Letters Cite This: ACS Chem. Biol. 2018, 13, 53−59
Design and Application of a DNA-Encoded Macrocyclic Peptide Library Zhengrong Zhu,*,† Alex Shaginian,† LaShadric C. Grady,† Thomas O’Keeffe,† Xiangguo E. Shi,† Christopher P. Davie,† Graham L. Simpson,‡ Jeffrey A. Messer,† Ghotas Evindar,† Robert N. Bream,‡ Praew P. Thansandote,‡ Naomi R. Prentice,‡ Andrew M. Mason,‡ and Sandeep Pal‡ †
GlaxoSmithKline, 200 Cambridge Park Dr., Cambridge, Massachusetts 02140, United States GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom
‡
S Supporting Information *
ABSTRACT: A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4−20 natural and non-natural amino acids. Affinitybased selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.
D
Using non-natural amino acids also widens chemical library diversity and thus increases the chance for successful identification of hits in biological assays.8 Among therapeutic target classes that peptide drugs have been applied to, protein−protein interactions (PPIs) are of particular interest. PPIs have long been a challenging therapeutic target class for drug discovery.9,10 Due to the flat and featureless interaction interface between two proteins, it has proven challenging to design small molecule drugs to fit in the interface and disrupt the interaction. Since many PPI targets are located in the cytoplasm or nucleus of cells, it is also very challenging to develop traditional biologic drugs. However, as macrocyclic peptides can often mimic protein interfaces effectively and can still migrate into cells, macrocyclic peptidebased drugs have achieved some success in recent years against this target class.11,12 In addition, these macrocyclic peptides can be valuable tools to study the protein surface, probe the interaction between proteins,13 and, in some cases, allow design of small molecule “peptidomimetics.”14,15 Encoded library technology (ELT) provides a strategy for identifying small molecule compounds that bind protein targets using DNA tagged combinatorial libraries.16−21 Each molecule in the ELT library comprises a drug-like warhead attached to a double stranded DNA coding region through an adapter module (the DNA headpiece). Each cycle of synthetic chemistry to construct the warhead is encoded by ligation of a short double-stranded DNA tag that identifies the building block added. Using split/mix methods, chemical diversity on
rug discovery falls into two major classes of therapeutics: small molecule chemical compounds (molecular weight in the range of 100 to 1000 Da) and biologics or proteins (molecular weight greater than 5000 Da). Each class has its pros and cons. Small molecule drugs are often suitable for oral delivery and may have good cell permeability and thus can be applied to many classes of therapeutic targets. However, small molecules are limited to protein targets with a lipophilic binding pocket and more prone to off-target side effects limiting their utility. Biologic drugs generally have fewer offtarget side effects, can be longer-acting in the body, and are more suited for certain cell surface receptors and protein− protein interactions. However, biologics such as monoclonal antibodies cannot be delivered orally and require a cold chain for their distribution, thus making them only available to developed countries. Since the molecular weight of peptides of 5−50 amino acids are between these two major categories, peptide drugs offer opportunity of combining advantages of both biologics and small molecules as therapeutics.1,2 Peptide drugs may have high specificity and potency similar to biologics but are chemically synthesized and, depending on their composition and molecular properties, can be cell permeable, gaining access to intracellular drug targets. Recently, cell permeable peptides have been discovered that can be used to carry other molecules into cells,3 as well as cyclic penetrant peptides with biological activity.4,5 However, linear peptides made from natural amino acids are subject to proteolysis and are rapidly degraded in vivo. Cyclization of peptides and incorporation of non-natural amino acids can stabilize their backbone, increasing their resistance to proteolysis.6,7 In addition, cyclization reduces the conformational freedom and forces peptides to form a more ordered secondary structure, often leading to higher binding affinity and higher specificity. © 2017 American Chemical Society
Received: September 28, 2017 Accepted: November 29, 2017 Published: November 29, 2017 53
DOI: 10.1021/acschembio.7b00852 ACS Chem. Biol. 2018, 13, 53−59
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Figure 1. DNA-encoded libraries (DELs). DEL-1 macrocycle formed by installation of azidoacetic acid and subsequent click cyclization. A linear control library (DEL-2) was prepared by acylation of the terminal amine with acetic anhydride. These libraries of six cycles contain 2.4 × 1012 members, with each member having 4−20 natural and non-natural amino acids (including two glycines at two termini).
