A tryptophan-based assay method to search regulatory compounds for transcriptionally controlled tumor protein.

Transcriptionally controlled tumor protein (TCTP) is a highly conserved protein performing a large number of cellular functions by binding with various partner proteins. The importance of its roles in many diseases requires an assay method to find regulatory compounds. However, the molecular characteristics of TCTP made it difficult to search for chemicals interacting with it. In this study, a tryptophan-based assay method was designed and Y151W mutant TCTP was constructed to search binding chemicals. Since there is no tryptophan in the native sequence of TCTP, the incorporation of tryptophan in the Y151W mutant was very effective to establish the method. A flavonoid library was employed to the assay with the method. With the native and Y151W mutant TCTPs, three flavonoids such as morin, myricetin and isobavachalcone have been found to interact with TCTP. Combined with native gel electrophoresis, the binding region of isobavachalcone was suggested to be the flexible loop of TCTP. This approach can be easily applicable to find binding compounds of proteins with similar molecular characteristics of TCTP.

Inhibition of d-glycero-ß-d-manno-heptose 1-phosphate adenylyltransferase from Burkholderia pseudomallei by epigallocatechin gallate and myricetin.

Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. BpHldC is one of the essential enzymes in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei. Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of BpHldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and ßG1P. Analyses with EGCG homologs could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei.

Dimerization Tendency of 3CLpros of Human Coronaviruses Based on the X-ray Crystal Structure of the Catalytic Domain of SARS-CoV-2 3CLpro.

3CLpro of SARS-CoV-2 is a promising target for developing anti-COVID19 agents. In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents.

A Study of Drug Repurposing to Identify SARS-CoV-2 Main Protease (3CLpro) Inhibitors.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wreaked havoc all over the world. Although vaccines for the disease have recently become available and started to be administered to the population in various countries, there is still a strong and urgent need for treatments to cure COVID-19. One of the safest and fastest strategies is represented by drug repurposing (DRPx). In this study, thirty compounds with known safety profiles were identified from a chemical library of Phase II-and-up compounds through a combination of SOM Biotech's Artificial Intelligence (AI) technology, SOMAIPRO, and in silico docking calculations with third-party software. The selected compounds were then tested in vitro for inhibitory activity against SARS-CoV-2 main protease (3CLpro or Mpro). Of the thirty compounds, three (cynarine, eravacycline, and prexasertib) displayed strong inhibitory activity against SARS-CoV-2 3CLpro. VeroE6 cells infected with SARS-CoV-2 were used to find the cell protection capability of each candidate. Among the three compounds, only eravacycline showed potential antiviral activities with no significant cytotoxicity. A further study is planned for pre-clinical trials.

A study of inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei as a potential antibiotic target.

d-Glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) is the fourth enzyme in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway producing a lipopolysaccharide core. Therefore, BpHldC is an anti-melioidosis target. Three ChemBridge compounds purchased from ChemBridge Corporation (San Diego, CA) were found to have an effective inhibitory activity on BpHldC. Interestingly, ChemBridge 7929959 was the most effective compound due to the presence of the terminal benzyl group. The enzyme kinetic study revealed that most of them show mixed type inhibitory modes against ATP and ßG1P. The induced-fit docking indicated that the medium affinity of ChemBridge 7929959 is originated from its benzyl group occupying the substrate-binding pocket of BpHldC. The inhibitory role of terminal aromatic groups was proven with ChemBridge 7570508. Combined with the previous study, ChemBridge 7929959 is found to work as a dual inhibitor against both HldC and HddC. Therefore, three ChemBridge compounds can be developed as a potent anti-melioidosis agent with a novel inhibitory concept.

A triple-targeting inhibitory activity of Rose Bengal on polysaccharide biosynthesis of Burkholderia pseudomallei.

Sugar nucleotidyltransferases (SNTs) participate in various biosynthesis pathways constructing polysaccharides in Gram-negative bacteria. In this study, a triple-targeting inhibitory activity of Rose Bengal against SNTs such as d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC), d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase (HldC), and 3-deoxy-d-manno-oct-2-ulosonic acid cytidylyltransferase (KdsB) from Burkholderia pseudomallei is provided. Rose Bengal effectively suppresses the nucleotidyltransferase activity of the three SNTs, and its IC50 values are 10.42, 0.76, and 5.31 µM, respectively. Interestingly, Rose Bengal inhibits the three enzymes regardless of their primary, secondary, tertiary, and quaternary structural differences. The experimental results indicate that Rose Bengal possesses the plasticity to shape its conformation suitable to interact with the three SNTs. As HddC functions in the formation of capsular polysaccharides and HldC and KdsB produce building blocks to constitute the inner core of lipopolysaccharide, Rose Bengal is a potential candidate to design antibiotics in a new category. In particular, it can be developed as a specific antimelioidosis agent. As the mortality rate of the infected people caused by B. pseudomallei is quite high, there is an urgent need for specific antimelioidosis agents. Therefore, a further study is being carried out with derivatives of Rose Bengal.

