Hydroxamate and thiosemicarbazone: Two highly promising scaffolds for the development of SARS-CoV-2 antivirals.

The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (Mpro) of SARS-CoV-2 is promising target for antiviral drugs, which plays a vital role for viral duplication. Development of the inhibitor against Mpro is an ideal strategy to combat COVID-19. In this work, twenty-three hydroxamates 1a-i and thiosemicarbazones 2a-n were identified by FRET screening to be the potent inhibitors of Mpro, which exhibited more than 94% (except 1c) and more than 69% inhibition, and an IC50 value in the range of 0.12-31.51 and 2.43-34.22 µM, respectively. 1a and 2b were found to be the most effective inhibitors in the hydroxamates and thiosemicarbazones, with an IC50 of 0.12 and 2.43 µM, respectively. Enzyme kinetics, jump dilution and thermal shift assays revealed that 2b is a competitive inhibitor of Mpro, while 1a is a time-dependently inhibitor; 2b reversibly but 1a irreversibly bound to the target; the binding of 2b increased but 1a decreased stability of the target, and DTT assays indicate that 1a is the promiscuous cysteine protease inhibitor. Cytotoxicity assays showed that 1a has low, but 2b has certain cytotoxicity on the mouse fibroblast cells (L929). Docking studies revealed that the benzyloxycarbonyl carbon of 1a formed thioester with Cys145, while the phenolic hydroxyl oxygen of 2b formed H-bonds with Cys145 and Asn142. This work provided two promising scaffolds for the development of Mpro inhibitors to combat COVID-19.

Thiosemicarbazones exhibit inhibitory efficacy against New Delhi metallo-ß-lactamase-1 (NDM-1).

The superbug infection caused by metallo-ß-lactamases (MßLs) carrying drug-resistant bacteria, specifically, New Delhi metallo-ß-lactamase (NDM-1) has become an emerging threat. In an effort to develop novel inhibitors of NDM-1, thirteen thiosemicarbazones (1a-1m) were synthesized and assayed. The obtained molecules specifically inhibited NDM-1, with an IC50 in the range of 0.88-20.2 µM, and 1a and 1f were found to be the potent inhibitors (IC50 = 1.79 and 0.88 µM) using cefazolin as substrate. ITC and kinetic assays indicated that 1a irreversibly and non-competitively inhibited NDM-1 in vitro. Importantly, MIC assays revealed that these molecules by themselves can sterilize NDM-producing clinical isolates EC01 and EC08, exhibited 78-312-fold stronger activities than the cefazolin. MIC assays suggest that 1a (16 µg ml-1) has synergistic antimicrobial effect with ampicillin, cefazolin and meropenem on E. coli producing NDM-1, resulting in MICs of 4-32-, 4-32-, and 4-8-fold decrease, respectively. These studies indicate that the thiosemicarbazide is a valuable scaffold for the development of inhibitors of NDM-1 and NDM-1 carrying drug-resistant bacteria.

Dipyridyl-substituted thiosemicarbazone as a potent broad-spectrum inhibitor of metallo-ß-lactamases.

To combat the superbug infection caused by metallo-ß-lactamases (MßLs), a dipyridyl-substituted thiosemicarbazone (DpC), was identified to be the broad-spectrum inhibitor of MßLs (NDM-1, VIM-2, IMP-1, ImiS, L1), with an IC50 value in the range of 0.021-1.08 µM. It reversibly and competitively inhibited NDM-1 with a Ki value of 10.2 nM. DpC showed broad-spectrum antibacterial effect on clinical isolate K. pneumonia, CRE, VRE, CRPA and MRSA, with MIC value ranged from 16 to 32 µg/mL, and exhibited synergistic antibacterial effect with meropenem on MßLs-producing bacteria, resulting in a 2-16-, 2-8-, and 8-fold reduction in MIC of meropenem against EC-MßLs, EC01-EC24, K. pneumonia, respectively. Moreover, mice experiments showed that DpC also had synergistic antibacterial action with meropenem. In this work, DpC was identified to be a potent scaffold for the development of broad-spectrum inhibitors of MßLs.

Ebsulfur and Ebselen as highly potent scaffolds for the development of potential SARS-CoV-2 antivirals.

The emerging COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised a global catastrophe. To date, there is no specific antiviral drug available to combat this virus, except the vaccine. In this study, the main protease (Mpro) required for SARS-CoV-2 viral replication was expressed and purified. Thirty-six compounds were tested as inhibitors of SARS-CoV-2 Mpro by fluorescence resonance energy transfer (FRET) technique. The half-maximal inhibitory concentration (IC50) values of Ebselen and Ebsulfur analogs were obtained to be in the range of 0.074-0.91 µM. Notably, the molecules containing furane substituent displayed higher inhibition against Mpro, followed by Ebselen 1i (IC50 = 0.074 µM) and Ebsulfur 2k (IC50 = 0.11 µM). The action mechanism of 1i and 2k were characterized by enzyme kinetics, pre-incubation and jump dilution assays, as well as fluorescent labeling experiments, which suggested that both compounds covalently and irreversibly bind to Mpro, while molecular docking suggested that 2k formed an SS bond with the Cys145 at the enzymatic active site. This study provides two very potent scaffolds Ebsulfur and Ebselen for the development of covalent inhibitors of Mpro to combat COVID-19.

