Drug Discovery DOI: 10.1002/anie.201202544 Application of Fragment Screening and Merging to the Discovery of Inhibitors of the Mycobacterium tuberculosis Cytochrome P450 CYP121** Sean A. Hudson, Kirsty J. McLean, Sachin Surade, Yong-Qing Yang, David Leys, Alessio Ciulli, Andrew W. Munro, and Chris Abell* The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) drives a critical need for new front-line tuberculosis (TB) drugs with a novel mode of action. [1] It is estimated that there are over 650000 cases of multidrug-resistant tuber- culosis emerging every year, and that 1.3 million cases will need to be treated by 2015, at a budgeted cost of over 16 billion US dollars. [2] The World Health Organization has declared this epidemic a global health emergency. [3] The success of drugs that inhibit biosynthetic cytochrome P450 enzymes (CYPs), such as abiraterone, letrozole, and voriconazole, has propelled research towards understanding the unusually high number of CYPs (20) found encoded in the Mtb H37Rv genome. [4] Of particular interest is the essential CYP121 isoform, which has recently come into focus as an enticing new anti-TB drug target. [4a–c, 5] This biosynthetic CYP appears to be exclusive to Mtb, and construction of an Mtb chromosomal CYP121 knock-out mutant was only possible when a complementary vector carrying CYP121 was pre- sent. [4b, 5b] CYP121 has also recently been shown to catalyze an unusual intramolecular C À C bond-forming reaction between the ortho-positions of two tyrosines in cyclodityrosine (cYY) to form mycocyclosin. [5a] While the physiological roles of cYY and mycocyclosin remain to be determined, we believe that the unique catalytic action of CYP121 will lead to selective inhibitors. Specific inhibitors could also be used as chemical probes to show how this pivotal enzyme relates to Mtb infection, growth and persistence. The only high-affinity ligands of CYP121 currently known are azole antifungals (traditional fungal CYP51 inhibitors, which act by way of type-II azole–heme coordination). [5b, 6] These compounds exhibit potent in vitro/in vivo antimyco- bacterial activity, where their MIC (minimal inhibitory concentration) values for Mtb H37Rv correlate with their binding affinities to CYP121. [5b, 7] However, the large flexible antifungals also have broad overlapping CYP inhibition profiles and are thus poor scaffolds for developing specific inhibitors and front-line TB drug candidates. [4a, 6b, 8] Those antifungals that are administered clinically, for example, fluconazole and voriconazole, also only bind weakly to CYP121. [5b, 6a, 8a] Furthermore, resistant Mtb mutants have already been isolated that show upregulation of a transmem- brane transporter protein believed to act as an azole efflux pump. [9] Fragment-based approaches represent a new method in the field of developing small-molecule ligands as chemical tools and leads for drug development. [10] This powerful method involves the structure-guided design and synthesis of potent ligands from weaker-binding low-molecular-weight fragment molecules (typically < 250 Da). [10] Herein, we report a fragment-based approach to targeting Mtb CYP121 in an attempt to identify new inhibitory molecules and to explore the active-site properties of the enzyme. Through an initial fragment-screening cascade involving thermal shift, NMR spectroscopy, and X-ray crystallography, four fragments were found to bind within the CYP121 active site, in two over- lapping groups. A direct fragment–fragment merging strategy was implemented, leading to the discovery of a novel type-II aminoquinoline inhibitor with high ligand efficiency (LE = ÀDG of binding/non-hydrogen atoms (NHA) in the ligand) and fourfold greater affinity than the natural CYP121 substrate cYY. This lead provides a pattern for CYP121- specific inhibition and confirms the potential druggability of CYP121. This study represents the first successful application of fragment-based approaches to a cytochrome P450. [*] S.A. Hudson, Dr. Y.-Q. Yang, [+] Dr. A. Ciulli, Prof. C. Abell Department of Chemistry, University of Cambridge Lensfield Road, Cambridge, CB2 1EW (UK) E-mail: ca26@cam.ac.uk Homepage: http://www-abell.ch.cam.ac.uk/ Dr. K. J. McLean, Prof. D. Leys, Prof. A. W. Munro Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester 131 Princess Street, Manchester, M1 7DN (UK) Dr. S. Surade Department of Biochemistry, University of Cambridge 80 Tennis Court Road, Cambridge, CB2 1GA (UK) [ + ] Present address: Shanghai ChemPartner Co. Ltd. Shanghai, 201203 (China) [**] We acknowledge funding from the EC (as part of the NM4TB project) and the BBSRC (grants BB/I019227/1 and BB/I019669/1 to C.A. and A.W.M., respectively). S.A.H. was supported by a Sir Mark Oliphant Cambridge Australia Scholarship awarded by the Cam- bridge Commonwealth Trust & Cambridge Overseas Trust, Uni- versity of Cambridge. We give thanks to: Dr. J. Goodman (Cam- bridge) for assisting with the in silico calculations; Dr. J. E. Davies (Cambridge) for determining the small molecule crystal structure of the 1,5-diphenoltriazole 7; Dr. A. Boodhun (Cambridge) for performing mass spectrometry on recombinant His 6 -tagged CYP121; Dr. C. Levy (Manchester) for help with synchrotron data collection; Prof. T. L. Blundell (Cambridge) for helpful discussions. Supporting information for this article (experimental details) is available on the WWW under http://dx.doi.org/10.1002/anie. 201202544. Crystal structures of the CYP121-ligand complexes are deposited in the Protein Data Bank (http://www.rcsb.org/pdb/) under the following accession codes : 1: 4G44; 2 : 4G45; 3 : 4G46; 4 : 4G47; 7: 4G2G; 10 : 4G48; 14 : 4G1X. A ngewandte Chemi e 1 Angew. Chem. Int. Ed. 2012, 51,1–7  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü