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

Sammanfattning: Human immunodeficiency virus type I (HIV-I), which causes the acquired immunodeficiencysyndrome (AIDS), begins its intracellular infection life cycle with reverse transcriptionof its plus-strand RNA genome into a double-stranded proviral DNA intemmediate whichis integrated into the host chromosome inducing a persistent infection. A virallyencoded enzyme, reverse transcriptase (RT), carries out the reverse transcriptionprocess by perfomming 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 ofthe reverse transcription process and possibilities to inhibit them. In order tounderstand the basis of viral resistance, several cloned recombinant RTs and RT mutants(genetic variants) that are associated with viral resistance to different classesof non-nucleoside RT inhibitors, have been prepared and characterised with respectto 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 displayedthe most retarded catalytic properties on heteropolymeric RNA and DNA templates. Emergence of drug-resistant variants is a major obstacle in the development ofanti-HlV agents. Different RTs were used as individual molecular targets in orderto characterise the inhibitory profiles of NNRTls. These RT inhibitors, representedby 9-CI-TIBO, nevirapine, L-697,661, displayed different pattems of inhibitory activityagainst the activity of HIV-I RT and mutant RTs. The inhibitory effects were characterisedby inhibition constants (Kis and Kii) in Michaelis & Menten enzyme kinetics.In general, inhibition of wild type RT exhibited exclusively non-competitive patternsgiving Kis = Kii. Pure non-competitive inhibition was not observed for mutant enzymesgiving Kis < Kii. A mixed inhibitory pattem was obtained reflecting a partialcontribution of a competitive inhibition at an allosteric NNRTI binding site. Access to NNRTI-associated resistant enzymes is of importance in the evaluationof new antiviral agents, to determine common properties of resistance and therebyassist in the development of new drugs. The profile of an NNRTI can be determinedrapidly by using different NNRTI resistant enzymes. A non-competitive pattern ofinhibition gives a common feature of Kis = Kii = IC50 and thereby a rapid assessmentof inhibitory potency. Feedback of biochemical and biological information to thechemical synthesis process was used extensively and led to the discovery of a newgeneration of PETT derivatives inhibitory to both wild type and mutant HIV-I RTsin 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 thatinhibition of RT by trovirdine was purely non competitive with regard to deoxynucleosidetriphosphates. The allosteric binding to HIV-I RT resulted in a decelerated rateof 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 (lOOLeu-Ile) has verified that trovidine shares common features with othergroups of non-nucleoside RT inhibitors havmg overlapping binding sites on RT. Combined chemotherapeutic approaches have been used extensively in order to reducedrug toxicity, possibly delaying development of viral resistance and achieving synergisticantiviral effects. This now represents the best anti-HIV strategy. Hence, it is importantto evaluate the combination profile of a new inhibitor. Combinations of trovirdinewith other RT inhibitors including AZT, ddC, ddI and their triphosphates, were studiedin 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-infectedMT-4 cells and by using different HIV-I RT mutants, as well as HIV-I drug resistantvariants known to be resistant to the inhibitory effects of trovirdine. These studiesindicate a potential for synergistic effects also in vivo when combining trovirdmeand several clinically used anti-HIV drugs. The difference in inhibition by FLG-TP between HIV-I RT and cellular DNA polymerasesmake FLG a potenbally useful HIV-I RT inhibitor. The emergence of resistant variantsduring in vitro selection was slower for FLG than for 3TC and similar to that observedwith AZT. FLG inhibited HIV-I resistant to AZT and to non nucleoside RT inhibitorsand showed some cross-resistance to virus resistant to 3TC. The viral resistancewas also manifest in HIV polymerase assays using virion-derived RT. The Ki valueof FLG-resistant virion RT showed a 10 fold decreased ability to compete with thenatural substrate. Chain elongation studies using M13mpl8 single-strand DNA showedthat FLG-TP temminated RT-catalysed transcription at a base-specific site and thisserved as a major inhibitory functionality. A greater potential for interupting the dynamic process of HIV replication canbe achieved by combined antiviral therapy. Selection of drug regimens should be basedon individual characteristics of the inhibitors in order to interrupt viral replication.Therefore, characterisation of different inhibitory mechanisms manuscrtipts and investigationof viral variants and associated enzyme properties in the optimisation of new inhibitorycompound plays a fundamental role in the struggle to combat this devastating humandisease. ISBN 91-628-2773-1