Pairs of thiol-substituted 1,2,4-triazole-based isomeric covalent inhibitors with tunable reactivity and selectivity

Covalent bioactive compounds are successfully used in clinic and attracted intense research efforts in the fundamental study as well as drug development.The advantageous effects of covalent compounds compared with non-covalent ones are highly dependent on electrophilic warheads.Hence,electrophilic warheads with tunable reactivity and selectivity are highly demanded in fields of medicinal chemistry and chemical biology.Herein,we report a novel electrophilic warhead,chloromethyl group activated by thiol-substituted 1,2,4-triazole.Interestingly,a pair of regioisomers could be simultaneously occurred in the step of alkylation during the synthesis of this unique motif.This is a rare example that the alkylation could simultaneously generate these two separable regioisomers of 1,2,4-triazole at the nitrogen or sulfur atom.The covalent-working mechanism of this new warhead is confirmed by various chemoproteomics experiments including target identification and binding site mapping.Importantly,the reactivity and selectivity of this new electrophilic warhead could be efficiently tuned by virtue of stereo effect.Interestingly,one pair of regioisomers(19S and 19X)induced distinct modes of cell death.Isomer 19S could induce apoptosis of colon cancer cells while 19X could induce both apoptosis and ferroptosis.Together,this study provides pairs of novel electrophilic warheads that could be useful not only in supporting the design of covalent compounds for drug discovery but also in providing chemical probes for the fundamental biological study.

Discovery and characterization of naturally occurring covalent inhibitors of SARS-CoV-2 M^(pro)from the antiviral herb Ephedra

The Chinese herb Ephedra(also known as Mahuang)has been extensively utilized for the prevention and treatment of coronavirus-induced diseases,including coronavirus disease 2019(COVID-19).However,the specific anti-SARS-CoV-2 compounds and mechanisms have not been fully elucidated.The main protease(M^(pro))of SARS-CoV-2 is a highly conserved enzyme responsible for proteolytic processing during the viral life cycle,making it a critical target for the development of antiviral therapies.This study aimed to identify naturally occurring covalent inhibitors of SARS-CoV-2 M^(pro)from Ephedra and to investigate their covalent binding sites.The results demonstrated that the non-alkaloid fraction of Ephedra(ENA)exhibited a potent inhibitory effect against the SARS-CoV-2 M^(pro)effect,whereas the alkaloid fraction did not.Subsequently,the chemical constituents in ENA were identified,and the major constituents'anti-SARS-CoV-2 M^(pro)effects were evaluated.Among the tested constituents,herbacetin(HE)and gallic acid(GA)were found to inhibit SARS-CoV-2 M^(pro)in a time-and dose-dependent manner.Their combination displayed a significant synergistic effect on this key enzyme.Additionally,various techniques,including inhibition kinetic assays,chemoproteomic methods,and molecular dynamics simulations,were employed to further elucidate the synergistic anti-M^(pro)mechanisms of the combination of HE and GA.Overall,this study deciphers the naturally occurring covalent inhibitors of SARS-CoV-2 M^(pro)from Ephedra and characterizes their synergistic anti-M^(pro)synergistic effect,providing robust evidence to support the anti-coronavirus efficacy of Ephedra.

Covalent inhibitor targets KRas^(G12C):A new paradigm for drugging the undruggable and challenges ahead

KRAS is one of the most commonly mutated oncogenes in cancers and therapeutics directly targeting the KRas have been challenging.Among the different known mutants,KRas^(G12C) has been proved to be successfully targeted recently.Several covalent inhibitors selectively targeting KRas^(G12C) have shown promising efficacy against cancers harboring KRAS G12C mutation in clinical trials and AMG510(sotorasib)has been approved for the treatment of KRAS G12C-mutated locally advanced or metastatic non-small cell lung cancer.However,the overall responsive rate of KRas^(G12C) inhibitors was around 50%in patients with non-small cell lung cancer and the efficacy in patients with colorectal cancer or appendiceal cancer appears to be less desirable.It is of great importance to discover biomarkers to distinguish patients who are likely benefitted.Moreover,adaptive resistance would occur inevitably with the persistent administration like other molecularly targeted therapies.Several combinatorial regimens have been studied in an effort to potentiate the efficacy of KRas^(G12C) inhibitors in preclinical settings.This review summarized the recent progress of covalent KRas^(G12C) inhibitors with a focus on identifying biomarkers to predict or monitor the efficacy and proposing rational drug combinations based on elucidation of the mechanisms of drug resistance.

Identification of a novel PHGDH covalent inhibitor by chemical proteomics and phenotypic profiling

The first rate-limiting enzyme of the serine synthesis pathway(SSP), phosphoglycerate dehydrogenase(PHGDH), is hyperactive in multiple tumors, which leads to the activation of SSP and promotes tumorigenesis. However, only a few inhibitors of PHGDH have been discovered to date, especially the covalent inhibitors of PHGDH. Here, we identified withangulatin A(WA), a natural small molecule,as a novel covalent inhibitor of PHGDH. Affinity-based protein profiling identified that WA could directly bind to PHGDH and inactivate the enzyme activity of PHGDH. Biolayer interferometry and LC-MS/MS analysis further demonstrated the selective covalent binding of WA to the cysteine 295 residue(Cys295)of PHGDH. With the covalent modification of Cys295, WA blocked the substrate-binding domain(SBD)of PHGDH and exerted an allosteric effect to induce PHGDH inactivation. Further studies revealed that with the inhibition of PHGDH mediated by WA, the glutathione synthesis was decreased and intracellular levels of reactive oxygen species(ROS) were elevated, leading to the inhibition of tumor proliferation.This study indicates WA as a novel PHGDH covalent inhibitor, which identifies Cys295 as a novel allosteric regulatory site of PHGDH and holds great potential in developing anti-tumor agents for targeting PHGDH.

Discovery of a subtype-selective, covalent inhibitor against palmitoylation pocket of TEAD3

The TEA domain(TEAD)family proteins(TEAD1-4)are essential transcription factors that control cell differentiation and organ size in the Hippo pathway.Although the sequences and structures of TEAD family proteins are highly conserved,each TEAD isoform has unique physiological and pathological functions.Therefore,the development and discovery of subtype selective inhibitors for TEAD protein will provide important chemical probes for the TEAD-related function studies in development and diseases.Here,we identified a novel TEAD 1/3 covalent inhibitor(DC-TEADin1072)with biochemical IC50 values of 0.61±0.02 and 0.58±0.12μmol/L against TEAD1 and TEAD3,respectively.Further chemical optimization based on DC-TEAD in 1072 yielded a selective TEAD3 inhibitor DCTEAD3 in03 with the IC_(50) value of 0.16±0.03μmol/L,which shows 100-fold selectivity over other TEAD isoforms in activity-based protein profiling(ABPP)assays.In cells,DC-TEAD3 in03 showed selective inhibitory effect on TEAD3 in GAL4-TEAD(1-4)reporter assays with the IC50 value of1.15μmol/L.When administered to zebrafish juveniles,experiments showed that DC-TEAD3 in03 reduced the growth rate of zebrafish caudal fins,indicating the importance of TEAD3 activity in controlling proportional growth of vertebrate appendages.

