pubs.acs.org/jmc Published on Web 12/30/2009 r 2009 American Chemical Society J. Med. Chem. 2010, 53, 1407–1411 1407 DOI: 10.1021/jm9013345 Characterization of Sirtuin Inhibitors in Nematodes Expressing a Muscular Dystrophy Protein Reveals Muscle Cell and Behavioral Protection by Specific Sirtinol Analogues Matthieu Y. Pasco, †,‡,§ Dante Rotili, ) Lucia Altucci, ^ Francesca Farina, †,‡ Guy A. Rouleau, § Antonello Mai,* , ) and Christian N eri* ,†,‡ † INSERM, Laboratory of Neuronal Cell Biology and Pathology, Center for Psychiatry and Neurosciences U894, ‡ Universit e Paris Descartes, Equipe d’accueil 4059, 75014 Paris, France, § University of Montreal, Department of Medicine and the Center for Excellence in Neuromics, Montreal, Qu ebec, Canada H2L 4M1, ) Pasteur Institute;Cenci-Bolognetti Foundation, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, 00185 Rome, Italy, and ^ Dipartimento di Patologia Generale, Facolt  a di Medicina e Chirurgia, Seconda Universit  a di Napoli, Vico L. De Crecchio 7, 80138 Napoli, Italy Received September 8, 2009 In oculopharyngeal muscular dystrophy (OPMD), a disease caused by polyalanine expansion in the nuclear protein PABPN1, the genetic inhibition of sirtuins and treatment with sirtuin inhibitors protect from mutant PABPN1 toxicity in transgenic nematodes. Here, we tested the SIRT1/2 inhibitors 1-12, bearing different degrees of inhibition, for protection against mutant PABPN1 toxicity in Caenorhabditis elegans. Compounds 2, 4, and 11 were the most efficient, revealing a potential therapeutic application for muscle cell protection in OPMD. Introduction Sirtuins are NAD þ a dependent deacetylases, also referred to as type III histone deacetylases, that modify the acetylation state of several intracellular messengers, thereby regulating downstream mechanisms associated with these substrates. 1 These deacetylases are able to integrate environmental cues and modulate several important physiological mechanisms such as inflammation, apoptosis, glucose homeostasis, life span, and neuroprotection. 2 The pharmacological manipula- tion of theses enzymes has therefore strong potential for the therapy of many human diseases such as cancer, metabolic disorders, and degenerative diseases. 3-5 Sirtuins (SIRT1-7 in mammals) are highly conserved enzymes. The most studied member of the sirtuin family is SIRT1. The SIRT1 protein is most related to the Caenorhab- ditis elegans sirtuin sir-2.1. Transgenic nematodes with in- creased sir-2.1 dosage can live up to 50% longer. 6 Recent studies indicated that, depending on the cellular context and type of sirtuin considered, either the activation or the inhibi- tion of sirtuins may protect against cell/tissue injury. 7 Whereas sir-2.1/SIRT1 activation may protect against neuro- nal dysfunction in simple models of HD pathogenesis 8 and models of ALS and Aβ toxicity, 9 the pharmacological inhibi- tion of SIRT2 may be neuroprotective against R-synuclein toxicity. 10 Additionally, SIRT1 inhibition may alleviate gene silencing in Fragile X mental retardation syndrome under treatment with nicotinamide (NAM) or splitomicin (7, Figure 1). 11 Sirtuin inhibition may also be involved in the beneficial effect of NAM observed in the triple-transgenic model of Alzheimer’s disease harboring PS1, APP, and tau mutations. 12 Finally, the genetic (loss-of-function; LOF) and pharmacological (sirtinol 1, Figure 1) inhibition of sir-2.1/ SIRT1 protects C. elegans transgenics from the toxicity of polyalanine expansion in the oculopharyngeal muscular dys- trophy (OPMD) protein PABPN1, an effect that requires the transcriptional factor and key longevity protein daf-16/ FoxO, 13 suggesting that sirtuin inhibitors may be protective in OPMD through cell survival mechanisms. Before evaluation of sirtuin modulators in mouse models of disease, it is important to identify potent lead compounds and to test whether they may be active in physiological conditions. Because PABPN1 nematodes show cellular (progressive loss of muscle cell nuclei) and behavioral (defective motility) phenotypes amenable to drug screening, 13 we hypothesized that these animals may allow to select for sirtuin inhibitors that best rescue the toxicity of mutant PABPN1 at the muscle cell and behavioral levels. In this study, we tested some known SIRT inhibitors, such as 1, 14,15 meta-sirtinol 2, 15 para-sirtinol 3, 15 salermide 4, 16 7, 17 EX-527 11, 18 and AGK-2 12, 10 prepared by us according to published procedures (Figure 1). In addition, we tested two new synthetic sirtinol analogues (5 and 6), a splitomicin derivative (the ketosplitomicin analogue 8), and the benzodeazaoxaflavines (BDF4s) 9 and 10, two totally different compounds recently identified in our lab (Figure 1). Compounds 1-12 were tested for their ability to inhibit the enzymatic activity of SIRT1 and SIRT2 in vitro and to modulate mutant PABPN1 toxicity in trans- genic nematodes. *To whom correspondence should be addressed. For C.N.: christian. neri@inserm.fr. For chemistry, A.M.: phone, þ39 064 991 3392; fax, þ39 064 91 491; E-mail, antonello.mai@uniroma1.it. a Abbreviations: ALS, amyotrophic lateral sclerosis; APP, amyloid precusor protein; Aβ, β-amyloid peptide; FoxO, forkhead box class O; GFP, green fluorescent protein; HD, Huntington’s disease; NAD þ , nicotinamide adenine dinucleotide; NAM, nicotinamide; OPMD, ocu- lopharyngeal muscular dystrophy; PABPN1, polyadenylate-binding protein, nuclear, 1; PS1, presenilin-1 transmembrane protein; RNAi, RNA interference; RT-PCR, reverse transcription-polymerase chain reaction.