Hot-spot guided design of macrocyclic inhibitors of the LSD1-CoREST1 interaction

Journal Pre-proof Hot-spot guided design of macrocyclic inhibitors of the LSD1-CoREST1 interaction Peter ‘t Hart, Joseph Openy, Adrian Krzyzanowski, Hélène Adihou, Herbert Waldmann PII:

S0040-4020(19)31060-9

DOI:

https://doi.org/10.1016/j.tet.2019.130685

Reference:

TET 130685

To appear in:

Tetrahedron

Received Date: 27 August 2019 Revised Date:

8 October 2019

Accepted Date: 10 October 2019

Please cite this article as: Hart Peter‘, Openy J, Krzyzanowski A, Adihou Héè, Waldmann H, Hot-spot guided design of macrocyclic inhibitors of the LSD1-CoREST1 interaction, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.130685. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

H N H N

AcHN O

HN N

O H N O

CoREST1 367-372

NH

O N H

H N O

LSD1-CoREST1 PPI-inhibitor

O NH2

Hot-spot guided design of macrocyclic inhibitors of the LSD1-CoREST1 interaction† Peter ‘t Hart1, Joseph Openy1, Adrian Krzyzanowski1, Hélène Adihou2,3, Herbert Waldmann1,4* 1

Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany

2

AstraZeneca MPI Satellite Unit, Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany

3

Medicinal Chemistry, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Pepparedsleden 1, 43183, Gothenburg, Sweden 4

Technical University Dortmund, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Strasse 6, 44227, Dortmund, Germany * Corresponding author: [email protected]

† This paper is dedicated to professor Steve Davies in recognition of his achievements in asymmetric synthesis. Abstract: New modalities such as cyclic peptides are attractive structures to inhibit challenging targets. The interaction between LSD1 and CoREST1 is required for histone demethylation and represents an attractive therapeutic target. The large interaction surface between these two proteins was analyzed by virtual alanine scanning using DrugScore PPI and a cluster of hot-spot residues was identified on CoREST1. The cluster was converted into a series of cyclic peptides and the inhibitory potency was optimized by stereochemical inversion at one of the amino acids alpha carbons combined with modification of amino acid side chains. Active peptides were further studied by variable temperature 1H-NMR and docking to evaluate the effect of conformation on binding. Potent inhibitors of the challenging PPI were obtained and will allow future optimization into more druglike structures. Keywords: LSD1, protein-protein interactions, macrocyclic peptides, new modalities. 1. Introduction Protein-protein interactions (PPIs) offer a large selection of novel therapeutic targets.[1] They are, however, hard to address with conventional small molecule type drugs and often require “beyond rule of 5” type molecules for potent inhibition. New modalities such as macrocycles and macrocyclic peptides are promising candidates for PPI inhibition.[2] Due to their higher molecular weight (> 500 Da) cyclic peptides can interact with larger surface areas which is required for strong interaction.[3–5] Such new modalities can be used to address challenging targets that were previously deemed undruggable.[2,3] Herein, we describe the design and synthesis of macrocyclic peptide inhibitors of the LSD1-CoREST1 interaction by using PPI hot-spots as a starting point. The binding energy of a given PPI is not equally distributed over the interaction surface areas, but typically mediated by a few critical amino acids hot-spots.[6,7] For successful inhibition of a PPI a significant number of hot-spot residues has to be targeted by the inhibitor.[5,8] The combination of new modalities (e.g. macrocyclic peptides with novel cyclization modes) with the analysis of PPIs through computational techniques is a powerful strategy to identify inhibitors of targets which could not be addressed previously.[9] Computational techniques such as virtual alanine scanning allow for rapid identification of critical residues in a given PPI without the cumbersome (and often impossible) isolation of each individual mutant protein.[9–11] Identification of critical residues allows them to be incorporated into the design

1

of creative novel peptide structures. Such peptides often benefit from cyclization as a strategy to improve potency through lowering the entropic penalty upon binding.[9,12]