Inhibition of SARS-CoV 3CL protease by flavonoids.

There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3' sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.

A study of Rose Bengal against a 2-keto-3-deoxy-d-manno-octulosonate cytidylyltransferase as an antibiotic candidate.

Frequent occurrences of multi-drug resistance of pathogenic Gram-negative bacteria threaten human beings. The CMP-2-keto-3-deoxy-d-manno-octulosonic acid biosynthesis pathway is one of the new targets for antibiotic design. 2-Keto-3-deoxy-d-manno-octulosonate cytidylyltransferase (KdsB) is the key enzyme in this pathway. KdsB proteins from Burkholderia pseudomallei (Bp), B. thailandensis (Bt), Pseudomonas aeruginosa (Pa), and Chlamydia psittaci (Cp) have been assayed to find inhibitors. Interestingly, Rose Bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) was turned out to be an inhibitor of three KdsBs (BpKdsB, BtKdsB, and PaKdsB) with promising IC50 values and increased thermostability. The inhibitory enzyme kinetics of Rose Bengal revealed that it is competitive with 2-keto-3-deoxy-manno-octulosonic acid (KDO) but non-competitive against cytidine 5'-triphosphate (CTP). Induced-fit docking analysis of PaKdsB revealed that Arg160 and Arg185 together with other interactions in the substrate binding site seemed to play an important role in binding with Rose Bengal. We suggest that Rose Bengal can be used as the scaffold to develop potential antibiotics.

Inhibition of African swine fever virus protease by myricetin and myricitrin.

African swine fever (ASF) caused by the ASF virus (ASFV) is the most hazardous swine disease. Since a huge number of pigs have been slaughtered to avoid a pandemic spread, intense studies on the disease should be followed quickly. Recent studies reported that flavonoids have various antiviral activity including ASFV. In this report, ASFV protease was selected as an antiviral target protein to cope with ASF. With a FRET (Fluorescence resonance energy transfer) method, ASFV protease was assayed with a flavonoid library which was composed of sixty-five derivatives classified based on ten different scaffolds. Of these, the flavonols scaffold contains a potential anti-ASFV protease activity. The most prominent flavonol was myricetin with IC50 of 8.4 µM. Its derivative, myricitrin, with the rhamnoside moiety was also showed the profound inhibitory effect on ASFV protease. These two flavonols apparently provide a way to develop anti-ASFV agents based on their scaffold.

Flavonoids with inhibitory activity against SARS-CoV-2 3CLpro.

Coronavirus disease 2019 (COVID-19) has been a pandemic disease of which the termination is not yet predictable. Currently, researches to develop vaccines and treatments is going on globally to cope with this disastrous disease. Main protease (3CLpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the good targets to find antiviral agents before vaccines are available. Some flavonoids are known to inhibit 3CLpro from SARS-CoV which causes SARS. Since their sequence identity is 96%, a similar approach was performed with a flavonoid library. Baicalin, herbacetin, and pectolinarin have been discovered to block the proteolytic activity of SARS-CoV-2 3CLpro. An in silico docking study showed that the binding modes of herbacetin and pectolinarin are similar to those obtained from the catalytic domain of SARS-CoV 3CLpro. However, their binding affinities are different due to the usage of whole SARS-CoV-2 3CLpro in this study. Baicalin showed an effective inhibitory activity against SARS-CoV-2 3CLpro and its docking mode is different from those of herbacetin and pectolinarin. This study suggests important scaffolds to design 3CLpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with SARS-CoV-2.

GTP Preference of d-Glycero-α-d-manno-Heptose-1-Phosphate Guanylyltransferase from Yersinia pseudotuberculosis.

d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) is the fourth enzyme synthesizing a building component of lipopolysaccharide (LPS) of Gram-negative bacteria. Since HddC is a potential new target to develop antibiotics, the analysis of the structural and functional relationship of the complex structure will lead to a better idea to design inhibitory compounds. X-ray crystallography and biochemical experiments to elucidate the guanine preference were performed based on the multiple sequence alignment. The crystal structure of HddC from Yersinia pseudotuberculosis (YPT) complexed with guanosine 5'-(ß-amino)-diphosphate (GMPPN) has been determined at 1.55 Å resolution. Meanwhile, the mutants revealed their reduced guanine affinity, instead of acquiring noticeable pyrimidine affinity. The complex crystal structure revealed that GMPPN is docked in the catalytic site with the aid of Glu80 positioning on the conserved motif EXXPLGTGGA. In the HddC family, this motif is expected to recruit nucleotides through interacting with bases. The crystal structure shows that oxygen atoms of Glu80 forming two hydrogen bonds play a critical role in interaction with two nitrogen atoms of the guanine base of GMPPN. Interestingly, the binding of GMPPN induced the formation of an oxyanion hole-like conformation on the L(S/A/G)X(S/G) motif and consequently influenced on inducing a conformational shift of the region around Ser55.

