By Sharon Kingman

Special To BioWorld Today

New insights into the way in which certain antibacterial compounds work could lead to a new class of antibiotics and even new anti-cancer drugs. Researchers at the University of Sheffield in the U.K. report that their discovery might also make it possible to overcome current problems of resistance with isoniazid, the drug commonly used to treat tuberculosis.

David Rice, professor of molecular biology and biotechnology at the Krebs Institute for Biomolecular Research, in Sheffield, said: “It is possible that this work could lead to a new specific antibiotic against tuberculosis, or form the basis for the development of a new broad-spectrum antibiotic. We just don’t know yet, but there is undoubtedly great potential for development of new drugs, including those against cancer and those which suppress graft rejection.“

Michael James, professor of biochemistry at the University of Alberta, Edmonton, Canada, described the findings, published in the Dec. 20 Science, as “very exciting. It is well known that the resistance of bacteria and viruses stays close behind the antibiotic and antiviral drugs that are constantly being developed. The promise of the research described in this paper gives yet another target against which we can hope to stay ahead of the multidrug-resistant pathogenic strains that are constantly emerging against their human hosts.“

Rice, together with colleagues in his own department and from the Institute of Molecular Biological Studies in Amsterdam, the Netherlands, the University of Durham, in Durham, U.K., and Zeneca Agrochemicals, in Bracknell, Berkshire, U.K., set out to study a bacterial enzyme called enoyl reductase (ENR). ENR has a key role in the pathway leading to fatty acid biosynthesis; without it the bacteria cannot make lipid molecules which are essential components of their plasma membranes and cell walls.

The scientists were interested in ENR because this enzyme is very different from the enzymes involved in fatty acid biosynthesis in humans. Exploiting such differences can lead to drugs which kill bacteria but which have few unpleasant side effects for humans.

ENR was already known to be the target for isoniazid and for a group of antibacterial compounds called diazaborines. The diazaborines cannot be used as drugs themselves, however, because some bacterial strains are already resistant to them. Furthermore, they contain boron, which is toxic to humans.

Using X-ray crystallography, the team determined the structure of ENR belonging to Escherichia coli, and how several of the diazaborines complexed with it. “We found that the diazaborines work by a novel mechanism. They bind to the enzyme surface adjacent to one of the other substrates of the enzyme, forming a covalent bond with that substrate,“ Rice said.

Earlier research by other groups had already shown that it was necessary for nicotinamide adenine dinucleotide (NAD⁺) to be present if diazaborines were to bind the ENR of E. coli. Rice and his colleagues have now shown that the diazaborine molecule makes a covalent bond with NAD⁺, forming a bisubstrate analog. The bond is between the 2’ hydroxyl of the nicotinamide ribose and the boron atom in the diazaborine. The NAD⁺ and the diazaborine — both flat molecules — stack up against each other.

A New Drug, Minus Toxicity

“This is the first report of such a finding for any NAD⁺ dependent enzyme,“ Rice said. “Its real interest stems from the fact that it is telling you how to make a bisubstrate analog. The covalent bond is through the boron. We can’t use boron in a drug because of its toxicity, but now that we know what the boron is doing, we can mimic it by chemically synthesizing a preformed bisubstrate analog where the chemical reactivity of the boron is no longer needed.“ The result would be a new drug which has the same antibacterial properties of the diazaborines, but without its toxicity.

Changing the size of the molecule might also make it possible to overcome problems of resistance, Rice said. The study found that the mutations in the ENR of E. coli which lead to resistance to the diazaborines, and those in the ENR of Mycobacterium tuberculosis which lead to resistance to isoniazid, were in the region of the site which binds NAD⁺. “Now that we know what the mechanism of resistance is,“ Rice said, “we may be able to change the shape of the drug to avoid this.“

Rice said that the most exciting implication of this research is that nucleotide dependent enzymes like ENR are common, and form the targets for many different drugs. For example, dihydrofolate reductase is the target for the anticancer agent methotrexate; steroid 5a reductase is the target for finasteride, used to treat benign prostatic hyperplasia; and inosine monophosphate dehydrogenase is the target for an immunosuppressive drug, mycophenolic acid (MPA).

Rice said all these nucleotides possess a chemical reactivity in one of their atoms, which is normally involved in transferring a hydride ion to and from other chemicals. In the past, other researchers have tried to link such nucleotides with other substrates with the help of these reactive atoms, to form bisubstrate analogs.

“So far, none of these studies have been successful,“ Rice said. “But our study shows how you might go about linking the substrate and the nucleotide in a way that has not yet been explored and which is totally unexpected.“

The group’s work is continuing with the aim of exploring the potential of their finding in the development of new antibiotics and in other areas of medicinal chemistry. Rice said the team is in the “late stages of negotiation“ with a biotechnology company which may be giving further backing to the work.