A targeted covalent inhibitor of p97 with proteome-wide selectivity

A resurging interest in targeted covalent inhibitors(TCIs)focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target.p97 is an emerging protein target for cancer therapy,viral infections and neurodegenerative diseases.Extensive efforts were devoted to the development of p97 inhibitors.The most promising inhibitor of p97 was in phase 1 clinical trials,but failed due to the off-target-induced toxicity,suggesting the selective inhibitors of p97 are highly needed.We report herein a new type of TCIs(i.e.,FL-18)that showed proteome-wide selectivity towards p97.Equipped with a Michael acceptor and a basic imidazole,FL-18 showed potent inhibition towards U87 MG tumor cells,and in proteome-wide profiling,selectively modified endogenous p97 as confirmed by in situ fluorescence scanning,label-free quantitative proteomics and functional validations.FL-18 selectively modified cysteine residues located within the D2 ATP site of p97.This covalent labeling of cysteine residue in p97 was verified by LC-MS/MS-based site-mapping and site-directed mutagenesis.Further structure-activity relationship(SAR)studies with FL-18 analogs were established.Collectively,FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity,thus providing a promising scaffold for cancer therapy.

Strategic design of lysine-targeted irreversible covalent NDM-1 inhibitors

New Delhi metallo-β-lactamase 1(NDM-1) can hydrolyze most β-lactam antibiotics, which is the major factor for drug resistance of Gram-negative bacteria. The binding of most reversible inhibitors to NDM-1 is relatively weak due to the shallow active pocket of NDM-1. Alternatively, irreversible covalent inhibitors can prevent their dissociation from the target, leading to permanent inactivation of the protein.Herein, we report a series of irreversible covalent inhibitors of NDM-1 targeting the conserved Lys211 in the active pocket. Several methods, including mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorescent labeling, and coumarin probe were used to demonstrate that pentafluorophenyl ester formed a covalent bond with Lys211. Moreover, our target inhibitor, in combination with meropenem, achieved an antibacterial effect on drug-resistant bacteria, along with an excellent safety profile. Our new strategy in designing lysine-targeted irreversible covalent NDM-1 inhibitors provides a potential option for the clinical treatment of Gram-negative bacteria.

Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyridazinone derivatives as covalent FGFR inhibitors

Fibroblast growth factor receptors (FGFRs) have emerged as promising targets for anticancer therapy.In this study,we synthesized and evaluated the biological activity of 66 pyrazolo[3,4-d]pyridazinone derivatives.Kinase inhibition,cell proliferation,and whole blood stability assays were used to evaluate their activity on FGFR,allowing us to explore structureàactivity relationships and thus to gain understanding of the structural requirements to modulate covalent inhibitors’selectivity and reactivity.Among them,compound 10h exhibited potent enzymatic activity against FGFR and remarkably inhibited proliferation of various cancer cells associated with FGFR dysregulation,and suppressed FGFR signaling pathway in cancer cells by the immunoblot analysis.Moreover,10h displayed highly potent antitumor efficacy (TGI Z 91.6%,at a dose of 50 mg/kg) in the FGFR1-amplified NCI-H1581 xenograft model.

Recent developments in the medicinal chemistry of single boron atom-containing compounds

Various boron-containing drugs have been approved for clinical use over the past two decades,and more are currently in clinical trials.The increasing interest in boron-containing compounds is due to their unique binding properties to biological targets;for example,boron substitution can be used to modulate biological activity,pharmacokinetic properties,and drug resistance.In this perspective,we aim to comprehensively review the current status of boron compounds in drug discovery,focusing especially on progress from 2015 to December 2020.We classify these compounds into groups showing anticancer,antibacterial,antiviral,antiparasitic and other activities,and discuss the biological targets associated with each activity,as well as potential future developments.

A bifunctional vinyl-sulfonium tethered peptide induced by thio-Michael-type addition reaction

The modification and functionalization of peptides is of great significance in modern biotechnology and drug development. Here we report a highly reactive Michael-type warhead for the covalently modification of cysteine on peptide and protein. By installing a vinyl group onto a methionine residue of peptide,the produced vinyl sulfonium can be efficiently nucleophilic added by appropriate cysteine residue of this peptide, and thus yield a cyclized peptide. This peptide cyclization strategy was proven to exhibit improved cell penetration and good stability. Moreover, a peptide ligand bearing vinyl sulfonium could covalently bind to the cysteine in the target protein, indicating the potential of vinyl sulfonium as a novel warhead for developing covalent peptide inhibitor.