Abstract
Enzymes govern biological processes as they are biocatalysts that increase the rate of chemical reactions within cells, which would not spontaneously occur. Kinases catalyze the transfer of phosphate groups from high-energy molecules to their substrates. Since these processes are crucial for many biological signaling processes, dysregulation of kinases is correlated with many human diseases. Thus, inhibition of kinases has been investigated as a means of disease treatment for many years, but mainly via orthosteric sites. However, orthosteric inhibition of kinases often presented non-specificity due to the conserved active site architecture of kinases. Furthermore, long-term clinical use of orthosteric inhibitors resulted in emergence of resistance mutations, incapacitating the treatments. Hence, allosteric inhibition has recently been highlighted as an alternative but effective way treating diseases.Recent break-through research, which combined the use of both orthosteric and allosteric drugs inhibiting a single target (double-drugging), has presented a means of effectively overcoming the resistance mutations in clinics. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric inhibitors is less understood. In this work, we quantitatively investigated the modulation of kinases, Aurora A kinase and Abelson kinase, with rational selection of the combination between orthosteric and allosteric modulators. We find that conformational equilibria shift is the main principle for the cooperativities during double-drugging. We quantify the combined inhibition effect double-drugging provides, such as how much the required concentration for orthosteric inhibition decreases in the presence of the allosteric inhibitor. Furthermore, our structural characterization of these double-drugged complexes provides insights into the atomistic mechanism underlying double-drugging’s cooperativities. Our results present a framework for future design and evaluation of double-drugging strategies.
Not only rationally designing drugs, but designing new enzymes has also garnered much interest in the past decades. Current approaches in enzyme design commonly involve multiple rounds of directed evolution mimicking the natural evolution. During this process, we expected that a more evolvable starting-point would provide wider genomic possibilities traveling the sequence (fitness) landscape, potentially achieving better end-points. However, biophysical properties affecting the evolution have not been fully elucidated despite decades of investigation by evolutionary biochemists. Guided by scientific intuition, we hypothesized that ancestral proteins are more evolvable than modern proteins due to their less exposure to evolutionary pressures. Indeed, our preliminary data suggest that ancestral adenylate kinases may be more evolvable than their extant forms, indicated by much diverse ancestral population after selection.
Collectively, we employed various methods, including coupled-enzyme assays, discontinuous assays, isothermal titration calorimetry, X-ray crystallography, Förster resonance energy transfer, and molecular biology techniques, to better understand how kinases work. We believe our data provide valuable knowledge into design and evaluation of future double-drugging candidates as well as new enzymes.