A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

Covalent Inhibition of the Human 20S Proteasome with Homobelactosin C Inquired by QM/MM Studies.

20S proteasome is a main player in the protein degradation pathway in the cytosol, thus intervening in multiple pivotal cellular processes. Over the years the proteasome has emerged as a crucial target for the treatment of many diseases such as neurodegenerative diseases, cancer, autoimmune diseases, developmental disorders, cystic fibrosis, diabetes, cardiac diseases, atherosclerosis, and aging. In this work, the mechanism of proteasome covalent inhibition with bisbenzyl-protected homobelactosin C (hBelC) was explored using quantum mechanics/molecular mechanics (QM/MM) methods. Molecular dynamic simulations were used to describe key interactions established between the hBelC and its unique binding mode in the primed site of the ß5 subunit. The free energy surfaces were computed to characterize the kinetics and thermodynamics of the inhibition process. This study revealed that although the final inhibition product for hBelC is formed according to the same molecular mechanism as one described for hSalA, the free energy profile of the reaction pathway differs significantly from the one previously reported for γ-lactam-ß-lactone containing inhibitors in terms of the height of the activation barrier as well as the stabilization of the final product. Moreover, it was proved that high stabilization of the covalent adduct formed between ß5-subunit and hBelC, together with the presence of aminocarbonyl side chain in the structure of the inhibitor which prevents the hydrolysis of the ester bond from taking place, determines its irreversible character.

Mechanism of inhibition of SARS-CoV-2 Mpro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity.

The SARS-CoV-2 main protease (Mpro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 Mpro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 Mpro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of Mpro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

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 ß5-subunit by dihydroeponemycin, an epoxyketone. The presence of a dual electrophilic center in this α,ß-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 Cß 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.