Zn 2+ -Chelating Motif-Tethered Short-Chain Fatty Acids as a Novel Class of Histone Deacetylase Inhibitors Qiang Lu, † Ya-Ting Yang, † Chang-Shi Chen, † Melanie Davis, ‡ John C. Byrd, ‡ Mark R. Etherton, § Asad Umar, § and Ching-Shih Chen* ,† Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and Division of Hematology-Oncology, The Arthur James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, and Laboratory of Biosystems & Cancer, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 Received July 31, 2003 Among various classes of histone deacetylase (HDAC) inhibitors, short-chain fatty acids exhibit the least potency, with IC 50 in the millimolar range. We rationalized that this weak potency was, in part, attributable to their inability to access the zinc cation in the HDAC active-site pocket, which is pivotal to the deacetylation catalysis. We thus explored the structural optimization of valproate, butyrate, phenylacetate, and phenylbutyrate by coupling them with Zn 2+ -chelating motifs (hydroxamic acid and o-phenylenediamine) through aromatic ω-amino acid linkers. This strategy has led to a novel class of Zn 2+ -chelating, motif-tethered, short- chain fatty acids that exhibited varying degrees of HDAC inhibitory potency. One hydroxamate- tethered phenylbutyrate compound, N-hydroxy-4-(4-phenylbutyrylamino)benzamide (HTPB), displayed nanomolar potency in inhibiting HDAC activity. Exposure of several cancer cell lines to HTPB at the submicromolar level showed reduced cell proliferation accompanied by histone hyperacetylation and elevated p21 WAF/CIP1 expression, which are hallmark features associated with intracellular HDAC inhibition. Introduction The acetylation status of core histones plays a pivotal role in regulating gene transcription through the modu- lation of nucleosomal packaging of DNA. 1-3 In a hy- poacetylated state, nucleosomes are tightly compacted, resulting in transcriptional repression due to restricted access of transcriptional factors to their targeted DNA. Conversely, histone acetylation leads to relaxed nucleo- somal structures, giving rise to a transcriptionally permissive chromatin state. The level of this posttrans- lational modification is maintained by a dynamic bal- ance between the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs), both of which are recruited to target genes in complexes with sequence-specific transcription activators. Aberrant regu- lation of this epigenetic marking system has been shown to cause inappropriate gene expression, a key event in the pathogenesis of many forms of cancer. 4-6 Moreover, evidence demonstrates that inhibition of HDAC triggers growth arrest, differentiation, and/or apoptosis in many types of tumor cells by reactivating the transcription of a small number of genes. 7-11 These in vitro findings have also been confirmed in xenograft models, suggest- ing that modulation of HDAC’s function might be targeted for the prevention and/or therapeutic interven- tion of cancer. To date, several structurally distinct classes of HDAC inhibitors have been reported, 7-11 including short-chain fatty acids (e.g., butyrate, valproate, phenylacetate, and phenylbutyrate), 12-15 benzamide derivatives (e.g., MS-27-275), 16,17 trichostatin A (TSA) and analogues, 18-20 hybrid polar compounds [e.g., suberoylanilide hydrox- amic acid (SAHA)], 21,22 cyclic tetrapeptides (e.g., apicidin), 23-26 and the depsipeptide FR901228. 26 Among these agents, short-chain fatty acids are the least potent inhibitors with IC 50 in the millimolar range, as com- pared to micromolar or even nanomolar for other types of HDAC inhibitors. Although the use of short-chain fatty acids in cancer treatment has been reported, their therapeutic efficacy has been limited by low antiprolif- erative activity, rapid metabolism, and nonspecific mode of action. Recently, X-ray crystallographic analysis of HDLP (histone deacetylase-like protein), a bacterial HDAC homologue, has revealed a distinctive mode of protein- ligand interactions whereby TSA and SAHA mediate enzyme inhibition. 27 The HDAC catalytic domain con- sists of a narrow, tubelike pocket spanning the length equivalent to four- to six-carbon straight chains. A Zn 2+ cation is positioned near the bottom of this enzyme pocket, which, in cooperation with two His-Asp charge- relay systems, facilitates the deacetylation catalysis. Accordingly, the structures of TSA and SAHA might be divided into three motifs, each of which interacts with a discrete region of the enzyme pocket. These include a Zn 2+ -chelating function (i.e., hydroxamic acid), an ali- phatic chain as linker, and a polar, planar cap group (i.e., dimethylaminophenyl and phenylamino functions, respectively). 27 To mediate the enzyme inhibition, the long aliphatic chain facilitates the insertion of the HDAC inhibitor into the tubelike enzyme pocket, per- mitting the hydroxamate group to reach the polar bottom of the pocket and coordinate with the Zn 2+ * To whom correspondence should be addressed. Address: Parks Hall, Rm 336, 500 West 12 th Avenue, The Ohio State University, Columbus, OH 43210-1291. Tel: (614) 688-4008. Fax: (614) 688-8556. E-mail: chen.844@osu.edu. † Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University. ‡ Division of Hematology-Oncology, The Ohio State University. § National Cancer Institute. 467 J. Med. Chem. 2004, 47, 467-474 10.1021/jm0303655 CCC: $27.50 © 2004 American Chemical Society Published on Web 12/11/2003 Downloaded by NATIONAL CHENG KUNG UNIV on July 16, 2009 Published on December 11, 2003 on http://pubs.acs.org | doi: 10.1021/jm0303655