On the Origin of the Different Reversible Characters of Salinosporamide A and Homosalinosporamide A in the Covalent Inhibition of the Human 20S Proteasome

Covalent inhibition of the 20S proteasome is one of the strategies used in the fight against cancer, Chagas’ disease, malaria, tuberculosis, or neurodegenerative disorders. In this work, studies based on quantum mechanics/molecular mechanics (M06-2X/AMBER) molecular dynamics simulations reveal, in agreement with experiments, that homosalinosporamide A (hSalA), contrary to the natural analogue, salinosporamide A (SalA), inhibits the β5 subunit in a reversible manner. The computed free energy landscapes indicate that either SalA or hSalA blocks the active site of the enzyme by a three-step process that includes two common chemical transformations. The last step of the inhibition with SalA, which is an intramolecular cyclization step that defines its irreversible character, was found to be energetically prohibited in the case of hSalA. In contrast, an additional chemical step was found that ensures neutralization of the negative charge accumulated during the β-lactone ring opening, thus providing a stable product of inhibition that agrees with X-ray diffraction studies. The weaker stabilization of this final product explains its reversible character that was further confirmed by exploring possible paths of hydrolysis of the covalent adduct. Finally, the rate-limiting step in both inhibitors corresponds to the nucleophilic attack of Thr1 on C1 carbon of the β-lactone. The identical activation free energies computed for SalA and hSalA agree with the undistinguishable experimentally measured values of IC50.We would like to thank the Spanish Ministerio de Ciencia e Innovación (grant PGC2018-094852-B-C21 and PID2019-107098RJ-I00), the Generalitat Valenciana (grant AICO/2019/195 and SEJI/2020/007), and Universitat Jaume I (grant UJI-B2020-03 and UJI-A2019-04). N.S.A. thanks the MINECO for the doctoral FPI grant (BES-2016-078029). Authors acknowledge computational resources from the Servei d’Informàtica of Universitat Jaume I. The authors thankfully acknowledge the computer resources at Pirineus and the technical support provided by the Consorci de Serveis Universitaris de Catalunya (RES-QSB-2020-3-0012)