combinatorial chemistry online volume 11 issue 3 march 2009

Combinatorial Chemistry - An Online Journal 11 (2009) 9–12 Contents lists available at ScienceDirect Combinatorial Chemistry - An Online Journal journal homepage: www.elsevier.com/locate/comche Combinatorial Chemistry Online Volume 11, Issue 3, March 2009 N K Terrett Ensemble Discovery Corp., Cambridge, MA 02139, USA Current literature highlights 1.1 Selective substrates of proteinase produced through the use of combinatorial chemistry Human neutrophil elastase, cathepsin G and proteinase are synthesised and stored in the azurophilic granules of polymorphonuclear neutrophils These members of the serine protease family are involved in several physiological processes such as degradation of the components of the extracellular matrix (e.g elastin, collagen), and the control of cytokine activity (e.g tumour necrosis factor-a) Recent work has revealed that only proteinase is involved in the regulation of cell proliferation, and thus this 30kDa enzyme is an excellent target for drug discovery However, due to high sequence identity and nearly identical primary specificity with human neutrophil elastase, it is currently difficult to discriminate between the effects of these two proteinases Published efforts to date that attempt to provide some discrimination have relied on the use of fluorogenic substrates to investigate the prime side of the enzyme catalytic site where the main structural differences between proteinase and elastase are located Based on the crystal structures of both enzymes, it is clear that the proteinase substrate binding site is much more polar than in the case of elastase Specifically, proteinase prefers a combination of charged and/or polar residues in the first three prime positions, whereas elastase has a preference for small aliphatic nonpolar amino acid residues in the same region In order to advance knowledge of how one might better discriminate between these proteinases, recent work has disclosed efforts on the synthesis and kinetic evaluation of a tripeptide library with general formula ABZ-X3-X2-X1-ANB-NH2 that has been designed to select fluorogenic substrates of proteinase 3.1 In this general formula, ABZ is an amino benzoic acid, ANB-NH2 is an amide of 5-amino-2-nitrobenzoic acid, X2 and X3 are the set of all proteinogenic amino acids except Cys, and X1 can be any of the amino acids Ala, Abu, Val, Nva, Ser, Thr, Ile, Leu, and Nle In this study, the ABZ and ANB-NH2 groups at the N- and C-termini of peptides synthesised served as donor and acceptor respectively, for fluorescence resonance energy transfer (FRET) within the peptides Sequences in the library that were selectively and efficiently hydrolysed by proteinase were detected by a loss of their FRET signal To determine the influence of modification, peptides with 5-amino-2-nitrobenzoic acid (ANB) and para-nitroanilide were synthe- E-mail: nterrett@ensemblediscovery.com doi:10.1016/j.comche.2009.02.001 sised and their activity as substrates for proteinase and human neutrophil elastase activity were evaluated After solid-phase synthesis of library members, followed by deconvolution, the isolated peptides were assayed for the ability to resist enzymatic hydrolysis by either proteinase or human neutrophil elastase Among peptides present in the library, the most active fluorogenic substrates of proteinase were selected and a determination of proteinase substrate specificity and human neutrophil elastase substrate activity of selected library members was carried out One of the peptides with the highest specificity constant for proteinase ( kcat/ KM =189 Â 103 M-1 s-1) was ABZ-Tyr-Tyr-Abu-ANB-NH2 This compound represents the first example of a short peptide that undergoes selective proteolysis by proteinase and displays no significant hydrolysis in the presence of human neutrophil elastase This approach provides methods for further investigation