Inhibition of Helicobacter pylori aminoacyl-tRNA amidotransferase by chloramphenicol analogs Christian Balg a , Maria De Mieri c , Jonathan L. Huot b , Sébastien P. Blais b , Jacques Lapointe b , Robert Chênevert a,⇑ a Département de Chimie, PROTEO, Faculté des Sciences et de Génie, Université Laval, Québec, Canada G1V 0A6 b Département de Biochimie et de Microbiologie, PROTEO, Faculté des Sciences et de Génie, Université Laval, Québec, Canada G1V 0A6 c Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università degli Studi di Milano, 20133 Milano, Italy article info Article history: Received 5 July 2010 Revised 14 September 2010 Accepted 18 September 2010 Available online 1 October 2010 Keywords: Chloramphenicol Amidotransferase Aminoacyl-tRNA Transamidation GatCAB Helicobacter pylori Puromycin abstract Genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthe- tase in many bacteria and all known archaea. In these microorganisms, glutaminyl-tRNA Gln (Gln-tRNA Gln ) and/or asparaginyl-tRNA Asn (Asn-tRNA Asn ) are synthesized via an indirect pathway involving side chain amidation of misacylated glutamyl-tRNA Gln (Glu-tRNA Gln ) and/or aspartyl-tRNA Asn (Asp-tRNA Asn ) by an amidotransferase. A series of chloramphenicol analogs have been synthesized and evaluated as inhibitors of Helicobacter pylori GatCAB amidotransferase. Compound 7a was identified as the most active compet- itive inhibitor of the transamidase activity with respect to Asp-tRNA Asn (K m =2 lM), with a K i value of 27 lM. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction The specific aminoacylation of tRNAs is essential for the faithful translation of the genetic information into proteins. Aminoacyl- tRNA synthetases (aaRS) are the enzymes that catalyze the esterifi- cation reaction between an amino acid and its cognate tRNA. 1,2 In the cytoplasm of eukaryotic cells, the pairing of glutamine and asparagine with their corresponding tRNA proceeds through a direct pathway involving a glutaminyl-tRNA synthetase (GlnRS) and an asparaginyl-tRNA synthetase (AsnRS), respectively (Fig. 1A). Geno- mic studies revealed the absence of GlnRS and/or AsnRS in all known archaea, in many bacteria, and in some organelles. The alternative indirect pathway for the formation of Gln-tRNA Gln and Asn-tRNA Asn involves the misacylation of tRNA Gln with Glu (or tRNA Asn with Asp) by a nondiscriminating aminoacyl-tRNA synthetase (ND-aaRS) fol- lowed by the transamidation of the misacylated aa-tRNA by an amidotransferase (AdT) (Fig. 1B). 3,4 Two types of AdT are found in nature, a heterotrimeric (GatCAB) and a heterodimeric protein (GatDE). GatCAB uses both Glu-tRNA Gln and Asp-tRNA Asn as substrates and is present in bac- teria, archaea and some organelles, while GatDE functions solely as a Glu-tRNA Gln amidotransferase and is found only in archaea. 5 Crystal structures of GatCAB and GatDE have been determined re- cently. 6–9 The transamidation of the misacylated Glu-tRNA Gln and/or Asp- tRNA Asn by the GatCAB-type aminoacyl-tRNA amidotransferase (AdT) involves three distinct events ( Fig. 2). Glutamine is hydrolyzed in the GatA subunit serine-based glutaminase site to generate ammonia, which reaches GatB through a molecular tunnel. In the GatB subunit, the side-chain carboxyl group of the misacylated- tRNA is activated by ATP to form a phosphoryl-Glu-tRNA Gln or a phosphoryl-Asp-tRNA Asn intermediate (kinase step). In the final transamidase step, the enzyme-bound ammonia reacts with the high-energy mixed anhydride to yield the corresponding amide side-chain (Gln-tRNA Gln or Asn-tRNA Asn ). The small GatC subunit 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.09.045 ⇑ Corresponding author. Tel.: +1 418 656 3283; fax: +1 418 656 7916. E-mail address: robert.chenevert@chm.ulaval.ca (R. Chênevert). Figure 1. Glutaminyl-tRNA and asparaginyl-tRNA biosynthetic pathways. ND- aaRS = nondiscriminating aminoacyl-tRNA synthetase; AdT = amidotransferase. Bioorganic & Medicinal Chemistry 18 (2010) 7868–7872 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc