The new class of drugs would have the important benefit of being active against other pathogenic bacteria while leaving the endogenous microflora of the human intestinal system intact.
Although the drugs are still in the earliest stages of development, officials at both VDDI Pharmaceuticals and the University of Alabama at Birmingham's Center for Biophysical Sciences and Engineering (UAB-CBSE, who jointly filed the patent, are optimistic about the future use of the compounds, the first of which appears to discriminate between Gram-positive and Gram-negative bacteria.
As director of the UAB-CBSE, Larry DeLucas chose to first develop compounds that were designed to block NAD synthetase, an enzyme critical to the bacteria's transition from its sporulated state to its potentially lethal vegetative form. The enzyme is one of several that could provide future drug targets for the anthrax bacterium, and was a natural first choice because the crystal structure had already been published.
The research is partially funded by the US Department of Defence, in hopes that the compounds could be used to protect potential victims of a biological attack with anthrax. Normally, when a spore is inhaled, DeLucas explains, it is engulfed by a macrophage. Once the spore encounters the right environment - "water, a few crucial amino acids, like alanine," - it begins to germinate. "The spore is harmless... it has to lose its outside core," said DeLucas. "Then it becomes a vegetative cell. It looks black; it begins to divide and multiply, you get so many in the lungs, and then they begin to release toxin." He sums up the consequences simply: "Then you have big problems."
The new compounds were designed to shut down the bacteria's emergence from its protective coating. DeLucas explains that the spore tries to become the bug and it eventually loses its shell; however, when NAD synthetase is blocked, the walls break apart and it looks like "a bomb went off, and you get no bug."
DeLucas stresses that the compounds have only just begun down the long road of drug design but, if successful, could be a powerful prophylactic against a biological attack. Even with a large number of spores in the air, they would just fall apart and infection would be prevented. The currently used antibiotic Cipro, in contrast, is only effective when anthrax exists in its vegetative state, which means that the victim is already becoming infected. Compounds that inactivate NAD synthetase would also be useful against the vegetative cell after the spore has germinated, adds DeLucas, because the enzyme is still vitally important to the organism.
Indeed, the enzyme is vital to any bacterium, so DeLucas was surprised when the new inhibitors showed "an absolute predilection for Gram-positive bacteria," while leaving Gram-negative bacteria, such as E. coli, intact. The researchers are hoping that this means that it is not going to kill all internal microflora, "but it is difficult to predict whether a compound will act with such specificity," said DeLucas; "it is likely to depend on subtle structural differences in the proteins."
Although researchers at the UAB-CBSE have not yet co-crystallized NAD synthetase with the bound inhibitor, they have compared the crystal structures of the enzyme from Bacillus subtilis and from E. coli, and they hypothesize that a small amino acid loop near the active site in the Gram-negative bacterial enzyme might prevent the compound from disabling NAD synthetase.
DeLucas and his group aimed to design compounds that would specifically disable their target by binding to the enzyme's active site. Structure-based drug design has many advantages, says DeLucas, primarily that such a drug is effective: "the enzyme just can't work," he said.
In addition, a compound that binds at a protein's active site is less likely to lead an organism to develop resistance. Amino acids on the surface of a protein could be easily mutated without affecting function but the active site is different, explains DeLucas. "If you look at any protein...there will be certain amino acids that are critical for it to function," he said. By making a drug that binds to those specific residues, you use an area of the protein that is less likely to naturally mutate and thereby confer resistance.
J. Todd Weber, of the National Center for Infectious Disease at the US Center for Disease Control (CDC), says that it is "clear that we do need new classes of antimicrobial drugs," because, notes, rates of resistance are increasing all the time. Although Weber is not familiar with the unpublished details of the current work, he noted, "any advance to create new drugs that get around the mechanisms of developing antibiotic resistance would certainly be valuable".
Currently, researchers must also consider a non-natural source of mutations that might lead to drug resistance: those engineered by potential bioterrorists. DeLucas suggests that by making the active site the drug target, you could protect the drug's efficacy. Vaccines, too, are subject to lose their effectiveness through natural protein mutations. "Does this mean we should not use vaccines" to protect people against a biological attack, DeLucas asked. "No. They present another hurdle for terrorists," and fighting biological warfare, he says, "should use a dual-pronged approach." DeLucas predicts that one day the newly developed compounds will be used in that fight.
This article was originally published in Drug Discovery Today.
26 March 2003
Original web page at BioMedNet