Structure-guided design of a-amino acid-derived Pin1 inhibitors Andrew J. Potter, Stuart Ray, Louisa Gueritz, Claire L. Nunns, Christopher J. Bryant, Simon F. Scrace, Natalia Matassova, Lisa Baker, Pawel Dokurno, David A. Robinson   , Allan E. Surgenor, Ben Davis, James B. Murray, Christine M. Richardson à , Jonathan D. Moore * Vernalis (R&D) Ltd, Granta Park, Great Abington, Cambridge CB21 6GB, United Kingdom article info Article history: Received 21 October 2009 Revised 16 November 2009 Accepted 17 November 2009 Available online 22 November 2009 Keywords: Pin1 Cancer Rotamase Isomerase Kinase signaling Crystal structure Small molecule inhibitor NMR Surface plasmon resonance abstract The peptidyl prolyl cis/trans isomerase Pin1 is a promising molecular target for anti-cancer therapeutics. Here we report the structure-guided evolution of an indole 2-carboxylic acid fragment hit into a series of a-benzimidazolyl-substituted amino acids. Examples inhibited Pin1 activity with IC 50 <100 nM, but were inactive on cells. Replacement of the benzimidazole ring with a naphthyl group resulted in a 10–50-fold loss in ligand potency, but these examples downregulated biomarkers of Pin1 activity and blocked pro- liferation of PC3 cells. Ó 2009 Elsevier Ltd. All rights reserved. Pin1 provides the great majority of peptidyl prolyl cis/trans isomerise (PPIase) activity capable of isomerising pSer/pThr-Pro bonds. 1 Isomerisation around such bonds can promote major con- formational changes within proteins, enabling Pin1 to influence dynamics and outcomes within pathways regulated by MAP kinases, cyclin-dependent kinases and GSK-3b. 2–6 Pin1 overexpression is only weakly oncogenic in itself, but enhances transformation by ErbB2 or activated Ras alleles. 5 Remarkably, cells from Pin1 deficient mice are resistant to transformation by Ras and ErbB2. 7 As Pin1-defi- cient mice are viable, 8 there are considerable grounds for hope that Pin1 inhibitors will have value for the therapy of cancer. The irreversible inhibitor, Juglone (1), has been widely used to probe the function of Pin1 inside cells, but is neither selective nor a suitable start point for drug discovery. 9–11 Several classes of presumably reversible small molecule inhibitors have since been reported. 12,13 Structural data confirming interactions of inhibitor with Pin1, however, has been restricted to peptide antagonists, 14,15 with the exception of a series of phenylalaninol phosphates, re- cently disclosed by Pfizer. 16 The same report revealed that when 10 6 compounds were screened for activity against Pin1 no hits were found whose binding to target could be verified by isothermal titration calorimetry or NMR. 16 This suggests high-throughput screening is not an effective way of finding start points for drug discovery programs targeting Pin1, but that alternative approaches should be sought. Vernalis have developed an NMR-based fragment screening platform (SeeDs) capable of identifying compounds that compete for binding to target with known competitor ligands. 17,18 Two competitor ligands had been identified whose binding to the Pin1 active site had been verified by crystallography: the D-peptide inhibitor 2, and the phenylalaninol phosphate 3. 15,16 To identify potential start points for drug discovery, a library of 1200 frag- ments was screened by NMR for their ability to compete with the binding of 2 and 3 to the PPIase domain of Pin1. This process identified five competitively-binding compounds, 19 of which the indole 2-carboxylic acid 4 (IC 50 16 lM) was the most potent inhib- itor of Pin1’s PPIase activity. 20 Pin1 crystals were readily obtained from an R14A (Arg14 mu- tated to Ala) variant of full length Pin1, 14 or a K77Q/K82Q mutated variant of the isolated peptidyl-prolyl isomerase domain. 16 Com- pound 4 was soaked into crystals of Pin1R14A protein. This en- abled the determination of a ligand-bound X-ray crystal structure (Fig. 1), in which the indole moiety fits snugly into a hydrophobic 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.11.090 * Corresponding author. Tel.: +44 1223 895555. E-mail address: j.moore@vernalis.com (J.D. Moore).   Present address: Division of Biological Chemistry & Drug Discovery, College of Life Sciences, James Black Centre, University of Dundee, Dundee DD1 5EH, UK. à Present address: Biofocus DPI, Chesterford Research Park, Saffron Walden, Essex CB10 1XL, UK. Bioorganic & Medicinal Chemistry Letters 20 (2010) 586–590 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl