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Design, Synthesis, and Biological Evaluation of Trisubstituted Piperazine Derivatives as Noncovalent Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors with Improved Antiviral Activity and Favorable Druggability
... Indeed, as mentioned before, PROTACs may achieve antiviral activity through the degradation of targets involved in virus replication differently from the target inhibition exploited by the anti-SARS-CoV-2 drugs licensed so far (molnupiravir, paxlovid) [32,33] or by the multitude of antivirals identified in record-breaking times by the research community [34][35][36][37]. In this context, small-molecule main protease (M pro ) inhibitors [38][39][40][41][42][43][44][45], papain-like protease inhibitors [46][47][48], RNA-dependent RNA polymerase inhibitors [49,50], entry targeting inhibitors [51], and non-structural proteins inhibitors [52] emerged as the most promising anti-SARS-CoV-2 strategies. ...
The versatile basic structure of piperazine allows for the development and production of newer bioactive molecules that can be used to treat a wide range of diseases. Piperazine derivatives are unique and can easily be modified for the desired pharmacological activity. The two opposing nitrogen atoms in a six‐membered piperazine ring offer a large polar surface area, relative structural rigidity, and more acceptors and donors of hydrogen bonds. These properties frequently result in greater water solubility, oral bioavailability, and ADME characteristics, as well as improved target affinity and specificity. Various synthetic protocols have been reported for piperazine and its derivatives. In this review, we focused on recently published synthetic protocols for the synthesis of the piperazine and its derivatives. The structure–activity relationship concerning different biological activities of various piperazine‐containing drugs has also been highlighted to provide a good understanding to researchers for future research on piperazines.
Exposure to ultraviolet (UV) rays is a known risk factor for skin cancer, which can be notably mitigated through the application of sun care products. However, escalating concerns regarding the adverse health and environmental impacts of synthetic anti-UV chemicals underscore a pressing need for the development of biodegradable and eco-friendly sunscreen ingredients. Mycosporine-like amino acids (MAAs) represent a family of water-soluble anti-UV natural products synthesized by various organisms. These compounds can provide a two-pronged strategy for sun protection as they not only exhibit a superior UV absorption profile but also possess the potential to alleviate UV-induced oxidative stresses. Nevertheless, the widespread incorporation of MAAs in sun protection products is hindered by supply constraints. Delving into the biosynthetic pathways of MAAs can offer innovative strategies to overcome this limitation. Here, we review recent progress in MAA biosynthesis, with an emphasis on key biosynthetic enzymes, including the dehydroquinate synthase homolog MysA, the adenosine triphosphate (ATP)-grasp ligases MysC and MysD, and the nonribosomal peptide synthetase (NRPS)-like enzyme MysE. Additionally, we discuss recently discovered MAA tailoring enzymes. The enhanced understanding of the MAA biosynthesis paves the way for not only facilitating the supply of MAA analogs but also for exploring the evolution of this unique family of natural sunscreens.
One-Sentence Summary
This review discusses the role of mycosporine-like amino acids (MAAs) as potent natural sunscreens and delves into recent progress in their biosynthesis.
The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CL pro ) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CL pro with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CL pro inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.
The main protease (M pro) plays a pivotal role in the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is considered a highly conserved viral target. Disruption of the catalytic activity of M pro produces a detrimental effect on the course of the infection, making this target one of the most attractive for the treatment of COVID-19. The current success of the SARS-CoV-2 M pro inhibitor Nirmatrelvir, the first oral drug for the treatment of severe forms of COVID-19, has further focused the attention of researchers on this important viral target, making the search for new M pro inhibitors a thriving and exciting field for the development of antiviral drugs active against SARS-CoV-2 and related coronaviruses.
The main protease (Mpro) of SARS‐CoV‐2 is a well‐characterized target for antiviral drug discovery. To date, most antiviral drug discovery efforts have focused on the S4–S1′ pocket of Mpro; however, it is still unclear whether the S1′–S3′ pocket per se can serve as a new site for drug discovery. In this study, the S1′–S3′ pocket of Mpro was found to differentially recognize viral peptidyl substrates. For instance, S3′ in Mpro strongly favors Phe or Trp, and S1′ favors Ala. The peptidyl inhibitor D‐4–77, which possesses an α‐bromoacetamide warhead, was discovered to be a promising inhibitor of Mpro, with an IC50 of 0.95 μM and an antiviral EC50 of 0.49 μM. The Mpro/inhibitor co‐crystal structure confirmed the binding mode of the inhibitor to the S1′–S3′ pocket and revealed a covalent mechanism. In addition, D‐4–77 functions as an immune protectant and suppresses SARS‐CoV‐2 Mpro‐induced antagonism of the host NF‐κB innate immune response. These findings indicate that the S1′–S3′ pocket of SARS‐CoV‐2 Mpro is druggable, and that inhibiting SARS‐CoV‐2 Mpro can simultaneously protect human innate immunity and inhibit virion assembly.
The main protease (Mpro) of SARS-CoV-2 is an attractive target in anti-COVID-19 therapy for its high conservation and major role in the virus life cycle. The covalent Mpro inhibitor nirmatrelvir (in combination with ritonavir, a pharmacokinetic enhancer) and the non-covalent inhibitor ensitrelvir have shown efficacy in clinical trials and have been approved for therapeutic use. Effective antiviral drugs are needed to fight the pandemic, while non-covalent Mpro inhibitors could be promising alternatives due to their high selectivity and favorable druggability. Numerous non-covalent Mpro inhibitors with desirable properties have been developed based on available crystal structures of Mpro. In this article, we describe medicinal chemistry strategies applied for the discovery and optimization of non-covalent Mpro inhibitors, followed by a general overview and critical analysis of the available information. Prospective viewpoints and insights into current strategies for the development of non-covalent Mpro inhibitors are also discussed.
COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro’s active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics. Effective antivirals are critical for combatting SARS-CoV-2 infections. Here, the authors develop two orally available small molecules, which specifically inhibit the activity of the SARS-CoV-2 main protease and potently block the infectivity and replication of various SARS-CoV-2 strains in cells and mice.
Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.
Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection in vitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.
The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 μM) and displays excellent antiviral activity (EC50 = 1.1 μM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 μM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 μM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.
Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 Mpro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of Mpro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed Mpro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.
COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment. The 3CL protease of SARS-CoV-2 is inhibited by PF-00835231 in vitro. Here, the authors show that the prodrug PF-07304814 has broad spectrum activity, inhibiting SARS-CoV and SARS-CoV-2 in mice and its ADME and safety profile support clinical development.
Novel therapies are urgently needed to improve global treatment of SARS-CoV-2 infection. Herein, we briefly provide a concise report on the medicinal chemistry strategies towards the development of effective SARS-CoV-2 inhibitors with representative examples in different strategies from the medicinal chemistry perspective.
Starting from our previous finding of 14 known drugs as inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19, we have redesigned the weak hit perampanel to yield multiple noncovalent, nonpeptidic inhibitors with ca. 20 nM IC50 values in a kinetic assay. Free-energy perturbation (FEP) calculations for Mpro-ligand complexes provided valuable guidance on beneficial modifications that rapidly delivered the potent analogues. The design efforts were confirmed and augmented by determination of high-resolution X-ray crystal structures for five analogues bound to Mpro. Results of cell-based antiviral assays further demonstrated the potential of the compounds for treatment of COVID-19. In addition to the possible therapeutic significance, the work clearly demonstrates the power of computational chemistry for drug discovery, especially FEP-guided lead optimization.
Targeting the SARS-CoV-2 main protease
Vaccines are an important tool in the fight against COVID-19, but developing antiviral drugs is also a high priority, especially with the rise of variants that may partially evade vaccines. The viral protein main protease is required for cleaving precursor polyproteins into functional viral proteins. This essential function makes it a key drug target. Qiao et al. designed 32 inhibitors based on either boceprevir or telaprevir, both of which are protease inhibitors approved to treat hepatitis C virus. Six compounds protected cells from viral infection with high potency, and two of these were selected for in vivo studies based on pharmokinetic experiments. Both showed strong antiviral activity in a mouse model.
