HIV-1 gp120: A novel, large-scale purification technique and thermodynamic characterization of the binding of two small-molecule inhibitors

As of December 2007 approximately 33 million individuals worldwide were infected with HIV/AIDS. Each year since the identification of AIDS in 1981 the number of people living with HIV worldwide has increased, and since 1981 HIV/AIDS has caused the death of approximately 25 million individuals. HAART (Highly Active Anti-Retroviral Therapy, or the simultaneous utilization of multiple drugs that inhibit multiple HIV targets) has demonstrated clinical utility against HIV/AIDS, but multi-drug resistant viral strains are emerging. Necessary to increasing and extending the efficacy of HAART will be the utilization of novel HIV targets such as the HIV-1 envelope protein gp120.;As described within this study we have incorporated, scaled-up, and optimized existing gp120 production techniques and developed novel, large-scale purification techniques. We have refined the characterization of the monomeric gp120 structure using differential scanning calorimetry and have refined the characterization of the binding of gp120 to soluble CD4 and chemokine coreceptor surrogate monoclonal antibody 17b using isothermal titration calorimetry. We have used these data not only to more fully investigate the structure of gp120 but also to develop thermodynamic and structural guidelines for development of competitive and allosteric inhibitors of gp120.;Further, building upon the discovery by other research groups of the pre-clinical gp120 binding compounds NBD-556 and BMS-806, we have thermodynamically characterized the interactions of NBD-556 and BMS-806 with gp120 and the effects of these interactions upon receptor binding. Through application of our thermodynamic guidelines we have determined that NBD-556 is a competitive inhibitor of gp120 and that BMS-806 is a gp120 inhibitor with allosteric inhibitory properties. Thermodynamic and mechanistic understanding of interactions of each of these compounds with gp120 will not only aid in the derivatization of these compounds but also generate a more complete understanding of the structure and mechanism of envelope-mediated cell entry. Thermodynamic and structural characterization of the interactions of gp120 inhibitors with gp120 and inhibitor-induced perturbations of receptor binding are critical tools towards optimization of these and future gp120 inhibitors.