the order of 106 to 109 drug-like warheads can be readily achieved, orders of magnitude more than other lead discovery technologies. The libraries are then screened by affinity-based selection. Binders are separated from nonbinders by multiple rounds of capture of target protein to affinity matrix and heat elution. Then chemical structures of binders are identified by translation of the DNA tagging sequences. Because of the high sensitivity and throughput of modern DNA sequencers, nanomole quantities of the input library, and microgram amounts of the target protein, are sufficient for selection experiments. The low material consumption allows multiple selection conditions to be tested in parallel. Since detection is based on affinity binding, no understanding of the mechanism of action is required. When making ELT libraries, chemical reactions used in ELT must be compatible with DNA tags, which include phosphodiesters, ribose with anomeric linkage, bases of nitrogenous heterocycles, and exocyclic amines. Moreover, all chemical reactions have to be carried out in water to adequately solvate the DNA tags. This does however allow convenient purification of the reaction products to be achieved using an
ethanol precipitation. Sequences of DNA tags are randomly selected to encode building blocks. In order to cover the diverse chemical space of macrocyclic peptides, a macrocyclic library with six cycles of chemistry was made with natural and non-natural amino acids, dipeptides, and tripeptides, resulting in a ring size of 4 to 20 amino acids and a library size of 2.4 × 1012. A linear peptide library of the same size was also made as a control. The size of this library is ∼1000 times larger than the largest that has been previously reported and necessitated a concomitant increase in DNA sequencing coverage of the selection output. The design and synthetic strategy for the macrocyclic DNA-encoded library (DEL-1) and the corresponding linear control DEL-2 is outlined in Figure 1. An affinity-based selection was performed to separate ELT molecules bound to the target protein from unbound. Then, the selection output was PCR amplified and sequenced. On the basis of sequence information on DNA tags, chemical structure information on encoded warheads was determined. Von Hippel−Lindau tumor suppressor (VHL) is an E3 ubiqutin ligase enzyme which is involved in the ubiquitination and degradation of a hypoxia-inducible factor (HIF), a 54
DOI: 10.1021/acschembio.7b00852 ACS Chem. Biol. 2018, 13, 53−59
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over linear peptides and with good predicted permeability and solubility were selected for resynthesis without a DNA tag. Synthesis of the peptides was carried out using FMOC-Rink Amide-Polystyrene resin using the corresponding FMOC− amino acids and cyclization using modified Cu(II)-click chemistry according to Figure 1. Their binding to RSV N was detected by an AS-MS (affinity selection−mass spectrometry) assay. In Table 1, macrocyclic off-DNA peptides showed better binding affinity than corresponding linear peptides, reproducing on-DNA peptide binding patterns in ELT selection. The difference in binding between macrocyclic and linear peptides may be due to reduced rotational freedom and more rigid structure by cyclization. In a TR-FRET (timeresolved fluorescent resonance energy transfer) assay detecting disruption of the binding interaction between RSV N and P proteins, functional activity of these macrocyclic peptides showed good correlation with binding affinity to RSV N (Figure 3). In this study, we have designed and synthesized a macrocyclic ELT library with 2.4 × 1012 cyclic peptides composed of 4−20 natural and non-natural amino acids. As far as we know, it is the largest DNA-encoded library ever made. Testing with two protein−protein interaction targets demonstrates that active macrocyclic peptides can be identified from this large macrocyclic library (confirmed by off-DNA synthesis and testing). This work points to a new direction in discovering active compounds for traditionally intractable targets and fills the gap between small molecule and biologics drug discovery.