Science , this issue p. 1374
The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies. The SARS-CoV-2 3CL main protease (3CL Mpro) is a chymotrypsin-like protease that facilitates the production of non-structural proteins, which are essential for viral replication and is therefore of great interest as a drug target. Here, the authors present the 2.30 Å room temperature crystal structure of ligand-free 3CL Mpro and compare it with the earlier determined low-temperature ligand-free and inhibitor-bound crystal structures.
Promising antiviral protease inhibitors
With no vaccine or proven effective drug against the virus that causes coronavirus disease 2019 (COVID-19), scientists are racing to find clinical antiviral treatments. A promising drug target is the viral main protease M pro , which plays a key role in viral replication and transcription. Dai et al. designed two inhibitors, 11a and 11b, based on analyzing the structure of the M pro active site. Both strongly inhibited the activity of M pro and showed good antiviral activity in cell culture. Compound 11a had better pharmacokinetic properties and low toxicity when tested in mice and dogs, suggesting that this compound is a promising drug candidate.
Science , this issue p. 1331
A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1–4. Currently there are no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these compounds inhibited Mpro with IC50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.
Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats1–4. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans5–7. Here we report the identification and characterization of a novel coronavirus (2019-nCoV) which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started from 12 December 2019, has caused 2,050 laboratory-confirmed infections with 56 fatal cases by 26 January 2020. Full-length genome sequences were obtained from five patients at the early stage of the outbreak. They are almost identical to each other and share 79.5% sequence identify to SARS-CoV. Furthermore, it was found that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. The pairwise protein sequence analysis of seven conserved non-structural proteins show that this virus belongs to the species of SARSr-CoV. The 2019-nCoV virus was then isolated from the bronchoalveolar lavage fluid of a critically ill patient, which can be neutralized by sera from several patients. Importantly, we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as SARS-CoV.
The SARS-CoV-2 main protease (Mpro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in Mpro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring Mpro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (kcat/Km < 10-fold change) while being resistant to nirmatrelvir (Ki > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that Mpro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in Mpro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.
Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with pyridine (3, 4), fluorine-substituted pyridine (5), phenyl (6, 7), and thiophene side chains (8, 9), for comparison with unsubstituted phenyl (1, 2) and thiophene side chain (10, 11) containing thieno[2,3-d]pyrimidine compounds. Compounds 3-9 inhibited proliferation of Chinese hamster ovary cells (CHO) expressing folate receptors (FRs) α or β but not the reduced folate carrier (RFC); modest inhibition of CHO cells expressing the proton-coupled folate transporter (PCFT) by 4, 5, 6, and 9 was observed. Replacement of the side-chain 1',4'-phenyl ring with 2',5'-pyridyl, or 2',5'-pyridyl with a fluorine insertion ortho to l-glutamate resulted in increased potency toward FR-expressing CHO cells. Toward KB tumor cells, 4-9 were highly active (IC50's from 2.11 to 7.19 nM). By metabolite rescue in KB cells and in vitro enzyme assays, de novo purine biosynthesis was identified as a targeted pathway (at 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase)). Compound 9 was 17- to 882-fold more potent than previously reported compounds 2, 10, and 11 against GARFTase. By targeted metabolomics and metabolite rescue, 1, 2, and 6 also inhibited mitochondrial serine hydroxymethyl transferase 2 (SHMT2); enzyme assays confirmed inhibition of SHMT2. X-ray crystallographic structures were obtained for 4, 5, 9, and 10 with human GARFTase. This series affords an exciting new structural platform for potent multitargeted antitumor agents with FR transport selectivity.
