Converting One-Face α-Helix Mimetics into ... - ACS Publications

Dec 11, 2015 - New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu 701-310, South Korea. ‡. Department of Chemistry ...
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Converting One-Face #-Helix Mimetics into Amphiphilic #-Helix Mimetics as Potent Inhibitors of Protein-Protein Interactions Ji Hoon Lee, Misook Oh, Hyun Soo Kim, Hui Sun Lee, Wonpil Im, and Hyun-Suk Lim ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.5b00080 • Publication Date (Web): 11 Dec 2015 Downloaded from http://pubs.acs.org on December 18, 2015

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Converting One-Face α-Helix Mimetics into Amphiphilic α-Helix Mimetics as Potent Inhibitors of Protein-Protein Interactions Ji Hoon Lee,‡,a Misook Oh,‡,b,c Hyun Soo Kim,b Huisun Lee,d Wonpil Im,d and Hyun-Suk Lim*,b,c a

New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu

701-310, South Korea b

Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang

790-784, South Korea c

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine,

Indianapolis, Indiana 46202, United States d

Department of Molecular Biosciences and Centre for Computational Biology, The University of

Kansas, Lawrence, Kansas 66047, United States ABSTRACT. Many biologically active α-helical peptides adopt amphiphilic helical structures that contain hydrophobic residues on one side and hydrophilic residues on the other side. Therefore, α-helix mimetics capable of mimicking such amphiphilic helical peptides should possess higher binding affinity and specificity to target proteins. Here we describe an efficient method for generating amphiphilic α-helix mimetics. One-face α-helix mimetics having

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hydrophobic side chains on one side was readily converted into amphiphilic α-helix mimetics by introducing appropriate charged residues on the opposite side. We also demonstrate that such two-face amphiphilic α-helix mimetics indeed show remarkably improved binding affinity to a target protein, compared to one-face hydrophobic α-helix mimetics. We believe that generating a large combinatorial library of these amphiphilic α-helix mimetics can be valuable for rapid discovery of highly potent and specific modulators of PPIs.

Keywords: amphiphilic α-helix mimetics, protein-protein interaction inhibitor, solid-phase synthesis, combinatorial library

Introduction α-Helices are the most commonly found form of protein secondary structures and often involved in protein-protein interactions (PPIs) as recognition motifs.1-4 A general feature of such α-helix-mediated PPIs is that short helical peptides spanning 2-3 helical turns make key contacts with interacting proteins, where side chain residues at i, i+3 or i+4, and i+7 positions are often crucial as recognition motifs. Since aberrant α-helix-mediated PPIs are linked to various disease states such as many cancers, there is great interest in developing modulators of such interactions as potential therapeutic candidates. Given the relatively compact PPI interfaces, it is conceivable to develop small-molecule inhibitors of α-helix-mediated PPIs. One such strategy is to design small molecule scaffolds decorated with appropriate functional groups, which can mimic spatial arrangement of critical residues at i, i+3 or i+4, and i+7 positions in α-helical segments.4-7 The most popular scaffold is a terphenyl-like structure developed by Hamilton and co-workers.5,8

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Over the last decade, a number of terphenyl-inspired α-helix mimetics have been developed, and their feasibility as PPI modulators has been successfully demonstrated.4,5,9-27 Most biologically active α-helical peptides adopt amphiphilic helical structures that contain hydrophobic residues on one side and hydrophilic residues on the other side (Figure 1A). Therefore, α-helix mimetics capable of mimicking such amphiphilic helical peptides should possess higher binding affinity and specificity to target proteins. Despite the importance of amphiphilic nature, there are only a few reports on amphiphilic α-helix mimetic scaffolds.10,28-33 Moreover, their biological activity has yet to be fully explored. Here we report an efficient strategy for generating a novel class of amphiphilic α-helix mimetics by converting one-face αhelix mimetics into two-face amphiphilic α-helix mimetics. We also demonstrate that such twoface amphiphilic α-helix mimetics indeed show remarkably improved binding affinity to a target protein, compared to one-face hydrophobic α-helix mimetics.

