Bicyclams, in which the cyclam (1,4,8,11-tetraazacyclotetradecane) moieties are tethered via an aliphatic bridge (i.e., propylene, as in JM2763) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) (E. De Clercq, N. Yamamoto, R. Pauwels, M. Baba, D. Schols, H. Nakashima, J. Balzarini, Z. Debyser, B. A. Murrer, D. Schwartz, D. Thornton, G. Bridger, S. Fricker, G. Henson, M. Abrams, and D. Picker, Proc. Natl. Acad. Sci. USA 89:5286-5290, 1992). We have now found that the bicyclam JM3100, in which the cyclam moieties are tethered by an aromatic bridge [i.e., phenylenebis(methylene)], inhibits the replication of various HIV-1 and HIV-2 strains in various cell lines at a 50% effective concentration (EC50) of 1 to 10 ng/ml, which is about 100-fold lower than the concentration required for JM2763 to inhibit HIV replication and at least 100,000-fold lower than the cytotoxic concentration (> 500 micrograms/ml). In primary T4 lymphocytes or primary monocytes, JM3100 proved inhibitory to HIV-1(IIIB) and several clinical HIV-1 isolates at an EC50 of less than 1 ng/ml. On the basis of time-of-addition experiments, JM3100 appeared to interact with a viral uncoating event, and this was further corroborated by an uncoating assay in which RNase sensitivity of [5-3H]uridine-labeled virions was monitored. In addition, but possibly mechanistically related, JM3100 blocks formation of infectious particles. JM3100 was also found to interfere directly with virus-induced syncytium formation, albeit at a higher concentration (1 to 2 microgram/ml) than that required for inhibition of viral replication. Following subcutaneous injection of 10 mg of JM3100 per kg of body weight to rabbits, anti-HIV activity was detected in serum corresponding to serum drug levels exceeding for at least 6 h by >100-fold the EC(50) required to inhibit HIV replication in vitro. When combined with either 3'-azido-2',3' -dideoxythymidine or 2',3' -dideoxyinosine, JM3100 achieved a additive inhibition of HIV replication, and when repeatedly subcultivated in the presence of JM3100, the virus remained sensitive to the compound for at least 30 passages (120 days) in cell culture.
(Me-Ile-4)cyclosporin (SDZ NIM 811) is a 4-substituted cyclosporin which is devoid of immunosuppressive activity but retains full capacity for binding to cyclophilin and exhibits potent anti-human immunodeficiency virus type 1 (HIV-1) activity. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line, and in HeLa T4 cells. Furthermore, its antiviral activity against laboratory strains and against clinical isolates from geographically distinct regions in primary T4 lymphocytes and in primary monocytes (50%o inhibitory concentration = 0.011 to 0.057 ,g/ml) was demonstrated. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, either free or bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virus-infected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs, and monkeys resulted in levels in blood considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than cyclosporine (formerly cyclosporin A). Thus, the potent and selective anti-HIV-1 activity of SDZ NIM 811 and its favorable pharmacokinetic behavior together with its lower nephrotoxicity than that of cyclosporine make this compound a promising candidate for development as an anti-HIV drug.
Bicyclams are a novel class of antiviral compounds which act as potent and selective inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2. They block an early step in the viral life cycle following adsorption to the CD4 receptor and preceding reverse transcription. To identify the molecular target of these compounds, we genetically analyzed variants of the HIV-1 molecular clone NL4-3, which developed resistance against two structurally related bicyclams, JM2763 and the more potent SID791. The resistant strains were obtained after long-term passaging in MT-4 cells in the presence of progressively increasing compound concentrations. Recombinants between selected genes of the resistant strains and the parental NL4-3 provirus were generated by adapting the marker rescue technique to MT-4 cells. The bicyclamresistant phenotype was rescued by transferring the envelope gp120 gene of bicyclam-resistant virus into the NL4-3 parental genetic background. In the gp120 genes of the resistant strains, we identified several mutations leading to amino acid substitutions in the V3 loop. Furthermore, two substitutions of highly conserved amino acids in close proximity to the disulfide bridges of the V3 and V4 loops were found in both SID791-and JM2763-resistant strains. Additional mutations in regions encoding V3, C4, V5, and C5 were present in SID791-resistant viruses. Recombination experiments with overlapping parts of the envelope gene indicated that most, if not all, of the mutations were necessary to develop the fully SID791 resistant phenotype. The mutations in the C-terminal part of gp120 downstream of the V3 loop sequence conferred partial resistance to JM2763 but did not significantly decrease susceptibility to SID791. The genetic data and the biological properties of the resistant viruses point to inhibition of entry and fusion as the mode of action of the HIV-inhibitory bicyclams. A possible mechanism of binding of bicyclams to gp120 leading to inhibition of unfolding of gp120 and its shedding from the gp41 fusion domain is discussed.
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