| dc.description.abstract | Several drugs inhibiting the replication of HIV-1, the causative agent of AIDS, are available today. However, HIV-1 treatment is hampered by side effects, poor adherence and development of resistance. Antiretroviral compounds must be combined to minimize development of resistance, and expanding the reservoir of drug classes would extend the combination possibilities. Hence, there is a need for new and safer antiretrovirals, especially with new modes of action.This thesis will present data showing that short synthetic peptides and their metabolites inhibit the replication of HIV-1 in a hitherto unexplored way: by interfering with capsid assembly. The thesis aims to determine the characteristics of these new compounds with focus on resistance, cytotoxicity, and combinations with licensed antiretroviral drugs.Short peptides corresponding to the V3 loop of gp120 were tested for their effect on HIV-1 replication in vitro. The tripeptide glycyl-prolyl-glycine-amide (GPG-NH2) was found to inhibit the replication of laboratory strains as well as clinical isolates of HIV-1, including most existing subtypes. The concentration that inhibited the replication by 50% (IC50) ranged between 2.7-37 muM. Clinical HIV-1 isolates with different drug resistance-related mutations were susceptible to GPG-NH2 regardless of mutations, indicating the lack of cross-resistance between GPG-NH2 and other antiretroviral compounds. It was also established that GPG-NH2 added to the effect of zidovudine, a reverse transcriptase inhibitor, and ritonavir, a protease inhibitor. Furthermore, the tripeptide inhibited HIV-1 of both the syncytia- and the nonsyncytia-inducing phenotypes. The antiretroviral effect was not due to inhibition of proliferation or of viability of cultured cells. After 30 consecutive passages in two parallel series, no resistance to GPG-NH2 could be detected. However, one mutation (T107I) in the p24 gene was found in both series. The metabolites from proteolytic cleavage of GPG-NH2 were tested for antiretroviral activity. Glycine-amide (G-NH2) inhibited the replication of both HIV-1 and HIV-2. No inhibitory effect could be found on the replication of a number of other viruses tested, including single-stranded and double-stranded RNA and DNA-viruses. The antiretroviral effect of G-NH2 was not due to inhibition of proliferation or of viability of cultured cells, and the IC50-values on laboratory strains as well as clinical isolates of HIV-1 ranged from 3.2-41 muM. Transmission electron microscopy studies revealed that the effect of G-NH2 on capsid morphology was equivalent to that of GPG-NH2 with disarranged core structures. As with GPG-NH2, resistance to G-NH2 was difficult to select for in vitro. The enzyme responsible for the conversion of GPG-NH2 to G-NH was shown to be CD26, a dipeptidyl peptidase present on T-cells and in serum. Purified CD26 efficiently cleaved GPG-NH2 to G-NH2 and GP-OH within a few hours, and inhibitors of CD26 abrogated the inhibitory effect of GPG-NH2. It was therefore concluded that GPG-NH2 is not antiretrovirally active as such, but acts as a prodrug requiring cleavage to G-NH2 to exert its effect. Unpublished data have shown that G-NH2 is metabolized to alpha-hydroxy-glycine-amide, alphaHGA, and this compound is planned to enter a clinical trial on HIV-1 infected patients in 2005. | en |