RESULTS AND DISCUSSION

Recently, we have reported the development of small-molecule α-helix mimetics based on a triazine-piperazine-triazine scaffold (Figure 1B).23 By screening a ~1,500-member combinatorial library of triazine-piperazine-triazines, we have discovered 1 (Figure 1C) as an inhibitor of the Mcl-1/BH3 interaction.23 Compound 1 bears three hydrophobic substituents (R1R3) to mimic the spatial orientations of three key residues (Leu, Val, and Val) at i, i+3, and i+7 positions in the BH3 helical peptide.34 Because this scaffold has two additional substitution sites (R4 and R5) on the opposite side of the R1-R3 positions (Figure 1B), we envisaged that incorporation of hydrophilic functionality at R4 or R5 position would convert 1 into amphiphilic

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α-helix mimetics (Figure 1C). In contrast to our previously reported scaffolds with three residues on a single face,23,24 this newly designed scaffold has five substituents on both sides, thereby enabling to reproduce functionality on both faces of amphiphilic helical peptides. Such two-face amphiphilic α-helix mimetics are expected to have enhanced binding affinity to target proteins due to additional interactions with hydrophilic residues on the target protein surface.

B

A i-2 i i+3

i-2 i

i+1

i+3

i+5

i+7

i+5

i+7

C Introduction of hydrophilic residues at R4 or R5

Amphiphilic α-helix mimetics

Figure 1. (A) X-ray crystal structure of an Mcl-1 binding helical peptide. (B) α-Helix mimetics based on a triazine-piperazine-triazine scaffold and overlay of an energy-minimized structure of a triazine-piperazine-triazine scaffold with α-helix. (C) Structure of 1, an Mcl-1 inhibitor and a strategy to generate amphiphilic α-helix mimetics.

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Table 1. Synthesized amphiphilic α-helix mimetics 1a-t for evaluation as Mcl-1 inhibitors. cpd

R4

R5

cpd

R4

R5

1a

(CH2)2OH

H

1k

Et

(CH2)2OH

1b

(CH2)2CO2H

H

1l

Et

(CH2)2CO2H

1c

(CH2)3CO2H

H

1m

Et

(CH2)3CO2H

1d

(CH2)4CO2H

H

1n

Et

(CH2)4CO2H

1e

(CH2)2NH2

H

1o

Et

(CH2)2NH2

1f

(CH2)3NH2

H

1p

Et

(CH2)3NH2

1g

(CH2)4NH2

H

1q

Et

(CH2)4NH2

1h

(CH2)2NHC(=NH)NH2

H

1r

Et

(CH2)2NHC(=NH)NH2

1i

(CH2)3NHC(=NH)NH2

H

1s

Et

(CH2)3NHC(=NH)NH2

1j

(CH2)4NHC(=NH)NH2

H

1t

Et

(CH2)4NHC(=NH)NH2

To test this idea, we synthesized a series of derivatives of 1 by introducing various side chains with different functional groups at R4 and R5 positions (Table 1). For the synthesis of R4substituted derivatives (1a-j), we modified our previous solid-phase synthesis route (Scheme 1).23 First, 4,6-dichloro-N-(4-phenoxyphenyl)-1,3,5-triazine-2-amine 2 was loaded on Rink amide MBHA resin. The resin-bound triazine 3 was coupled with 2-benzyl-1-Ns-piperazine 4, and the Nosyl (Ns) group was removed by 2-mercaptoethanol. Next, the resin-bound piperazine 5 was reacted with 4-(2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)ethyl)phenol 6 to provide 7. Replacement of chloride on 7 with various amines (R4NH2) followed by cleavage reaction with TFA gave R4-modified derivatives 1a-g. Guanidine derivatives 1h-j were prepared by

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guanylation of resin-bound amines 8e-g in the presence of 1,3-di-Boc-2-methylisothiourea and subsequent cleavage reaction.

