MPI8 is Potent against SARS‐CoV‐2 by Inhibiting Dually and Selectively the SARS‐CoV‐2 Main Protease and the Host Cathepsin L**

Abstract

A number of inhibitors have been developed for the SARS‐CoV‐2 main protease (M Pro ) as potential COVID‐19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed M Pro inhibitors including MPI1‐9, GC376, 11a, 10–1, 10–2, and 10–3. MPI1‐9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the M Pro active site cysteine and 10–1, 10–2 and 10–3 contain a labile ester to exchange with the M Pro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC 50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular M Pro inhibition potency and anti‐SARS‐CoV‐2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS‐CoV‐2 cell entry. By selectively inhibiting both SARS‐CoV‐2 M Pro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID‐19.

Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity

“…The majority of the reported M pro inhibitors contain the aldehyde reactive warhead, which is known to have nonspecific reactivity toward host proteins. 16 − 19 It should be noted that both of the Pfizer M pro inhibitors that are currently in clinical trials do not contain the aldehyde warhead. 9 , 10 As such, we are interested in developing SARS-CoV-2 M pro inhibitors with high target specificity.…”

Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro/3CLpro) and Human Cathepsin-L

“…An additional site to optimize the selectivity of dual Mpro/Cathepsin-L inhibitors is the cysteine reactive warhead as recently discussed by Ma et al [52]. Substitutions of the aldehyde group with other reversible groups, such as a-keto derivatives or nitriles can be considered.…”

Inhibition of SARS-CoV-2 by Targeting Conserved Viral RNA Structures and Sequences

“…PF-07321332 (Paxlovoid) was developed as an oral drug targeting M pro and is being tested in a Phase 3 clinical trial ( ClinicalTrials.gov Identifier: NCT04960202) ( Owen et al, 2021 ). Other reported M pro inhibitors such as an FDA-approved drug, bepridil, and a peptoid MPI8 were demonstrated to have efficacy in virus-infected cells ( Ma et al, 2021 ; Vatansever et al, 2021 ). RdRP inhibitors remdesivir and molnupiravir, which impede the RNA replication/transcription processes, both showed clinical improvement in the COVID-19 patients ( Wang et al, 2020 ; Fischer et al, 2021 ).…”