GPR88 is an orphan G protein-coupled receptor mainly expressed in the brain, whose endogenous ligand has not yet been identified. To elucidate GPR88 functions, our group has developed RTI-13951-33 (1b) as the first in vivo active GPR88 agonist, but its poor metabolic stability and moderate brain permeability remain to be further optimized. Here, we report the design, synthesis, and pharmacological characterization of a new series of RTI-13951-33 analogues with the aim of improving pharmacokinetic properties. As a result, we identified a highly potent GPR88 agonist RTI-122 (30a) (cAMP EC50 = 11 nM) with good metabolic stability (half-life of 5.8 h) and brain permeability (brain/plasma ratio of >1) in mice. Notably, RTI-122 was more effective than RTI-13951-33 in attenuating the binge-like alcohol drinking behavior in the drinking-in-the-dark paradigm. Collectively, our findings suggest that RTI-122 is a promising lead compound for drug discovery research of GPR88 agonists.
ConspectusSARS-CoV-2 is the etiological pathogen of the COVID-19 pandemic, which led to more than 6.5 million deaths since the beginning of the outbreak in December 2019. The unprecedented disruption of social life and public health caused by COVID-19 calls for fast-track development of diagnostic kits, vaccines, and antiviral drugs. Small molecule antivirals are essential complements of vaccines and can be used for the treatment of SARS-CoV-2 infections. Currently, there are three FDA-approved antiviral drugs, remdesivir, molnupiravir, and paxlovid. Given the moderate clinical efficacy of remdesivir and molnupiravir, the drug-drug interaction of paxlovid, and the emergence of SARS-CoV-2 variants with potential drug-resistant mutations, there is a pressing need for additional antivirals to combat current and future coronavirus outbreaks.In this Account, we describe our efforts in developing covalent and noncovalent main protease (Mpro) inhibitors and the identification of nirmatrelvir-resistant mutants. We initially discovered GC376, calpain inhibitors II and XII, and boceprevir as dual inhibitors of Mpro and host cathepsin L from a screening of a protease inhibitor library. Given the controversy of targeting cathepsin L, we subsequently shifted the focus to designing Mpro-specific inhibitors. Specifically, guided by the X-ray crystal structures of these initial hits, we designed noncovalent Mpro inhibitors such as Jun8-76-3R that are highly selective toward Mpro over host cathepsin L. Using the same scaffold, we also designed covalent Mpro inhibitors with novel cysteine reactive warheads containing di- and trihaloacetamides, which similarly had high target specificity. In parallel to our drug discovery efforts, we developed the cell-based FlipGFP Mpro assay to characterize the cellular target engagement of our rationally designed Mpro inhibitors. The FlipGFP assay was also applied to validate the structurally disparate Mpro inhibitors reported in the literature. Lastly, we introduce recent progress in identifying naturally occurring Mpro mutants that are resistant to nirmatrelvir from genome mining of the nsp5 sequences deposited in the GISAID database. Collectively, the covalent and noncovalent Mpro inhibitors and the nirmatrelvir-resistant hot spot residues from our studies provide insightful guidance for future work aimed at developing orally bioavailable Mpro inhibitors that do not have overlapping resistance profile with nirmatrelvir.
The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 μM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 μM), similar to that of remdesivir (EC50 = 2.27 μM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.
Two years after its emergence, SARS-CoV-2 still represents a serious and global threat to human health. Antiviral drug development usually takes a long time and, to increase the chances of success, chemical variability of hit compounds represents a valuable source for the discovery of new antivirals. In this work, we applied a platform of variably oriented virtual screening campaigns to seek for novel chemical scaffolds for SARS-CoV-2 main protease (Mpro) inhibitors. The study on the resulting 30 best hits led to the identification of a series of structurally unrelated Mpro inhibitors. Some of them exhibited antiviral activity in the low micromolar range against SARS-CoV-2 and other human coronaviruses (HCoVs) in different cell lines. Time-of-addition experiments demonstrated an antiviral effect during the viral replication cycle at a time frame consistent with the inhibition of SARS-CoV-2 Mpro activity. As a proof-of-concept, to validate the pharmaceutical potential of the selected hits against SARS-CoV-2, we rationally optimized one of the hit compounds and obtained two potent SARS-CoV-2 inhibitors with increased activity against Mpro both in vitro and in a cellular context, as well as against SARS-CoV-2 replication in infected cells. This study significantly contributes to the expansion of the chemical variability of SARS-CoV-2 Mpro inhibitors and provides new scaffolds to be exploited for pan-coronavirus antiviral drug development.