Scheme 1. Solid phase synthesis of R4-substituted compounds 1a-j.

For the preparation of R5-modified derivatives (1k-t), we used Pal aldehyde resin (Scheme 2). Ethylamine was coupled to resin by reductive amination reaction to give 9. Next, piperazinesubstituted triazine 10 was introduced, and the chloride on 11 was replaced with 4-(2aminoethyl)phenyl group by reacting with tyramine. After deprotecting Ns group, 4,6-dichloroN-(4-phenoxyphenyl)-1,3,5-triazin-2-amine 2 was coupled to resin-bound piperazine 13.

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Subsequent amination reaction with different primary amines (R5NH2) gave 14k-q. Using the same procedure described in Scheme 1, guanidine derivatives 14r-t were obtained from amine derivatives 14o-q. Cleavage reaction with TFA afforded final products 1k-t (Table 1). The cleaved products were purified by HPLC (Figure S1).

Scheme 2. Solid phase synthesis of R5-substituted compounds 1k-t.

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3 µM 30 µM 100

Mcl-1/BH3 Binding (%)

75

50

25

1t

1r 1s

1p 1q

1n 1o

1l 1m

1j 1k

1h 1i

1f 1g

1d 1e

1b 1c

1 1a

M SO

0 D

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Figure 2. Inhibitory effect of the synthesized compounds 1a-t on the interaction between Mcl1172−320 and a TAMRA-labeled Bak-BH3 peptide. Fluorescence polarization was measured at 3 µM (white bars) and 30 µM (gray bars).

The synthesized compounds (1a-t) were examined for their inhibitory effects on the interaction between Mcl-1172-320 (a deletion construct of human Mcl-1 with amino acids 172-320) and TAMRA-labeled Bak-BH3 peptide (TAMRA-Abu-KALETLRRVGDGVQRNHETAF-NH2) by using a fluorescence polarization (FP) assay in 3 µM and 30 µM concentrations (For details,

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see SI) (Figure 2). For R4-modified derivatives (1a-j), the compounds bearing COOH (1b-c) displayed notably improved antagonizing activity. Particularly, 1c (n = 2) showed best results at both 3 µM and 30 µM, while 1d possessing a longer linker length (n = 4) had no effect. These results suggested that a negatively charged side chain at R4 position make additional interaction with Mcl-1 protein surface. Our computational docking study also predicted that the COOH moiety of 1b is overlaid with Glu7 on the BH3 peptide bound by a hydrophobic pocket on Mcl-1 (Figure S2A). In contrast, the compounds substituted with amino group (1e-f) and guanidino group (1h-j) showed considerably decreased inhibitory activity, indicating that positively charged residues at R4 position interfere with the binding of 1 to Mcl-1 surface. A similar pattern was observed in R5-modified compounds (1k-t) (Figure 2). The carboxylic acid residue at R5 position in 1l was predicted to overlay with Asp14 of the BH3 peptide (Figure S2B). This is in good agreement with a previous X-ray study result showing that the carboxylic acid of Asp14 at i+5 position on the BH3 peptide interacts with Arg263 residue in Mcl-1 through the electrostatic interactions.35,36 In this series of derivatives, the positively charged residues at R5 position (1o-t) had no significant effect in contrast to the corresponding R4-modified derivatives. Based on the screening results (Figure 2), we chose 1b, 1c, and 1l as the best compounds and determined their binding affinity. First, we conducted fluorescence anisotropy assays to determine the binding affinity of the TAMRA-labeled Bak-BH3 peptide to Mcl-1172-320 (Figure S3). The KD value of the peptide was determined to be 375.6 nM, which was comparable with the previous result.23,34 Next, we performed a competitive fluorescence anisotropy assays to examine the capability of the compounds to disrupt the interaction between Mcl-1172-320 and TAMRA-Bak-BH3 peptide. Compared to the original compound 1 (Ki = 10.28 µM), 1b, 1c, and 1l were shown to have improved activity (Ki = 3.83, 4.86, and 5.14 µM, respectively), which are