transcription factor that plays a central role in the regulation of gene expression by oxygen. VHL is associated with many diseases.22−24 Ligands of VHL can be used in proteolysis targeting chimera technology (PROTAC), a new approach for therapeutic intervention.25 It is known that peptides containing hydroxyproline are binders to VHL.26 Analysis of the macrocyclic peptide ELT library showed that 1.4% of the library members contained the hydroxyproline monomer. Encouragingly, following ELT selection, 92% of hits contained the hydroxyproline monomer, which validated this newly made peptide ELT library, though no preference for any specific sequences. Two putative hits are shown in Figure 2 as
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METHODS
Building Block Validation. A total of 352 Fmoc−amino acid building blocks (BBs) were validated as described herein, and product yield was one of the key factors in selecting the 276 that were incorporated into library synthesis. Fmoc−amino acids (including Fmoc-protected di- and tripeptides BBs) were validated using AOPheadpiece-L-propargylglycine as the substrate. Di- and tripeptide BBs underwent a two-step validation that included (1) acylation of the BB onto AOP-headpiece-L-propargylglycine using standard DMTMM conditions and then (2) Fmoc-deprotection. Product yields of the 40 di- and nine tripeptide BBs incorporated into the library ranged from 65 to 100% yield based on negative ion mass spectrometry (HPLC/ESI-MS; Thermo Fisher Scientific LCQ Advantage or Bruker uTOF). The remaining BBs underwent a four-step validation that included (1) acylation of the BB onto AOP-headpiece-L-propargylglycine using standard DMTMM conditions, (2) Fmoc-deprotection, (3) acylation of the product with Fmoc-Phe using standard DMTMM conditions, and (4) Fmoc deprotection. Four-step product yields of the 227 BBs incorporated into the library ranged from 50 to 95% based on negative ion mass spectrometry. BB validation results are summarized in Supporting Information (SI) Table 1. DNA Tag Validation. Sequences of DNA tags are randomly selected to encode building blocks (SI Table 2). All DNA tags were purchased from Biosearch Technologies (Novato, CA). Average purity of the DNA tags is 80% (minimum purity for QC: 60%) determined by the UPLC/MS method. After library synthesis was completed, PCR was performed to generate DNA sequences compatible with Illumina sequencing flowcells from DNA-encoded molecules in the library. The PCR output was purified using Agencourt AMPure XP SPRI beads according to the manufacturer’s instructions and then quantitated on an Agilent BioAnalyzer using a high sensitivity DNA kit. The final concentration of amplicons for each sample was between 3 and 40 nM. Portions of this material were loaded to generate 20 million clusters on an Illumina GAII or HiSeq platform. The resulting sequence information was used to ensure all DNA tags in the library were intact and evenly represented (what we refer to as “dilute library sequencing” (DLS) QC).
Figure 2. Two examples of macrocyclic peptides containing hydroxyproline bound to VHL identified through ELT selection.
