NMR Screening Applied to the Fragment-Based Generation of Inhibitors of Creatine Kinase Exploiting a New Interaction Proximate to the ATP Binding Site Anne-Sophie Bretonnet, ² Anne Jochum, ‡ Olivier Walker, ² Isabelle Krimm, ² Peter Goekjian, ‡ Olivier Marcillat, § and Jean-Marc Lancelin* ,² Laboratoire de RMN et Spectrome ´ trie de Masse Biomole ´ culaires, UniVersite ´ Claude Bernard Lyon 1, UMR CNRS 5180 Sciences Analytiques, ESCPE Lyon, Laboratoire de Chimie Organique II-Glycochimie, UniVersite ´ Claude Bernard Lyon 1, UMR CNRS 5181 Me ´ thodologie de Synthe ` se et Mole ´ cules Bio-ActiVes, ESCPE Lyon, and Biomembranes et Enzymes Associe ´ s, UMR CNRS 5246, UniVersite ´ Claude Bernard Lyon 1, 69622 Villeurbanne, France ReceiVed December 21, 2006 Using an in-house fragment NMR library, we identified a set of ligands that bind rabbit muscular creatine kinase, an enzyme involved in key ATP-dependent processes. The ligands docked to the crystal structures of creatine kinase indicated that a phenylfuroic acid could enter into a pocket adjacent to the nucleotide binding site. This fragment served as an anchor to develop in silico a series of potential inhibitors which could partly access the nucleotide binding site. The short synthesis of only four derivatives provided entirely novel hit compounds that reversibly inhibit creatine kinase at micromolar concentrations with a mixed ATP- competitive/noncompetitive mechanism in agreement with the structural model of the inhibited enzyme. These initial biologically active compounds are novel and modular and exploit a new interaction proximate to the ATP binding site. Introduction Identifying an initial biologically active compound for a new target protein (hit compound) 1 either for drug discovery or as a pharmacological tool remains a troublesome step. Considerable progress has been made in the route from hit to clinical candidate, including early integration of ADMET parameters, 1 new computational and chemical methods have already been successfully tested, 2 and a few NMR-based methods have raised particular interest along this line. 3-5 However, widely used approaches for hit generation, including high-throughput screen- ing (HTS), natural product leads, structural analogy to existing inhibitors, or rational design, are not appropriate in a number of situations. Small molecular fragments have therefore gained widespread popularity as a tool for drug design and hit generation over the past decade. Fragments have simple features, which differ from those of the more elaborate “leadlike” or “druglike” properties of combinatorial compounds. 6 Fragments are thus more readily amenable to chemical modifications, and have a higher prob- ability of binding protein targets. 7 Hence, fragment libraries can be much smaller than HTS libraries while sampling a chemical space inaccessible to the latter, sometimes disclosing binding “needles” where HTS would fail to find hits. 8 A set of empirical rules have been established that define the proper features for a fragment to allow tractable optimiza- tion. 9 Those limits, known as the “rule of three” 10 (M w e 300 g‚mol -1 , H bond donors and acceptors <3, rotatable bonds <3, and polar surface area e60 Å 2 ), usually restrict the affinities of fragments within the micro- to millimolar range. These affinities are unsuitable for detection by classical HTS methods, but are typical of those dealt with by NMR methods 11,12 or other structural techniques. The combined power of both structural methods and fragment concepts makes the approach particularly viable. The capacity to generate an initial biologically active “hit” compound with a minimal number of biological tests is attractive in cases which are not amenable to broad screening. Furthermore, some structural information may be obtained which can be valuable for guiding further inhibitor design. Finally, the hits generated by fragment-based design are of high quality: the inhibitors are modular, water-soluble, and simple, and are thus more readily developed into lead compounds or pharmacological tools. Diverse collections of compounds (from a few dozen 13 to hundreds 14,15 or even thousands 16,17 ) have previously been gathered in accordance with various criteria to perform NMR screening. 18 Importantly, known drug scaffolds can be used as fragment templates to minimize toxicity and untractability issues in further drug design. 19,20 This underpinned a pioneering work by Fejzo et al. where a 100-compound library called SHAPES was used to identify several ligands of the p38 MAP kinase and inosine-5′-monophosphate dehydrogenase. 15 Using an approach similar to SHAPES, we developed an in- house NMR library of just 53 compounds and tested it with established NMR screening protocols on rabbit muscle creatine kinase (RMCK; EC 2.7.3.2). 21 This enzyme plays multiple roles in the cellular energy distribution network 22 by catalyzing the reversible transfer of a phosphate group between adenosine triphosphate (ATP) and creatine (Cr; Scheme 1). It is interesting as a model system for phosphagen kinases such as brain creatine kinase and arginine kinase, both potential targets for new drugs, 23,24 because RMCK is commercially available and compatible with NMR requirements (buffer, temperature, ex- * To whom correspondence should be addressed. E-mail: lancelin@ hikari.cpe.fr. Phone/fax: 0033 472 431 395. ² Laboratoire de RMN et Spectrome ´trie de Masse Biomole ´culaires. ‡ Laboratoire de Chimie Organique II-Glycochimie. § Biomembranes et Enzymes Associe ´s. Scheme 1. Interconversion of Creatine to Phosphocreatine Catalyzed by Creatine Kinase in the Presence of MgATP 1865 J. Med. Chem. 2007, 50, 1865-1875 10.1021/jm061460r CCC: $37.00 © 2007 American Chemical Society Published on Web 03/22/2007