Background:  PA-457 is the lead drug candidate in a new class of antiretrovirals that inhibit HIV replication by disrupting virus maturation. PA-457 blocks a late step in Gag processing that results in defective core condensation and the release of non-infectious virus particles. Specifically, PA-457 disrupts the conversion of the capsid precursor, p25 to mature CA protein, p24. PA-457’s mechanism of action is distinct from that of protease inhibitors in that it appears to directly target the Gag precursor protein.

Methods:  We selected for PA-457-resistant virus by continuous culture in the presence of compound. Genotyping of resistant virus and preparation of molecular clones with resistance-conferring mutations were carried out using standard methods. PA-457 was tested in variety of in vitro test systems to characterize its metabolic profile and was administered orally and intravenously to rats and marmosets to characterize its in vivo disposition.    

Results:  In vitro selection generated PA-457-resistant virus. Consistent with our mechanism of action studies these mutations map to residues flanking the Gag CA-SP1 cleavage site. In studies employing liver microsomes from several species including human, only rat liver microsomes caused significant metabolism. Inhibition of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) was measured using model substrates and human liver microsomes. The IC50 for inhibition for all isoforms, was >100 μM except for CYP2C9 which was ~10 μM. Interaction with the human p-glycoprotein (PGP) was assessed using porcine kidney-derived, LLC-PK₁ cells expressing human PGP cDNA. No interaction with PGP was observed and the mean P_app value was 2.6 x 10^-6 cm/sec, indicating moderate permeability. Following intravenous and oral administration to rats, the oral bioavailability was ~50% with a half-life of ~2 hours. In contrast, the oral bioavailability in marmosets was ~60% with a half-life of ~8 hours. 

Conclusions:  These results extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage. Determinants that confer resistance to PA-457 map to the Gag CA-SP1 domain. PA-457 exhibits good oral bioavailability in rats and marmosets, is not oxidatively metabolized by human liver microsomes and does not inhibit the cytochrome P450 system or interact with human PGP. This novel mechanism of action coupled with a promising preclinical profile support continued development of PA-457.

Keywords: therapeutics; novel; maturation inhibitor