examples. Since it is well-known that peptides containing hydroxyproline are binders to VHL, these peptides were not synthesized off-DNA to confirm activity but potentially could serve as useful tools to probe the VHL:HIF protein−protein interaction. Interaction between respiratory syncytial virus (RSV) Nprotein and P-protein plays a vital role in the replication of RSV.27 Compounds disrupting this protein−protein interaction may be a treatment for RSV infection.28 An ELT selection with both macrocyclic and linear peptide ELT libraries was run against RSV N protein. Data analyses were performed based on ELT selection data and predicted cell permeability and solubility.29 Due to concern that the cyclization reaction for some macrocyclic peptides may not be complete, four macrocyclic peptides with a 10-fold preference for cyclized 55
DOI: 10.1021/acschembio.7b00852 ACS Chem. Biol. 2018, 13, 53−59
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Table 1. Structure, Binding Affinity to RSV N Protein, and Inhibition of RSV N−P Binding of Macrocyclic and Linear Peptides
Synthesis of Macrocyclic Peptide DNA-Encoded Library. As shown in Figure 1, following installation of Fmoc-L-propargylglycine (Fmoc-Pra−OH) onto the AOP-headpiece, six encoded cycles incorporating Fmoc−amino acids, Fmoc-protected di- and tripeptides, as well as encoded nulls (deletions) in cycles 2−5 were carried out using DMTMM-promoted acylation and Fmoc-deprotection conditions analogous to what has been previously published16 (scale ranging from 5 μmol per building block for cycle 1 to 0.5 μmol per building block for cycle 6). A portion of this postcycle 6 material was converted to macrocyclic DEL-1 via acylation with azidoacetic acid and subsequent intramolecular click cyclization, and DEL-2, the noncyclized control library, was prepared by acetylation of the terminal amine with acetic anhydride. Standard click macrocyclization conditions used in the synthesis of DEL-1: To a microtube containing a solution of on-DNA alkyne/azide substrate (1 mM in 250 mM pH 9.4 sodium borate buffer) was added 4 equiv of CuSO4·5H20 (200 mM in water), followed by 4 equiv of sodium ascorbate (200 mM in water). The reaction mixture was briefly vortexed and then heated at 60 °C for 30 min. When azidoacetic acid was attached to the Cterminal amide group, we found side chains of Asn and Gln did not need to be protected. ELT Selection. ELT selection was performed as previously described with some modifications.16,30,31 HIS-tagged VHL or RSV N protein (5 μg) was immobilized on 5 μL of IMAC resins (Phynexus). The DEL1 or DEL2 library (2.5 nmol,
Figure 3. Correlation between binding to RSV N protein and inhibition of RSV N−P binding (at 10 μM peptide) for macrocyclic and linear peptides. Binding to RSV N protein was determined by ASMS assay and inhibition of RSV N−P binding was determined by TRFRET assay.
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ACS Chemical Biology 100 copies per member in this library of 2.4 × 1012 members) in 60 μL of selection buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20, 0.1 mg mL−1 sheared salmon sperm DNA (Ambion)) was incubated with the immobilized protein for 1 h at RT, then washed five times with 100 μL of selection buffer to remove unbound DEL molecules. To elute bound molecules, resins were incubated in 60 μL of selection buffer at 80 °C for 10 min. The eluent was incubated with 5 μL of IMAC resins for 22 min to remove denatured protein before the next round of selection. This process was repeated two additional times. The same procedure was followed for no target control, except naked IMAC resins were used instead of IMAC resins with protein immobilized. After the copy number of selection output was determined by qPCR, the appropriate number of PCR cycles was