Identification of novel antistaphylococcal hit compounds.

Staphylococcus aureus is one of the most common nosocomial biofilm-forming pathogens worldwide that has developed resistance mechanisms against majority of the antibiotics. Therefore, the search of novel antistaphylococcal agents with unexploited mechanisms of action, especially with antibiofilm activity, is of great interest. Seryl-tRNA synthetase is recognized as a promising drug target for the development of antibacterials. We have carried out molecular docking of compounds with antistaphycoccal activity, which were earlier found by us using phenotypic screening, into synthetic site of S. aureus SerRS and found seven hit compounds with low inhibitory activity. Further, we have performed search of S. aureus SerRS inhibitors among compounds which were previously tested by us for inhibitory activity toward S. aureus ThrRS, that belong to the same class of aminoacyl-tRNA synthetases. Among them six hits were identified. We have selected four compounds for antibacterial study and found that the most active compound 1-methyl-3-(1H-imidazol-1-methyl-2-yl)-5-nitro-1H-indazole has MIC values toward S. aureus multidrug-resistant clinical isolates ranging from 78.12 to 156.2 µg/ml. However, this compound precipitated during anti-biofilm study. Therefore, we used 3-[N'-(2-hydroxy-3-methoxybenzylidene)hydrazino]-6-methyl-4H-[1,2,4]triazin-5-one with better solubility (ClogS value = 2.9) among investigated compounds toward SerRS for anti-biofilm study. It was found that this compound has a significant inhibitory effect on the growth of planktonic and biofilm culture of S. aureus 25923 with MIC value of 32 µg ml-1. At the same time, this compound does not reveal antibacterial activity toward Esherichia coli ATCC 47076. Therefore, this compound can be proposed as effective antiseptic toward multidrug-resistant biofilm-forming S. aureus isolates.

Pharmacophore modeling, docking and molecular dynamics simulation for identification of novel human protein kinase C beta (PKCß) inhibitors.

Protein kinase Cß (PKCß) is considered as an attractive molecular target for the treatment of COVID-19-related acute respiratory distress syndrome (ARDS). Several classes of inhibitors have been already identified. In this article, we developed and validated ligand-based PKCß pharmacophore models based on the chemical structures of the known inhibitors. The most accurate pharmacophore model, which correctly predicted more than 70% active compounds of test set, included three aromatic pharmacophore features without vectors, one hydrogen bond acceptor pharmacophore feature, one hydrophobic pharmacophore feature and 158 excluded volumes. This pharmacophore model was used for virtual screening of compound collection in order to identify novel potent PKCß inhibitors. Also, molecular docking of compound collection was performed and 28 compounds which were selected simultaneously by two approaches as top-scored were proposed for further biological research. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02075-y.

Probing interactions of aminoacyl-adenylate with Mycobacterium tuberculosis methionyl-tRNA synthetase through in silico site-directed mutagenesis and free energy calculation.

Methionyl-tRNA synthetase (MetRS) is an attractive molecular target for antibiotic discovery. Recently, we have developed several classes of small-molecular inhibitors of Mycobacterium tuberculosis MetRS possessing antibacterial activity. In this article, we performed in silico site-directed mutagenesis of aminoacyl-adenylate binding site of M. tuberculosis MetRS in order to identify crucial amino acid residues for substrate interaction. The umbrella sampling algorithm was used to calculate the binding free energy (ΔG) of these mutated forms with methionyl-adenylate analogue. According to the obtained results, the replacement of Glu24 and Leu293 by alanine leads to the most significant decrease in the binding free energy (ΔG) for adenylate analogue with methionyl-tRNA synthetase indicating increasing of the affinity, which in turn causes the loss of compounds inhibitory activity. Therefore, these amino acid residues can be proposed for further experimental site-directed mutagenesis to confirm binding mode of inhibitors and should be taken into account during chemical optimization to overcome resistance due to mutations.Communicated by Ramaswamy H. Sarma.

Identification of dual-targeted Mycobacterium tuberculosis aminoacyl-tRNA synthetase inhibitors using machine learning.

Background: The most serious challenge in the treatment of tuberculosis is the multidrug resistance of Mycobacterium tuberculosis to existing antibiotics. As a strategy to overcome resistance we used a multitarget drug design approach. The purpose of the work was to discover dual-targeted inhibitors of mycobacterial LeuRS and MetRS with machine learning. Methods: The artificial neural networks were built using module nnet from R 3.6.1. The inhibitory activity of compounds toward LeuRS and MetRS was investigated in aminoacylation assays. Results: Using a machine-learning approach, we identified dual-targeted inhibitors of LeuRS and MetRS among 2-(quinolin-2-ylsulfanyl)-acetamide derivatives. The most active compound inhibits MetRS and LeuRS with IC50 values of 33 µm and 23.9 µm, respectively. Conclusion: 2-(Quinolin-2-ylsulfanyl)-acetamide scaffold can be useful for further research.