to broaden the SAR in this series, as well as progress towards more drug-like compounds 1.2 Screening galectin ligands from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy The lectin family of galectins have been implicated in causing undesired biological interactions As they are involved in processes such as angiogenesis, tumour spread and tumour invasion, the human galectins and are attractive targets for drug discovery In particular, interactions mediated by lectin–glycan recognition have become a target for inhibitor development Recently, library screening approaches to detect sugar-based inhibitors have been undertaken Galectins themselves are involved in both carbohydrate–protein interactions as well as protein–protein interactions, and thus glycopeptide ligands may prove to be versatile in the development of synthetic inhibitors Glycopeptides may be particularly useful in modulating galectin binding because the carbohydrate moiety provides specificity, whereas the peptide backbone may actively participate in binding by hydrogen bonding or hydrophobic interactions Glycopeptides can be generated in a library format via a combinatorial approach Several such libraries have been generated via this strategy to date, and have allowed the identification of effective mimics More recently, the systematic examination of the capacity of human galectins 1- and 3-binding glycopeptides to interfere with lectin–glycan interaction by surface plasmon resonance has been reported.2 Several solid-phase libraries were synthesised on PEGA1900 resin (0.2 mmol/g loading, 300–500 lM) and Wang resin (0.68 mmol/g loading, 200–400 mesh, pre-functionalised with a Rink amide linker Compounds were synthesised by the split-and-mix 10 N K Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 9–12 methodology and screened at room temperature The beads (approximately 25 mg, approximately 10,500 in number) were incubated with the fluorescently labelled galectin at a concentration of 1.5 lM The most fluorescent beads were manually collected and analysed by matrix-assisted laser desorption/ionisation time-offlight mass spectrometry (MALDI–TOF MS) To systematically define the inhibitory potency of a selection of resynthesised glycopeptides, a surface plasmon resonance-based inhibition assay with immobilised asialofetuin was used For active mixtures, re-synthesis and screening as singletons followed From this approach, a number of active compounds were obtained One of the most active glycopeptides was (iPIF(Lac-)TRR) which possessed an IC50 of 25 lM for inhibition of galectin binding In this glycopeptide, P denotes the amino acid Pro, I is Ile, F is Phe, T is Thr, R is Arg, and Lac is the sugar moiety Lac(b1-O)-coupled to the threonine Compounds synthesised via this library chemistry have revealed the beneficial effects of presenting the carbohydrate ligand in a distinct hexapeptide context A summary of the papers in this month’s issue 2.1 Solid-phase synthesis An efficient approach has been developed for the immobilisation of a series of analogues of aloisine A, an in vitro inhibitor of protein kinases, to polymeric supports via a [3 + 2] cycloaddition reaction.3 The efficiency of the pyruvoyl group as an amino protecting group in solid phase peptide synthesis has been investigated Both Na-fluorenylmethoxycarbonyl and (Fmoc)-Ne-pyruvoyl-lysine were synthesised and introduced into peptides and glycopeptides by the ordinary Fmoc-based solid phase peptide synthesis The pyruvoyl peptide could be condensed with a peptide thioester, and was easily removed by o-phenylenediamine treatment without significant side reactions.4 Seven- and 10-membered cyclic thioenamino peptides have been synthesised by both solid and solution-phase methods, and the structure of one compound has been identified by X-ray diffraction analysis of its TFA salt.5 Substituted hydrazines are precursors for many compounds of