selected to add DNA sequences compatible with Illumina sequencing flowcells. PCR output is purified using Agencourt AMPure XP SPRI beads according to the manufacturer’s instructions and then quantitated on an Agilent BioAnalyzer using a high sensitivity DNA kit. The final concentration of amplicon for each sample is between 3 and 40 nM. Portions of products were loaded to generate 20 million clusters on an Illumina GAII or HiSeq platform. On the basis of sequence information on DNA tags, chemical structures were obtained. Analysis of ELT Selection Data. Enriched features were identified as described previously.32 Background subtraction was performed by removing any feature from DEL1 or DEL2 selections that contained one or more copies enriched in the no target control. Full macrocycle structures were selected that had three or more copies with a 10-fold preference for cyclized. Further selection of peptides was done by 3D conformational search using Low MoDE MD (CCG MOE software) and summarized in SI Table 3 and SI Figure 1. The solvation energy is calculated using Poisson−Boltzmann calculations with two different dielectric constants, 80 and 4, to mimic the water and the membrane environment. The 3D conformation of a peptide with the lowest penalty in diffusing from the water to the membrane environment was chosen among all the 3D conformations as given by LowMode MD (svmChromLogD 4−6).33 The peptides were also ranked based on solvation energy and the more traditional LogD/CMR permeability measurements as well.34 Aimed to identify active peptides with a balance between permeability and solubility, cyclic peptides in Table 1 were selected based on following criteria: (1) SVMchromlogD range between 4 and 633 (2) most compounds nearer to the discrimination line34 (3) solvation energy is less than −40 kcal/mol so that compounds are predicted to be water-soluble34 (4) most of the compounds are between the chromlogD/cmr and chromlogD/solubility line (the golden triangle)34 On the basis of consideration of chemical diversity, four cyclic peptides were selected for synthesis. There was minor modification of the structure due to the availability of building blocks and concern of feasibility of chemical synthesis. In addition, their linear peptides were synthesized as a control (Table 1). Synthesis of Off-DNA Linear and Macrocyclic Peptides. Linear and cyclic peptides 3−10 (Table 1) were synthesized using the general method: Linear peptides were synthesized using the Liberty 1 Microwave Peptide Synthesizer using Rink-AM resin (200−400 mesh, Merck Millipore, 0.2 mmol scale) and standard FMOC−amino acid coupling conditions with FMOC-AA (0.5 mmol, 0.2 M in NMP), HCTU (0.5 mmol, 0.5 M in DMF), and DIPEA (1 mmol, 1 M in DMF). Half of the peptide material was cleaved from the resin using TFA-TIPS-H2O (2 mL, 95:2.5:2.5) to provide the linear peptides 4, 6, 8, and 10 as the trifluoroacetate salts after purification using RP-HPLC (0.1%TFA, MeCN/H2O). The remaining resin was transferred to a microwave vial and cyclized by adding a solution of water/tert-butanol (1:1, 3 mL), copper(II) sulfate pentahydrate (1.500 mmol), and sodium L-ascorbate (1.500 mmol). The resin was filtered and washed with water, DCM, and DMF and dried and the peptide material cleaved using TFA-TIPS-H2O (2 mL, 95:2.5:2.5). The macrocyclic peptides 3, 5, 7, and 9 were provided after purification as the
