Ligand efficiency has proven to be a valuable concept for optimization of leads in the early stages of drug design. Taking this one step further, group efficiency (GE) evaluates the binding efficiency of each appendage of a molecule, further fine-tuning the drug design process. Here, GE analysis is used to systematically improve the potency of inhibitors of Mycobacterium tuberculosis pantothenate synthetase, an important target in tuberculosis therapy. Binding efficiencies were found to be distributed unevenly within a lead molecule derived using a fragment-based approach. Substitution of the less efficient parts of the molecule allowed systematic development of more potent compounds. This method of dissecting and analyzing different groups within a molecule offers a rational and general way of carrying out lead optimization, with potential broad application within drug discovery.Acknowledgements: This research was supported by the Bill & Melinda Gates Foundation[“Integrated Methods for TB Drug Development(IMTB)” Accelerator Grant], the UK Biotechnology and Biological Sciences Research Council (Grant BB/D006104/1), the Fundação para a Ciência e Tecnologia (PhD sponsorship to H.L.S.), and Homerton College (Junior Research Fellowship to A.C.).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/cmdc.20150041

Abell, Chris

Blundell, Tom

Boland, Jennifer

Ciulli, Alessio

George, Guillaume PC

Hung, Alvin W

Silvestre, H Leonardo

Wen, Shijun

Blundell, Tom L

Apollo (Cambridge)

