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PDF Inhibitor: Rare Success with Rational Drug Design

Actinonin (left) and VRC3324 (right). VRC3324 is one of more than 1,000 compounds modeled on actinonin's structure and has potent antibacterial activity.

The development of several bacterial peptide deformylase (PDF) inhibitors represents a "new class of antibacterial agentsand a rare example of rational design [leading to] orally active compounds with antibiotic activity," says Zhengyu Yuan of Versicor Inc. in Freemont, Calif., who described how these recent developments trace from observations made on actinonin, a natural product discovered nearly four decades ago. He participated in the colloquium, "New Metabolites from Actinomycetes, with Novel Physiologic Functions," held during the 101 ASM General Meeting in Orlando Fla., 20-24 May 2001.

The natural product actinonin, which was isolated in 1962, was recognized for its ability in vitro to inhibit gram-positive bacteria, according to Yuan. Promising though it appeared, it failed to show in vivo activity. Later researchers realized that this natural product inhibits metalloproteases.

On a separate track, investigators studying the bacterial peptide deformylase that removes formyl-methionine residues from nascent polypeptides realized that PDF is a metalloenzyme—but one that proved frustratingly unstable and, for many years, forestalled all efforts to isolate and purify this widely distributed protein, Yuan says. However, molecular cloning techniques enabled researchers to purify the enzyme in 1993 and, several years later, to learn that it contains ferrous iron rather than zinc, which is more widely distributed among metalloenzymes. Indeed, by substituting nickel for the ferrous ion, investigators found a convenient means for stabilizing this otherwise highly oxygen-sensitive—and, hence, (usually) readily inactivated—enzyme.

While these understandings were being worked out, Yuan and his collaborators embarked on a "rational design" of deformylase inhibitors, reasoning that chelators capable of removing required ferrous ions from this enzyme were a good bet. "We constructed chemical libraries, screened for deformylase activity, and also looked at antimicrobial activity," he says. "Later, we realized that actinonin possessed many of the features of the compounds we selected. When we tested it, we found that it's a potent competitive inhibitor of this enzyme." Several other tests indicate that it inhibits bacterial growth by blocking this enzyme but, as the tests from many years earlier also suggested, "it has no activity in mice."

"So we went back to constructing libraries," Yuan continues. This second round of rational design of inhibitors proved more promising than the earlier round. It led Yuan and his collaborators to produce more than 1,000 compounds modeled roughly on actinonin, including some with "very potent antibacterial activity at levels of less than 1 m g per ml against both gram-positive and gram-negative bacteria," he says. These compounds are "bacteriostatic and with no cross-resistance to existing agents. Bacteria can develop resistance to [these compounds] in vitro, but they pay a big price in terms of virulence."

Even better, some of these compounds are active in vivo. For instance, the current lead compound shows potent activity against Staphylococcus aureus when administered orally to infected mice, according to Yuan. ``We are moving full speed ahead toward better inhibitors and preclinical trials.''

Jeffrey L. Fox
Jeffrey L. Fox is the ASM News Current Topics and Features Editor.