Design, synthesis and biological evaluation of peptidomimetic benzothiazolyl ketones as 3CLpro inhibitors against SARS-CoV-2.

A series of peptidomimetic compounds containing benzothiazolyl ketone and [2.2.1] azabicyclic ring was designed, synthesized and evaluated in the hope of obtaining potent oral 3CLpro inhibitors with improved pharmacokinetic properties. Among the target compounds, 11b had the best enzymatic potency (IC50 = 0.110 µM) and 11e had the best microsomal stability (t1/2 > 120 min) and good enzyme activity (IC50 = 0.868 µM). Therefore, compounds 11b and 11e were chosen for further evaluation of pharmacokinetics in ICR mice. The results exhibited that the AUC(0-t) of 11e was 5143 h*ng/mL following single-dose oral administration of 20 mg/kg, and the F was 67.98%. Further structural modification was made to obtain compounds 11g-11j based on 11e. Among them, 11j exhibited the best enzyme inhibition activity against SARS-CoV-2 3CLpro (IC50 = 1.646 µM), the AUC(0-t) was 32473 h*ng/mL (20 mg/kg, po), and the F was 48.1%. In addition, 11j displayed significant anti-SARS-CoV-2 activity (EC50 = 0.18 µM) and low cytotoxicity (CC50 > 50 µM) in Vero E6 cells. All of the above results suggested that compound 11j was a promising lead compound in the development of oral 3CLpro inhibitors and deserved further research.

Design, synthesis and biological evaluation of covalent peptidomimetic 3CL protease inhibitors containing nitrile moiety.

In this paper, a series of peptidomimetic SARS-CoV-2 3CL protease inhibitors with new P2 and P4 positions were synthesized and evaluated. Among these compounds, 1a and 2b exhibited obvious 3CLpro inhibitory activities with IC50 of 18.06 nM and 22.42 nM, respectively. 1a and 2b also showed excellent antiviral activities against SARS-CoV-2 in vitro with EC50 of 313.0 nM and 170.2 nM, respectively, the antiviral activities of 1a and 2b were 2- and 4-fold better than that of nirmatrelvir, respectively. In vitro studies revealed that these two compounds had no significant cytotoxicity. Further metabolic stability tests and pharmacokinetic studies showed that the metabolic stability of 1a and 2b in liver microsomes was significantly improved, and 2b had similar pharmacokinetic parameters to that of nirmatrelvir in mice.

Synthesis, cytotoxicity, and pharmacokinetic evaluations of niclosamide analogs for anti-SARS-CoV-2.

Niclosamide, an oral anthelmintic drug, could inhibit SARS-CoV-2 virus replication through autophagy induction, but high cytotoxicity and poor oral bioavailability limited its application. Twenty-three niclosamide analogs were designed and synthesized, of which compound 21 was found to exhibit the best anti-SARS-CoV-2 efficacy (EC50 = 1.00 µM for 24 h), lower cytotoxicity (CC50 = 4.73 µM for 48 h), better pharmacokinetic, and it was also well tolerated in the sub-acute toxicity study in mice. To further improve the pharmacokinetics of 21, three prodrugs have been synthesized. The pharmacokinetics of 24 indicates its potential for further research (AUClast was 3-fold of compound 21). Western blot assay indicated that compound 21 could down-regulate SKP2 expression and increase BECN1 levels in Vero-E6 cells, indicating the antiviral mechanism of 21 was related to modulating the autophagy processes in host cells.

Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2.

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2. Here we report that nelfinavir, an FDA approved drug for the treatment of HIV infection, is effective against SARS-CoV-2 and COVID-19. Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2 (IC50 = 8.26 µM), while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93 µM (EC50). In comparison with vehicle-treated animals, rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals. At necropsy, nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude. A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center, which were randomized (1:1) to nelfinavir and control groups, showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days (9.0 vs. 14.5 days, P = 0.055) and the duration of fever time by 3.8 days (2.8 vs. 6.6 days, P = 0.014) in mild/moderate COVID-19 patients. The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients, together with its well-established good safety profile in almost all ages and during pregnancy, indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target.

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.

Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CLpro) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CLpro. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CLpros, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.