Structure-Based Design of Potent Peptidomimetic Inhibitors Covalently Targeting SARS-CoV-2 Papain-like Protease.

The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. Here, we report the structure-guide design of novel peptidomimetic inhibitors covalently targeting SARS-CoV-2 PLpro. The resulting inhibitors demonstrate submicromolar potency in the enzymatic assay (IC50 = 0.23 µM) and significant inhibition of SARS-CoV-2 PLpro in the HEK293T cells using a cell-based protease assay (EC50 = 3.61 µM). Moreover, an X-ray crystal structure of SARS-CoV-2 PLpro in complex with compound 2 confirms the covalent binding of the inhibitor to the catalytic residue cysteine 111 (C111) and emphasizes the importance of interactions with tyrosine 268 (Y268). Together, our findings reveal a new scaffold of SARS-CoV-2 PLpro inhibitors and provide an attractive starting point for further optimization.

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.

Unraveling the catalytic mechanism of SARS-CoV-2 papain-like protease with allosteric modulation of C270 mutation using multiscale computational approaches.

Papain-like protease (PLpro) is a promising therapeutic target against SARS-CoV-2, but its restricted S1/S2 subsites pose an obstacle in developing active site-directed inhibitors. We have recently identified C270 as a novel covalent allosteric site for SARS-CoV-2 PLpro inhibitors. Here we present a theoretical investigation of the proteolysis reaction catalyzed by the wild-type SARS-CoV-2 PLpro as well as the C270R mutant. Enhanced sampling MD simulations were first performed to explore the influence of C270R mutation on the protease dynamics, and sampled thermodynamically favorable conformations were then submitted to MM/PBSA and QM/MM MD simulations for thorough characterization of the protease-substrate binding and covalent reactions. The disclosed proteolysis mechanism of PLpro, as characterized by the occurrence of proton transfer from the catalytic C111 to H272 prior to the substrate binding and with deacylation being the rate-determining step of the whole proteolysis process, is not completely identical to that of the 3C-like protease, another key cysteine protease of coronaviruses. The C270R mutation alters the structural dynamics of the BL2 loop that indirectly impairs the catalytic function of H272 and reduces the binding of the substrate with the protease, ultimately showing an inhibitory effect on PLpro. Together, these results provide a comprehensive understanding at the atomic level of the key aspects of SARS-CoV-2 PLpro proteolysis, including the catalytic activity allosterically regulated by C270 modification, which is crucial to the follow-up inhibitor design and development.

Discovery of Polyphenolic Natural Products as SARS-CoV-2 Mpro Inhibitors for COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the development of direct-acting antiviral drugs due to the coronavirus disease 2019 (COVID-19) pandemic. The main protease of SARS-CoV-2 is a crucial enzyme that breaks down polyproteins synthesized from the viral RNA, making it a validated target for the development of SARS-CoV-2 therapeutics. New chemical phenotypes are frequently discovered in natural goods. In the current study, we used a fluorogenic assay to test a variety of natural products for their ability to inhibit SARS-CoV-2 Mpro. Several compounds were discovered to inhibit Mpro at low micromolar concentrations. It was possible to crystallize robinetin together with SARS-CoV-2 Mpro, and the X-ray structure revealed covalent interaction with the protease's catalytic Cys145 site. Selected potent molecules also exhibited antiviral properties without cytotoxicity. Some of these powerful inhibitors might be utilized as lead compounds for future COVID-19 research.

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.

Bioactive compounds from Huashi Baidu decoction possess both antiviral and anti-inflammatory effects against COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health concern, and effective antiviral reagents are urgently needed. Traditional Chinese medicine theory-driven natural drug research and development (TCMT-NDRD) is a feasible method to address this issue as the traditional Chinese medicine formulae have been shown effective in the treatment of COVID-19. Huashi Baidu decoction (Q-14) is a clinically approved formula for COVID-19 therapy with antiviral and anti-inflammatory effects. Here, an integrative pharmacological strategy was applied to identify the antiviral and anti-inflammatory bioactive compounds from Q-14. Overall, a total of 343 chemical compounds were initially characterized, and 60 prototype compounds in Q-14 were subsequently traced in plasma using ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Among the 60 compounds, six compounds (magnolol, glycyrrhisoflavone, licoisoflavone A, emodin, echinatin, and quercetin) were identified showing a dose-dependent inhibition effect on the SARS-CoV-2 infection, including two inhibitors (echinatin and quercetin) of the main protease (Mpro), as well as two inhibitors (glycyrrhisoflavone and licoisoflavone A) of the RNA-dependent RNA polymerase (RdRp). Meanwhile, three anti-inflammatory components, including licochalcone B, echinatin, and glycyrrhisoflavone, were identified in a SARS-CoV-2-infected inflammatory cell model. In addition, glycyrrhisoflavone and licoisoflavone A also displayed strong inhibitory activities against cAMP-specific 3',5'-cyclic phosphodiesterase 4 (PDE4). Crystal structures of PDE4 in complex with glycyrrhisoflavone or licoisoflavone A were determined at resolutions of 1.54 Å and 1.65 Å, respectively, and both compounds bind in the active site of PDE4 with similar interactions. These findings will greatly stimulate the study of TCMT-NDRD against COVID-19.