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comparable with that of a known BH3 peptide (Ki = 1.99 µM) (Figure 3A). Given the significant contribution of COOH moiety either at R4 or R5 position, we anticipated that doubly substituted compounds would have even more increased activity. To test this, we synthesized such derivatives (1u and 1v) (Figure 3B) substituted with two carboxylic acid-containing side chains at both R4 and R5 (Scheme S1). Not surprisingly, 1u and 1v exhibited a remarkably increased antagonizing activity with Ki values of 880 nM and 530 nM, which are >11-fold improvement over the parent compound 1. Further biological studies on these amphiphilic helix mimetics are underway.

A

B

Ki (μM) 10.3 1 3.83 1b 4.86 1c 5.14 1l 0.88 1u 0.53 1v BH3 peptide 1.99

C

0.14

Anisotropy

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0.12 0.10 0.08 0.06 0.1

1

10

100

[Compound],µ µM

Figure 3. (A) Fluorescence anisotropy competition curves for the inhibition of the interaction between a fluorescein-labeled BH3 peptide and Mcl-1172−320 by selected compounds. Error bars represent standard deviation from two independent experiments. (B) Structure of 1u and 1v. (C) A predicted binding mode of 1v in the binding sites of the Mcl-1.

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In summary, we have described an efficient method for generating amphiphilic α-helix mimetics. One-face α-helix mimetics having hydrophobic side chains on one side was readily converted into two-face amphiphilic α-helix mimetics by introducing appropriate charged residues on the opposite side. Three functional groups (R1- R3) on the triazine-piperazine-triazine are anticipated to reproduce spatial arrangement of three hydrophobic amino acid side chains at i, i+3, and i+7 positions, while two additional functional groups (R4 and R5) on the other side would represent hydrophilic side chains at i-2 and i+5 positions of amphiphilic helical peptides. We then demonstrated that such amphiphilic α-helix mimetics indeed showed remarkably improved antagonizing activity on the Mcl-1/BH3 interaction, compared to one-face hydrophobic α-helix mimetics. Taken together, we believe that our scaffold with five functional groups (R1- R5) as amphiphilic α-helix mimetics is expected to serve as an excellent source of highly potent and selective modulators of many PPIs mediated by amphiphilic α-helices. We are currently creating a large combinatorial library of such amphiphilic α-helix mimetics by using structurally diverse building blocks.

EXPERIMENTAL PROCEDURES General Procedure for the Solid-Phase Synthesis. For the synthesis of R4-substituted derivatives (1a-j), Rink amide MBHA resin (100 mg, 75 µmol) was swelled in DMF (2 mL) for 1 h. The Fmoc was deprotected by treating with 20 % piperidine in DMF (2 × 10 min). Beads were drained and thoroughly washed with DMF (3×), MeOH (2×), CH2Cl2 (2×), and DMF (3×). 4,6-Dichloro-N-(4-phenoxyphenyl)-1,3,5-triazin-2-amine 2 was loaded onto the resin in DIPEA (5 equiv) and DMF (1 mL) at room temperature overnight. Next, 2-benzyl-1-((4nitrophenyl)sulfonyl)piperazine 4 (5 equiv) was coupled to the resin-bound triazine derivatives 3