Path to another drug against COVID-19
The rapid development of vaccines has been crucial in battling the ongoing COVID-19 pandemic. However, access challenges remain, breakthrough infections occur, and emerging variants present increased risk. Developing antiviral therapeutics is therefore a high priority for the treatment of COVID-19. Some drug candidates in clinical trials act against the viral RNA-dependent RNA polymerase, but there are other viral enzymes that have been considered good targets for inhibition by drugs. Owen et al . report the discovery and characterization of a drug against the main protease involved in the cleavage of polyproteins involved in viral replication. The drug, PF-07321332, can be administered orally, has good selectivity and safety profiles, and protects against infection in a mouse model. In a phase 1 clinical trial, the drug reached concentrations expected to inhibit the virus based on in vitro studies. It also inhibited other coronaviruses, including severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, and could be in the armory against future viral threats. —VV
Indomethacin (INM), a well-known non-steroidal anti-inflammatory drug, has recently gained attention for its antiviral activity demonstrated in drug repurposing studies against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Although the mechanism of action of INM is not yet fully understood, recent studies have indicated that it acts at an early stage of the coronaviruses (CoVs) replication cycle. In addition, a proteomic study reported that the anti-SARS-CoV-2 activity of INM could be also ascribed to its ability to inhibit human prostaglandin E synthase type 2 (PGES-2), a host protein which interacts with the SARS-CoV-2 NSP7 protein. Although INM does not potently inhibit SARS-CoV-2 replication in infected Vero E6 cells, here we have explored for the first time the application of the Proteolysis Targeting Chimeras (PROTACs) technology in order to develop more potent INM-derived PROTACs with anti-CoV activity. In this study, we report the design, synthesis, and biological evaluation of a series of INM-based PROTACs endowed with antiviral activity against a panel of human CoVs, including different SARS-CoV-2 strains. Two PROTACs showed a strong improvement in antiviral potency compared to INM. Molecular modelling studies support human PGES-2 as a potential target of INM-based antiviral PROTACs, thus paving the way toward the development of host-directed anti-CoVs strategies. To the best of our knowledge, these PROTACs represent the first-in-class INM-based PROTACs with antiviral activity and also the first example of the application of PROTACs to develop pan-coronavirus agents.
The ongoing pandemic of a new human coronavirus, SARS-CoV-2, has generated enormous global concern. We and others in China were involved in the initial genome sequencing of the virus. Herein, we describe what genomic data reveal about the emergence SARS-CoV-2 and discuss the gaps in our understanding of its origins.
-methoxynicotinoyl)-N-(thiophen-2-ylmethyl)piperazine-2-carboxamide (GC-66). Yellow solid, 63% yield, melting point 117−119°C. 1 H NMR (600 MHz
- Hz
Hz, 1H), 4.67−4.37 (m, 2H), 4.35−4.13 (m, 2H), 3.90−3.66 (m,
2H), 3.58 (ddd, J = 12.5, 8.8, 4.0 Hz, 1H), 3.55−3.35 (m, 2H). 13 C
NMR (150 MHz, DMSO-d 6 ) δ 170.26, 166.18, 152.03, 149.56,
147.60, 142.54, 132.60, 132.18, 132.03, 130.93 (2 × C), 127.02 (2 ×
C), 125.61, 125.43, 119.77, 115.63, 114.55, 58.67, 47.63, 43.03, 41.79,
37.86. HRMS (ESI) m/z [M + H] + calcd for C 23 H 19 Cl 2 F 3 N 4 O 2 S,
542.0558; found 543.0631. HPLC purity: 96.32%.
