The Johns Hopkins Gazette: May 29, 2001
May 29, 2001
VOL. 30, NO. 36


Proteins in African HIV Interact Differently With Drugs

By Michael Purdy
Johns Hopkins Gazette Online Edition

Naturally occurring genetic variations in HIV-A and HIV-C, the two subtypes of HIV prevalent in Africa, make it harder for inhibitory drugs to bind to the protease, a key protein involved in viral maturation, according to a new report by biologists in the Krieger School of Arts and Sciences.

Ernesto Freire, the Henry Walters Professor of Biology, emphasizes that the new findings, published in the Proceedings of the National Academy of Sciences, are based on in vitro tests of basic biochemical properties and, therefore, cannot be used to assess the effectiveness of inhibitor drugs in patient treatments.

Ernesto Freire, seated in his lab, with four of his students, Irene Luque, Azin Nezami, Adrian Velazquez and Sonia Vega.

Instead, Freire suggests that the results add new support to the argument that scientists need to broaden the focus of HIV drug development, which has been almost exclusively centered on HIV-B.

"More than two-thirds of all AIDS cases today are in Africa, and those cases are predominantly HIV-A and HIV-C. "Those different subtypes can vary genetically from the B subtype as much as 10 percent to 30 percent along their entire genome, and this new report proves that variation can affect interactions between drugs and HIV proteins at a very basic biochemical level. We need to broaden drug development efforts to include these subtypes."

For the research, Freire's group created recombinant forms of the proteases from HIV-A and HIV-C, using information from viral gene databases (GenBank) in Africa to recreate the proteins. HIV-A dominates in the northern part of sub-Saharan Africa, while HIV-C is prevalent in southern regions.

Freire's lab measured the proteases' efficiency and biochemical fitness through factors related to catalysis, a chemical term for a process where one substance (the protease) accelerates a chemical reaction in another substance (the substrate) without being changed itself. The catalytic action of the protease is vital in viral maturation, which has made it a key target for drug development.

Measurements of the catalytic factors were taken in the absence and in the presence of indinavir, ritonavir, saquinavir and nelfinavir, four protease inhibitor drugs that are part of the drug "cocktails" currently used to control AIDS.

Proteases from the A and C subtype consistently scored better than the normal B subtype, meaning that they were better able to perform their catalytic functions in the presence of inhibitory drugs. Researchers also tested the structural stability of the various proteases, and found that A and C proteases could remain stable at higher temperatures than the B protease.

Freire's lab has long specialized in studies of the effects of genetic variations on the structures of proteins, and the links between such variations and drug resistance in disease-causing microorganisms. In addition to work with HIV, Azin Nezami, a graduate student in the lab, is involved in the development of new drugs against malaria, one of the world's deadliest diseases.

"The malaria parasite afflicts close to 300 million people annually, mostly in underdeveloped countries," Freire says. "The parasite is a plasmodium. It relies on proteaselike proteins known as plasmepsins, which chew up the hemoglobin in red blood cells that the parasite feeds on. Unlike HIV, though, plasmodium has several versions of plasmepsin in its genome. Each of these versions has slight genetic variations."

Genetic variations cause amino acid polymorphisms--differences in the sequence of amino acids that are put together to make up a protein. Sometimes, as in the case of the different HIV subtypes and the different proteases available in plasmodium, the polymorphisms occur naturally; at other times, polymorphisms result when microorganisms exposed to inhibitory drugs mutate.

Either way, Freire notes, polymorphisms can alter the structure of important proteins. The changes are typically fairly subtle; major changes might make the protein unable to do its job for the microorganism. But even subtle changes can still cause problems for inhibitory drugs.

"Many of today's drugs are designed using the lock-and-key paradigm," Freire explains. "Researchers identify a target--a binding site in an important protein in a microorganism. They validate the target, and then design a drug whose shape complements that of the binding site like the shape of a key complements a lock."

Genetic variations in microorganisms change the shape of the locks, making the keys work less well. For example, Freire's lab has identified a compound that inactivates one of the most important plasmepsins. However, to be clinically effective, a drug also needs to inactivate the other plasmepsins, which are slightly different. In HIV, both the polymorphisms naturally present in different subtypes, and, more important, drug-resistant mutations can disrupt shape complementarity.

"An additional cause for concern regarding HIV is the fact that we're starting to get some initial reports of drug-resistant mutations in HIV-A and HIV-C," Freire says. "This will lead to a combination of natural variations with drug-resistant mutations, and we don't know yet how those are going to interact."

To help cope with such challenges, Freire has been developing techniques for creating drugs whose structures include built-in flexibility. He and one of his postdoctoral associates, Irene Luque, recently received a patent on one such technique.

"Drugs produced by these novel techniques should work a bit more like master keys--good for a family of locks, instead of just one lock," Freire says. "Our techniques should help scientists design molecules that can more easily adapt to changes in the structure of the target molecule."

Other authors on the PNAS paper are Adrian Velazquez-Campoy, a postdoctoral fellow; Matthew Todd, an associate research scientist now at 3D Pharmaceuticals; and Sonia Vega, a visiting scientist. The research was supported by the National Institutes of Health and the National Science Foundation. Velazquez-Campoy, the lead author, was partially supported by a postdoctoral fellowship from the Universidad de Granada, Spain.