Articles Novel Effect of C75 on Carnitine Palmitoyltransferase I Activity and Palmitate Oxidation † Assia Bentebibel, ‡ David Sebastia ´n, ‡ Laura Herrero, ‡ Eduardo Lo ´pez-Vin ˜as, § Dolors Serra, ‡ Guillermina Asins, ‡ Paulino Go ´mez-Puertas, § and Fausto G. Hegardt* ,‡ Department of Biochemistry and Molecular Biology, School of Pharmacy, UniVersity of Barcelona, E-08028 Barcelona, Spain, and Centro de Biologia Molecular “SeVero Ochoa”, UniVersidad Autonoma de Madrid, Consejo Superior de InVestigaciones Cientı ´ficas, Cantoblanco, E-28049 Madrid, Spain ReceiVed October 26, 2005; ReVised Manuscript ReceiVed January 23, 2006 ABSTRACT: C75 is a potential drug for the treatment of obesity. It was first identified as a competitive, irreversible inhibitor of fatty acid synthase (FAS). It has also been described as a malonyl-CoA analogue that antagonizes the allosteric inhibitory effect of malonyl-CoA on carnitine palmitoyltransferase I (CPT I), the main regulatory enzyme involved in fatty acid oxidation. On the basis of MALDI-TOF analysis, we now provide evidence that C75 can be transformed to its C75-CoA derivative. Unlike the activation produced by C75, the CoA derivative is a potent competitive inhibitor that binds tightly but reversibly to CPT I. IC 50 values for yeast-overexpressed L- or M-CPT I isoforms, as well as for purified mitochondria from rat liver and muscle, were within the same range as those observed for etomoxiryl-CoA, a potent inhibitor of CPT I. When a pancreatic INS(823/13), muscle L6E9, or kidney HEK293 cell line was incubated directly with C75, fatty acid oxidation was inhibited. This suggests that C75 could be transformed in the cell to its C75-CoA derivative, inhibiting CPT I activity and consequently fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 in mice produced short-term inhibition of CPT I activity in mitochondria from the liver, soleus, and pancreas, indicating that C75 could be transformed to its C75- CoA derivative in these tissues. Finally, in silico molecular docking studies showed that C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibiting mechanism based on mutual exclusion. Overall, our results describe a novel role for C75 in CPT I activity, highlighting the inhibitory effect of its C75-CoA derivative. C75 is a chemically stable synthetic inhibitor of fatty acid synthase (FAS). 1 Structurally, it is a cell-permeable R-meth- ylene-γ-butyrolactone, designed to be less reactive and potentially safer than cerulenin, a natural product obtained from the fungus Cephalosporium caerulens. C75 lacks the reactive epoxide present in cerulenin, which enhances † This study was supported by Grant SAF2004-06843-C03 from the Ministerio de Educacio ´n y Ciencia, Grant C3/08 from the Fondo de Investigacio ´n Sanitaria of the Instituto de Salud Carlos III, Red de Centros en Metabolismo y Nutricio ´n (RCMN) from the Ministerio de Educacio ´ n y Ciencia, Research Prize 2004 from Fundacio ´ n Puleva, and the Ajut de Suport als Grups de Recerca de Catalunya (2001SGR- 00129), Spain. A.B. and L.H. are recipients of fellowships from the Ministerio de Educacio ´n y Ciencia, and D. Sebastian is a recipient of a fellowship from the University of Barcelona. * To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Diagonal 643, E-08028 Barcelona, Spain. Phone: +34 93 4024523. Fax: +34 93 4024520. E-mail: fgarciaheg@ub.edu and dserra@ub.edu. ‡ University of Barcelona. § Consejo Superior de Investigaciones Cientı ´ficas. © Copyright 2006 by the American Chemical Society Volume 45, Number 14 April 11, 2006 10.1021/bi052186q CCC: $33.50 © 2006 American Chemical Society Published on Web 03/14/2006