U-81749

Highly purified, recombinant preparations of the viral-encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to (1) their specificities toward nonviral protein and synthetic peptide substrates, and (2) their inhibition by several P₁-P₁' pseudodipeptidyl-modified substrate analogs.Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qual. the same for HIV-2 protease as published earlier for the HIV-1 enzyme.However, the rates of cleavage at these 2 sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond.The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides.Hydrolysis by the 2 proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity.For example, while the 2 proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template.Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P₁ and P₁', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions.In addition to these comparisons of the 2 proteases with respect to substrate specificity, inhibitor structure-activity data for the HIV-2 protease is presented.Relative to P₁-P₁' statine or Pheψ[CH₂N]Pro-modified pseudopeptidyl inhibitors, compounds having Xaaψ[CH(OH)CH₂]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding 10-100 times stronger to HIV-1 protease than to the HIV-2 enzyme.Mol. modeling comparisons based upon the sequence homol. of the 2 enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.In addition, energy-based modeling of a peptide fragment patterned after one of the observed substrates indicated that class 3 substrates may be interacting with charged protease side chains clustered at the ends of the binding cleft.

A synthetic peptidemimetic substrate of the human immunodeficiency virus 1 (HIV-1) protease with a nonhydrolyzable pseudodipeptidyl insert at the protease cleavage site was prepared. The peptide U-81749 inhibited recombinant HIV-1 protease in vitro (inhibition constant Ki of 70 nanomolar) and HIV-1 replication in human peripheral blood lymphocytes (inhibitory concentration IC 50 of 0.1 to 1 micromolar). Moreover, 10 micromolar concentrations of U-81749 significantly inhibited proteolysis of the HIV-1 gag polyprotein (p55) to the mature viral structural proteins p24 and p17 in cells infected with a recombinant vaccinia virus expressing the HIV-1 gag-pol genes. The HIV-1 like particles released from inhibitor-treated cells contained almost exclusively p55 and other gag precursors, but not p24. Incubation of HIV-like particles recovered from drug-treated cultures in drug-free medium indicated that inhibition of p55 proteolysis was at least partially reversible, suggesting that U-81749 was present within the particles.