Figure 1: LSD1-CoREST1 interaction. LSD1 is shown in green (tower domain in light green) and CoREST1 in gray. PDB ID: 2UXN. Hot-spot residues on CoREST are indicated. Residues with a ΔΔG 1.52 are shown in blue, residues with ΔΔG > 2 are shown in red and residues making electrostatic contacts are shown in orange. The insert depicts the hot-spot cluster used for the design of inhibitory peptides. Lysine-specific demethylase 1 (LSD1) removes mono- and dimethyl marks on the lysine residues 4 and 9 of histone 3 in a flavin-dependent manner.[13,14] LSD1 is part of the CoREST and NuRD complexes and its demethylation chemistry typically leads to the repression of target genes, although activation has been observed as well.[15–17] It has attracted significant attention due to its therapeutic potential in the treatment of cancer.[18,19] Most of the histone modifying proteins are found as part of multi-protein complexes containing a variety of subunits that interact with different chromatin components (i.e. modified histone tails, methylated DNA).[20] By incorporating the catalytically active protein into different complexes selective regulation of gene expression is achieved. Within the CoREST complex the histone demethylase activity of LSD1 depends on its interaction with either CoREST1, -2 or -3, suggesting that inhibition of this interaction would lead to reduced activity of LSD1.[21,22] Mattevi and coworkers further refined a model where CoREST1 is required to displace the H3 tail to make it accessible for LSD1 demethylation.[23] Extensive crystal structure information is available on the LSD1-CoREST1 interaction showing the unique LSD1 tower domain as the interaction site of CoREST1 (see Fig. 1). The close analogue LSD2 lacks this tower domain and this interaction is therefore unique to LSD1. Although small molecule inhibitors of LSD1 have been reported, it is challenging to achieve selectivity between LSD1 and other monoamine oxidases (including LSD2) due to high homology of the catalytic domains.[18,24,25] Besides demethylation of histones there are several non-histone targets that undergo demethylation by LSD1.[26,27] An inhibitor of the interaction between LSD1 and the CoREST proteins will increase selectivity by allowing LSD1 to demethylate certain targets while avoiding histones. 2. Results and Discussion The availability of several crystal structures of the LSD1-CoREST1 complex made the interaction a good starting point for inhibitor design. As shown in Figure 1 the interaction surface consists of long 2

α-helical segments and would typically be considered a difficult to target PPI due to the many contacts between the two proteins.[1,6] To evaluate whether the interaction was mediated by “hotspots” we subjected the LSD1-CoREST1 crystal structure (2UXN) to virtual alanine scanning using the DrugScore PPI server.[10] By applying a cut-off at a ΔΔG of 1.5 kcal/mol we identified several hot-spot residues (see Fig. 1 and supplemental Table 3). To validate the computational analysis several fluorescently labeled CoREST1 fragments (1-5, supplemental Fig. 2) were prepared and tested for binding to recombinant LSD1 by fluorescence polarization. Synthesis of all fragments, including 63 amino acid fragment 1, was done entirely by linear solid-phase synthesis (see supplemental Fig. 1). Combining both microwave irradiation and incorporation of backbone modified amino acids such as Dmb-protected glycine and pseudoproline dipeptides allowed us to obtain all peptides in good yields.[28] All peptides were N-terminally modified with a short spacer and a fluorescein isothiocyanate (FITC) fluorophore. The results of the fluorescence polarization measurements using purified LSD1 are listed in Table 1 and supplemental Figure 3. The affinity of the longest fragment was found to be 3.0 nM which corresponded to the previously reported affinity of CoREST1 293-380 which was found to be 7.78 nM by isothermal titration calorimetry (ITC).[29] The Kd values stayed similar upon truncation of residues 313-336 (1-3), which corresponded to the observations in the virtual alanine scan as there were no hot-spot residues found in this area. The smallest fragment to show significant binding was the sequence 344-375 (4) and the affinity was lost when 7 more amino acids were truncated (5). Although this fragment has lost several hot-spot residues, the >1000 fold reduction of affinity could also be induced by a loss of preorganization of the α-helical section. Table 1: Affinity of truncated CoREST fragments measured by FP.

Peptide 1 2 3 4 5

Amino acids 313-375 330-375 337-375 345-375 351-375

Kd (nM) 3.0 ± 1.6 4.0 ± 0.5 3.1 ± 0.02 75.3 ± 7.8 >2000

The sequence CoREST1 367-372 contains two hot-spots with ΔΔG > 2 kcal/mol and an arginine (371) residue which makes a salt bridge with aspartic acid 495 of LSD1. The short sequence was therefore chosen as the starting point for inhibitor design. To generate peptides with a high affinity one can reduce the entropic penalty upon binding by preorganization of the binding conformation through macrocyclization. The crystal structure indicated an intramolecular salt bridge between residues glutamic acid 368 and arginine 371 (Fig. 2A) and we therefore selected these for a side-chain to sidechain cyclization strategy. Since arginine 371 also forms a salt-bridge with LSD1 we chose to include the guanidine as part of the macrocycle to maintain this interaction (Fig. 2A and B). Several analogues with a variety of ring-sizes were designed to explore the influence of macrocycle size on the binding of the cyclic peptide (Fig. 2B). Furthermore, both saturated and unsaturated structures were synthesized to induce specific conformational constraints.

3

A

B

C

D

Figure 2: A) Fragment of CoREST1 residues 367-372. A cyclic structure is formed by a salt bridge between residues glutamic acid 368 and arginine 371. The backbone of the peptide is depicted in cyan and side chains in orange. Intramolecular hydrogen bonds are shown as well as the electrostatic interactions made by arginine 371. PDB ID: 2UXN. B) Schematic representation of the hexapeptide

