The survival of living organisms relies on the rapid reactions of biological pathways which are catalyzed by enzymes. Aberrant enzyme activities are linked with most diseases. Small molecule enzyme inhibitors therefore are sought after as lead compounds for further pharmaceutical development into commercial drugs. A routine inhibitor discovery scheme begins by measuring the inhibitory activity of random compounds against the enzyme of interest. In these measurements, enzymes are isolated away from the cellular environments and placed in slightly salted water namely dilute solutions. The non-ideality of this testing condition is its lack of representation of the crowdedness inside the cell. Cellular volume is occupied by macromolecules such proteins, nucleic acids, polysaccharides, leaving limited space for enzymatic movement. The crowded condition is referred to as “molecular crowding” which can be generated in test tubes by adding inner synthetic polymers, termly “molecular crowders”. Past studies revealed that enzymes have strikingly different profiles under molecular crowding including their stability, conformation, self-association rates and molecular interactions. Surprisingly, given the pharmaceutical importance of enzyme inhibitors, there is very limited knowledge about the macromolecular crowding effects on enzyme inhibition. We investigated the effect of macromolecular crowding, generated by 20% Ficoll70, on the inhibitory ability of various inhibitors against a protease enzyme, trypsin. We found that the inhibitors generally bind to trypsin with lower affinity in the crowded media than in the dilute media. However, the magnitude of the affinity change differs widely, from 1.5 fold decrease to 10-fold decrease, which is dependent on the hydrophobicity of the inhibitors. To the best of our knowledge, this is the first time the molecular crowding effects on enzyme inhibitors are studied and compared.

Exploring the macromolecular crowding effects on enzyme inhibition

254th National Meeting (2017)

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