(S)-1-(3,4-Dichlorophenyl)-4-(6-methoxynicotinoyl)-N-(thiophen-2-ylmethyl)piperazine-2-carboxamide (GC-66). Yellow solid,
63% yield, melting point 117−119°C. 1 H NMR (600 MHz, DMSOd 6 ) δ 8.72 (s, 1H), 8.21 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 6.1 Hz, 1H),
HRMS (ESI) m/z [M + H] + calcd for C 23 H 22 Cl 2 N 4 O 3 S, 504.0790; found 505.0862. HPLC purity: 97.06%. (S)-1-(3,4-Dichlorophenyl)-4-(6-hydroxynicotinoyl)-N-(thiophen-2-ylmethyl)piperazine-2-carboxamide (GC-68)
- Dmso Mhz
MHz, DMSO-d 6 ) δ 174.21, 165.27, 161.49, 152.53, 149.90, 132.00,
130.91, 127.06, 125.63, 125.43, 125.35, 122.05, 119.66, 115.36,
114.33, 107.80, 56.35, 46.85, 44.12, 43.07, 41.40, 37.86. HRMS (ESI)
m/z [M + H] + calcd for C 23 H 22 Cl 2 N 4 O 3 S, 504.0790; found 505.0862.
HPLC purity: 97.06%.
(S)-1-(3,4-Dichlorophenyl)-4-(6-hydroxynicotinoyl)-N-(thiophen-2-ylmethyl)piperazine-2-carboxamide (GC-68). White solid, 55%
yield, melting point >206°C. 1 H NMR (600 MHz, DMSO-d 6 ) δ
11.89 (s, 1H), 8.81 (dt, J = 30.3, 6.1 Hz, 1H), 7.52 (d, J = 2.8 Hz,
1H), 7.44−7.38 (m, 2H), 7.37 (ddd, J = 8.3, 5.1, 1.3 Hz, 1H), 6.98
(dd, J = 28.3, 3.0 Hz, 1H), 6.92 (ddd, J = 8.5, 5.1, 3.4 Hz, 1H), 6.88−
6.79 (m, 1H), 6.79−6.70 (m, 1H), 6.35 (d, J = 10.2 Hz, 1H), 4.51−
4.43 (m, 1H), 4.34 (dd, J = 15.7, 5.4 Hz, 2H), 4.23 (s, 1H), 3.76−
3.63 (m, 1H), 3.57 (dd, J = 7.8, 4.4 Hz, 2H), 3.35 (s, 1H), 2.96 (d, J =
-nitronicotinoyl)-N-(thiophen-2-ylmethyl)piperazine-2-carboxamide (GC-75). White solid, 61% yield
- Dmso Mhz
MHz, DMSO-d 6 ) δ 170.42, 167.35, 155.39, 149.63, 142.56, 140.07,
139.02, 132.38, 132.02, 130.93, 127.05, 125.58, 125.42, 119.32,
115.41, 114.37, 58.87, 56.21, 47.59, 42.99, 41.74, 37.90. HRMS (ESI)
m/z [M + H] + calcd for C 23 H 22 Cl 2 N 4 O 3 S, 504.0790; found 505.086.
HPLC purity: 97.25%.