4

derived from CoREST residues 367-372 (6) and the conversion into the first generation of covalently linked macrocycles. C) General synthesis route A. D) General synthesis route B. A previously reported guanidine forming peptide cyclization method was employed with slight modifications.[30] The route started with linear SPPS of the acetylated hexapeptide on solid support (Fig. 2C). Next, the alloc protected amine was deprotected selectively using Pd(PPh3)4 and converted to the isothiocyanate using a convenient method employing CS2 and PyBOP.[31] Removal of the Mttgroup was facilitated by mild acidic conditions (AcOH/TFE/CH2Cl2, 1:2:7) followed by cyclization of the peptide upon treatment with base. The formed thiourea was then converted to the desired guanidine in a two-step procedure. First, the thiourea is activated by treatment with methyl iodide followed by reaction with ammonium acetate and N-methylmorpholine. Compounds with a double bond in the macrocycle required an alternative route starting with a dipeptide where the N-terminal amino acid is an alloc-protected ornithine (Fig. 2D). After alloc removal the side chain is converted to the thiourea using Pbf-NCS. The thiourea was converted to a substituted guanidine using amines with terminal alkenes. Next, the linear synthesis was continued and included allyl glycine at position 2. Macrocyclization was facilitated by ring-closing metathesis under previously described conditions.[32] Final cleavage and deprotection by TFA yielded the desired products. The inhibitors were evaluated in a competitive fluorescence polarization assay using the FITC labelled CoREST1 344 – 375 (4) fragment as a tracer. The results (Table 2) showed that the acetylated version of CoREST 344-375(4Ac) was able to compete off the tracer with an IC50 of 1.71 µM under the assay conditions. All of the designed compounds showed weak inhibition as partial curves were observed (see supplemental Fig. 4). Linear peptide 6 showed the strongest inhibition although an exact IC50 could not be determined.

Figure 3: Design of optimized macrocycles by stereochemical inversion at the α-position of residue 2 (denoted by *). A single isomer was obtained for compound 18. We hypothesized that the cyclization strategy induced an unfavorable conformation on the cyclic peptides. The chosen cyclization strategy forces the side chain at position 2 to fold around to mimic the conformation of the glutamic acid 368 in the crystal structure. Although such a conformation can be obtained in the full length CoREST1 protein, it is possible that it distorts the macrocycle conformation in our covalent analogues. We therefore hypothesized that inverting the stereochemistry at position 2 from L to D would be favorable for macrocycle conformation (Fig. 3). We synthesized three analogues containing either D-diaminobutyric acid, D-ornithine or D-lysine to link the position 2 backbone to the guanidine and explore the right macrocycle size (Fig. 3). Gratifyingly, the D-ornithine containing peptide 14 showed a significant improvement in IC50 to 39.9 µM (Table 2, supplemental Fig. 5). The inactivity of all other variants indicates that macrocycle size and possibly conformation is critical for proper binding to LSD1. Table 2: IC50 values for compounds 4-Ac, 6-20. Compound 4-Ac

IC50 (µM) 1.71 ± 1.21 5

>500 >500 >500 >500 >500 >500 >500 >500 39.9 ± 11.5 >500 >500 >500 >500 65.8 ± 12.7 430.6 ± 57.7

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 4: Structures of derivatives of peptide 14 with modifications at position 3. The importance of the conformation of the macrocycle was further studied by modification of proline 3 (Fig. 4). Several variants of peptide 14 were prepared including the “ring-opened” alanine (17) and N-methyl alanine (18) analogues as well as a four and six-membered ring proline analogue (19/20). The results in Table 2 (supplemental Fig. 6) show that opening of the proline ring led to a complete loss of affinity, further indicating that conformation is a key factor in the binding of the peptide to LSD1 as the proline side chain does not make any contacts with the LSD1 surface. The azetidine analogue (19) maintained binding affinity albeit at an approximately 2-fold higher IC50 (65.8 µM), while the pipecolic acid analogue (20) was less active (430.6 μM). These results indicated that the proline induced the most favorable conformation and the original design (14) was used for further variation. Table 3: Modifications of 14 and their IC50 values.

R3

R4

IC50 (µM)

