References

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DISCUSSION

Colston: You have been looking at drugs such as ethambutol and isoniazid that have similar targets, and now you're saying that by using this target, which you identified by using isoniazid, you can look for other drugs. Aren't you going to run into problems with cross-resistance? Isn't it the general principle to look for novel targets rather than concentrate on known targets of existing drugs?

Miesel: We believe that inhibitors of InhA, a target for isoniazid, can be used for developing new drugs without having problems ofcross resistance. Inhibitors that do not require chemical activation and that bind differently to the InhA active site may be effective against isoniazid-resistant strains.

Davies: Have you selected for revertants of your temperature-sensitive strains and demonstrated whether they are now sensitive to isoniazid?

Miesel: I have taken the ndh mutants that are isoniazid resistant and temperature sensitive and selected for temperature-resistant revertants. Most ofthese revertants are sensitive to isoniazid, although a small fraction are isoniazid resistant. These isoniazid-resistant revertants may have a partial restoration of NADH dehydrogenase activity that is sufficient to restore growth at higher temperatures. A small defect in NADH dehydrogenase activity is sufficient to confer isoniazid resistance.

Davies: Would this be at a second site?

Miesel: Some of these strains may have mutations in other genes that suppress the defect of ndh mutations. Second-site suppressor mutations may activate other NADH oxidation systems (e.g. fermentation systems) or increase the expression of the defective NADH dehydrogenase enzyme.

Davies: Other members of the actinomycetes and streptomycetes have InhA analogues, and yet they're not sensitive to isoniazid. Is your argument that these organisms are insensitive to isoniazid because isoniazid cannot get in or because these organisms have other backup systems for balancing the NAD/NADH system?

Miesel: The actinomycetes and most other bacteria also have enoyl acyl carrier protein (ACP) reductases that are similar to InhA. These enzymes are involved in making the fatty acids that comprise the phospholipids of the cytoplasmic membrane. This differs from InhA, which we believe elongates long fatty acids (> C 16) as part of a mycolic acid synthesis pathway.

The question is, why are mycobacteria sensitive to isoniazid when other bacteria are not? One possibility is that the activated form of isoniazid specifically inhibits InhA and may fail to inhibit the enoyl reductases involved in phospholipid synthesis. Alternatively, isoniazid may not be activated in other bacteria due to differences in the catalase peroxidase enzyme or to other metabolic differences such as the NADH/NAD+ ratio.

'Duncan: I would like your comments on the work that Cliff Barry presented at the ASM Conference 'Tuberculosis: past, present and future' held in Copper Mountain, Colorado in July 1997. His group showed that at concentrations close to the isoniazid MIC, a ^-ketoacyl synthase and an acyl carrier protein are cross-linked by isoniazid, thus shutting down mycolate synthesis and resulting in bacterial growth inhibition.

Miesel: Based on biochemical evidence, Cliff Barry proposes that isoniazid covalently attaches to the phosphopantetheine moiety of the ACP and that the isoniazid ACP inhibits fatty acid elongation by forming a complex between ACP and ^-ketoacyl ACP synthase.

Isoniazid may have targets other than InhA, and this may explain why isoniazid is such an effective drug. However, the possibility of other targets does not weaken the evidence that InhA is a primary target for isoniazid. Mutations in the promoter region of inhA are frequently found in isoniazid-resistant tuberculosis isolates; amino acid substitutions are also found. These mutations must confer a selective advantage during isoniazid treatment because they are never found in isoniazid-sensitive strains. We find that in Mycobacterium smegmatis, inhA mutations cause low level isoniazid resistance: about a 10-fold increase in the minimal inhibitory concentration. The sensitivity in these strains may be due to inhibition of other targets.

Brennan: There is a structural relationship between isoniazid and nicotinamide, and there is a compensatory effect ofmalate dehydrogenase and NADH. In view of that, it is difficult to compare your results with those of Cliff Barry. He is saying that isoniazid causes the accumulation of a saturated 24-.fatty-acyl ACP in M. tuberculosis, and that the target is a A-4 desaturase; in other words, an enzyme that creates an unsaturated group. The free hydrogen could then go on to NAD. This seems to be the reverse of what you're saying. One alternative is that the target is InhA, but isoniazid causes an accumulation of the G24 fatty-acyl ACP downstream. His evidence is based solely on the accumulation of a product, and I'm inferring that the target is the subsequent step, without any direct evidence for a NADH requirement.

Young: I would like to take the opportunity here of broadening this discussion into a general discussion by asking Patrick Brennan to talk about genetics and the biosynthesis of cell wall molecules.

Novartis 217: Genetics and Tuberculosis.

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