HIV-1 reverse transcriptase as a target in the development of specific enzyme inhibitors

Abstract

Human immunodeficiency virus type I (HIV-I), which causes the acquired immunodeficiency syndrome (AIDS), begins its intracellular infection life cycle with reverse transcription of its plus-strand RNA genome into a double-stranded proviral DNA intermediate which is integrated into the host chromosome inducing a persistent infection. A virally encoded enzyme, reverse transcriptase (RT), carries out the reverse transcription process by performing all three enzymatic activities, i.e. RNA-directed DNA synthesis, DNA-directed DNA synthesis and hydrolysis of the RNA strand from RNA-DNA hybrids. Hence, it is a major target for chemotherapy of HIV infections.

The aims of the present studies are focused on the major intrinsic features of the reverse transcription process and possibilities to inhibit them. In order to understand the basis of viral resistance, several cloned recombinant RTs and RT mutants(genetic variants) that are associated with viral resistance to different classes of non-nucleoside RT inhibitors, have been prepared and characterised with respect to their catalytic properties. When comparing the catalytic specificity (kcal/Km)of wild type (parental) enzyme in steady state enzyme kinetics to different mutants, mutant RT with the double amino acid changes of Leu100-lle and Tyrl88-His displayed the most retarded catalytic properties on heteropolymeric RNA and DNA templates.

Emergence of drug-resistant variants is a major obstacle in the development of anti-HlV agents. Different RTs were used as individual molecular targets in order to characterise the inhibitory profiles of NNRTls. These RT inhibitors, represented by 9-CI-TIBO, nevirapine, L-697,661, displayed different patterns of inhibitory activity against the activity of HIV-I RT and mutant RTs. The inhibitory effects were characterised by inhibition constants (Kis and Kii) in Michaelis & Menten enzyme kinetics. In general, inhibition of wild type RT exhibited exclusively non-competitive patterns giving Kis = Kii. Pure non-competitive inhibition was not observed for mutant enzymes giving Kis < Kii. A mixed inhibitory pattern was obtained reflecting a partial contribution of a competitive inhibition at an allosteric NNRTI binding site.

Access to NNRTI-associated resistant enzymes is of importance in the evaluation of new antiviral agents, to determine common properties of resistance and thereby assist in the development of new drugs. The profile of an NNRTI can be determined rapidly by using different NNRTI resistant enzymes. A non-competitive pattern of inhibition gives a common feature of Kis = Kii = IC50 and thereby a rapid assessment of inhibitory potency. Feedback of biochemical and biological information to the chemical synthesis process was used extensively and led to the discovery of a new generation of PETT derivatives inhibitory to both wild type and mutant HIV-I RTs in the nanomolar range.

The prototype PETT compound, trovirdine, inhibited HIV-I RT with an IC50 of 7nM, when employing heteropolymeric RNA template. Enzyme kinetic studies showed that inhibition of RT by trovirdine was purely non-competitive with regard to deoxynucleoside triphosphates. The allosteric binding to HIV-I RT resulted in a decelerated rate of DNA polymerisation and has been implicated as a major inhibitory functionality. The cross resistance profile and inhibitory mechanism on mutant RTs (181Tyr-Cys) and RT (100 Leu-Ile) has verified that trovirdine shares common features with other groups of non-nucleoside RT inhibitors having overlapping binding sites on RT.

Combined chemotherapeutic approaches have been used extensively in order to reduce drug toxicity, possibly delaying development of viral resistance and achieving synergistic antiviral effects. This now represents the best anti-HIV strategy. Hence, it is important to evaluate the combination profile of a new inhibitor. Combinations of trovirdine with other RT inhibitors including AZT, ddC, ddI and their triphosphates, were studied in both cell-free HIV-I polymerase assays and HIV-I-infected MT-4 cell cultures. Synergistic and additive effects were observed both by using RT and HIV-I-infected MT-4 cells and by using different HIV-I RT mutants, as well as HIV-I drug resistant variants known to be resistant to the inhibitory effects of trovirdine. These studies indicate a potential for synergistic effects also in vivo when combining trovirdine and several clinically used anti-HIV drugs.

The difference in inhibition by FLG-TP between HIV-I RT and cellular DNA polymerases make FLG a potentially useful HIV-I RT inhibitor. The emergence of resistant variants during in vitro selection was slower for FLG than for 3TC and similar to that observed with AZT. FLG inhibited HIV-I resistant to AZT and to non-nucleoside RT inhibitors and showed some cross-resistance to virus resistant to 3TC. The viral resistance was also manifest in HIV polymerase assays using virion-derived RT. The Ki value of FLG-resistant virion RT showed a 10 fold decreased ability to compete with the natural substrate. Chain elongation studies using M13mpl8 single-strand DNA showed that FLG-TP terminated RT-catalysed transcription at a base-specific site and this served as a major inhibitory functionality.

A greater potential for interrupting the dynamic process of HIV replication can be achieved by combined antiviral therapy. Selection of drug regimens should be based on individual characteristics of the inhibitors in order to interrupt viral replication. Therefore, characterisation of different inhibitory mechanisms manuscripts and investigation of viral variants and associated enzyme properties in the optimisation of new inhibitory compound plays a fundamental role in the struggle to combat this devastating human disease.