considerable scientific value The synthesis of N’-substituted 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz) protected hydrazines has been described, and using these materials, a general approach for the solid phase synthesis of aza-peptides has been developed.6 2.2 Solution-phase synthesis No papers this month 2.3 Scaffolds and synthons for combinatorial libraries A simple and convenient synthesis of orthogonally protected multi-tethered, optically pure 2-ketopiperazine, diketopiperazine, 2-ketodiazepane and 3-aminopyrrolidone scaffolds for Fmoc combinatorial chemistry has been developed The approach uses accessible chiral amino acid precursors, sequentially applying reductive alkylation, dipeptide coupling and regioselective ring formation as key steps These scaffolds provide expansion of the range of privileged building blocks, and can introduce valuable drug-like properties in three independent directions to any medicinally relevant piperazine-, diazepane- and pyrrolidone-based motif.7 2.4 Solid-phase supported reagents In addition to their high affinity for inorganic cations, crown ethers have been shown to efficiently sequester ammonium ions, forming a stable adduct via hydrogen bonding The successful im- mobilisation of an 18-crown-6 ether derivative onto a solid support has been reported and these have been incorporated into a continuous flow reactor whereby the sequestration of a series of primary amine salts has been demonstrated.8 Copper (I), copper (II) and scandium (III) triflate complexes of BOX and PYBOX ligands have been electrostatically immobilised on silica The performance of the immobilised catalysts in two carbonyl-ene reactions has been studied, and it compares very well with that of their homogeneous equivalents.9 2.5 Novel resins, linkers and techniques Combinatorial peptide libraries prepared by split-and-mix synthesis on solid support can be decoded by amino acid analysis (AAA) using the TAGSFREE method, which assigns variable amino acids to ‘unique pair’ positions The method has been used to investigate on-bead cyclisation in a library of 15,625 octapeptides X8X7X6X5X4-Lys-X2-glu(b-Ala-b-Ala-TentaGel Macrobead)-OAllyl, anchored via the side-chain carboxylate of the d-glutamate Cyclisation was carried out by amide bond formation between the free N-terminus and the a-carboxyl group of d-glutamate after selective removal of the Fmoc and allyl protecting groups It was found that fast-cyclising sequences often contained a turn element, and gave higher preparative yields of cyclic peptides.10 2.6 Library applications High-throughput screening of a library of diverse molecules has identified the 1,4-naphthoquinone scaffold as a new class of Hsp90 inhibitors The synthesis and evaluation of a rationally-designed library of analogues containing the naphthoquinone core scaffold has provided key structure-activity relationships for these compounds The most active inhibitors exhibited potent in vitro activity with low micromolar IC50 values in anti-proliferation and Her2 degradation assays, and some compounds induced the degradation of oncogenic Hsp90 client proteins, a hallmark of Hsp90 inhibition.11 Matrix metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodelling processes and a molecular marker of some metastatic cancers A combination of computational chemistry, parallel synthesis and biochemical screening has been used to develop new technetium-99m radiolabelled probes that are sensitive to protease activity These have potential in studying the role of MMP-14 in pathologic processes and may serve as guides for the development of targeted molecular therapies.12 A novel 1,4-benzoxazin-3-one derivative with inhibitory activities against tyrosine kinases has been designed and synthesised The method has been used for the preparation of a novel 1,4-benzoxazin-3-one chemical library which has been used to determine inhibitory activities against the