21

NH

AcNH

131.5 ± 40.4

22

NH

AcNH

125.5 ± 34.9

Compound

R1

R2

6

23

NH

AcNH

>500

24

NH

AcNH

255.9 ± 131.1

25

NH

AcNH

70.2 ± 33.0

26

NH

AcNH

>500

27

NH

AcNH

112.7 ± 40.5

28

NH

AcNH

>500

29

NH

AcNH

>500

30

NH

AcNH

>500

31

NH

AcNH

34.5 ± 8.4

32

NH

AcNH

97.3 ± 30.5

33

NH

AcNH

21.9 ± 7.6

34

NH

AcNH

56.5 ± 8.4

35

NH

AcNH

>250

36

NH

AcNH

>250

37

NH

AcNH

>500

38

NH

AcNH

164.9 ± 73.3

39

NH

AcNH

>125

40

S

AcNH

>500

41

N-Me

AcNH

>500

7

42

N-Et

43 44

NH

45

NH

46

NH

AcNH

>500

AcNH

>500

H

>500 92.3 ± 21.3

O HO

N H

>500

Next, we explored variations of the amino acids making direct contacts with LSD1. The tyrosine sidechain does not fully occupy its shallow pocket suggesting alternative side-chains could be explored. A variety of analogues was synthesized (21-33) and the observed IC50 values show a distinct pattern for this residue (see Table 3 and supplemental Fig. 7-9). For single ring side chains, only the close analogue bearing a methoxytyrosine (25) showed similar potency. Smaller hydrophobic rings were still able to bind, albeit with reduced affinity, whereas the introduction of electron withdrawing substituents typically caused a decrease of potency. Fully saturated rings or heterocycles also were not tolerated. Improved potency was observed for bicyclic systems such as the tryptophan indole (31) or a napthyl group (32) with IC50 values of 34.5 and 21.9 µM, respectively, further improving peptide 14. The remaining two residues that could potentially be modified where those at position 1 (isoleucine) and position 6 (leucine). The isoleucine residue was found to have a high degree of shape complementarity with its binding pocket, correlating with the identification of the residue as a hotspot, and was therefore left intact. Contrary to isoleucine, the leucine residue has a more spacious binding pocket, possibly accommodating alternative structures. Although it scored relatively high in the virtual alanine scan, it was not considered a hot-spot with a ΔΔG of 1.27 kcal/mol. The corresponding electron density was found to be poorly defined indicating flexibility in the side chain orientation. Several analogues were synthesized (34-39) to explore the possibilities around this binding pocket including amino acids with hydrophobic side-chains that could potentially fit in the available extra space (Table 3, supplemental Fig. 10). Only modification to isoleucine (34) or norleucine (39) maintained binding albeit at higher IC50 values (56.5 and 164.9 µM, respectively). Bulkier side chains such as tert-butyl and cyclic structures could not be accommodated in the pocket and abolished binding (35 and 37). Next, we explored modifications on the periphery of the macrocycle instead of amino acid sidechains. We started with modification of the guanidine that closes the macrocycle. When the guanidine was modified with a small methyl, ethyl, or hydroxyethyl group (41-43) the binding affinity was completely lost indicating a clash with the LSD1 aspartic acid 495 (Table 3, supplemental Fig. 11). The importance of the salt-bridge was further illustrated by the inactivity of 40, the thiourea analogue of 14. To reduce the peptidic character of the molecule we attempted to truncate the N-terminus by synthesizing an analogue that lacked the acetamide by coupling (S)-methyl-pentanoic acid (44). We explored two more variants where we extended the N-terminus with a hydroxyacetyl group (46) or N-acetylglycine (45). The extended compounds were designed to form a new hydrogen bond to the 8

LSD1 tyrosine 494 hydroxyl group. Truncation of the N-terminal acetamide led to complete loss of activity even though this group does not participate in binding to LSD1 (Table 3, supplemental Fig. 12). It is therefore likely that it is important in proper positioning of the isoleucine side-chain. Extension of the N-terminus also did not improve the inhibitory potency, with 45 maintaining activity at 92.3 µM while 46 was inactive.

Figure 5: General synthetic route C. Synthesis of C-terminally modified peptides. A similar truncation strategy was applied to the C-terminus requiring an alternative synthetic route (Fig. 5). We used a recently described method based on the Dbz-linker that allows simultaneous cleavage and substitution of the C-terminus using amine nucleophiles.[33] The described room temperature conditions to convert the linker to the benzotriazole and the following nucleophilic substitution were found to be insufficient for our macrocycles. However, a simple adjustment by increasing the temperature to 60 °C and using microwave irradiation allowed us to obtain the desired compounds. To avoid having to further deprotect the peptides after cleavage we chose to substitute the tyrosine for the previously optimized naphtylalanine (33). The method allows for introduction of a variety of amines and both aliphatic or ring structures were explored (Table 4, supplemental Fig. 13 and 14). Interestingly, the isopentyl amine analogue (47) corresponding to a deamidated version of 33 has a similar IC50 value (23.9 µM) as its parent compound. Larger cyclic substituents (48/49) could not be accommodated therefore more aliphatic variations were explored. Compounds 50-56 showed that various linear, branched and cyclic aliphatic chains can be accommodated without affecting the affinity as the IC50 values were almost identical to 47. The requirement for a hydrophobic chain was confirmed by compound 52 possessing a terminal hydroxyl group which resulted in a 3.5 fold increased IC50. A slight improvement in the IC50 was observed for the cyclobutyl analogue 56. Table 4: C-terminal modifications of 33 and their IC50 values.

Compound

R5

IC50 (µM) 9

47

23.9 ± 3.6

48

101.4 ± 14.0

49

386.0 ± 11.8

50

44.6 ± 14.6

51

26.8 ± 3.9

52

84.7 ± 9.7

53

24.8 ± 4.3

54

50.9 ± 12.4

55

41.8 ± 20.9

56

16.2 ± 4.1

Figure 6: Intrinsic FAD fluorescence measured upon denaturation of either LSD1 alone (Tm = 48.0 °C), LSD1 in the presence of 33 (Tm = 48.0 °C), complex (LSD1-CoREST1, Tm = 52.0 °C) or complex in the presence of 33 (Tm = 51.0 °C). To further evaluate our inhibitors we employed the previously described ThermoFAD assay, which relies on the intrinsic fluorescence from the Flavin adenine dinucleotide (FAD) cofactor that is bound by LSD1.[34] During the assay the protein is exposed to a gradient of increasing temperature and upon denaturation the FAD cofactor is released which results in a measurable fluorescence signal. As previously shown an increase in the Tm of LSD1 was observed in the presence of CoREST1 (Fig. 6).[34] Initially we tested whether 33 stabilizes LSD1 upon binding, which was not the case. Therefore, we incubated the LSD1-CoREST1 complex with 33 and measured whether destabilization due to disruption of the complex was observed. The stability of the intact complex was decreased by 1 °C 10

