ABSTRACT
In this study we show that the detergent Triton X-100, which is widely used in screening campaigns, significantly decreases the binding affinities of some known specific inhibitors of HIV-1 protease and the well-established model protease endothiapepsin in a fluorescence-based assay. Surprisingly, other structurally related inhibitors remain entirely unaffected. As a consequence, those compounds that were affected would most likely have been misclassified as unspecific binders, although they are actually true positives, and thus could be considered excellent starting points for further hit optimization.
Subject(s)
Detergents/metabolism , Enzyme Assays/methods , HIV Protease/metabolism , HIV-1/enzymology , Octoxynol/metabolism , Artifacts , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Cholic Acids/metabolism , False Negative Reactions , HIV Infections/virology , HIV Protease Inhibitors/pharmacology , HumansABSTRACT
Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases.