kinases, KDR and ABL.13 The prolactin receptor is involved in normal lactation and reproduction However, excessive prolactin levels can cause various reproductive disorders such as prolactinomas Small-molecule antagonists against the human prolactin receptor (hPRLr) have potential clinical applications and may serve as useful molecular probes in biomedical research A large, support-bound cyclic peptide library (theoretical diversity of 1.2 Â 107) has been prepared on 90-lm TentaGel beads and screened against the extracellular domain of hPRLr, resulting in 20 hits Two of these were selected for further analysis and were shown to bind to hPRLr with dissociation constants of 2–3 lM.14 Resveratrol (4,30 ,50 -trihydroxystilbene) is a naturally occurring antioxidant that inhibits cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and the transcription factor NF-jB A 78-membered library of resveratrol analogues in which the substituents on the two aryl rings and alkene were varied has been synthesised using N K Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 9–12 a solid-phase Wittig olefination reaction The library contained inhibitors against all three proteins that were more potent than resveratrol itself, and preliminary structure–activity relationships were also obtained from these data that permitted the derivation of pharmacophore models for each of the three targets.15 The Ellman group has been one of the most influential in the development and widespread adoption of combinatorial chemistry techniques for biomedical research Their work has included substantial methodological development for library synthesis with a particular focus on new scaffolds rationally targeted to biomolecules of interest and biologically relevant natural products This review analyses a representative set of libraries from this group with respect to their biological and biomedical relevance in comparison to existing drugs and probe compounds.16 Proteases are a family of naturally occurring enzymes in the body whose dysregulation has been implicated in numerous diseases and cancers This review focuses on the development of activity-based methodologies to characterise proteases, and in particular, the use of positional scanning, synthetic combinatorial libraries (PS-SCL’s), and substrate activity screening (SAS) assays.17 Maslinic acid has been coupled at C-28 with several a- and xamino acids by using solution- and solid-phase synthetic procedures Twelve derivatives with a single amino acid residue were prepared in solution phase, whereas a dipeptide, a tripeptide, and a series of conjugate dipeptides have been synthesised on solid support The anti-HIV activity of these compounds has been assessed on MT-2 cells infected with viral clones carrying the luciferase gene as a reporter.18 The solid-phase parallel synthesis and SAR of 4-amidofuran-3one inhibitors of cathepsin S have been described Highly potent inhibitors have been described, and the synthesis and structure– activity relationship of the compounds with a sulphonamide moiety in the P3 position have been presented Several members of the series show sub-nanomolar inhibition of the target enzyme, and molecular modelling described interactions in the extended S3 pocket and the observed selectivity towards cathepsin K.19 References Wysocka, M et al Anal Biochem 2008, 378 (2), 208–215 Maljaars, C E P et al Anal Biochem 2008, 378 (2), 190–196 Haddoub, R et al Tetrahedron Lett 2009, 50 (7), 741–744 Katayama, H et al Tetrahedron Lett 2009, 50 (7), 818–821 Goren, L et al Tetrahedron Lett 2009, 50 (9), 1048–1050 Freeman, N S et al Tetrahedron 2009, 65 (8), 1737–1745 Gellerman, G et al Tetrahedron 2009, 65 (7), 1389–1396 Wild, G P et al Tetrahedron 2009, 65 (8), 1618–1629 McDonagh, C.; O’Leary, P Tetrahedron Lett 2009, 50 (9), 979–982 10 Fluxa, V S.; Reymond, J.-L Bioorg Med Chem 2009, 17 (3), 1018–1025 11 Hadden, M K et al Bioorg Med Chem 2009, 17 (2), 634–640 12 Watkins, G A et al Bioorg Med Chem 2009, 17 (2), 653–659 13 Honda, T et al Bioorg Med Chem 2009, 17 (2), 699–708 14 Liu, T et al Bioorg Med Chem 2009, 17 (3), 1026–1033 15 Kang, S S et al Bioorg Med Chem 2009, 17 (3), 1044–1054 16 Shelat, A A.; Guy, R K Bioorg Med Chem 2009, 17 (3), 1088–1093 17 Lim, M D.; Craik, C S Bioorg Med Chem 2009, 17 (3), 1094–1100 18 Parra, A et al Bioorg Med Chem 2009, 17 (3), 1139–145 19 Ayesa, S et al Bioorg Med Chem 2009, 17 (3), 1307–1324 Further reading Papers on combinatorial chemistry or solid-phase synthesis from other journals Chatterjee, J.; Gilon, C.; Homan, A.; Kessler, H N-methylation of peptides: a new perspective in medicinal chemistry Accounts of Chemical Research 2008, 41 (10), 1331–1342 Hackenberger, C P R.; Schwarzer, D Chemoselective ligation and modification strategies for peptides and proteins Angewandte Chemie, International Edition 2008, 47 (52), 10030–10074 Laurent, N.; Haddoub, R.; Voglmeir, J.; Wong, S C C.; Gaskell, S J.; Flitsch S L SPOT synthesis of peptide arrays on self-assembled monolayers and their evaluation as enzyme substrates Chembiochem: a European Journal of Chemical Biology 2008, (16), 2592–2596 11 Roof, R A.; Sobczyk-Kojiro, K.; Turbiak, A J.; Roman, D L.; Pogozheva, I D.; Blazer, L L.; Neubig, R R.; Mosberg, H I Novel peptide ligands of RGS4 from a focused onebead, one-compound library Chemical Biology & Drug Design 2008, 72 (2), 111– 119 Morgan, R E.; Ahn, S.; Nzimiro, S.; Fotie, J.; Phelps, M A.; Cotrill, J.; Yakovich, A J.; Sackett, D L.; Dalton, J T.; Werbovetz, K A Inhibitors of tubulin assembly identified through screening a compound library Chemical Biology & Drug Design 2008, 72 (6), 513–524 Kwon, Y.-U.; Kodadek, T Encoded combinatorial libraries for the construction of cyclic peptoid microarrays Chemical Communications (Cambridge, England) 2008, (44), 5704–5706 Besenius, P.; Cormack, P A G.; Liu, J.; Otto, S.; Sanders, J K M.; Sherrington, D C Tailored polymer-supported templates in dynamic combinatorial libraries: simultaneous selection, amplification and isolation of synthetic receptors Chemistry – A European Journal 2008, 14 (29), 9006–9019 Lipkin, M J.; Stevens, A P.; Livingstone, D J.; Harris, C J How large does a compound screening collection need to be? Combinatorial Chemistry & High Throughput Screening 2008, 11 (6), 482–493 Hansen, M H.; Blakskjaer, P.; Petersen, L K.; Hansen, T H.; Hoejfeldt, J W.; Gothelf, K V.; Hansen, N J V A yoctoliter-scale DNA reactor for small-molecule evolution Journal of the American Chemical Society 2009, 131 (3), 1322–1327 Albada, H B.; Liskamp, R M J TAC-scaolded tripeptides as artificial hydrolytic receptors: a combinatorial approach toward esterase mimics Journal of Combinatorial Chemistry 2008, 10 (6), 814–824 Wang, N.; Xiang, J.; Ma, Z.; Quan, J.; Chen, J.; Yang, Z A concise and diversity-oriented approach to the synthesis of SAG derivatives Journal of Combinatorial Chemistry 2008, 10 (6), 825–834 Bogolubsky, A V; Ryabukhin, S V; Plaskon, A S; Stetsenko, S V; Volochnyuk, D M; Tolmachev, A A Dry HCl in parallel synthesis of fused pyrimidin-4-ones Journal of Combinatorial Chemistry 2008, 10 (6), 858–862 Treu, M.; Karner, T.; Kousek, R.; Berger, H.; Mayer, M.; McConnell, D B; Stadler, A Microwave-assisted parallel synthesis of fused heterocycles in a novel parallel multimode reactor Journal of Combinatorial Chemistry 2008, 10 (6), 863–868 Verdie, P.; Subra, G.; Averland-Petit, M.-C.; Amblard, M.; Martinez, J Solid-phase synthesis of 4-methylcarboxy-1,4-benzodiazepine-2,5-diones Journal of Combinatorial Chemistry 2008, 10 (6), 869–874 Soural, M.; Bouillon, I.; Krchnak, V Combinatorial libraries of bis-heterocyclic compounds with skeletal diversity Journal of Combinatorial Chemistry 2008, 10 (6), 923–933 Cho, C.