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 IC50 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.

The activity of the human immunodeficiency virus (HIV) protease is essential for processing of the gag-pol precursor proteins and maturation of infectious virions. We have prepared a peptidomimetic inhibitor, U-75875, that inhibited HIV-1 gag-pol protein processing in an essentially irreversible manner. Noninfectious virus particles produced in the presence of the drug contained gag precursors and were morphologically immature. In human peripheral blood mononuclear cells and in a continuous cell line, U-75875 completely blocked HIV replication; in the latter case, no spread occurred over a period of 4 weeks. U-75875, on a molar basis, was as potent as 3'-azido-3'-deoxythymidine in blocking HIV-1 replication in human lymphocytes and also inhibited HIV-2 and simian immunodeficiency virus proteases, demonstrating that it has broad activity. These results provide further evidence for the therapeutic potential of protease inhibitors in HIV infection.The expanding AIDS epidemic and the relentless nature of the disease have created a desperate need for effective anti-viral therapies to control the replication of the human immunodeficiency virus (HIV) in infected patients. One attractive target for specific anti-viral therapy is the protease, encoded by the pol gene of HIV (1, 2). This aspartic protease (3-9) cleaves the viral p55 gag precursor into the four structural proteins of the virion core (p17, p24, p8, and p7); additionally, protease activity is required for cleavage of the p160 gag-pol precursor, which yields protease itself, reverse transcriptase (RT), and endonuclease as well as structural proteins (10). Such processing of the HIV gag and gag-pol precursor polyproteins is essential for the maturation of infectious virions (11)(12)(13)(14) 7472The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci. USA 87 (1990) 7473 filtered culture supernatant from H9/HTLVIIIB cells were added to quadruplicate wells. After 6 days, the MT-4 cultures were screened for the presence of RT. The infectivity titers of the H9/HTLVIIIB supernatants were expressed as tissue culture 50% infective dose (TCID50) per ml with 1 TCID50 corresponding to the amount of supernatant required to infect 50% of the replicate MT-4 cultures.Infection of Peripheral Blood Mononuclear Cells (PBMCs) with HIV-1. Ficoll/Hypaque-isolated PBMCs were stimulated for 3 days in RPMI/FCS containing phytohemagglutinin (5 ,ug/ml). The cells were washed and suspended at 107 cells per ml in RPMI/FCS, and HIV-lLAV was added at the multiplicity of 0.005 TCID50 per cell. After a 2-hr adsorption period, the volume was raised 20-fold with RPMI/FCS supplemented with 10% (vol/vol) interleukin 2-containing conditioned medium (Boehringer Mannheim). The cells were seeded in 24-well tissue culture plates plus drug additions (2.5x 105 cells/1.25 x 10i TCID50 of HIVLAV in a total volume of 1 ml...

A number of potential HIV protease inhibitory peptides that contain the dihydroxyethylene isostere were prepared and evaluated for their enzyme binding affinity and antiviral activity in cell cultures. From the template of a previously reported active peptide A, modifications at the N-and C-terminal groups were assessed for potential maintenance of good inhibitory activity of the resulting peptides. Among the active peptides found, peptide XVIII exhibited potent enzyme inhibitory activity. Interestingly, the previously reported, effective l(S)-amino-2(i?)-hydroxyindan C-terminal group for the preparation of very active HIV protease inhibitory peptides could not be applied to the template of peptide XVIII. Molecular modeling of peptide XVIII was studied using the X-ray crystal structure of peptide A as a starting point in order to study the likely conformation of peptide XVIII in the active-site cleft. Relative binding conformations of peptide A and XVIII were obtained, although the reason for poor binding affinity for a number of congeneric peptides in this report was not straightforwardly apparent. More importantly, however, peptide XVIII was found to exhibit more effective antiviral activity in the HIV-l/PBMC assay than the reference peptide A which was previously reported to be approximately equal in efficacy to the reverse transcriptase inhibitor AZT in this assay.

The outstanding limitations to the oligopeptide as a therapeutic agent are poor oral availability and rapid biliary clearance. To address these concerns a series of eight peptidic HIV-1 protease inhibitors containing the structural segment of the vitamin biotin have been prepared. These have been evaluated with regard to the hypothesis that this vitamin would cloak the peptidic character of these oligopeptides, and thus impart to these inhibitors the potential for absorption and distribution via biotin transporters and receptors. By iterative optimization about a-Cha^[CH-(OH)CH(OH)]Val-core inhibitory insert, three particularly potent inhibitors (K\ < 10 nM) of the HIV-1 protease were obtained. Although excellent cell culture antiviral activity is observed for other peptidic protease inhibitors of comparable affinity, none in this series exhibited satisfactory antiviral activity. This failure is attributed to the incompatibility of the hydrophilic and hydrogen-bonding biotin segment, with the facile membrane permeability and intracellular access presumably required for antiviral activity. The ability of the biotin to cloak the peptide, and thus render the overall appearance of the conjugate as that of a vitamin, was evaluated. Four of this series were evaluated for recognition by the Caco-2 cell intestinal biotin transporter. None inhibited competitively biotin uptake, indicating a lack of recognition. A vitamin may bind to a specific protein carrier, and thus attain an improved serum profile (by resistance to biliary clearance) and advantageous delivery to cells. Therefore, the serum concentrations of three were evaluated following an iv bolus in a rat model for serum clearance. One of the three protease inhibitors (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-2-(l-methylethyl)-5-[[3-methyl-l-oxo-2-[[5-(hexahydro-2-oxo-lif-thieno [3,4-d] imidazol-4-yl)-1-oxopentyl] amino] butyl] amino]-N-[2-methyl-1-[ [ (2-pyridinylmeth-yl) amino] carbonyl] butyl]-, [3aS-[3aa,4/3(lR* ,2ñ*,3fi*),6a]]-) sustained a more than 5-fold increase in serum concentration at all time points relative to the benchmark structure. The remaining two had serum concentrations at least equal to the benchmark, suggestive of improved resistance to clearance. One (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-5-[[2-[[5-(hexahydro-2-oxo-lH-thieno-[3,4-d]imidazol-4-yl)pentyl]thio]benzoyl]amino]-2-(l-methylethyl)-N-[2-methyl-l-[[(2-pyridinyl-methyl)amino]carbonyl]butyl]-, [3aS-[3aa,4/3(lE*,2fi*),6aa]]-) was prepared as a complex with the biotin-binding protein avidin. Avidin may resemble an endogenous serum biotin carrier protein. The antiviral activity (evaluated in an H9-HTLVnre acute HIV-1 infection assay) of the inhibitor and the avidin complex was identical. This suggests that the avidin-inhibitor complex is capable of cell internalization. Although the weak antiviral activity of these biotinylated inhibitors precludes consideration as practical HIV therapeutics, the overall data remain suggestive of vitamin cloaking of oligopeptides as a strategy of poten...