem. Soc., 2014, 136, 7877.
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