Amentoflavone, a potent natural matrix metalloproteinase 2 inhibitor.

Gelatinase A (MMP-2) has been studied and proven to play a vital role in the intrusion and metastasis of cancer. Flavonoids influence on molecular and cellular functions of MMP-2 and thus a systematic investigation of flavonoids against the metalloproteolytic activity of MMP-2 has been performed in this study. A fluorescence resonance energy transfer method was used to investigate the inhibitory activities of various flavonoids. Flavone, flavonol and isobavachalcone derivatives showed their inhibitory activity against MMP-2. Surprisingly, the most effective inhibitor was Amentoflavone and its blocking function was superior to other flavonoids. Its IC50 value was 0.689 µM. An induced-fit docking study was carried out to survey its extraordinary activity. The binding mode of Amentoflavone is quite similar to that of (2 ∼ {S})-2-[2-[4-(4-methoxyphenyl) phenyl] sulfanylphenyl] pentanedioic acid complexed with MMP-9. Amentoflavone interacts with the functional zinc and catalytic residue, Glu202. Therefore, the docking study reasonably confirmed the strong inhibitory activity of Amentoflavone.

A Study of 3CLpros as Promising Targets against SARS-CoV and SARS-CoV-2.

The outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), results in serious chaos all over the world. In addition to the available vaccines, the development of treatments to cure COVID-19 should be done quickly. One of the fastest strategies is to use a drug-repurposing approach. To provide COVID-19 patients with useful information about medicines currently being used in clinical trials, twenty-four compounds, including antiviral agents, were selected and assayed. These compounds were applied to verify the inhibitory activity for the protein function of 3CLpros (main proteases) of SARS-CoV and SARS-CoV-2. Among them, viral reverse-transcriptase inhibitors abacavir and tenofovir revealed a good inhibitory effect on both 3CLpros. Intriguingly, sildenafil, a cGMP-specific phosphodiesterase type 5 inhibitor also showed significant inhibitory function against them. The in silico docking study suggests that the active-site residues located in the S1 and S2 sites play key roles in the interactions with the inhibitors. The result indicates that 3CLpros are promising targets to cope with SAR-CoV-2 and its variants. The information can be helpful to design treatments to cure patients with COVID-19.

A Study of a Potent Inhibitor Against a GDP-6-Deoxy-α-d-Manno-Heptose Biosynthesis Pathway as Antibiotic Candidates.

The GDP-6-deoxy-α-d-manno-heptose is a key building block molecule in constructing lipopolysaccharide of Gram-negative bacteria. Therefore, blockage of the biosynthesis pathway of GDP-6-deoxy-α-d-manno-heptose is lethal or increases antibiotics susceptibility to pathogens. In this study, we assayed d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) from Yersinia pseudotuberculosis (Yp) using an efficient assay method supplying its natural substrate. Using the method, 102 chemical compounds were tested to search inhibitory compounds and electrospray ionization mass spectrometry was used to detect the HddC from Y. pseudotuberculosis (YpHddC) reaction product, GDP-d-glycero-α-d-manno-heptose. Interestingly, one promising lead, ethyl 5-({[(5-benzyl-1, 3, 4-oxadiazol-2-yl) thio] acetyl} amino)-4-cyano-3-methyl-2-thiophenecarboxylate (Chembridge 7929959), was discovered. The inhibitory activity of the lead compound against YpHddC has been proven by blocking its nucleotidyltransferase activity transferring the GMP moiety to α-d-mannose-1-phosphate (αM1P). Chembridge 7929959 shows that the half maximal inhibitory concentration (IC50) is 0.222 µM indicating its affinity with αM1P.

Characteristics of flavonoids as potent MERS-CoV 3C-like protease inhibitors.

Middle East respiratory syndrome-coronavirus (MERS-CoV) is a zoonotic virus transmitted between animals and human beings. It causes MERS with high mortality rate. However, no vaccine or specific treatment is currently available. Since antiviral activity of some flavonoids is known, we applied a flavonoid library to probe inhibitory compounds against MERS-CoV 3C-like protease (3CLpro). Herbacetin, isobavachalcone, quercetin 3-ß-d-glucoside and helichrysetin were found to block the enzymatic activity of MERS-CoV 3CLpro. The binding of the four flavonoids was also confirmed independently using a tryptophan-based fluorescence method. The systematic comparison of the binding affinity of flavonoids made it possible to infer their scaffolds and functional groups required to bind with MERS-CoV 3CLpro. An induced-fit docking analysis revealed that S1 and S2 sites play a role in interaction with flavonoids. The experimental and computational study showed that flavonol and chalcone are favourite scaffolds to bind with the catalytic site of MERS-CoV 3CLpro. It was also deduced that some flavonoid derivatives with hydrophobic or carbohydrate attached to their core structures have a good inhibitory effect. Therefore, we suggest that flavonoids with these characteristics can be used as templates to develop potent MERS-CoV 3CLpro inhibitors.