trifluoroacetate salts using RP-HPLC (0.1% TFA, MeCN/H2O). Characterization of synthesized peptides are summarized in SI Table 4. AS-MS Assay. We have developed an AS-MS assay in house, which was similar to SpeedScreen and SEC-TID technologies reported in the literature.35,36 Mixtures containing peptides and RSV N protein were prepared by combining 2 μL of 100 μM of peptide solution with 20 μL of 10 μM of RSV N protein in a 384-well assay plate. Duplicate samples thus prepared were incubated for 60 min at RT, then chilled to 4°C prior to AS-MS analysis. Control experiments were conducted for each compound or mixture to confirm that any unbound ligand was trapped by the stationary phase, and only the protein-bound ligand is eluted for analysis (i.e., no chromatographic breakthrough is occurring). Bio-Rad P10 resins were swelled in water at 4 °C over 12 h. The 384-well filter plates (cat. MZHVN0W10; EMD-Millipore, Billerica, MA) were loaded with 130 μL of P10 resin slurry per well. The plates were spun at 1000g for 2 min. Then, the plates were rinsed with selection buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.4) three times. The sample volume transferred was 18 μL. Samples were transferred from the assay plate using a Biomek FX (Beckman Coulter) equipped with a 384-well head. SEC assemblies (SEC plate and collection plate) were then spun for 2 min at 1000g. Then, 9 μL of a 1:1 mixture of DMSO and acetonitrile was added to each well in the collection plate. Total volume in each well was 27 μL. SEC plates were discarded, and collection plates were prepped for LC-MS detection. Then, ligands were dissociated from the complex and were desalted and eluted into a Thermo LTQ linear trap mass spectrometer (Thermo, Billerica, MA). The sample volume injected was 6 μL. Chromatography consisted of a C18 guard column and an analytical column (Kinetex C18, 1.7u, 100A, 2.1 × 50 mm, Phenomenex, Torrance, CA) eluted with a gradient of A (0.1% formic acid in water) versus B (100% methanol) at a 1 mL/min flow rate. The molecule peak area, obtained from the mass spectrometer, represented a signal. MS analysis was performed with positive mode ionization occurring from a standard nebulized ESI source with the capillary at 3.5 kV, a desolvation temperature of 180 °C, a source temperature of 100 °C, and a 30 V “cone” and 3 V extraction lens settings. TR-FRET Assay. The assay was run in buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 2 mM CHAPS, 15 mM DTT, and 0.05% BSA. In a Greiner Black FIA 384-well Plate (cat. 784076) containing compound (100 nl in 100% DMSO), 5 μL of 24 nM RSV N protein/14 nM d2-anti-His Ab (CisBio cat. 61HisDLB) was added; then 5 μL of 6 nM Biotin-RSV P/6 nM Eu-Streptavidin (PerkinElmer cat. AD0063) was added. After incubation for 1 h at RT, plates were read on a PerkinElmer Viewlux, using the following settings: measurement time = 20 s, excitation filter = 340/10 nm, emissions filters = 618/8 nm and 671/8 nm, delay time = 50 μs, and read time = 354 μs. Data were reported as the ratio of the D2 counts (at 660 nm)/ Eu counts (610 nm). The IC50 value was determined by using ActivityBase of the XC50 module (SI Figure 2).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00852.
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SI Figures 1 and 2, SI Tables 1−4 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Zhengrong Zhu: 0000-0001-6766-8927 Jeffrey A. Messer: 0000-0001-6636-9642 Notes
The authors declare no competing financial interest. 57
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(18) Arico-Muendel, C. (2016) From haystack to needle: finding value with DNA encoded library technology at GSK. MedChemComm 7, 1898−1909. (19) Zimmermann, G., and Neri, D. (2016) DNA-encoded chemical libraries: foundations and applications in lead discovery. Drug Discovery Today 21, 1828−1834. (20) Yuen, L. H., and Franzini, R. M. (2017) Achievements, Challenges, and Opportunities in DNA-Encoded Library Research: An Academic Point of View. ChemBioChem 18, 829−836. (21) Goodnow, R. A., Dumelin, C. E., and Keefe, A. D. (2016) DNAencoded chemistry: enabling the deeper sampling of chemical space. Nat. Rev. Drug Discovery 16, 131−147. (22) Maher, E. R., Neumann, H. P., and Richard, S. (2011) von Hippel-Lindau disease: a clinical and scientific review. Eur. J. Hum. Genet. 