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in the presence of DIPEA (5 equiv) in NMP at 80 ºC overnight, and N-Ns group was removed by treating with 2-mercaptoethanol (20 equiv) and DBU (10 equiv) in DMF at room temperature for 2 hr to provide 5. Next, the resin-bound piperazine 5 was reacted with 4-(2-((4,6-dichloro-1,3,5triazin-2-yl)amino)ethyl)phenol 6 in DIPEA (5 equiv) and NMP at room temperature to afford triazine-piperazine-triazine 7. Chloride on 7 was replaced with various amines (R4NH2, 20 equiv) in NMP at 80 ºC for 12 hr to give tetrasubstituted triazine-piperazine-triazine 8a-g. Guanidine derivatives 1h-j, amino group on 8e-g was guanylated in the presence of 1,3-di-boc-2methylisothiourea (10 equiv) in THF at 50 ºC for 4hr. Cleavage reaction with trifluoroacetic acid (TFA) furnished final products 1a-j. For the synthesis of R5-substituted derivatives (1k-t), ethyl amine (5 equiv, 2M solution in THF) was loaded onto the PAL aldehyde resin (100 mg, 90 µmol) in anhydrous THF (2 mL) containing acetic acid (15 equiv) at room temperature. The reaction mixture was then treated with NaBH(OAc)3 (7 equiv) for 2 hr at room temperature to afford 9. 4-(3-benzyl-4-(4,6dichloro-1,3,5-triazin-2-yl)piperazin-1-yl)-3-nitrobenzenesulfonic acid 10 (5 equiv) was coupled to resin-bound ethylamine 9 in DIPEA (5 equiv) and NMP (1 mL) at 60 ºC overnight. Next, the chloride on 11 was displaced with tyramine (20 equiv) in DIPEA (20 equiv) in NMP at 80 ºC overnight. After deprotecting N-Ns group, 4,6-dichloro-N-(4-phenoxyphenyl)-1,3,5-triazin-2amine 2 (5 equiv) was introduced in DIEA (5 equiv) and NMP at 80 ºC overnight. The Subsequent amination reaction with different primary amines (R5NH2, 20 equiv) in DIPEA and NMP at 80 ºC for 12 hr gave 14k-q. Guanidine derivatives 14r-t were obtained from amine derivatives 14o-q as described above. Cleavage reaction with TFA provided final products 1k-t. 1u and 1v were synthesized on Pal aldehyde resin by the same procedure for the synthesis of 1k-t, except for using β-alanine t-butylester (5 equiv) instead of ethylamine (Scheme

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S1). The cleaved products were purified by reverse-phase HPLC and characterized by 1H-NMR, 13

C-NMR, and HRMS. Protein Purification. The plasmid expressing BH3-binding domain of human Mcl-1172-

320 (amino

acids 172-320) tagged with GST were transformed into BL21 (DE3) E. coli bacterial

cells, and protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. Cell pellets were resuspended in lysis buffer (20 mM Tris, pH 7.2, 250 mM NaCl, complete protease tablet (Roche)) and lysed by sonication. After centrifugation, cell lysates were applied to a 5-mL GSTrap HP column (GE Life Sciences) according to the manufacturer’s instructions. Mcl-1172-320 was obtained by thrombin cleavage reaction, another round of GSTrap HP column, and size exclusion chromatography. Fluorescence Anisotropy Assays. For screening of synthesized compounds 1a-t, 10 nM of TAMRA-labeled Bak-BH3 peptide (TAMRA-Abu-KALETLRRVGDGVQRNHETAF-NH2) was incubated with 800 nM of Mcl-1172-320, in binding buffer (50 mM Tris, 100 mM NaCl, 20 nM of bovine serum albumin, pH 8.0) of a final volume of 60 µL in black Costar 384-well plates at room temperature for 30 min in the dark. Varying concentrations of 1 or 1a-v in 40 µL of binding buffer were added to the mixture. After incubation for 15 min at room temperature, the fluorescence polarization values were measured on a SpectraMax® M5 Multi-Mode Microplate Reader (Molecular Devices). Excitation wavelength was 485 nm, and emission was detected at 535 nm. For competition fluorescence anisotropy assays, first the binding affinity of TAMRABak-BH3 peptide to Mcl-1172-320 was determined by titrating Mcl-1172-320 into 80 nM TAMRABak-BH3 peptide in binding buffer at room temperature for 30 min in the dark. The binding affinity (KD) was determined with non-linear regression and fitting to the following equation in GraphPad Prism v5.0 (San Diego, CA): Y = Bmax × X/(KD + X). Bmax was determined to be

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0.08969. The affinity of TAMRA-Bak-BH3 peptide for Mcl-1 was determined to be 375.6 ± 62.4 nM, which is comparable to the previous value.23 For competition assays, 80 nM of TAMRAlabeled Bak-BH3 peptide was incubated with 800 nM of Mcl-1172-320 in binding buffer of a final volume of 60 µL at room temperature for 30 min. Varying concentrations of selected compounds in 40 µL of binding buffer were added. After incubation for 30 min at room temperature, the fluorescence anisotropy values were measured. The IC50 values of the selected compounds in the competition assays were determined with nonlinear regression and fitting using the following equation in GraphPad Prism v5.0: Y = Bottom + (Top – Bottom)/(1 + 10X-LogIC50). Ki values were calculated by using the following equation: Ki = I50/(L50/KD + P0/KD +1), I50: the concentration of the free inhibitor at 50% inhibition, L50: the concentration of the free labeled ligand at 50% inhibition, P0: the concentration of the free protein at 0% inhibition, KD: the dissociation constant of the protein-ligand complex.37

ASSOCIATED CONTENT Supporting Information Materials and general methods, characterization, LC/MS traces, 1H and 13C NMR spectral data of final products. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E mail: [email protected].

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Author Contribution J.H.L. and M.O. contributed equally Funding This research was supported by the National Science Foundation (NSF CHE-1212720, H.-S.L) and the National Research Foundation of Korea (NRF-2014M3A9D9069711 and NRF2012R1A1A2007768). This research was also supported by XSEDE resources (TGMCB070015N, W.I.). Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank Dr. Loren Walensky (Harvard University) for the plasmid expressing Mcl-1.

REFERENCES (1) Davis, J. M.; Tsou, L. K.; Hamilton, A. D. Synthetic non-peptide mimetics of alpha-helices. Chem. Soc. Rev. 2007, 36, 326-334. (2) Henchey, L. K.; Jochim, A. L.; Arora, P. S. Contemporary strategies for the stabilization of peptides in the alpha-helical conformation. Curr. Opin. Chem. Biol. 2008, 12, 692-697. (3) Bullock, B. N.; Jochim, A. L.; Arora, P. S. Assessing helical protein interfaces for inhibitor design. J. Am. Chem. Soc. 2011, 133, 14220-14223. (4) Azzarito, V.; Long, K.; Murphy, N. S.; Wilson, A. J. Inhibition of alpha-helixmediated protein-protein interactions using designed molecules. Nat. Chem. 2013, 5, 161-173. (5) Cummings, C. G.; Hamilton, A. D. Disrupting protein-protein interactions with non-peptidic, small molecule alpha-helix mimetics. Curr. Opin. Chem. Biol. 2010, 14, 341-346. (6) Lanning, M.; Fletcher, S. Recapitulating the alpha-helix: nonpeptidic, lowmolecular-weight ligands for the modulation of helix-mediated protein-protein interactions. Future. Med. Chem. 2013, 5, 2157-2174.