(S)-1-(3,4-Dichlorophenyl)-4-(6-nitronicotinoyl)-N-(thiophen-2-ylmethyl)piperazine-2-carboxamide (GC-75). White solid, 61%
yield, melting point 97−100°C. 1 H NMR (600 MHz, DMSO-d 6 ) δ
6 mmol) in the mixture of THF/MeOH (100 mL, v/v = 1/1) the newly prepared 1 M LiOH aqueous solution (76.2 mL, 76.2 mmol) was (S)-1-(3,4-Dichlorophenyl)-4-nicotinoyl-N-(thiophen-2-ylsulfonyl)piperazine-2-carboxamide (GC-14a)
- Acid
Acid (15). To a stirred solution of methyl ester 14 (3.0 g, 7.6 mmol)
in the mixture of THF/MeOH (100 mL, v/v = 1/1) the newly
prepared 1 M LiOH aqueous solution (76.2 mL, 76.2 mmol) was
(S)-1-(3,4-Dichlorophenyl)-4-nicotinoyl-N-(thiophen-2-ylsulfonyl)piperazine-2-carboxamide (GC-14a). White solid, 65%
yield, melting point 181−183°C. 1 H NMR (600 MHz, DMSO-d 6 ) δ
12.29 (s, 1H), 8.64 (s, 1H), 8.49 (d, J = 43.8 Hz, 1H), 7.75 (s, 1H),
7.65 (s, 1H), 7.54−7.36 (m, 2H), 7.30 (d, J = 7.1 Hz, 1H), 6.97 (s,
1H), 6.89 (s, 1H), 6.72 (dd, J = 9.1, 3.0 Hz, 1H), 4.41−4.22 (m, 1H),
DMSO-d 6 ) δ 8.67 (d, J = 6.6 Hz, 2H), 8.57 (s, 2H)
- H Nmr
H NMR (600 MHz, DMSO-d 6 ) δ 8.67 (d, J = 6.6 Hz, 2H), 8.57 (s,
2H), 8.12 (s, 1H), 7.76 (dt, J = 7.8, 1.9 Hz, 2H), 7.50 (dd, J = 7.9, 4.8
Hz, 1H), 4.00 (s, 2H), 3.87 (d, J = 15.5 Hz, 1H), 3.76 (d, J = 14.2 Hz, 2H), 3.67−3.64 (m, 2H), 3.50 (d, J = 15.0 Hz, 1H). 13 C NMR (150 MHz, DMSO-d 6 )
- Hz
Hz, 2H), 7.42 (d, J = 9.1 Hz, 2H), 7.02 (d, J = 2.9 Hz, 2H), 6.81 (dd,
J = 9.1, 3.0 Hz, 2H), 4.70−4.49 (m, 1H), 4.29 (s, 1H), 4.24 (d, J =
14.2 Hz, 1H), 4.00 (s, 2H), 3.87 (d, J = 15.5 Hz, 1H), 3.76 (d, J =
14.2 Hz, 2H), 3.67−3.64 (m, 2H), 3.50 (d, J = 15.0 Hz, 1H). 13 C
NMR (150 MHz, DMSO-d 6 ) δ 170.00, 167.76, 151.00, 149.81,
148.14, 136.71, 135.30, 132.07 (2 × C), 131.90, 131.25, 130.99,
129.46, 125.89, 125.71 (2 × C), 125.18, 123.99, 119.87, 115.61,
114.55, 68.85, 68.00, 67.76, 58.30, 48.03, 43.23, 41.68. HRMS (ESI)
m/z [M + H] + calcd for C 28 H 26 Cl 2 FeN 4 O 4, 576.0782; found
577.0841. HPLC purity: 99.93%.
(S)-1-(3,4-Dichlorophenyl)-N-methyl-4-nicotinoyl-N-(thiophen-2-ylmethyl)piperazine-2-carboxamide (GC-14c). White powder,
52% yield, melting point 94−96°C. 1 H NMR (600 MHz, DMSOd 6 ) δ 8.68 (dd, J = 4.9, 1.7 Hz, 1H), 8.61−8.43 (m, 1H), 7.74 (d, J =
-methoxyphenyl)-4-nicotinoylpiperazine-2-carboxamide (GC-14f). White solid, 55% yield
- Dmso Mhz
MHz, DMSO-d 6 ) δ 170.69, 167.71, 151.03, 148.11, 144.82, 135.26,
132.03, 131.84, 130.93, 130.01, 126.51 (2 × C), 123.93, 119.82,
115.47, 114.46, 110.47, 58.77, 47.75, 43.04, 41.77, 38.14. HRMS
(ESI) m/z [M + H] + calcd for C 22 H 19 BrCl 2 N 4 O 2 S, 551.9789; found
552.9867. HPLC purity: 98.36%.
(S)-1-(3,4-Dichlorophenyl)-N-(4-methoxyphenyl)-4-nicotinoylpiperazine-2-carboxamide (GC-14f). White solid, 55% yield, melting
point 98−100°C. 1 H NMR (600 MHz, DMSO-d 6 ) δ 9.96 (d, J =
]pyrimidines as folate receptor-selective anticancer agents that inhibit cytosolic and mitochondrial one-carbon metabolism
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