Diaryl-substituted thiosemicarbazone: A potent scaffold for the development of New Delhi metallo-ß-lactamase-1 inhibitors.

The superbug infection caused by New Delhi metallo-ß-lactamase (NDM-1) has become an emerging public health threat. Inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. A potent scaffold, diaryl-substituted thiosemicarbazone, was constructed and assayed with metallo-ß-lactamases (MßLs). The obtained twenty-six molecules specifically inhibited NDM-1 with IC50 0.038-34.7 µM range (except 1e, 2e, and 3d), and 1c is the most potent inhibitor (IC50 = 0.038 µM). The structure-activity relationship of synthetic thiosemicarbazones revealed that the diaryl-substitutes, specifically 2-pyridine and 2-hydroxylbenzene improved inhibitory activities of the inhibitors. The thiosemicarbazones exhibited synergistic antimycobacterial actions against E. coli-NDM-1, resulted a 2-512-fold reduction in MIC of meropenem, while 1c restored 16-256-, 16-, and 2-fold activity of the antibiotic on clinical isolates ECs, K. pneumonia and P. aeruginosa harboring NDM-1, respectively. Also, mice experiments showed that 1c had a synergistic antibacterial ability with meropenem, reduced the bacterial load clinical isolate EC08 in the spleen and liver. This work provided a highly promising scaffold for the development of NDM-1 inhibitors.

Hydroxamic acid with benzenesulfonamide: An effective scaffold for the development of broad-spectrum metallo-ß-lactamase inhibitors.

Given that ß-lactam antibiotic resistance mediated by metallo-ß-lactamases (MßLs) seriously threatens human health, we designed and synthesized nineteen hydroxamic acids with benzenesulfonamide, which exhibited broad-spectrum inhibition against four tested MßLs ImiS, L1, VIM-2 and IMP-1 (except 6, 13 and 18 on IMP-1, and 18 on VIM-2), with an IC50 value in the range of 0.6-9.4, 1.3-27.4, 5.4-43.7 and 5.2-49.7 µM, respectively, and restored antibacterial activity of both cefazolin and meropenem, resulting in a 2-32-fold reduction in MIC of the antibiotics. Compound 17 shows reversible competitive inhibition on L1 with a Ki value of 2.5 µM and significantly reduced the bacterial load in the spleen and liver of mice infected by E. coli expressing L1. The docking studies suggest that 17 tightly binds to the Zn(Ⅱ) of VIM-2 and CphA by the oxygen atoms of sulfonamide group, but coordinates with the Zn(II) of L1 through the oxygen atoms of hydroxamic acid group. These studies reveal that the hydroxamic acids with benzenesulfonamide are the potent scaffolds for the development of MßL inhibitors.

Identification of Cisplatin and Palladium(II) Complexes as Potent Metallo-ß-lactamase Inhibitors for Targeting Carbapenem-Resistant Enterobacteriaceae.

The emergence and prevalence of carbapenem-resistant bacterial infection have seriously threatened the clinical use of almost all ß-lactam antibacterials. The development of effective metallo-ß-lactamase (MßL) inhibitors to restore the existing antibiotics efficacy is an ideal alternative. Although several types of serine-ß-lactamase inhibitors have been successfully developed and used in clinical settings, MßL inhibitors are not clinically available to date. Herein, we identified that cisplatin and Pd(II) complexes are potent broad-spectrum inhibitors of the B1 and B2 subclasses of MßLs and effectively revived Meropenem efficacy against MßL-expressing bacteria in vitro. Enzyme kinetics, thermodynamics, inductively coupled plasma atomic emission spectrometry (ICP-AES), matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), and site-directed mutation assays revealed that these metal complexes irreversibly inhibited NDM-1 through a novel inhibition mode involving binding to Cys208 and displacing one Zn(II) ion of the enzyme with one Pt(II) containing two NH3's or one Pd(II) ion. Importantly, the combination therapy of Meropenem and metal complexes significantly suppressed the development of higher-level resistance in bacteria producing NDM-1, also effectively reduced the bacterial burden in liver and spleen of mice infected by carbapenem-resistant Enterobacteriaceae producing NDM-1. These findings will offer potential lead compounds for the further development of clinically useful inhibitors targeting MßLs.

Disulfiram as a potent metallo-ß-lactamase inhibitor with dual functional mechanisms.

