"PA-824, now in early stage clinical trials, holds promise for shortening the TB treatment regimen, which is currently cumbersome and lengthy," says NIAID Director Anthony S. Fauci, M.D. "This new finding will allow a streamlined approach for making improved versions of the drug."
"Previously, we were flying blind in trying to optimize PA-824 in a rational way because we didn't know which M. tb protein was the target of PA-824's action," says NIAID scientist Clifton Barry, III, Ph.D., who headed the research team.
In preclinical testing, PA-824 showed evidence of being effective against both actively dividing and slow-growing M. tb, giving rise to optimism that the compound may be useful in treating both active and latent TB. (For information about the first clinical trial of PA-824, see June 14, 2005, NIAID press release: http://www3.niaid.nih.gov/news/newsreleases/2005/tb_pa_824.htm.)
PA-824 must be chemically activated in the bacterium before it exerts its anti-tubercular effect, notes Dr. Barry. Earlier research had sketched out the first few steps in this process, but Dr. Barry and his colleagues wanted to pinpoint the precise protein that binds PA-824 and transforms it into a lethal molecule for TB.
The scientists approached the problem indirectly by searching for M. tb mutants that resisted the killing power of PA-824. The team confirmed previous research suggesting that resistance usually occurs when M. tb lacks components called FGD1 and F420, neither of which interacts directly with the drug.
Next, the investigators screened for PA-824-resistant M. tb that retained sensitivity to a close relative of PA-824. Within this subgroup of PA-824-resistant bacteria, the team identified those mutant strains with FGD1 and F420. The investigators reasoned that resistance to PA-824 in mutants possessing FGD1 and F420 must be due to a mutation in the M. tb protein that directly interacts with PA-824.
But determining exactly which of M. tb's thousands of proteins was changed in these mutants proved difficult, says Dr. Barry. Conventional genetic techniques for comparing normal and mutant strains of M. tb failed, so the team turned to a specially modified microarray-based technique, called comparative genome sequencing, developed by NimbleGen Systems, Inc. (Madison, WI). This was the first time the technique has been used to identify a protein involved in TB drug resistance, notes Dr. Barry.
Using the NimbleGen technique, which effectively re-sequences the entire genome of the bacterium, the scientists quickly pinpointed the protein altered in the PA-824-resistant mutant strains of M. tb. In the past, such a complete genome comparison might have taken many months of work; this new technology enables scientists to zero in on the specific genetic difference between mutant and normal bacterial strains in just days, says Dr. Barry.
The scientists found a total of four PA-824-resistant mutant strains: two lacked the newly described M. tb protein altogether, while the remaining two mutants evidently acquired resistance to PA-824 through a mutation that made the protein unable to bind to the drug, Dr. Barry says.
With the discovery of the specific protein that interacts with PA-824, Dr. Barry and colleagues, including researchers at the Novartis Institute for Tropical Diseases in Singapore, have information they can use to produce improved PA-824 relatives and accel erate the pace of new TB drug development.