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(7) Jayatunga, M. K.; Thompson, S.; Hamilton, A. D. alpha-Helix mimetics: outwards and upwards. Bioorg. Med. Chem. Lett. 2014, 24, 717-724. (8) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. Toward proteomimetics: terphenyl derivatives as structural and functional mimics of extended regions of an alpha-helix. J. Am. Chem. Soc. 2001, 123, 5382-5383. (9) Moon, H.; Lim, H. S. Synthesis and screening of small-molecule alpha-helix mimetic libraries targeting protein-protein interactions. Curr. Opin. Chem. Biol. 2015, 24C, 3847. (10) Volonterio, A.; Moisan, L.; Rebek, J., Jr. Synthesis of pyridazine-based scaffolds as alpha-helix mimetics. Org. Lett. 2007, 9, 3733-3736. (11) Maity, P.; Konig, B. Synthesis and structure of 1,4-dipiperazino benzenes: chiral terphenyl-type peptide helix mimetics. Org. Lett. 2008, 10, 1473-1476. (12) Shaginian, A.; Whitby, L. R.; Hong, S.; Hwang, I.; Farooqi, B.; Searcey, M.; Chen, J.; Vogt, P. K.; Boger, D. L. Design, synthesis, and evaluation of an alpha-helix mimetic library targeting protein-protein interactions. J. Am. Chem. Soc. 2009, 131, 5564-5572. (13) Anderson, L.; Zhou, M.; Sharma, V.; McLaughlin, J. M.; Santiago, D. N.; Fronczek, F. R.; Guida, W. C.; McLaughlin, M. L. Facile iterative synthesis of 2,5terpyrimidinylenes as nonpeptidic alpha-helical mimics. J. Org. Chem. 2010, 75, 4288-4291. (14) Kazi, A.; Sun, J.; Doi, K.; Sung, S. S.; Takahashi, Y.; Yin, H.; Rodriguez, J. M.; Becerril, J.; Berndt, N.; Hamilton, A. D.; Wang, H. G.; Sebti, S. M. The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner. J. Biol. Chem. 2011, 286, 9382-9392. (15) Lee, J. H.; Zhang, Q.; Jo, S.; Chai, S. C.; Oh, M.; Im, W.; Lu, H.; Lim, H. S. Novel pyrrolopyrimidine-based alpha-helix mimetics: cell-permeable inhibitors of proteinprotein interactions. J. Am. Chem. Soc. 2011, 133, 676-679. (16) Lee, J. H.; Lim, H. S. Solid-phase synthesis of tetrasubstituted pyrrolo[2,3d]pyrimidines. Org. Biomol. Chem. 2012, 10, 4229-4235. (17) Ravindranathan, P.; Lee, T. K.; Yang, L.; Centenera, M. M.; Butler, L.; Tilley, W. D.; Hsieh, J. T.; Ahn, J. M.; Raj, G. V. Peptidomimetic targeting of critical androgen receptorcoregulator interactions in prostate cancer. Nat. Commun. 2013, 4, 1923. (18) Cao, X.; Yap, J. L.; Newell-Rogers, M. K.; Peddaboina, C.; Jiang, W.; Papaconstantinou, H. T.; Jupitor, D.; Rai, A.; Jung, K. Y.; Tubin, R. P.; Yu, W.; Vanommeslaeghe, K.; Wilder, P. T.; MacKerell, A. D., Jr.; Fletcher, S.; Smythe, R. W. The novel BH3 alpha-helix mimetic JY-1-106 induces apoptosis in a subset of cancer cells (lung cancer, colon cancer and mesothelioma) by disrupting Bcl-xL and Mcl-1 protein-protein interactions with Bak. Mol. Cancer 2013, 12, 42. (19) Naduthambi, D.; Bhor, S.; Elbaum, M. B.; Zondlo, N. J. Synthesis of a tetrasubstituted tetrahydronaphthalene scaffold for alpha-helix mimicry via a MgBr2-catalyzed Friedel-Crafts epoxide cycloalkylation. Org. Lett. 2013, 15, 4892-4895. (20) Peters, M.; Trobe, M.; Tan, H.; Kleineweischede, R.; Breinbauer, R. A modular synthesis of teraryl-based alpha-helix mimetics, part 1: Synthesis of core fragments with two electronically differentiated leaving groups. Chem. Eur. J. 2013, 19, 2442-2449. (21) Lao, B. B.; Grishagin, I.; Mesallati, H.; Brewer, T. F.; Olenyuk, B. Z.; Arora, P. S. In vivo modulation of hypoxia-inducible signaling by topographical helix mimetics. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 7531-7536.