Discovery of Polyphenolic Natural Products as SARS-CoV-2 Mpro Inhibitors for COVID-19

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the development of direct-acting antiviral drugs due to the coronavirus disease 2019 (COVID-19) pandemic. The main protease of SARS-CoV-2 is a crucial enzyme that breaks down polyproteins synthesized from the viral RNA, making it a validated target for the development of SARS-CoV-2 therapeutics. New chemical phenotypes are frequently discovered in natural goods. In the current study, we used a fluorogenic assay to test a variety of natural products for their ability to inhibit SARS-CoV-2 Mpro. Several compounds were discovered to inhibit Mpro at low micromolar concentrations. It was possible to crystallize robinetin together with SARS-CoV-2 Mpro, and the X-ray structure revealed covalent interaction with the protease's catalytic Cys145 site. Selected potent molecules also exhibited antiviral properties without cytotoxicity. Some of these powerful inhibitors might be utilized as lead compounds for future COVID-19 research.

Ginkgolic acids inhibit SARS-CoV-2 and its variants by blocking the spike protein/ACE2 interplay.

Targeting the interaction between the spike protein receptor binding domain (S-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and angiotensin-converting enzyme 2 (ACE2) is a potential therapeutic strategy for treating coronavirus disease 2019 (COVID-19). However, we still lack small-molecule drug candidates for this target due to the missing knowledge in the hot spots for the protein-protein interaction. Here, we used NanoBiT technology to identify three Ginkgolic acids from an in-house traditional Chinese medicine (TCM) library, and they interfere with the S-RBD/ACE2 interplay. Our pseudovirus assay showed that one of the compounds, Ginkgolic acid C17:1 (GA171), significantly inhibits the entry of original SARS-CoV-2 and its variants into the ACE2-overexpressed HEK293T cells. We investigated and proposed the binding sites of GA171 on S-RBD by combining molecular docking and molecular dynamics simulations. Site-directed mutagenesis and surface plasmon resonance revealed that GA171 specifically binds to the pocket near R403 and Y505, critical residues of S-RBD for S-RBD interacting with ACE2. Thus, we provide structural insights into developing new small-molecule inhibitors and vaccines against the proposed S-RBD binding site.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Flavonoids from the roots and rhizomes of Sophoratonkinensis and their in vitro anti-SARS-CoV-2 activity.

Acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had caused a global pandemic since 2019, and posed a serious threat to global health security. Traditional Chinese medicine (TCM) has played an indispensable role in the battle against the epidemic. Many components originated from TCMs were found to inhibit the production of SARS-CoV-2 3C-like protease (3CLpro) and papain-like protease (PLpro), which are two promising therapeutic targets to inhibit SARS-CoV-2. This study describes a systematic investigation of the roots and rhizomes of Sophora tonkinensis, which results in the characterization of 12 new flavonoids, including seven prenylated flavanones (1-7), one prenylated flavonol (8), two prenylated chalcones (9-10), one isoflavanone (11), and one isoflavan dimer (12), together with 43 known compounds (13-55). Their structures including the absolute configurations were elucidated by comprehensive analysis of MS, 1D and 2D NMR data, and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC50 values of 34.89 and 19.88 µmol·L-1, repectively while compounds 9, 43 and 47 exhibited inhibitory effects against PLpro with IC50 values of 32.67, 79.38, and 16.74 µmol·L-1, respectively.

Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2.

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 µM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS‐CoV‐2 Papain‐Like Protease**

The coronavirus papain‐like protease (PLpro) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS‐CoV‐2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS‐CoV‐2 PLpro and provid a novel site for allosteric inhibitors design. [ FROM AUTHOR]

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS-CoV-2 Papain-Like Protease.

The coronavirus papain-like protease (PLpro ) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS-CoV-2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS-CoV-2 PLpro and provid a novel site for allosteric inhibitors design.

In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2.

