Affinity and Selectivity Assessment of Covalent Inhibitors by Free Energy Calculations.

Covalent inhibitors have been gaining increased attention in drug discovery due to their beneficial properties such as long residence time, high biochemical efficiency, and specificity. Optimization of covalent inhibitors is a complex task that involves parallel monitoring of the noncovalent recognition elements and the covalent reactivity of the molecules to avoid potential idiosyncratic side effects. This challenge calls for special design protocols, including a variety of computational chemistry methods. Covalent inhibition proceeds through multiple steps, and calculating free energy changes of the subsequent binding events along the overall binding process would help us to better control the design of drug candidates. Inspired by the recent success of free energy calculations on reversible binders, we developed a complex protocol to compute free energies related to the noncovalent and covalent binding steps with thermodynamic integration and hybrid quantum mechanical/molecular mechanical (QM/MM) potential of mean force (PMF) calculations, respectively. In optimization settings, we examined two therapeutically relevant proteins complexed with congeneric sets of irreversible cysteine targeting covalent inhibitors. In the selectivity paradigm, we studied the irreversible binding of covalent inhibitors to phylogenetically close targets by a mutational approach. The results of the calculations are in good agreement with the experimental free energy values derived from the inhibition and kinetic constants (Ki and kinact) of the enzyme-inhibitor binding. The proposed method might be a powerful tool to predict the potency, selectivity, and binding mechanism of irreversible covalent inhibitors.