Peptide Deformylase Inhibitors as Potent Antimycobacterial Agents<sup><a href="#fn3" rid="fn3" class=" fn">▿</a></sup> <sup><a href="#fn2" rid="fn2" class=" fn">†</a></sup>

Jeanette Teo

Pamela Thayalan

David Beer

Amelia Yap

Veronique Dartois+9 authors

Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, Singapore 138670, Republic of Singapore,^{Central New York Research Corporation, New York, New York,}^{Novartis Institutes for Biomedical Research, Inc., Infectious Disease Area, 100 Technology Square, Cambridge, Massachusetts 02139}³

^{Corresponding author. Mailing address: Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, Singapore 138670, Republic of Singapore. Phone: 65-67222923. Fax: 65-67222917. E-mail:}moc.sitravon@eejrehkum.ilokak.

^{Present address: National University Hospital, Department of Laboratory Medicine, 5 Lower Kent Ridge Road, Main Building, Level 3, Singapore 119 074, Republic of Singapore.}

Received 2006 May 4; Revised 2006 Jun 6; Accepted 2006 Aug 17.

Abstract

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC₅₀) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC₉₀ values of <1 μM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of ≤5 × 10ⁱⁿM. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents.

Abstract

Mycobacterium tuberculosis, a pathogen causing 2 million deaths and 8 million new cases of tuberculosis (TB) each year, is a leading health threat. This is further fueled by the human immunodeficiency virus epidemic and the appearance of multidrug-resistant TB (17, 18, 46). Infections involving multidrug-resistant TB are harder to treat and have limited the efficacy of TB control programs (29). This has necessitated the search for new antituberculosis drugs with a novel mode of action.

In prokaryotes and in certain organelles, like plastids and mitochondria of eukaryotes, nascent proteins carry an N-formylated methionine (40). After translational initiation, peptide deformylase (PDF) (EC 3.5.1.27) removes the N-formyl group from the nascent proteins (42). Deformylated proteins undergo further N-terminal processing to give mature proteins (61). PDF is an attractive candidate for the discovery of antibacterial agents, as the essentiality of the gene (def) encoding PDF has been shown for Escherichia coli and Streptococcus pneumoniae (8, 36). The enzyme has been characterized as a highly unstable metallopeptidase that uses Fe^{as the catalytic metal (}51). Oxidation of Fe^{inactivates the enzyme, and purification of active bacterial PDF has been a challenge (}51). So far, Ni^{, Co}^{, and Mn}^{have been described as cations that can stably replace Fe}^{while retaining high enzymatic activity (}25, 49).

Naturally occurring antibiotics, like actinonin, inhibit the activity of the PDF enzyme (2). Based on structural (7, 39, 41) and mechanistic design, as well as high-throughput screening, several classes of PDF inhibitors (PDF-I) have been studied previously (64). Thus far, only compounds having hydroxamic acid-chelating or N-formyl hydroxylamine-chelating groups show potent enzyme inhibition, good antibacterial activity, and in vivo efficacy, including oral activity (9, 12, 27).

In this work, we validate def (gene encoding PDF) as a drug target for TB by proving its essentiality in Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned and expressed heterologously in E. coli. Recombinant His-tagged M. tuberculosis PDF enzyme was purified, and biochemical characterization was carried out. Activities of a novel class of PDF-I, the N-alkyl urea reverse hydroxamates (27), were determined by both M. tuberculosis PDF enzyme and cell-based assays for antimycobacterial activity. A potent lead, PDF-611 (LBK-611) (15), was identified as a novel antimycobacterial agent.

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Acknowledgments

We thank Sabine Daugelat for providing advice on the knockout procedure and Marty Pavelka for pYUB657. We thank Brigitte Gicquel for providing pJEM15, which was used for overexpression studies.

Acknowledgments

Footnotes

^{Published ahead of print on 11 September 2006.}

^{Supplemental material for this article may be found at}http://aac.asm.org/.

Footnotes

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