FB2001 is a drug candidate that targets the main protease of SARS-CoV-2 via covalently binding to cysteine 145. In this study, we evaluated the inhibitory activities of FB2001 against several SARS-CoV-2 variants in vitro and in vivo (in mice), and we also evaluated the histopathological analysis and immunostaining of FB2001 on lung and brain which have been rarely reported. The results showed that FB2001 exhibited potent antiviral efficacy against several current SARS-CoV-2 variants in Vero E6 cells, namely, B.1.1.7 (Alpha): EC50 = 0.39 ± 0.01 µM, EC90 = 0.75 ± 0.01 µM; B.1.351 (Beta): EC50 = 0.28 ± 0.11 µM, EC90 = 0.57 ± 0.21 µM; B.1.617.2 (Delta): EC50 = 0.27 ± 0.05 µM, EC90 = 0.81 ± 0.20 µM; B.1.1.529 (Omicron): EC50 = 0.26 ± 0.06 µM and EC50 = 0.042 ± 0.007 µM (in the presence of a P-glycoprotein inhibitor). FB2001 remained potent against SARS-CoV-2 replication in the presence of high concentrations of human serum, which indicating that human serum had no significant effect on the in vitro inhibitory activity. Additionally, this inhibitor exhibited an additive effect against SARS-CoV-2 when combined with Remdesivir. Furthermore, FB2001 significantly reduced the SARS-CoV-2 copy numbers and titers in the lungs and brains in vivo, and alleviated the pathological symptoms. In addition, FB2001 could alleviated local bleeding, erythrocyte overflow, edema, and inflammatory cell infiltration in brain tissue, and inhibitors reducing viral titers and improving inflammation in the brain have been rarely reported. A physiologically based pharmacokinetic model was established and verified to predict the FB2001 concentration in human lungs. When FB2001 was administered at 200 mg twice a day for 5 days, the observed Ctrough ss in plasma and predicted Ctrough ss of lung total concentration were 0.163 and 2.5 µg/mL, which were approximately 9 and 132-fold higher than the EC50 of 0.019 µg/mL (0.042 µM) against Omicron variant. Taken together, our study suggests that FB2001 is a promising therapeutic agent in COVID-19 treatment and can be combined with remdesivir to achieve improved clinical outcomes. Owing to its good safety and tolerability in healthy human (NCT05197179 and NCT04766931), FB2001 has been approved for Phase II/III clinical trial (NCT05445934).

Small-Molecule Thioesters as SARS-CoV-2 Main Protease Inhibitors: Enzyme Inhibition, Structure-Activity Relationships, Antiviral Activity, and X-ray Structure Determination.

The main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure-activity relationships of novel small-molecule thioesters as SARS-CoV-2 Mpro inhibitors. Compounds 3w and 3x exhibited excellent SARS-CoV-2 Mpro inhibition with kinac/Ki of 58,700 M-1 s-1 (Ki = 0.0141 µM) and 27,200 M-1 s-1 (Ki = 0.0332 µM), respectively. In Calu-3 and Vero76 cells, compounds 3h, 3i, 3l, 3r, 3v, 3w, and 3x displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of 3w and 3af with SARS-CoV-2 Mpro was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the Mpro of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.

In silico screening-based discovery of novel covalent inhibitors of the SARS-CoV-2 3CL protease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease (3CLpro) has been regarded as an extremely promising antiviral target for the treatment of coronavirus disease 2019 (COVID-19). Here, we carried out a virtual screening based on commercial compounds database to find novel covalent non-peptidomimetic inhibitors of this protease. It allowed us to identify 3 hit compounds with potential covalent binding modes, which were evaluated through an enzymatic activity assay of the SARS-CoV-2 3CLpro. Moreover, an X-ray crystal structure of the SARS-CoV-2 3CLpro in complex with compound 8, the most potent hit with an IC50 value of 8.50 µM, confirmed the covalent binding of the predicted warhead to the catalytic residue C145, as well as portrayed interactions of the compound with S1' and S2 subsites at the ligand binding pocket. Overall, the present work not merely provided an experiment-validated covalent hit targeting the SARS-CoV-2 3CLpro, but also displayed a prime example to seeking new covalent small molecules by a feasible and effective computational approach.

Cocktail polysaccharides isolated from Ecklonia kurome against the SARS-CoV-2 infection.

Previous researches suggested that polysaccharides from brown algae had anti-virus activity. We hypothesized that nature polysaccharide from marine plants might have the effect on anti-SARS-CoV-2 activity. By high throughput screening to target 3CLpro enzyme using polysaccharides library, we discover a crude polysaccharide 375 from seaweed Ecklonia kurome blocked 3CLpro enzymatic activity and shows good anti-SARS-CoV-2 infection activity in cell. Further, we show that homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro well and disturb spike protein binding to ACE2 receptor. The structure characterization uncovers that 37502 is alginate. These results imply that the bioactivities of 375 on SARS-CoV-2 may target multiple key molecules implicated in the virus infection and replication. The above results suggest that 375 may be a potential drug candidate 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.