Because of the emergence of drug-resistant strains and the cumulative toxicities associated with current therapies, demand remains for new inhibitors of HIV-1 replication. The HIV-1 matrix protein (MA) is an essential viral component with established roles in the assembly of the virus. Using a combination of virtual screening, surface plasmon resonance analysis, and antiviral testing, we were the first to identify small drug- like molecules that bind to the HIV-1 MA protein and that possess broad-range anti-HIV properties. Combining computer-aided drug design techniques and medicinal chemistry, we have designed and synthesized next generation compounds that interact with HIV-1 MA and inhibit the virus. Our current lead compound, MTI-14 binds to HIV-1 MA (as judged by SPR and NMR), has low toxicity, and has mid-micromolar potency. Moreover, MA mutant viruses display reduced sensitivity to MTI-14, demonstrating that the antiviral action of the compound is mediated through disruption of the functions of MA. Excitingly, preliminary mechanism-of-action studies also suggest that MTI-14 functions by blocking HIV-1 Env incorporation into new viral particles. We propose to further optimize this MA-targeted inhibitory compound to potencies that are clinically relevant, while using these inhibitors as molecular tools to dissect the biology of the MA protein in the HIV-1 replication cycle. The goals of the proposed studies will be accomplished by three discrete but highly integrated specific aims.
In Aim 1, the leads will be optimized through structure-guided design and medicinal chemistry approaches, their kinetic properties including on- and off-rates and binding affinities (KD) will be measured, and their binding site verified by chemical shift NMR and competitive fluorescent polarization assays. In addition, novel chemotypes will be sought using ligand-based screening approaches.
In Aim 2, the antiviral potency and breadth of the leads will be assessed. Evaluation of their toxicity will be performed in parallel.
In Aim 3, we will perform detailed mechanism-of-action studies, generate resistance mutants, and determine the structures of the new inhibitors in complex with MA using crystallographic and nuclear magnetic resonance methods. The data from these studies will be used in designing potent next-generation MA-targeted inhibitors with high genetic barriers and unique modes of action. Such compounds will also serve as novel molecular probes for HIV-1 MA functions.
Despite the success of antiretroviral drug regimens, new targets for inhibition of HIV-1 replication are highly desirable, and new drugs are needed to combat the rising problem of drug resistance. The studies proposed in this application will optimize and characterize a new class of compounds that are targeted at the HIV-1 matrix (MA) protein and that act via novel mechanisms. The compounds generated within this proposal have the potential to serve as templates for novel anti-HIV agents while also providing unique reagents with which to dissect the basic biology of HIV-1.