indicating 33 is able to disrupt the LSD1-CoREST1 interaction. The moderate effect can partially be explained by the use of the full LSD1 binding domain of CoREST1 (residues 286-482) which has a higher affinity (Kd = 15.9 nM by ITC)[29] than the tracer (4, residues 345-375) used in the competition fluorescence polarization experiments (Kd = 75.3 nM).

Figure 7: 1H-NMR in H2O/D2O (9:1) of compounds 14 (left) and 7 (right) Table 5: VT-NMR results for 14 and 7. Numbers in brackets indicate shifts for minor conformer. Compound 14 Compound 7a Residue ppb/K Residue ppb/K Ile1 8.5 (7.7) Ile1 11.7 Orn2 6.7 (8.7) Lys2 9.3 Tyr4 2.5 (11.2) Tyr4 8 Orn5 2 (3) Orn5 3.5 Leu6 7.4 (s.o.b) Leu6 4.7 a Only a single conformer was observed for compound 7. b spectral overlap Peptides 14 and 33 were analyzed by 2D-NMR techniques and for both peptides two sets of peaks could be observed (see supplemental Fig. 16 and 22). These results indicated that the peptides have two distinct conformations in solution in an approx. 9:1 ratio (A:B, estimated by integration of 1HNMR peaks). We further analyzed these peptides by variable temperature 1H-NMR (VT-NMR) experiments. In a VT-NMR experiment increasing temperatures will cause backbone amide hydrogen signals to shift upfield but those involved in intramolecular hydrogen bonds will do so to a lesser extent.[35] A shift of >4ppb/K indicates a solvent exposed NH while shifts <4ppb/K indicate a NH which is shielded from the solvent and therefore potentially implicated in a hydrogen bond.[35,36] We compared the hydrogen bond pattern to the pattern observed in the crystal structure which the peptides are derived from (Fig. 2A). In the crystal structure two intramolecular hydrogen bonds are formed, one between the backbone NH of tyrosine 370 to the backbone carbonyl of isoleucine 367 and a second one between the backbone NH of arginine 371 and the carbonyl of glutamic acid 368. Indeed, in the major conformer A of peptide 14 we observe hydrogen bond formation from the backbone NH of tyrosine 4 and that of ornithine 5 (corresponding to tyrosine 370 and arginine 371 in CoREST1, Fig. 7 and Table 5). However, in the alternative minor conformation B the amide hydrogen of tyrosine shifts dramatically with 12 ppb/K indicating strong solvent exposure and a possible unfavorable conformation. An identical pattern and ratio is observed for compound 33 (supplemental Fig. 27 and supplemental Table 8) suggesting the conformational properties are the same as for 14. To further study whether the conformation is one of the main driving factors for potency we also analyzed inactive peptide 7. Peptide 7 was observed to be in an almost entirely 11

single conformation with very weak signals for an alternative conformation. The VT-NMR analysis of this peptide showed that the conformation contains only a single intramolecular hydrogen bond. The absence of the second hydrogen bond indicates that this molecule has the potentially unfavorable conformation similar to the minor conformer of compound 14.

Figure 8: Docking of 14 with LSD1. 14 is represented in green while the original CoREST1 fragment is shown in orange. To study the binding mode of peptide 14 we performed computational docking by using the Molecular Operating Environment (MOE) software suite. After the generation of a peptide placements set following the protein-protein docking mode, the pose with the lowest score and conformational energy was docked into LSD1. The binding mode observed partially mimics that of the CoREST1 fragment which was used as a template for its design (Fig. 8, rmsd: 0.792Å). The isoleucine is found to be in an almost identical position however a slight twist of the side chains connected to the guanidine is observed. The shift is required for the guanidine to form a salt bridge to Asp 495 of LSD1 and also shifts tyrosine 4 and leucine 6 in their binding modes. The tyrosine is shifted less deep into its pocket but a more dramatic difference is observed for the leucine residue. The leucine side chain has rotated so that only one of the terminal methyl groups contacts LSD1 while the original crystal structure shows both methyl groups to interact. Furthermore, its C-terminal carbonyl group now makes a hydrogen bond interaction with the imidazole nitrogen of histidine 484 of LSD1. Such an interaction is not possible in the original CoREST1 structure due to the flanking amino acids of the leucine residue. 3. Conclusion To modulate the “undruggable” PPI between LSD1 and CoREST1, we set out to use a structure-based strategy to design new modality inhibitors. We started our design process for PPI derived inhibitors by virtual alanine scanning identification of hot-spot residues. Inclusion of original amino acid side chain functionality in the cyclization linker maintained critical interactions while also rigidifying the peptide. Structural information combined with SAR information allowed optimization of the macrocycle by stereochemical inversion of the amino acid in position 2. Combining this approach with macrocycle size scanning identified 14 as an inhibitor of the LSD1-CoREST1 interaction. Further optimization of side chain residues identified napthylalanine in position 4 to improve the affinity. Cterminal truncation of the amide maintained binding affinity while reducing the peptidic nature of the compound. The described research shows that even a challenging target like the LSD1-CoREST1 12

