BB-3497, a peptide deformylase inhibitor, is active against Mycobacterium tuberculosis

Abstract

Sir, Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalysing the removal of the N-terminal formyl group from newly synthesized proteins. PDF is essential in prokaryotes and appears to be conserved throughout the eubacteria. Thus, it may be suitable as a target for new chemotherapeutic agents. Various PDF inhibitors have in vitro activities against several pathogens including Escherichia coli, Haemophilus influenzae and Staphylococcus aureus.1–3 In view of their wide spectrum of activity, six PDF inhibitors were initially screened against two isolates of Mycobacterium tuberculosis. BB-84416, BB-3497, BB-83857, BB-83815, BB84518 and BB-83698 were provided by British Biotech Pharmaceuticals Ltd (Oxford, UK). Isoniazid and rifampicin, used as control drugs in these experiments, were obtained from Sigma Chemical Company (St Louis, MI, USA). Stock solutions of these compounds were diluted in modified 7H10 broth to produce serial two-fold dilutions, 0.015–8 mg/L. Isolates were grown at 37°C in modified 7H10 broth (malachite green and agar omitted) with 10% OADC enrichment (oleic acid– albumin–dextrose–catalase) and 0.05% Tween 80, pH 6.6. The inoculum was prepared by dilution in modified 7H10 broth yielding a final concentration of ∼2 × 104 cfu/mL (range: 2 × 103–7.1 × 104 cfu/mL). The size of the inoculum was measured by titration and counting the duplicate 7H10 agar plates supplemented with 10% OADC. The MIC of a drug was defined as the lowest concentration that yielded no visible turbidity. The incubation time was ∼14 days. Statistical analysis was conducted with Minitab 13.2 (College Park, PA, USA). Since the sample sizes were small, non-parametric statistics were used. Initial testing showed that three of the six compounds had MICs greater than 2 mg/L. The remaining three drugs, BB-3497, BB-84518 and BB-83698 had MICs in the range 0.06–2 mg/L. Rifampicin, the control drug, had a median MIC of 0.06 mg/L. Expanded testing focused on the three most promising compounds with an additional 17 isolates of M. tuberculosis (Table 1). BB-3497 was the most active PDF inhibitor with a median MIC of 0.25 mg/L. Isoniazid, the control drug for this experiment, had a median MIC of 0.031 mg/L. The difference of 0.22 mg/L was statistically significant (Mann– Whitney test, P < 0.001), implying that BB-3497 was less potent than isoniazid. Additionally, the MICs of BB-3497 did not correlate with those of isoniazid (r = –0.0032; P = 0.99: Pearson’s rank order), implying that there was no relationship between the susceptibility of an isolate to BB-3497 and to isoniazid. The broad-spectrum activity of BB-3497 and other PDF inhibitors led us to construct a phylogenetic tree using the neighbour-joining method to compare protein sequences of M. tuberculosis, Mycobacterium leprae, Yersinia pestis, E. coli and Homo sapiens.4 The purpose of this comparison was to determine the degree of homology between the various amino acid sequences of the PDF proteins of these organisms. If the degree of homology was high between two organisms, and a drug effected one organism at a low concentration, then it was also likely that it would be very effective against the other organism at a low concentration. The neighbour-joining method was chosen as the method of analysis because it is an established, well known and robust mathematical algorithm for determining the phylogenic relationships of various sequences.5 A bootstrap model placed M. tuberculosis and M. leprae in one group since the alignment scores between these organisms was 83. It also suggested that any PDF drug that inhibited M. tuberculosis would also inhibit M. leprae. E. coli and Y. pestis were classified into another group with an alignment score of 89, suggesting that PDF inhibitors would be effective against Y. pestis since they are effective against E. coli.1 It was found that the relationship between H. sapiens PDF and the other organisms does not appear to be close. The alignment scores for human PDF and PDFs from other organisms vary from a low value of Table 1. MICs of various peptide deformylase inhibitors against 19 strains of M. tuberculosis