19, 617−623. (23) Moch, H. (2008) Von-Hippel-Lindau (VHL) protein function by initiation and progression of renal cancer. Pathologe 29, 149−152. (24) Czyzyk-Krzeska, M. F., and Meller, J. (2004) von Hippel-Lindau tumor suppressor: not only HIF’s executioner. Trends Mol. Med. 10, 146−149. (25) Toure, M., and Crews, C. M. (2016) Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew. Chem., Int. Ed. 55, 1966−1973. (26) Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G., and Pavletich, N. P. (2002) Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science 296, 1886−1889. (27) Oliveira, A. P., Simabuco, F. M., Tamura, R. E., Guerrero, M. C., Ribeiro, P. G. G., Libermann, T. A., Zerbini, L. F., and Ventura, A. M. (2013) Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells. Virus Res. 177, 108−112. (28) Bird, G. H., Boyapalle, S., Wong, T., Opoku-Nsiah, K., Bedi, R., Crannell, W. C., Perry, A. F., Nguyen, H., Sampayo, V., Devareddy, A., Mohapatra, S., Mohapatra, S. S., and Walensky, L. D. (2014) Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection. J. Clin. Invest. 124, 2113−2124. (29) Thansandote, P., Harris, R. M., Dexter, H. L., Simpson, G. L., Pal, S., Upton, R. J., and Valko, K. (2015) Improving the passive permeability of macrocyclic peptides: Balancing permeability with other physicochemical properties. Bioorg. Med. Chem. 23, 322−327. (30) Kollmann, C. S., Bai, X., Tsai, C.-H., Yang, H., Lind, K. E., Skinner, S. R., Zhu, Z., Israel, D. I., Cuozzo, J. W., Morgan, B. A., Yuki, K., Xie, C., Springer, T. A., Shimaoka, M., and Evindar, G. (2014) Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists. Bioorg. Med. Chem. 22, 2353−2365. (31) Arico-Muendel, C., Zhu, Z., Dickson, H., Parks, D., Keicher, J., Deng, J., Aquilani, L., Coppo, F., Graybill, T., Lind, K., Peat, A., and Thomson, M. (2015) Encoded library technology screening of hepatitis C virus NS4B yields a small-molecule compound series with in vitro replicon activity. Antimicrob. Agents Chemother. 59, 3450− 3459. (32) Wu, Z., Graybill, T. L., Zeng, X., Platchek, M., Zhang, J., Bodmer, V. Q., Wisnoski, D. D., Deng, J., Coppo, F. T., Yao, G., Tamburino, A., Scavello, G., Franklin, G. J., Mataruse, S., Bedard, K. L., Ding, Y., Chai, J., Summerfield, J., Centrella, P. A., Messer, J. A., Pope, A. J., and Israel, D. I. (2015) Cell-based selection expands the utility of DNA-encoded small-molecule library technology to cell surface drug targets: Identification of novel antagonists of the NK3 tachykinin receptor. ACS Comb. Sci. 17, 722−731. (33) Thansandote, P., Harris, R. M., Dexter, H. L., Simpson, G. L., Pal, S., Upton, R. J., and Valko, K. (2015) Improving the passive permeability of macrocyclic peptides: Balancing permeability with other physicochemical properties. Bioorg. Med. Chem. 23, 322−327. (34) Johnson, T. W., Dress, K. R., and Edwards, M. (2009) Using the Golden Triangle to optimize clearance and oral absorption. Bioorg. Med. Chem. Lett. 19, 5560−5564. (35) Zehender, H., and Mayr, L. M. (2007) Application of highthroughput affinity-selection mass spectrometry for screening of
ACKNOWLEDGMENTS We thank C. Phelps and C. Arico-Muendel for helpful insights, K. Valko and H. Dexter for help with peptide synthesis, and A. Goetz for help with assay data.
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REFERENCES