interaction can be addressed through accurate design of peptides derived from one of the binding partners. Conformation is likely to be one of the main driving forces for recognition of shallow binding pockets such as the LSD1 target described here and should be considered during the design of macrocyclic PPI inhibitors. Although further optimization is required to obtain a molecule with the desired potency and cell-permeability properties the strategy can serve as a good way to start the search for inhibitors of difficult PPIs. 4. Experimental section 4.1. General synthetic route A Synthesis typically took place at 0.05 mmol scale on Rink Amide AM resin (low loading, 0.4 mmol/g). Linear synthesis of the peptide was done by using 4 eq amino acid, 4 eq PyBOP and 8 eq of DIPEA in 1.25 mL of DMF for each coupling reaction. Fmoc deprotection was done by adding 1.25 mL of 20% piperidine in DMF and shaking for 5 min followed by addition of fresh reagents and shaking for 10 min. The N-terminus was acetylated by addition of 10 eq Ac2O and 10 eq of DIPEA in 1.25 mL DMF for 30 min. At this stage the method was largely adapted from [37]. With the linear synthesis completed the alloc group was removed by washing the resin with anhydrous CH2Cl2 (2 x 1.25 mL) and resuspending the resin in anhydrous CH2Cl2 (1.25 mL) followed by addition of PhSiH3 (154 μL; 1.24 mmol; 24.8 eq) and Pd(PPh3)4 (14.4 mg; 0.0125 mmol; 0.25 eq). After shaking for 1 h under inert atmosphere, fresh reagents were added and the reaction repeated. The resin was washed with CH2Cl2 (2 x 1.25 mL), DMF (2 x 1.25 mL), 0.5% sodium diethyldithiocarbamate in DMF (4 x 1.25 mL) and DMF (2 x 1.25 mL). The liberated amine was then converted to the isothiocyanate by addition of CS2 (1.0 mL) and DMF (0.5 mL) followed by PyBOP (104 mg; 0.2 mmol; 4 eq) and DIPEA (104.5 μL; 0.6 mmol; 12 eq). After shaking for 30 min the resin was washed with DMF (4 x 1.25 mL) and CH2Cl2 (5 x 1.25 mL). Mtt removal was performed by treating the resin with AcOH/TFE/ CH2Cl2 (1:2:7, 1.25 mL) 6 times for 20 min. The resin was washed with CH2Cl2 (4 x 1.25 mL) and DMF (2 x 1.25 mL) followed by resuspension in DMF (1.25) and addition of DIPEA (51.7 μL; 0.4 mmol; 8 eq) to form the thiourea. The mixture was bubbled through with argon for 1 h and then washed with DMF (4 x 1.25 mL). The resin was then suspended in 0.2 M methyliodide in DMF (1.25 mL) and shaken for 20 min. The treatment was repeated for a total of five times and then washed with DMF (2 x 1.25 mL), CH2Cl2 (2 x 1.25 mL) and diethyl ether (2 x 1.25 mL) followed by drying under vacuum. The dry resin was transferred to a roundbottom flask and DMSO (2 mL) was added. After stirring for 15 min ammonium acetate (154 mg; 2 mmol; 40 eq) and N-methylmorpholine (219 µL; 2 mmol; 40 eq) were added and the mixture was stirred at 80 °C for 16 h. After completion of the solid-phase synthesis the peptides were cleaved using a TFA/TIPS/H2O (95:2.5:2.5, 1 mL) and precipitated in cold diethyl ether (20 mL). The precipitate was collected by centrifugation (3500 rpm, 4 °C, 5 min) and washed twice more with cold ether. The pellet was then lyophilized to yield the crude peptide. Crude peptides were purified using a C18 column eluting with a gradient of 15-60% buffer B at a flow of 20 mL/min (buffer A: H2O + 0.1% TFA, Buffer B: MeCN + 0.1% TFA). Product containing fractions were pooled and lyophilized. 4.2. General synthetic route B Synthesis typically took place at 0.05 mmol scale on Rink Amide AM resin (low loading, 0.4 mmol/g). Linear synthesis of the peptide was done by using 4 eq amino acid, 4 eq PyBOP and 8 eq of DIPEA in 1.25 mL of DMF for each coupling reaction. Fmoc deprotection was done by adding 1.25 mL of 20% piperidine in DMF and shaking for 5 min followed by addition of fresh 13

