Biosensor-based screening and characterization of HIV-1 inhibitor interactions with Sap 1, Sap 2, and Sap 3 from Candida albicans.
A surface plasmon resonance (SPR) biosensor-based strategy for identification and characterization of compounds has been devised as a tool for the discovery of specific drugs for treatment of Candida albicans infections. Three secreted aspartic proteases (Saps 1-3) from C. albicans were used as parallel targets. The stepwise procedure involved screening of 104 HIV-1 pro-tease inhibitors at a single concentration for binding to the targets. Twenty-four compounds that appeared to interact with the targets were identified in the screen. False positives and compounds with low affinities or very fast dissociation rates could be removed after a series of additional measurements of these compounds at 3 different concentrations. Kinetic characterization was performed with 13 compounds, giving information about the interaction mechanism and interaction kinetic parameters (k(on), k(off), and K(D)). The pH dependence of the interaction and the inhibitory effect of a final small set of compounds were also evaluated. The strategy resulted in the identification of ritonavir as the compound generally exhibiting the highest affinity for the Candida enzymes. It had similar interaction kinetic characteristics for Sap 1 and Sap 2 but a lower affinity for Sap 3 due to a slower association rate. Several additional compounds with high affinity and/or slow dissociation rates for the targets were identified, revealing 2 other structural scaffolds for Sap inhibitors. In addition, important differences in the specificity for these types of compounds by the Saps were identified. The stepwise biosensor-based strategy was consequently efficient for identification and characterization of new lead compounds for 3 important drug targets.
Backman
Dan
D
Department of Biochemistry, Uppsala University, Uppsala, Sweden.
Monod
Michel
M
Danielson
U Helena
UH
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002176:Candida albicans / Q000201:enzymology*; D004347:Drug Interactions; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D008958:Models, Molecular; D010436:Pepstatins / Q000737:chemistry; D019438:Ritonavir / Q000494:pharmacology*
D005656:Fungal Proteins; D017320:HIV Protease Inhibitors; D007527:Isoenzymes; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D016282:Aspartic Acid Endopeptidases; C086390:SAP1 protein, Candida albicans; C090753:SAP2 protein, Candida; D019438:Ritonavir