-H.; Neuenswander, B.; Lushington, G H; Larock, R C Parallel synthesis of a multi-substituted benzo[b]furan library Journal of Combinatorial Chemistry 2008, 10 (6), 941–947 Roskov, K E.; Epps, T H.; Berry, B C.; Hudson, S D.; Tureau, M S.; Fasolka, M J Preparation of combinatorial arrays of polymer thin films for transmission electron microscopy analysis Journal of Combinatorial Chemistry 2008, 10 (6), 966–973 Messeguer, J.; Cortes, N.; Garcia-Sanz, N.; Navarro-Vendrell, G.; Ferrer-Montiel, A.; Messeguer, A Synthesis of a positional scanning library of pentamers of Nalkylglycines assisted by microwave activation and validation via the identification of trypsin inhibitors Journal of Combinatorial Chemistry 2008, 10 (6), 974–980 Scott, W L.; O’Donnell, M J Distributed drug discovery, part 1: Linking academia and combinatorial chemistry to find drug leads for developing world diseases Journal of Combinatorial Chemistry 2009, 11 (1), 3–13 Scott, W L.; Alsina, J.; Audu, C O.; Babaev, E.; Cook, L.; Dage, J L.; Goodwin, L A.; Martynow, J G.; Matosiuk, D.; Royo, M.; et al Distributed drug discovery, part 2: Global rehearsal of alkylating agents for the synthesis of resin-bound unnatural amino acids and virtual D3 catalog construction Journal of Combinatorial Chemistry 2009, 11 (1), 14–33 Scott, W L.; Audu, C O.; Dage, J L.; Goodwin, L A.; Martynow, J G.; Platt, L K.; Smith, J G.; Strong, A T.; Wickizer, K.; Woerly, E M.; et al Distributed drug discovery, part 3: using D3 methodology to synthesize analogs of an anti-melanoma compound Journal of Combinatorial Chemistry 2009, 11 (1), 34–43 Yermolayev, S A; Gorobets, N Y.; Desenko, S M Rapid three-step one-pot microwave-assisted synthesis of 2,5-dioxo-1,2,5,6,7,8-hexahydro-3-quinolinecarbonitrile library Journal of Combinatorial Chemistry 2009, 11 (1), 44–46 Roberge, J Y.; Harikrishnan, L S.; Kamau, M G.; Ruan, Z.; Van Kirk, K.; Liu, Y.; Cooper, C B.; Poss, M A.; Dickson, J K., Jr.; Gavai, A V.; et al Design and synthesis of a Gprotein-coupled receptor antagonist library of aryloxyalkanolamines using a polymer-supported acyclic acetal linker Journal of Combinatorial Chemistry 2009, 11 (1), 72–82 Baek, H G.; Liu, R.; Lam, K S Development of hydrogel TentaGel shell-core beads for ultrahigh throughput solution-phase screening of encoded OBOC combinatorial small molecule libraries Journal of Combinatorial Chemistry 2009, 11 (1), 91–102 Ryba, T D.; Depew, K M.; Marcaurelle, L A Large scale preparation of siliconfunctionalized SynPhase Polystyrene lanterns for solid-phase synthesis Journal of Combinatorial Chemistry 2009, 11 (1), 110–116 Green, D M; Goljer, I.; Andraka, D S; Chengalvala, M.; Shanno, L.; Hurlburt, W.; Pelletier, J C Parallel synthesis of 2-aryl-4-aminobenzimidazoles and their evaluation as gonadotropin releasing hormone antagonists Journal of Combinatorial Chemistry, 2009, 11 (1), 117–125 Porcheddu, A.; Giacomelli, G.; Piredda, I Parallel synthesis of trisubstituted formamidines: a facile and versatile procedure Journal of Combinatorial Chemistry 2009, 11 (1), 126–130 Muller, A.; Brinz, T.; Simon, U Preparation and measurement of combinatorial screen printed libraries for the electrochemical analysis of liquids Journal of Combinatorial Chemistry 2009, 11 (1), 138–142 12 N K Terrett / Combinatorial Chemistry - An Online Journal 11 (2009) 9–12 Jean, M.; Renault, J.; Camelin, J.-C.; Levoin, N.; Danvy, D.; Stark, H.; Capet, M.; Uriac, P A solid phase parallel synthesis of diverse amides as dopamine D3 receptor ligands Journal of Enzyme Inhibition and Medicinal Chemistry 2008, 23 (5), 588–592 Di Maro S.; Pong R.-C.; Hsieh J.-T.; Ahn J.-M Efficient solid-phase synthesis of FK228 analogues as potent antitumoral agents Journal of Medicinal Chemistry 2008, 51 (21), 6639–6641 Dixon, M J.; Nathubhai, A.; Andersen, O A.; van Aalten, D M F.; Eggleston, I M Solid-phase synthesis of cyclic peptide chitinase inhibitors: SAR of the argifin scaold Organic & Biomolecular Chemistry 2009, (2), 259– 268 Oishi, T.; 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