(1) Danho, W., Swistok, J., Khan, W., Chu, X. J., Cheung, A., Fry, D., Sun, H., Kurylko, G., Rumennik, L., Cefalu, J., Cefalu, G., and Nunn, P. (2009) Opportunities and challenges of developing peptide drugs in the pharmaceutical industry. Adv. Exp. Med. Biol. 611, 467−469. (2) Vlieghe, P., Lisowski, V., Martinez, J., and Khrestchatisky, M. (2010) Synthetic therapeutic peptides: science and market. Drug Discovery Today 15, 40−56. (3) Jafari, S., Maleki Dizaj, S., and Adibkia, K. (2015) Cellpenetrating peptides and their analogues as novel nanocarriers for drug delivery. BioImpacts 5, 103−111. (4) Upadhyaya, P., Qian, Z., Selner, N. G., Clippinger, S. R., Wu, Z., Briesewitz, R., and Pei, D. (2015) Inhibition of Ras signaling by blocking Ras-effector interactions with cyclic peptides. Angew. Chem., Int. Ed. 54, 7602−7606. (5) Hewitt, W. M., Leung, S. S. F., Pye, C. R., Ponkey, A. R., Bednarek, M., Jacobson, M. P., and Lokey, R. S. (2015) Cellpermeable cyclic peptides from synthetic libraries inspired by natural products. J. Am. Chem. Soc. 137, 715−721. (6) Marsault, E., and Peterson, M. L. (2011) Macrocycles are great cycles: applications, opportunities, and challenges of synthetic macrocycles in drug discovery. J. Med. Chem. 54, 1961−2004. (7) Giordanetto, F., and Kihlberg, J. (2014) Macrocyclic drugs and clinical candidates: what can medicinal chemists learn from their properties? J. Med. Chem. 57, 278−295. (8) Stevenazzi, A., Marchini, M., Sandrone, G., Vergani, B., and Lattanzio, M. (2014) Amino acidic scaffolds bearing unnatural side chains: an old idea generates new and versatile tools for the life sciences. Bioorg. Med. Chem. Lett. 24, 5349−5356. (9) Wells, J. A., and McClendon, C. L. (2007) Reaching for highhanging fruit in drug discovery at protein-protein interfaces. Nature 450, 1001−1009. (10) Drews, J. (2000) Drug discovery: a historical perspective. Science 287, 1960−1964. (11) Giordanetto, F., and Kihlberg, J. (2014) Macrocyclic drugs and clinical candidates: What can medicinal chemists learn from their properties? J. Med. Chem. 57, 278−295. (12) Doak, B. C., Zheng, J., Dobritzsch, D., and Kihlberg, J. (2016) How beyond rule of 5 drugs and clinical candidates bind to their targets. J. Med. Chem. 59, 2312−2327. (13) Cardote, T. A. F., and Ciulli, A. (2016) Cyclic and macrocyclic peptides as chemical tools to recognise protein surfaces and probe protein-protein interactions. ChemMedChem 11, 787−794. (14) Fischer, P. M. (2006) Peptide, peptidomimetic, and smallmolecule antagonists of the p53-HDM2 protein-protein interaction. Int. J. Pept. Res. Ther. 12, 3−19. (15) Bajwa, N., Liao, C., and Nikolovska-Coleska, Z. (2012) Inhibitors of the anti-apoptotic Bcl-2 proteins: a patent review. Expert Opin. Ther. Pat. 22, 37−55. (16) Clark, M. A., Acharya, R. A., Arico-Muendel, C. C., Belyanskaya, S. L., Benjamin, D. R., Carlson, N. R., Centrella, P. A., Chiu, C. H., Creaser, S. P., Cuozzo, J. W., Davie, C. P., Ding, Y., Franklin, G. J., Franzen, K. D., Gefter, M. L., Hale, S. P., Hansen, N. J. V., Israel, D. I., Jiang, J., Kavarana, M. J., Kelley, M. S., Kollmann, C. S., Li, F., Lind, K., Mataruse, S., Medeiros, P. F., Messer, J. A., Myers, P., O’Keefe, H., Oliff, M. C., Rise, C. E., Satz, A. L., Skinner, S. R., Svendsen, J. L., Tang, L., Van Vloten, K., Wagner, R. W., Yao, G., Zhao, B., and Morgan, B. A. (2009) Design, synthesis and selection of DNAencoded small-molecule libraries. Nat. Chem. Biol. 5, 647−654. (17) Kleiner, R. E., Dumelin, C. E., and Liu, D. R. (2011) Smallmolecule discovery from DNA-encoded chemical libraries. Chem. Soc. Rev. 40, 5707−5717. 58
DOI: 10.1021/acschembio.7b00852 ACS Chem. Biol. 2018, 13, 53−59
Letters
ACS Chemical Biology chemical compound libraries in lead discovery. Expert Opin. Drug Discovery 2, 285−294. (36) Salcius, M., Bauer, A. J., Hao, Q., Li, S., Tutter, A., Raphael, J., Jahnke, W., Rondeau, J.-M., Bourgier, E., Tallarico, J., and Michaud, G. A. (2014) SEC-TID: A label-free method for small-molecule target identification. J. Biomol. Screening 19, 917−927.
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DOI: 10.1021/acschembio.7b00852 ACS Chem. Biol. 2018, 13, 53−59
