Towards a New Generation of Anti-HIV Drugs Interaction Kinetic Analysis of Enzyme Inhibitors Using SPR-biosensors

Abstract: As of today, there are 25 drugs approved for the treatment of HIV and AIDS. Nevertheless, HIV continues to infect and kill millions of people every year. Despite intensive research efforts, both a vaccine and a cure remain elusive and the long term efficacy of existing drugs is limited by the development of resistant HIV strains. New drugs and preventive strategies that are effective against resistant virus are therefore still needed. In this thesis an enzymological approach, primarily using SPR-based interaction kinetic analysis, has been used for identification and characterization of compounds of potential use in next generation anti-HIV drugs.By screening of a targeted non-nucleoside reverse transcriptase inhibitor (NNRTI) library, one novel and highly potent NNRTI was identified. The inhibitor was selected with respect to resilience to drug resistance and for high affinity and slow dissociation – a kinetic profile assumed to be suitable for inhibitors used in topical microbicides. In order to confirm the hypothesis that such a kinetic profile would result in an effective preventive agent with long-lasting effect, the correlation between antiviral effect and kinetic profile was investigated for a panel of NNRTIs. The kinetic profiles revealed that NNRTI efficacy is dependent on slow dissociation from the target, although the induced fit interaction mechanism prevented quantification of the rate constants.To avoid cross-resistance, the next generation anti-HIV drugs should be based on chemical entities that do not resemble drugs in clinical use, either in structure or mode-of-action. Fragment-based drug discovery was used for identification of structurally new inhibitors of HIV-enzymes. One fragment that was effective also on variants of HIV RT with resistance mutations was identified. The study revealed the possibility of identifying structurally novel NNRTIs as well as fragments interacting with other sites of the protein.The two compounds identified in this thesis represent potential starting points for a new generation of NNRTIs. The applied methodologies also show how interaction kinetic analysis can be used as an effective and versatile tool throughout the lead discovery process, especially when integrated with functional enzymological assays.