Tracy Handel, UC San Diego

Estimates from the United Nations AIDS program indicate that approximately 40 million people are currently living with HIV. This high number is due to the introduction of therapies which have resulted in a dramatic improvement in survival rate. Nevertheless, the infection rate continues to grow. In 2003 there were 5 million new infections, more than any other previous year. 3 million people died, and of these, 500,000 were children under the age of 15. These statistics underscore the fact that prevention and treatment must be at the heart of every comprehensive AIDS strategy. This proposal is directed towards developing new treatments, which could have an impact on prevention as well.

The main therapies currently in use target HIV enzymes that are essential to its life cycle. While they have proven effective in significantly extending life span, the quest for alternative treatments continues because of limitations such as toxicity, the emergence of drug resistant strains because of the ability of the virus to mutate, and the ensuing suboptimal response in drug-treated individuals. An alternative approach to reduce viral load would be to prevent the virus from entering the host cells altogether. HIV requires chemokine receptors found on macrophages and T-cells for fusion with the host cell membrane; and indeed, blocking the interaction between these receptors and HIV reduces viral load. The mutability of the virus provides additional rationale for targeting host proteins because the host proteins won't mutate in response to drug challenge. Multiple therapies aimed at different events in the viral life cycle will then enable switching of treatments to combat resistant viruses. However, there are no current drugs in use that block viral entry. While this strategy is considered a promising addition to current therapies, drug discovery efforts have been plagued by problems of specificity and toxicity, and beg for structural studies to aid these efforts.

Our ultimate goal is to conduct structural studies of receptors with small molecule lead compounds, in order to aid the development of more potent and specific therapies that inhibit viral fusion. Unfortunately, chemokine receptors are membrane proteins, which have generally proven recalcitrant structural targets. The most difficult barrier has been the ability to express large quantities of receptor because of toxicity to the cell due to insertion of receptors into the cell membrane. Thus the immediate aim of this proposal is to use recently developed cell-free expression methods to express CCR5, the most important receptor in the infection and initial phase of AIDS.

Cell-free technologies utilize extracts from organisms that contain most of the necessary factors for expression in the absence of a viable cell. This is considered to be one of the most promising technologies for membrane protein expression because the absence of the membrane precludes toxicity. Additionally other agents are readily added that can facilitate folding, functional reconstitution and structural analysis. Successful expression will then form the basis for long term structural studies by NMR and crystallography. As cell-free expression is exceptionally powerful for NMR and can provide important information in the absence of a complete structure, as well as information that can facilitate crystallization, we outline some initial NMR experiments that will provide information such as the receptor-binding pocket for drugs.