Theoretical study of the inhibition mechanism of human 20S proteasome by dihydroeponemycin.

Proteasome deregulation has been related with several human diseases and, consequently, a detailed knowledge of its inhibition is essential for the design of efficient and selective drugs. The present paper is focused on the inhibition mechanism of proteasome 20S on the β5-subunit by dihydroeponemycin, an epoxyketone. The presence of a dual electrophilic center in this α,β-epoxyketone allows its irreversible bind to the active site by formation of two strong covalent bonds with the N-terminal threonine residue. Free energy surfaces for all possible mechanisms have been generated in terms of potentials of mean force (PMFs) within hybrid QM/MM potentials, with the QM subset of atoms described at semiempirical (AM1) and DFT (M06-2X) level. Two alternative reaction pathways, differentiated by reversing the order of chemical steps in full catalytic process and the product species, were explored. The resulting activation free energy barriers (ΔG) indicate that the most favourable mechanism is the one in which the reaction starts with epoxide-ring opening and finishing with 1,4-oxazepane product formation. This result is in agreement with the seven-membered product of inhibition recently determined by X-ray crystallography. Finally, calculations of primary kinetic isotope effects (1º-KIEs) on Cα and Cβ of epoxide and secondary 2º-KIE on C1 reveal their possible application in distinguishing between the formation of six- and seven-membered product and verifying the reaction mechanism proposed in the present work.