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(22) Barnard, A.; Long, K.; Martin, H. L.; Miles, J. A.; Edwards, T. A.; Tomlinson, D. C.; Macdonald, A.; Wilson, A. J. Selective and potent proteomimetic inhibitors of intracellular protein-protein interactions. Angew. Chem. Int. Ed. 2015, 54, 2960-2965. (23) Oh, M.; Lee, J. H.; Wang, W.; Lee, H. S.; Lee, W. S.; Burlak, C.; Im, W.; Hoang, Q. Q.; Lim, H. S. Potential pharmacological chaperones targeting cancer-associated MCL-1 and Parkinson disease-associated alpha-synuclein. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 1100711012. (24) Moon, H.; Lee, W. S.; Oh, M.; Lee, H.; Lee, J. H.; Im, W.; Lim, H. S. Design, solid-phase synthesis, and evaluation of a phenyl-piperazine-triazine scaffold as alpha-helix mimetics. ACS Comb. Sci. 2014, 16, 695-701. (25) Trobe, M.; Peters, M.; Grimm, S. H.; Breinbauer, R. The Development of a Modular Synthesis of Teraryl-Based alpha-Helix Mimetics as Potential Inhibitors of ProteinProtein Interactions. Synlett 2014, 25, 1202-1214. (26) Azzarito, V.; Miles, J. A.; Fisher, J.; Edwards, T. A.; Warriner, S. L.; Wilson, A. J. Stereocontrolled protein surface recognition using chiral oligoamide proteomimetic foldamers. Chem. Sci. 2015, 6, 2434-2443. (27) Becerril, J.; Hamilton, A. D. Helix mimetics as inhibitors of the interaction of the estrogen receptor with coactivator peptides. Angew. Chem. Int. Ed. 2007, 46, 4471-4473. (28) Marimganti, S.; Cheemala, M. N.; Ahn, J. M. Novel amphiphilic alpha-helix mimetics based on a bis-benzamide scaffold. Org. Lett. 2009, 11, 4418-4421. (29) Thompson, S.; Hamilton, A. D. Amphiphilic alpha-helix mimetics based on a benzoylurea scaffold. Org. Biomol. Chem. 2012, 10, 5780-5782. (30) Jung, K. Y.; Vanommeslaeghe, K.; Lanning, M. E.; Yap, J. L.; Gordon, C.; Wilder, P. T.; Mackerell, A. D., Jr.; Fletcher, S. Amphipathic alpha-Helix Mimetics Based on a 1,2-Diphenylacetylene Scaffold. Org. Lett. 2013, 15, 3234-3237. (31) Lanning, M. E.; Wilder, P. T.; Bailey, H.; Drennen, B.; Cavalier, M.; Chen, L.; Yap, J. L.; Raje, M.; Fletcher, S. Towards more drug-like proteomimetics: two-faced, synthetic alpha-helix mimetics based on a purine scaffold. Org. Biomol. Chem. 2015, 13, 8642-8646. (32) Lao, B. B.; Drew, K.; Guarracino, D. A.; Brewer, T. F.; Heindel, D. W.; Bonneau, R.; Arora, P. S. Rational design of topographical helix mimics as potent inhibitors of proteinprotein interactions. J. Am. Chem. Soc. 2014, 136, 7877-7888. (33) Barnard, A.; Long, K.; Yeo, D. J.; Miles, J. A.; Azzarito, V.; Burslem, G. M.; Prabhakaran, P.; T, A. E.; Wilson, A. J. Orthogonal functionalisation of alpha-helix mimetics. Org. Biomol. Chem. 2014, 12, 6794-6799. (34) Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer. Nat. Chem. Biol. 2010, 6, 595-601. (35) Czabotar, P. E.; Lee, E. F.; van Delft, M. F.; Day, C. L.; Smith, B. J.; Huang, D. C.; Fairlie, W. D.; Hinds, M. G.; Colman, P. M. Structural insights into the degradation of Mcl-1 induced by BH3 domains. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6217-6222. (36) Zhang, Z.; Li, X.; Song, T.; Zhao, Y.; Feng, Y. An anthraquinone scaffold for putative, two-face Bim BH3 alpha-helix mimic. J. Med. Chem. 2012, 55, 10735-10741. (37) Nikolovska-Coleska, Z.; Wang, R. X.; Fang, X. L.; Pan, H. G.; Tomita, Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.; Wang, S. M. Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal. Biochem. 2004, 332, 261-273.

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