Potent Inhibition of HIV-1 Replication by Novel Non-peptidyl Small Molecule Inhibitors of Protease Dimerization

Abstract

Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transferbased HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 M and blocked HIV-1 replication with IC 50 values of 0.0002-0.48 M. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1.

Absorption, Metabolism, and Excretion of Darunavir, a New Protease Inhibitor, Administered Alone and with Low-Dose Ritonavir in Healthy Subjects

“…Its molecular formula is C 27 H 37 N 3 O 7 S ⅐ C 2 H 5 OH and molecular weight is 593.73. Darunavir binds tightly to HIV protease with a dissociation constant (K d ) of 4.5 ϫ 10 Ϫ12 M (King et al, 2004) and is highly active against wild-type and resistant strains of the virus (De Meyer et al, 2005), inhibiting dimerization (Koh et al, 2007) and catalytic activity of HIV-1 protease. It selectively inhibits the cleavage of HIV-encoded gag-pol polyproteins in virus-infected cells, preventing the formation of mature infectious virus particles.…”

Non-Cleavage Site Gag Mutations in Amprenavir-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) Predispose HIV-1 to Rapid Acquisition of Amprenavir Resistance but Delay Development of Resistance to Other Protease Inhibitors

“…The PCR products obtained as described above were digested with two of the three enzymes BssHII, ApaI, and SmaI, and the obtained fragments were introduced into pHIV-1 NLSma , designed to have a SmaI site by changing two nucleotides (2590 and 2593) of pHIV-1 NL4-3 (15,19). To generate HIV-1 clones carrying the mutations, site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was performed, and the mutation-containing genomic fragments were introduced into pHIV-1 NLSma .…”

New Validated Rp-HPLC Method for the Estimation of Darunavir in Bulk and Its Dosage Form

“…Darunavir is an inhibitor of Dimerisation and the catalytic activity of the HIV-1 protease. It selectively inhibits the cleavage of HIV encoded Gag-Pol polyproteins in the virus infected cells, thereby preventing the formation of infectious virus particles 4,5 . Proposed method is validated as per ICH guideline for Analytical Procedures 6 .…”