Adenosine Mimetics as Inhibitors of NAD + -Dependent Histone Deacetylases, from Kinase to Sirtuin Inhibition Johannes Trapp, ² Anne Jochum, ‡ Rene Meier, § Laura Saunders, | Brett Marshall, | Conrad Kunick, ⊥ Eric Verdin, | Peter Goekjian, ‡ Wolfgang Sippl, § and Manfred Jung ², * Institute of Pharmaceutical Sciences, Albert-Ludwigs-UniVersita ¨t Freiburg, Albertstrasse 25, 79104 Freiburg, Germany, LCO2-Glycochimie, UMR 5181 Me ´ thodologie de Synthe ` se et Mole ´ cules BioactiVes, UniVersite ´ Claude Bernard Lyon 1, 43 Bd du 11 NoVembre 1918, 69622 Villeurbanne Cedex, France, Department of Pharmaceutical Chemistry, Martin-Luther UniVersita ¨t Halle-Wittenberg, Wolfgang-Langenbeckstrasse 4, 06120 Halle/Saale, Germany, Gladstone Institute of Virology and Immunology, UniVersity of California, 1650 Owens Street, San Francisco, California 94158, and Institut fu ¨r Pharmazeutische Chemie, Technische UniVersita ¨t Braunschweig, BeethoVenstrasse 55, 38106 Braunschweig, Germany ReceiVed February 3, 2006 NAD + -dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD + has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD + - dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets. Introduction Histone deacetylases (HDACs) a are enzymes that deacetylate histones and certain nonhistone proteins, thereby altering their conformational state or activity. 1 Three classes of histone deacetylases have been recognized in humans: class I and II are zinc-dependent amidohydrolases of which 11 subtypes have been discovered (HDAC1-11). Class III enzymes depend on NAD + for catalysis, and produce O-acetyl ADP ribose and nicotinamide (1) as a consequence of the acetyl transfer. Due to homology with the yeast histone deacetylase Sir2p, the NAD + -dependent deacetylases are also termed sirtuins, and seven members (SIRT1-7) are known in humans. 2 In the past few years a considerable amount of knowledge has accumulated on the biological activities of sirtuins. 2 They are linked to aging, and overexpression leads to an increased lifespan in yeast. 3 On the other hand, there are indications that sirtuins play a role in the pathogenesis of viral diseases 4 and cancer. 5-7 While class I and II HDAC inhibitors are already investigated as new anticancer agents in clinical studies 8 much less is known about inhibitors of class III histone deacetylases. Only a few sirtuin inhibitors are available, and several of them do not inhibit human subtypes (Chart 1). 9,10 Nicotinamide (1) is the physi- ological sirtuin inhibitor. The first synthetic inhibitor discovered was sirtinol (2) 11 (Chart 1), and its hydrolysis product, 2-hy- droxynaphthaldehyde, also shows some activity. At least in certain cases, precipitation of the sirtuin by sirtinol contributes to enzyme inhibition. 12 Structure-activity relationships of sirtinol analogues have been reported. 13 Another inhibitor, splitomicin (3a), was discovered as an inhibitor of yeast sirtuins 14 and is basically inactive on human subtypes. HR73 (3b) was derived from splitomicin by our group as the first selective (20-fold for SIRT1 over SIRT2) and potent inhibitor (IC 50 < 5 µM) of human sirtuins 4 (See Chart 1). Two other inhibitors of SIRT2 were discovered using a virtual screening approach. 15 Sirtuin inhibitors with half-inhibitory concentrations as low as 98 nM have been reported recently. 16 Suramin and several related adenosine receptor antagonists inhibit sirtuins as well. 17 This prompted us to start a systematic investigation of sirtuin inhibition by drugs that target enzymes or receptors that bind adenosine-containing cofactors or ligands to identify lead structures for sirtuin inhibitors. Among these enzymes are kinases (using ATP) and dehydrogenases (using NAD + ). Certain kinase inhibitors, namely members of the paullone cyclin-dependent kinase (CDK) inhibitor series (e.g., kenpaullone), also inhibit NAD + -dependent mitochondrial malate dehydrogenase (mMDH). 18 Thus, we tested a com- * To whom correspondence should be addressed. E-mail: manfred.jung@ pharmazie.uni-freiburg.de; Tel: +49-761-203-4896; Fax: +49-761-203- 6321. ² Albert-Ludwigs-Universita ¨t Freiburg. ‡ Universite ´ Claude Bernard Lyon 1. § Martin-Luther Universita ¨t Halle-Wittenberg. | University of California. ⊥ Technische Universita ¨t Braunschweig. a Abbreviations: HDAC: histone deacetylase; mMDH: mitochondrial malate dehydrogenase; CDK: cyclin-dependent kinase; BIM: bisindolyl- maleimide; PKC: protein kinase C. Chart 1. Known Inhibitors for Sirtuins 7307 J. Med. Chem. 2006, 49, 7307-7316 10.1021/jm060118b CCC: $33.50 © 2006 American Chemical Society Published on Web 11/10/2006