Very Important PaperOptimization of Inhibitors of Mycobacterium tuberculosisPantothenate Synthetase Based on Group EfficiencyAnalysisAlvin W. Hung,[a] H. Leonardo Silvestre,[b] Shijun Wen,[a] Guillaume P. C. George,[a]Jennifer Boland,[a] Tom L. Blundell,[b] Alessio Ciulli,[a] and Chris Abell*[a]Ligand efficiency has proven to be a valuable concept for opti-mization of leads in the early stages of drug design. Taking thisone step further, group efficiency (GE) evaluates the bindingefficiency of each appendage of a molecule, further fine-tuningthe drug design process. Here, GE analysis is used to systemati-cally improve the potency of inhibitors of Mycobacterium tuber-culosis pantothenate synthetase, an important target in tuber-culosis therapy. Binding efficiencies were found to be distribut-ed unevenly within a lead molecule derived using a fragment-based approach. Substitution of the less efficient parts of themolecule allowed systematic development of more potentcompounds. This method of dissecting and analyzing differentgroups within a molecule offers a rational and general way ofcarrying out lead optimization, with potential broad applica-tion within drug discovery.The concept of ligand efficiency (LE)[1–3] has been used as animportant guiding principle for lead optimization in early stagedrug design. Subsequently, the idea of group efficiency (GE)[4]was proposed, and this has been applied to identify hotpotson proteins and to analyze parts of a ligand that make impor-tant contributions to binding.[5–9] Herein, we describe the useof GE to optimize a fragment-derived inhibitor of Mycobacteri-um tuberculosis pantothenate synthetase, an attractive targetfor developing new drugs against tuberculosis.[10–12]Pantothenate synthetase catalyzes the ATP-dependent for-mation of an amide bond between pantoate and b-ala-nine.[11–12] We have previously reported the identification offragments 1 and 2 (see Schemes 1 and 2) from biophysicalscreens using thermal shift and NMR methods.[13–14] The step-wise growing of indole fragment 1 led to the generation oflead compound 5 (Scheme 1; see also, Figure S1 in the Sup-porting Information).[13] In a parallel study, linking of fragments1 and 2 afforded compounds 6–9 (Scheme 2; see also, Fig-ure S2 in the Supporting Infor-mation).[13,15] Both fragmentgrowing and linking approachesrapidly led to relatively potentinhibitors against pantothenatesynthetase (5 : KD=1.5 mm and 9 :KD=0.9 mm).Based solely on the KD valuesand LE data obtained for com-pounds 5 and 8, it was not obvi-ous how to identify areas andvectors for further optimizationof the compounds. Therefore, in an effort to improve bindingpotency while maintaining LE,[16–17] a GE approach was used toanalyze compounds 5 and 8. Following a similar Free–Wilsonanalysis[18] as proposed by Saxty and co-workers,[6] compounds5 and 8 were dissected into component parts, and the bindingcontributions (DDG) from these individual building blocks cal-culated. These data are summarized in Figure 1 (for detailedcalculations of GE, see the section entitled “Calculations for GEanalysis” in the Supporting Information). This GE analysisaround 5 and 8 quickly revealed inefficient binding compo-nents within the molecules, suggesting straightforward ap-proaches to fragment modifications without indiscriminatelyincreasing the inhibitor size.As shown in Figure 1, the majority of the binding energyresides in the original indole fragment (GE=0.75). A similarobservation has been observed in other fragment elaborationScheme 1. A fragment-growing approach applied against Mycobacterium tuberculosis pantothenate synthetase,generating lead compound 5.[a] Dr. A. W. Hung,+ Dr. S. Wen, Dr. G. P. C. George, Dr. J. Boland, Dr. A. Ciulli,++Prof. C. AbellDepartment of Chemistry, University of CambridgeLensfield Rd, Cambridge CB2 1EW (UK)E-mail : ca26@cam.ac.uk[b] Dr. H. L. Silvestre, Prof. T. L. BlundellDepartment of Biochemistry, University of Cambridge80 Tennis Court Rd, Cambridge CB2 1GA (UK)[+] Present address: Experimental Therapeutic Centre, A-STAR, 11 Biopolis Way,Singapore 138667 (Singapore)[++] Present address : Division of Biological Chemistry & Drug Discovery, Collegeof Life Sciences, James Black Centre, University of Dundee, Dow Street,Dundee, DD1 5EH (UK)Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cmdc.201500414.Ó 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.This is an open access article under the terms of the Creative CommonsAttribution License, which permits use, distribution and reproduction inany medium, provided the original work is properly cited.ChemMedChem 2016, 11, 38 – 42 Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim38CommunicationsDOI: 10.1002/cmdc.201500414strategies[6] and is mainly due to the high intrinsic binding en-ergies required for fragments to be detected (DG=4.2 kcalmol¢1[19] is added to the binding of indole group 1 to compen-sate for the free energy associated with loss of fragment rigid-body entropy during binding).Similarly, the charged acetate side chain in both 5 and 8also contributes significantly to the overall binding, possessinghigh GE values of 0.43 and 0.35, respectively. Conversely, GEanalysis highlights the limited contribution to binding of theacyl sulfonamide, methyl pyridine and the benzofuran groups,all of which have calculated GE values of 0.16–0.17 (the GE ofthe sulfamoyl group is based on the N-(methylsulfonyl)acet-amide group).Previous work on fragment growing and linking[13] haveshown