An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection.

Abstract

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...

Inhibition of Human Endogenous Retrovirus-K10 Protease in Cell-free and Cell-based Assays

“…Similar results obtained with viral RNA isolation from the particulate material of NCCIT cell culture medium and its subsequent quantification with RT-PCR amplification support this observation (data not shown). These data are consistent with the observation that HIV-1 protease inhibitors block the processing of Gag and Gag-Pol precursor polyproteins in HIV-1-infected cells but do not markedly alter either the number of particles released from the infected cells (44,45) or the amount of packaged viral RNA (46,47). In addition to using antigenspecific immunoblotting, we verified the identity of NCCIT released virions by checking the nucleotide sequence of packaged RNA.…”

Human immunodeficiency virus type‐1 reverse transcriptase and ribonuclease h as substrates of the viral protease

“…This sequence occurs in the middle of a-helix A (Davies et al, 1991), and may support the view that the helix is present in the partially unfolded RNase H at pH 4.0 but absent in p15. It should also be noted that the potent HIV-1 protease inhibitor U-75875 (Ashorn et al, 1990) with K j < 1 nM, blocked all of the cleavages documented in this work. …”

Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event.

“…Other, nonnucleoside inhibitors ofthe HIV-1 RT have been recently identified (3)(4)(5) that directly interact with the enzyme at a specific target site, provisionally designated as the TIBO {tetrahydroimidazo [4,5,1- In addition to the RT, several other stages in the HIV replicative cycle, starting from the virion binding to the cells to the ultimate release (budding) of the virus particles from the cells, have been envisaged as targets for therapeutic intervention (7)(8)(9)(10). One such target is the HIV protease, and several HIV-1 protease inhibitors have been designed that appear to inhibit the enzyme, and consequently virus replication, with high specificity (11)(12)(13)(14)(15)(16). The viral protease is essential for proper assembly of mature, fully infectious HIV particles.…”