HIV-1 disease progression is characterized by high levels of continuous viral replication at the expense of CD4(+)T cells that normally participate in host defense against pathogens. Highly specific drugs that target the HIV-1 reverse transcriptase and protease enzymes are currently being utilized clinically to retard the in vivo replication of HIV-1. Unfortunately, these antiviral targets undergo extensive mutation without gross loss of function. Future treatments of HIV-1 infection must therefore focus on therapeutic targets that will interfere with HIV-1 replication, but to which the virus will have limited potential for mutational escape. A potentially effective approach involves the identification of retroviral structures that demonstrate mutational intolerance and are absolutely essential to viral replication. Such a conserved motif would be less likely to mutate rapidly towards resistance when presented with the selective pressures inherent in any antiviral therapy. The highly conserved HIV-1 retroviral nucleocapsid zinc finger protein (NCP7) meets the aforementioned criteria, and thus serves as a prime target against which to develop antiviral drugs.


The 3D structure of NCp7 rendered as a tube tracing. The orange spheres represent zinc, whilst the yellow and blue coordinating ligands represent cysteines and histidines respectively. The structure is based on the amino acid sequence

H2N-MQRGNFRNQRKIIKCFNCGKEGHIAKN
CRAPRKRGCWKCGKEGHQMKDCTERQAN-COOH,

with the zinc coordinating ligands indicated in bold type and the amino acid linker between the two fingers indicated in italics.

The HIV-1 NCP7 is a small basic protein with two copies of a highly conserved non-classical C-X2-C-X4-H-X4-C (CCHC) sequence (where X is a less conserved amino acid) known as a zinc finger. Each of the two zinc finger domains tightly coordinates one zinc stoichiometrically with three cysteine thiols and a histidine imidazole group, and folds into a stable structure. Mutation or modification of either the conserved Zn chelating or non-chelating residues results in loss of NCP7 mediated activities and renders the HIV non-infectious. Findings from mutational studies on virus infectivity highlight the participation of the NCP7 in multiple activities during both early (reverse transcription and integration) and late (protease processing and genomic RNA selection) stages of HIV-1 replication. Thus, the essential roles of the retroviral zinc fingers in the HIV-1 replication cycle make them choice antiviral targets.

As a consequence of the central and essential roles played by the NCP7 in HIV replication, attempts have been made to design compounds that might inhibit its activity through the use of drug prototypes that can covalently modify its structure. Recent reports demonstrate that a variety of electrophilic reagents mediate electrophilic attack on the CCHC zinc fingers, resulting in covalent modification of the cysteine sulfur atoms and ejection of zinc. In vitro, these compounds have been observed to inactivate cell-free virions, blocking their ability to reverse transcribe, inhibit production of infectious virus from chronically infected cells, and cause intermolecular cross-linking between zinc fingers of adjacent Gag precursors during virus assembly and maturation, thus preventing the normal processing of Gag precursors.

However, despite the promise shown by these first generation NCP7 inhibitors in vitro, their further development has been hampered by problems associated with toxicity, and lack of reactivity and effectiveness in vivo. Although the results to date have been somewhat disappointing, these studies serve to illustrate the important fact that significant differences in zinc finger protein reactivity exist to make the targeting of retroviral zinc finger proteins a therapeutic strategy well worth further exploration.

To aid in the design of the small molecule nucleocapsid protein inhibitors, we have been using a number of computational techniques. This has called for the use of several molecular modeling programs-each enabling us to add additional layers of refinement to our lead compound structures. As a consequence, we have made significant progress in the design of small molecule inhibitors, and this has enabled us to advance to the stage where the compounds are being synthesized in the laboratory. Completion of the syntheses will allow us to test our ‘first generation’ inhibitors, whose structures we will further refine as the results of their ability to inhibit nucleocapsid protein functions are acquired.

 


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