reagents and shaking for 10 min. Resin containing Fmoc-Orn(Alloc)-Leu-Rink was washed with anhydrous CH2Cl2 (2 x 1.25 mL) and resuspending in anhydrous CH2Cl2 (1.25 mL) followed by addition of PhSiH3 (154 μL; 1.24 mmol; 24.8 eq) and Pd(PPh3)4 (14.4 mg; 0.0125 mmol; 0.25 eq). After shaking for 1 h under inert atmosphere, fresh reagents were added and the reaction repeated. The resin was washed with CH2Cl2 (2 x 1.25 mL), DMF (2 x 1.25 mL), 0.5% sodium diethyldithiocarbamate in DMF (4 x 1.25 mL) and DMF (2 x 1.25 mL). The resin was suspended in anhydrous CH2Cl2 and Pbf-NCS[38] (46.7 mg; 0.15 mmol; 3 eq) was added followed by DIPEA (52.2 uL; 0.3 mmol; 6 eq). The reaction vessel was sealed and shaken for 3 h followed by washing with CH2Cl2 (4 x 1.25 mL). The resin was suspended in anhydrous CH2Cl2 (1.25 mL) and allylamine (18.8 µL; 0.25 mmol; 5 eq) was added followed by EDCI (47.9 mg; 0.25 mmol; 5 eq). The vessel was purged with argon, sealed and shaken for 1 h. The resin was then washed with CH2Cl2 (4 x 1.25 mL). Linear peptide synthesis was continued until the acetylated hexapeptide was obtained. Ring-closing metathesis was performed using Hoveyda-Grubbs 2nd generation catalyst using a method described by Chapman et al.[32] The resin was transferred to a microwave vial and suspended in a solution of the catalyst in DCE (2 mg/mL) so that the final catalyst loading was 10%. The mixture was irradiated at 120 °C for 5 min after which the resin was washed, fresh reagents were added and the reaction repeated. The resin was washed with CH2Cl2 (4 x 1.25 mL) and cleavage and deprotection was performed the same as for method A. 4.3. General synthetic route C Synthesis of C-terminally modified peptides was performed using a method based on that described by Selvaraj et al.[33] Briefly, the Rink Amide AM resin was loaded with 4-amino-3nitrobenzoic acid followed by reduction of the nitro group using SnCl2. The first amino acid was coupled to the resin using and the synthesis of the macrocycle is performed as described for method A but omitting the TFA cleavage. The resin was suspended in DMF (1 mL) and amylnitrite (67.2 µL; 0.5 mmol; 10 eq) was added followed by microwave irradiation at 60 °C for 15 min. The resin was washed with DMF (4 x 1.25 mL) and a solution of the nucleophile (10 eq) and DIPEA (69.7 µL; 0.4 mmol; 8 eq) in DMF (1 mL) which was previously sparged with argon for 20 min was added. The mixture was irradiated in the microwave at 60 °C for 15 min. The resin was drained and the liquid was collected. The resin was washed with CH2Cl2 (4 x 1.25 mL) and all washes were combined with the reaction liquid and evaporated to dryness. The crude material was immediately purified as described for method A. 5. Acknowledgements P. ‘t Hart was supported by a fellowship from the Alexander von Humboldt foundation. H. Waldmann was supported by the Max Planck Society. A. Krzyzanowski thanks Aventis Foundation and Stiftung Stipendien-Fonds of the Verbandes der Chemischen Industrie (VCI) for the financial support through the Hoechst Doctoral Scholarship. The Dortmund protein facility is kindly acknowledged for expressing and purifying the proteins used in this project. Bernhard Griewel is kindly acknowledged for running NMR experiments. Declaration of interest: H. Adihou is employed by AstraZeneca. Other authors have no competing interests.

14

[1]

M. Pelay-Gimeno, A. Glas, O. Koch, T. N. Grossmann, Angew. Chem. Int. Ed. 2015, 54, 8896– 8927.

[2]

E. Valeur, S. M. Guéret, H. Adihou, R. Gopalakrishnan, M. Lemurell, H. Waldmann, T. N. Grossmann, A. T. Plowright, Angew. Chem. Int. Ed. 2017, 56, 10294–10323.

[3]

B. C. Doak, J. Zheng, D. Dobritzsch, J. Kihlberg, J. Med. Chem. 2016, 59, 2312–2327.

[4]

A. K. Yudin, Chem. Sci. 2015, 6, 30–49.

[5]

M. Egbert, A. Whitty, G. M. Keserű, S. Vajda, J. Med. Chem. 2019, acs.jmedchem.8b01732.

[6]

D. E. Scott, A. R. Bayly, C. Abell, J. Skidmore, Nat. Rev. Drug Discov. 2016, 15, 533–550.

[7]

A. Metz, E. Ciglia, H. Gohlke, Curr. Pharm. Des. 2012, 18, 4630–4647.

[8]

E. A. Villar, D. Beglov, S. Chennamadhavuni, J. A. Porco, D. Kozakov, S. Vajda, A. Whitty, Nat. Chem. Biol. 2014, 10, 723–731.

