Analysis of the interaction between HIV-1 protease and inhibitors : Applications for drug discovery

Detta är en avhandling från Uppsala : Acta Universitatis Upsaliensis

Sammanfattning: The aims of this study were to identify and characterize inhibitors of HIV-l protease aspotential leads in a drug discovery process, and to develop improved methods for suchcharacterization. More than 300 inhibitors, including linear and cyclic peptidomimetictransition state analogs and Cu2+, were analyzed.The inhibition of HIV-l protease mutants conferring viral resistance by two cyclicurea inhibitors of similar structure, was determined and differences observed.Both binding and the inhibitory effect of Cu2+ were analyzed and non active-siteamino acid residues influencing the interaction (among them His-69) were identifiedby selected point mutations.Biosensor based methods were developed for screening and characterization of lowmolecular weight inhibitors (Mw = 200 - 1000): The sensor surface with immobilizedHIV- 1 protease was found to be catalytically active and its affinity for inhibitorscorrelated with their Ki-values. By using a simple experimental design and dataanalysis technique ~100 samples per day could be screened. The association anddissociation rates were characteristic for different compounds although a correlationwith their structural class was observed. The interaction kinetic constants weredetermined for the clinically used inhibitors saquinavir, ritonavir, indinavir andnelfinavir. Saquinavir was found to have the slowest dissociation and highest affinity(koff = 2.8 x 10-4 s-1, KD = 0.33 nM) , while ritonavir had the fastest association (kon =2.4 x 106 M-1s-1). These parameters revealed differences between the compounds thatwere not distinguished by inhibition studies and that may be of greater relevance forthein vivo efficiency of the inhibitors.

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