Abstract
The potential of fluorescence-based methods and kinetic analysis in the screening and molecular-scale mechanistic investigation of PPI modulation by small molecules is discussed through several representative examples collected and commented. These experimental approaches take advantage of a variety of observables. Changes in the protein aggregation pattern have been monitored through fluorescence properties such as spectra, intensities (related to quantum yields), time-decays, and anisotropies of intrinsic protein fluorophores, of extrinsic fluorescent tags and, even, of the same small molecules added to modulate PPIs, as well as through bimolecular excited-state processes such as static and collisional quenching, including electron and excitation-energy transfer, or exciton interaction, whose efficiencies are crucially structure dependent. Besides allowing for qualitative and quantitative information on the small-molecule induced PPI modulation, these approaches can take advantage from the sensitivity of fluorescence observables on fine structural details to shed light on the molecular-scale mechanisms of action and their functional consequences. Direct investigation of the latter by kinetic inhibition analysis represents a useful change in perspective whenever PPI are relevant for enzyme activity. Dissociative inhibition, that is, the ability of some small molecules to inhibit enzymes by disrupting their active oligomeric assembly is shortly reviewed.