[9]

J. Gavenonis, B. A. Sheneman, T. R. Siegert, M. R. Eshelman, J. A. Kritzer, Nat. Chem. Biol. 2014, 10, 716–722.

[10]

D. M. Krüger, H. Gohlke, Nucleic Acids Res. 2010, 38, W480–W486.

[11]

L. Laraia, G. McKenzie, D. R. Spring, A. R. Venkitaraman, D. J. Huggins, Chem. Biol. 2015, 22, 689–703.

[12]

P. G. Dougherty, Z. Qian, D. Pei, Biochem. J. 2017, 474, 1109–1125.

[13]

Y. Shi, F. Lan, C. Matson, P. Mulligan, J. R. Whetstine, P. A. Cole, R. A. Casero, Y. Shi, Cell 2004, 119, 941–953.

[14]

F. Forneris, C. Binda, M. A. Vanoni, A. Mattevi, E. Battaglioli, FEBS Lett. 2005, 579, 2203–2207.

[15]

A. Maiques-Diaz, T. C. Somervaille, Epigenomics 2016, 8, 1103–1116.

[16]

Y.-J. Shi, C. Matson, F. Lan, S. Iwase, T. Baba, Y. Shi, Mol. Cell 2005, 19, 857–864.

[17]

A. P. Barrios, A. V. Gomez, J. E. Saez, G. Ciossani, E. Toffolo, E. Battaglioli, A. Mattevi, M. E. Andres, Mol. Cell. Biol. 2014, 34, 2760–2770.

[18]

G.-J. Yang, P.-M. Lei, S.-Y. Wong, D.-L. Ma, C.-H. Leung, Molecules 2018, 23, 3194.

[19]

E. M. Michalak, M. L. Burr, A. J. Bannister, M. A. Dawson, Nat. Rev. Mol. Cell Biol. 2019, DOI 10.1038/s41580-019-0143-1.

[20]

R. DesJarlais, P. J. Tummino, Biochemistry 2016, 55, 1584–1599.

[21]

M. G. Lee, C. Wynder, N. Cooch, R. Shiekhattar, Nature 2005, 437, 432–435.

[22]

M. Yang, C. B. Gocke, X. Luo, D. Borek, D. R. Tomchick, M. Machius, Z. Otwinowski, H. Yu, Mol. Cell 2006, 23, 377–387.

[23]

S. Pilotto, V. Speranzini, M. Tortorici, D. Durand, A. Fish, S. Valente, F. Forneris, A. Mai, T. K. Sixma, P. Vachette, et al., Proc. Natl. Acad. Sci. 2015, 112, 2752–2757.

[24]

C. Marabelli, B. Marrocco, A. Mattevi, Curr. Opin. Struct. Biol. 2016, 41, 135–144.

[25]

X. Fu, P. Zhang, B. Yu, Future Med. Chem. 2017, 9, 1227–1242.

[26]

T. B. Nicholson, T. Chen, Epigenetics 2009, 4, 129–132.

[27]

B. Majello, F. Gorini, C. Saccà, S. Amente, Cancers (Basel). 2019, 11, 324. 15

[28]

R. Behrendt, P. White, J. Offer, J. Pept. Sci. 2016, 22, 4–27.

[29]

S. Hwang, A. A. Schmitt, A. E. Luteran, E. J. Toone, D. G. McCafferty, Biochemistry 2011, 50, 546–557.

[30]

Y. Touati-Jallabe, E. Bojnik, B. Legrand, E. Mauchauffée, N. N. Chung, P. W. Schiller, S. Benyhe, M. C. Averlant-Petit, J. Martinez, J. F. Hernandez, J. Med. Chem. 2013, 56, 5964–5973.

[31]

U. Boas, H. Gertz, J. B. Christensen, P. M. H. Heegaard, Tetrahedron Lett. 2004, 45, 269–272.

[32]

R. N. Chapman, P. S. Arora, Org. Lett. 2006, 8, 5825–5828.

[33]

A. Selvaraj, H.-T. Chen, A. Ya-Ting Huang, C.-L. Kao, Chem. Sci. 2018, 9, 345–349.

[34]

F. Forneris, R. Orru, D. Bonivento, L. R. Chiarelli, A. Mattevi, FEBS J. 2009, 276, 2833–2840.

[35]

H. Kessler, Angew. Chem. Int. Ed. 1982, 21, 512–523.

[36]

S. D. Appavoo, T. Kaji, J. R. Frost, C. C. G. Scully, A. K. Yudin, J. Am. Chem. Soc. 2018, 140, 8763–8770.

[37]

Y. Touati-Jallabe, L. Chiche, A. Hamzé, A. Aumelas, V. Lisowski, D. Berthomieu, J. Martinez, J. F. Hernandez, Chem. - A Eur. J. 2011, 17, 2566–2570.

[38]

A. Alliband, F. A. Meece, C. Jayasinghe, D. H. Burns, J. Org. Chem. 2013, 78, 356–362.

16

Highlights: • • • •

Conversion of a CoREST1 derived peptide provided a LSD1-CoREST1 inhibitor A fluorescence polarization assay using synthetic CoREST1 fragments was developed Optimization of the macrocycle by conversion of side chain stereochemistry and functionality led to a low micromolar inhibitor NMR experiments indicate the conformation of the macrocycles is key to affinity

Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

H. Adihou is employed by AstraZeneca.