that the acyl sulfonamide groups not only contributedto additional binding energy by forming additional hydrogenbonds between the sulfone oxygen and both the backboneamide of Met40 and the sidechain of His47, but more impor-tantly also served as an effectivefunctional group for directingthe correct vectors towards boththe P1 and P2 pockets withoutclashing with the side of theactive site formed by Met40 (Fig-ure S4 in the Supporting Infor-mation). Based on these observa-tions, the initial optimizationstrategy was focused on replace-ment of the methyl pyridine andthe benzofuran groups ratherthan the acyl sulfonamide linkerin both compounds 5 and 8.Compounds were designedwith the objective of optimizinginteractions at the P1 or P2 sites of panthothenate synthetase.Consequently, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDCI)-mediated coupling reactions were employed to gener-ate a series of indole acyl sulfonamide compounds (10–19 ; thesyntheses are described in the Supporting Information).The binding of compounds 10–19 against M. tuberculosispantothenate synthetase was determined by isothermal titra-tion calorimetry (ITC), and the structure–affinity relationship(SAR) results are summarized in Table 1; ITC binding data for allcompounds are presented in the Supporting Information). Re-placing the methyl pyridine/benzofuran groups in 5 and 8generated a series of sub-micromolar inhibitors (10–14).Thesubstitution of the methyl pyridine ring (5) by a more electron-rich toluene group (10 : KD=340 nm, LE=0.32) resulted in ap-proximately a fourfold improvement in affinity towards theenzyme. Compound 10 was the most ligand efficient com-pound tested. The GE value for the toluene group in 10 isScheme 2. A fragment-linking approach applied against Mycobacterium tuberculosis pantothenate synthetase gen-erating lead compounds 6–9. (X–Y–Z represents the approximate three-atom length of the linker.)Figure 1. A) Group efficiency (GE) analysis of compound 5 estimates the contributions of the binding efficiencies from different functional groups and quickly re-veals inefficient binding groups in the molecule for further optimization of potency. B) A similar GE analysis applied towards compound 8. *DDG=DG¢DGrigid,DGrigid=4.2 kcalmol¢1.[19] The cross-sectional view of the X-ray crystal structure of the active pocket of pantothenate synthetase is shown in green with inhibitors5 and 8 bound. (The cross-sectional view was generated by removing residues from one half of the active pocket of pantothenate synthetase using the DS visu-alizer software. The surface on the other half of the protein was generated using PyMol v.0.99[20]). DG values of the compounds were determined from titrationexperiments using ITC. The GE value is subsequently calculated by dividing the DG contribution from each group by the number of heavy atoms in the group.ChemMedChem 2016, 11, 38 – 42 www.chemmedchem.org Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim39Communications0.35—a marked improvement over the GE values of 0.17 and0.16 for the methyl pyridine and benzofuran groups in 5 and8, respectively.Gratifyingly, the addition of a bulkier and more electronega-tive trifluromethyl group to the indole sulfonamide core gaverise to 11, the most potent compound of this series (KD=200 nm, LE=0.30, GE=0.27). Likewise, the installation of hy-drophobic and larger groups like tert-butylbenzene (12) andnaphthalene (13) gave rise to compounds that bind to pan-tothenate synthetase with improved affinity (KD=460 nm and610 nm, respectively) compared with parent compounds 5 and8. Interestingly, the more hydrophilic indole acyl sulfonamides15–19 (cLogP=1.0–3.2) bind with lower affinities (KD=2–17 mm) compared with more lipophilic compounds 10–14(cLogP=3.6–5.0).This step of the optimization process was achieved in largepart based on GE and SAR analysis without much consider-ation of structure. However, it did assume that the new com-pounds bind at the active site of pantothenate synthetase ina similar way to the original lead compounds, 5 and 8. In orderto establish this, the structures of the four most potent inhibi-tors (10–13) bound to the enzyme were solved using proteinX-ray crystallography (Figure 2).The X-ray crystal structures of 10–13 bound to pantothenatesynthetase show binding at the active site, with a conservedbinding mode for the indole sulfonamide fragment core. Lessobviously, the substituted groups on all four compounds wereseen to bind in the P1 pocket of the enzyme (see Figure 1B).The P1 pocket binds the alkyl groups of the pantoate substrateand is primarily lipophilic, surrounded by the hydrophobic resi-dues Pro38, Met40, Val 143, Leu146 and Phe157 (Figure S5 inthe Supporting Information). In contrast, the P2 site binds thephosphates of ATP and is relatively hydrophilic. As can be seenin Figure 2, the binding orientations of the added groups areall similar, and no new hydrogen bonds are formed. The de-tailed binding interactions of the most potent compound (11)with the P1 pocket residues are shown in Figure S5 in the Sup-porting Information. In addition to binding assays and X-raycrystallography studies, an inhibition study was carried outthat demonstrated that compound 11 inhibits pantothenatesynthetase with an IC50 value of 5.7 mm (see the SupportingInformation).Table 1. Indole sulfonamide analogues 10–19 derived from SAR considerations around 5 and 8.Compd R KD [mm][a] LE (GE)[b] cLogP[c] Compd R KD [mm][a] LE (GE)[b] cLogP[c]5 1.5 0.28 (0.17) 3.0 14 0.80 0.25 (0.15) 4.28 1.8 0.26 (0.16) 3.7 15 3.5 0.28 (0.17) 1.410 0.34 0.32 (0.35) 3.6 16 17 0.22 (0.01) 1.011 0.2 0.30 (0.27) 4.4 17 4 0.25 (0.17) 1.612 0.46 0.28 (0.22) 5.0 18 6 0.23 (0.07) 2.213 0.61 0.27 (0.21) 4.3 19 2 0.27 (0.17) 3.2[a] KD values were determined from titration experiments using ITC. [b] Ligand efficiency (LE) and group efficiency (GE) were calculated based on DGvalues derived from ITC and the number of heavy atoms associated with the corresponding groups/compounds. [c] cLogP values were derived fromChemDraw.ChemMedChem 2016, 11, 38 – 42 www.chemmedchem.org Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim40CommunicationsThe structural data on compounds 10–13 provided the im-petus for further elaboration of the series, with a view tomaking a compound that probes more deeply into the P1 site.It was rationalized that the introduction of a methylene groupbetween the aromatic and sulfonyl groups should allow the ar-omatic group to slide below Met40 and push a para substitu-ent to the back of the P1 pocket(Figure 3; for detailed bindinginteractions of 11 with the P1pocket, see also Figure S5 in theSupporting Information). To testthis hypothesis, compound 20was synthesized, using a trifluor-omethyl-substituted benzylsulfo-namide as a new coupling sub-strate.The X-ray crystal structure of20 bound to M. tuberculosis pan-tothenate synthetase (Figure 3)showed the hoped for bindingwith the trifluoromethyl grouppicking up favorable hydropho-bic interactions with Val139,Val 142 and Val143. Compound20 was shown to inhibit the en-zymatic reaction with a signifi-cantly improved IC50 value of250 nm as compared with 11.Furthermore, a cell-based assayagainst M. tuberculosis showedon-target inhibitory activityleading to cell death.[21]As the use of fragment-basedmethods expands, the need forsubsequent lead optimization offragment-derived compoundsbecomes increasingly important. The work presented heredemonstrates the use of GE analysis to critically and thorough-ly examine the binding distribution of a lead compound and il-lustrates the practicality of applying GE analysis to modifyparts of a molecule that are not making efficient contributionsto binding. In this case, it led to the generation of a relativelyFigure 2. The X-ray crystal structures of four of the most potent compounds (10–13) bound to Mycobacterium tuberculosis pantothenate synthetase (PDBcode: 4MQ6, 4MUE, 4MUF, 4MUL, respectively). The ligands are shown as sticks with carbon atoms in light blue, nitrogen atoms in dark blue, oxygen atomsin red, and sulfur atoms in yellow. The cross-sectional area of the active pocket of pantothenate synthetase is shown in green. All figures were generated andrendered with PyMOL v.0.99.[20]Figure 3. Compound 20 was found to inhibit Mycobacterium tuberculosis pantothenate synthetase with an IC50value of 253 nm (LE=0.28 based on IC50). The X-ray crystal structure of 20 bound to the active pocket of theenzyme shows the hydrophobic trifluoromethyl benzene group buried deep in the P1 site, surrounded by lipo-philic residues Gln72, Val142 and Val143 (PDB code: 4MUK).ChemMedChem 2016, 11, 38 – 42 www.chemmedchem.org Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim41Communicationspotent and bactericidal inhibitor of M. tuberculosis pantothen-ate synthetase.Experimental SectionSyntheses and characterization of organic molecules, biochemical,X-ray crystallography and isothermal titration calorimetry methodsare described in the Supporting Information. Additionally, NMRspectra related to this publication are also available at the Universi-ty of Cambridge data repository (www.repository.cam.ac.uk/handle/1810/249084).Protein X-ray crystallography structures of compounds 10–13 and20 bound to M. tuberculosis pantothenate synthetase are availablevia the RCSB Protein Data Bank via PDB codes: 4MQ6 (10), 4MUE(11), 4MUF (12), 4MUL (13), and 4MUK (20).AcknowledgementsThis research was supported by the Bill & Melinda Gates Founda-tion [“Integrated Methods for TB Drug Development (IMTB)” ac-celerator grant] and the UK Biotechnology and Biological Sci-ences Research Council (grant BB/D006104/1). The authors alsoacknowledge funding from the FundaÅ¼o para a CiÞncia e Tec-nologia (FCT), Portugal (PhD sponsorship of H. L. S.), HomertonCollege, Cambridge (Junior Research Fellowship to A. C.), andthe Agency for Science, Technology and Research (A*STAR),Singapore (PhD sponsorship of A. W. H.). The authors acknowl-edge the technical support staff at the Synchrotron Facilitiesfor their assistance.Keywords: drug design · fragment-based screening · groupefficiency · Mycobacterium tuberculosis · pantothenatesynthetase[1] A. L. Hopkins, C. R. Groom, A. Alex, Drug Discovery Today 2004, 9, 430.[2] I. D. Kuntz, K. Chen, K. A. Sharp, P. A. Kollman, Proc. Natl. Acad. Sci. USA1999, 96, 9997.[3] A. L. Hopkins, G. M. Keseru, P. D. Leeson, D. C. Rees, C. H. Reynolds, Nat.Rev. Drug Discovery 2014, 13, 105.[4] M. L. Verdonk, D. C. Rees, ChemMedChem 2008, 3, 1179.[5] A. Ciulli, G. Williams, A. G. Smith, T. L. Blundell, C. Abell, J. Med. Chem.2006, 49, 4992.[6] G. Saxty, S. J. Woodhead, V. Berdini, T. 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Optimization of Inhibitors of Mycobacterium tuberculosis Pantothenate Synthetase Based on Group Efficiency Analysis.

Optimization of Inhibitors of Mycobacterium tuberculosis Pantothenate Synthetase Based on Group Efficiency Analysis.

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