Dennis M. Lambert

SYNTHESIS AND ANTIVIRAL ACTIVITY OF A NOVEL CLASS OF HIV-1 PROTEASE INHIBITORS CONTAINING A HETEROCYCLIC P1'-P2' AMIDE BOND ISOSTERE

A novel series of hydroxyethylene-based peptidomimetics that contain 2-substituted nitrogen heterocycles as P1′-P2′ amide bond isosteres has been prepared and evaluated as inhibitors of HIV-1 protease and in vitro HIV-1 replication. Many of these compounds exhibit inhibition constants in the low to subnanomolar range. Structure-activity relationships are discussed.

Synergistic drug interactions of an HIV-1 protease inhibitor with AZT in different in vitro models of HIV-1 infection

Synthetic peptide mimetic inhibitors of HIV-1 protease effectively block spread of infectious virus in acutely infected T-cells. These compounds also inhibit production of infectious virions from chronically infected T-cell lines. In order to determine the potential for drug interaction effects on antiviral activity, an HIV-1 protease inhibitor (SK&F 108922) and AZT were studied in three different in vitro models of HIV-1 infection of T-cell lines, specifically, (1) acutely infected cells infected at low multiplicity, (2) HIV-1 chronically-infected cells and (3) co-cultivations of chronically infected with non-infected cells. Upon co-treatment, these compounds demonstrated synergy in Molt4 or H9 cells acutely infected with HIV-1 strain IIIB. Either compound alone was a potent inhibitor of HIV-1 in co-cultivations of uninfected and chronically infected cells. In combination treatments of co-cultures, SK&F 108922 demonstrated strong synergy with AZT. Treatment of H9/IIIB chronically infected cells demonstrated no inhibitory effect by AZT treatment (EC50 = > 100 microM) whereas SK&F 108922 was inhibitory (EC50 = 3 microM). Upon co-treatment of H9/IIIB chronically infected cultures with both compounds, the antiviral activity was similar to that of the protease inhibitor alone suggesting no drug interaction. In the co-cultivation experiments, AZTs antiviral effect was most likely due to blocking spread of acute infection to uninfected cells in the culture. No antagonistic effects were observed with AZT and SK&F 108922 co-treatments. These results clearly demonstrate that an HIV-1 protease inhibitor can exert a potent antiviral effect on chronically infected T-cells in contrast to AZT and is capable of potent synergy with AZT in acute and co-culture in vitro infection models.

The chronically infected cell as a target for the treatment of HIV infection and AIDS

Infection of the T lymphocyte with HIV results in a cytopathic effect and cell death that has been linked to a selective loss of the helper T-lymphocyte function of the immune system. In addition to acute infection, which leads to cell death, a chronic or persistent infection also occurs. The persistence of these viral reservoirs has been implicated in the progression of HIV infection and AIDS. Rational drug discovery targeted to late-stage events in HIV replication has the potential to yield antiviral agents capable of blocking virus spread by inhibiting the production of infectious virions from these chronic reservoirs. Steve Petteway and colleagues discuss antiviral strategies that target the chronically infected cell, with a focus on HIV protease inhibitors.

Effects of SKF 108922, an HIV-1 protease inhibitor, on retrovirus replication in mice.

Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polypeptides in cultures of human T lymphocytes infected with human immunodeficiency virus type 1 (HIV-1) and therefore suppress the infectivity of HIV-1 in vitro. We have previously reported the antiviral activity in vitro of HIV-1 protease inhibitors against the C-type retrovirus Rauscher murine leukemia virus (RMuLV) and the lentivirus simian immunodeficiency virus (SIV). The same compounds which blocked the infectivity of HIV-1 also inhibited the infectivity of RMuLV and SIV in vitro. This report extends these findings by testing the antiviral activity of HIV-1 protease inhibitors in vivo in the RMuLV model. RMuLV-infected mice were treated twice a day (bid) with either an active (SKF 108922) or inactive (SKF 109273) compound for fourteen days by the intraperitoneal (i.p.) route. Compared with excipient control, SKF 108922, formulated with hydroxypropyl-beta-cyclodextrin (HPB), reduced virus-induced splenomegaly, viremia, and serum reverse transcriptase (RT) levels, while SKF 109273 was inactive. The HPB vehicle by itself enhanced replication of RMuLV. The effects of changing the formulation and the route of administration were examined. SKF 108922, formulated in HPB, had similar antiviral activity when administered by the i.p. or subcutaneous (SC) routes. However, SKF 108922 administered as a colloidal suspension in cholesterol sulfate (CS) had no detectable antiviral effect. Measurements of the circulating levels of the protease inhibitor in plasma explained this result. Plasma concentrations of SKF 108922 exceeded 1000 nM within 10 min after SC administration of the compound solubilized in HPB, but SKF 108922 was not detected in plasma after SC administration of the same dose formulated with CS. Information on optimal conditions for administering these agents should prove useful in guiding their clinical application Therefore, RMuLV should provide a good model for the preclinical evaluation and development of this class of agents for the treatment of HIV.

Ultrastructural and Cytochemical Analysis of the Loss of Envelope Glycoproteins (gp120) on Budding and Mature HIV‐1 following Treatment with Soluble T4 (sT4)

The human immunodeficiency virus (HIV) infects cells that express the surface antigen CD4 (T4), a 55 kDa transmembrane glycoprotein expressed on the surface of CD4+ T lymphocytes and cells of the mononuclear phagocyte lineage.’.* HIV is believed to infect CD4+ cells following binding of the viral envelope glycoprotein gp120 to cellular CD4.3-4 Viral gp120 is noncovalently bound to gp41 on the surface of HIV-1 but can be visualized on the surface of HIV as 9 nm X 15 nm spikes by the tannic acid-lead citrate method of Gelderblom’ or by immunocytochemical staining. These spikes, however, appear to be “spontaneously” shed from the virion after it has budded from the infected celL5 Soluble T4 (sT4) is a recombinant soluble form of CD4 that inhibits both viral infectivity6 and syncytia formation.’ The mechanism of sT4 inhibition is not completely understood. Soluble CD4 constructs bind to gp120 and thus are believed to interfere with viral binding to CD4+ cells.” We attempted to demonstrate the binding of sT4 to gp120 on HIV using electron microscopic cytochemistry. At concentrations of sT4 that inhibit infectivity and syncytia formation, however, we were unable to detect binding of horseradish peroxidaseand colloidal gold-labeled sT4 to HIV-1, strain IIIB virions. To examine this apparent lack of binding and to evaluate the loss of gp120 spikes from virions, we examined the spike distribution on CEM cells infected with HIV-1, strain IIIB before and after treatment with sT4. On budding virions, distribution of spikes was highly periodic (FIG. 1A). After treatment with sT4 (10 pg/mL, FIG. lB), there was an apparent decrease in the number of spikes. Morphometric analysis (FIG. 2A) of the distribution of spikes revealed a

Synthesis of a C-Terminal Fluorescein-Labeled 36-mer Peptide

Biomedical researchers routinely incorporate fluorescently labeled peptides into assays to enhance detection sensitivity and/or to study receptor binding mechanisms associated with cellular physiology. While there are many methods available that allow a researcher to fluorescently label a peptide at the N-terminus, or on a lysine residue, there are no strategies for C-terminal labeling with fluorescein derivatives that utilize the carboxylic acid of amino acids [1,2].