We report a promising NDM-1 inhibitor, disulfiram, which can covalently bind to NDM-1 by forming an S-S bond with the Cys208 residue. Its copper-containing metabolite in vivo, Cu(DTC)2, also inactivated NDM-1 through oxidizing the Zn(ii) thiolate site of the enzyme, therefore exhibiting dual functional inhibitory potential against B1 and B2 subclass MßLs.

meta-Substituted benzenesulfonamide: a potent scaffold for the development of metallo-ß-lactamase ImiS inhibitors.

Metallo-ß-lactamase (MßL) ImiS contributes to the emergence of carbapenem resistance. A potent scaffold, meta-substituted benzenesulfonamide, was constructed and assayed against MßLs. The twenty-one obtained molecules specifically inhibited ImiS (IC50 = 0.11-9.3 µM); 2g was found to be the best inhibitor (IC50 = 0.11 µM), and 1g and 2g exhibited partially mixed inhibition with K i of 8.0 and 0.55 µM. The analysis of the structure-activity relationship revealed that the meta-substitutes improved the inhibitory activity of the inhibitors. Isothermal titration calorimetry (ITC) assays showed that 2g reversibly inhibited ImiS. The benzenesulfonamides exhibited synergistic antibacterial effects against E. coli BL21 (DE3) cells with ImiS, resulting in a 2-4-fold reduction in the MIC of imipenem and meropenem. Also, mouse experiments showed that 2g had synergistic efficacy with meropenem and significantly reduced the bacterial load in the spleen and liver after a single intraperitoneal dose. Tracing the ImiS in living E. coli cells by RS at a super-resolution level (3D-SIM) showed that the target was initially associated on the surface of the cells, then there was a high density of uniform localization distributed in the cytosol of cells, and it finally accumulated in the formation of inclusion bodies at the cell poles. Docking studies suggested that the sulfonamide group acted as a zinc-binding group to coordinate with Zn(ii) and the residual amino acid within the CphA active center, tightly anchoring the inhibitor at the active site. This study provides a highly promising scaffold for the development of inhibitors of ImiS, even the B2 subclasses of MßLs.

3-Bromopyruvate as a potent covalently reversible inhibitor of New Delhi metallo-ß-lactamase-1 (NDM-1).

The bacteria, harboring metallo-ß-lactamases (MßLs), become resistant on most ß-lactam antibiotics, specifically New Delhi metallo-ß-lactamase-1 (NDM-1), which hydrolyzes almost all ß-lactam antibiotics leading to bacterial multiple-drug resistance. It is highly desirable to develop effective NDM-1 inhibitors in reviving the efficacy of existing antibiotics. Here, we report a potent covalently reversible scaffold, 3-Bromopyruvate (3BP) to target the NDM-1 in vitro and in vivo. Enzymatic kinetic studies revealed that 3BP is capable of inhibiting the B1 and B2 MßLs and exhibited the best inhibition on NDM-1 with an IC50 of 2.57 µM, also, it was found to be a dose- and time-dependent inhibitor. The study of inhibition mechanism suggested that 3BP reversibly inactivate NDM-1, and may form a dynamic reversible covalent bond with cysteine at active site of the enzyme. Besides, 3BP effectively restored the activity of five ß-lactam antibiotics on three clinical strains expressing NDM-1, resulting in 2-8-fold reduction in MIC. Moreover, the toxicity evaluation of 3BP against L929 mouse fibroblastic cells indicated that 3BP had low cytotoxicity, implying it may be used as lead molecule for future drug candidate.

Dithiocarbamate as a Valuable Scaffold for the Inhibition of Metallo-ß-Lactmases.

The 'superbug' infection caused by metallo-ß-lactamases (MßLs) has grown into an emergent health threat. Given the clinical importance of MßLs, a novel scaffold, dithiocarbamate, was constructed. The obtained molecules, DC1, DC8 and DC10, inhibited MßLs NDM-1, VIM-2, IMP-1, ImiS and L1 from all three subclasses, exhibiting an IC50 < 26 µM. DC1 was found to be the best inhibitor of ImiS (IC50 < 0.22 µM). DC1-2, DC4, DC8 and DC10 restored antimicrobial effects of cefazolin and imipenem against E. coli-BL21, producing NDM-1, ImiS or L1, and DC1 showed the best inhibition of E. coli cells, expressing the three MßLs, resulting in a 2-16-fold reduction in the minimum inhibitory concentrations (MICs) of both antibiotics. Kinetics and isothermal titration calorimetry (ITC) assays showed that DC1 exhibited a reversible, and partially mixed inhibition, of NDM-1, ImiS and L1, with Ki values of 0.29, 0.14 and 5.06 µM, respectively. Docking studies suggest that the hydroxyl and carbonyl groups of DC1 form coordinate bonds with the Zn (II) ions, in the active center of NDM-1, ImiS and L1, thereby inhibiting the activity of the enzymes. Cytotoxicity assays showed that DC1, DC3, DC7 and DC9 have low toxicity in L929 mouse fibroblastic cells, at a dose of up to 250 µM. These studies revealed that the dithiocarbamate is a valuable scaffold for the development of MßLs inhibitors.