Identification of novel small-molecular inhibitors of Staphylococcus aureus sortase A using hybrid virtual screening.

Staphylococcus aureus is one of the most dangerous pathogens commonly associated with high levels of morbidity and mortality. Sortase A is considered as a promising molecular target for the development of antistaphylococcal agents. Using hybrid virtual screening approach and FRET analysis, we have identified five compounds able to decrease the activity of sortase A by more than 50% at the concentration of 200 µM. The most promising compound was 2-(2-amino-3-chloro-benzoylamino)-benzoic acid which was able to inhibit S. aureus sortase A at the IC50 value of 59.7 µM. This compound was selective toward sortase A compared to other four cysteine proteases - cathepsin L, cathepsin B, rhodesain, and the SARS-CoV2 main protease. Microscale thermophoresis experiments confirmed that this compound bound sortase A with KD value of 189 µM. Antibacterial and antibiofilm assays also confirmed high specificity of the hit compound against two standard and three wild-type, S. aureus hospital infection isolates. The effect of the compound on biofilms produced by two S. aureus ATCC strains was also observed suggesting that the compound reduced biofilm formation by changing the biofilm structure and thickness.

Rational Design of Hit Compounds Targeting Staphylococcus aureus Threonyl-tRNA Synthetase.

Staphylococcus aureus is one of the most dangerous nosocomial pathogens which cause a wide variety of hospital-acquired infectious diseases. S. aureus is considered as a superbug due to the development of multidrug resistance to all current therapeutic regimens. Therefore, the discovery of antibiotics with novel mechanisms of action to combat staphylococcal infections is of high priority for modern medicinal chemistry. Nowadays, aminoacyl-tRNA synthetases are considered as promising molecular targets for antibiotic development. In the present study, we used for the first time S. aureus threonyl-tRNA synthetase (ThrRS) as a molecular target. Recombinant S. aureus ThrRS was obtained in the soluble form in a sufficient amount for inhibitor screening assay. Using the molecular docking approach, we selected 180 compounds for investigation of inhibitory activity toward ThrRS. Among the tested compounds, we identified five inhibitors from different chemical classes decreasing the activity of ThrRS by more than 70% at a concentration of 100 µM. The most active compound 2,4-dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol has an IC50 value of 56.5 ± 3.5 µM. These compounds are not cytotoxic toward eukaryotic cells HEK293 (EC50 > 100 µM) and can be useful for further optimization and biological research.

Discovery of novel antituberculosis agents among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives targeting aminoacyl-tRNA synthetases.

Antibiotic resistance is a major problem of tuberculosis treatment. This provides the stimulus for the search of novel molecular targets and approaches to reduce or forestall resistance emergence in Mycobacterium tuberculosis. Earlier, we discovered a novel small-molecular inhibitor among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazoles targeting simultaneously two enzymes-mycobacterial leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS), which are promising molecular targets for antibiotic development. Unfortunately, the identified inhibitor does not reveal antibacterial activity toward M. tuberculosis. This study aims to develop novel aminoacyl-tRNA synthetase inhibitors among this chemical class with antibacterial activity toward resistant strains of M. tuberculosis. We performed molecular docking of the library of 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives and selected 41 compounds for investigation of their inhibitory activity toward MetRS and LeuRS in aminoacylation assay and antibacterial activity toward M. tuberculosis strains using microdilution assay. In vitro screening resulted in 10 compounds active against MetRS and 3 compounds active against LeuRS. Structure-related relationships (SAR) were established. The antibacterial screening revealed 4 compounds active toward M. tuberculosis mono-resistant strains in the range of concentrations 2-20 mg/L. Among these compounds, only one compound 27 has significant enzyme inhibitory activity toward mycobacterial MetRS (IC50 = 148.5 µM). The MIC for this compound toward M. tuberculosis H37Rv strain is 12.5 µM. This compound is not cytotoxic to human HEK293 and HepG2 cell lines. Therefore, 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives can be used for further chemical optimization and biological research to find non-toxic antituberculosis agents with a novel mechanism of action.

Novel isoniazid derivative as promising antituberculosis agent.

Background: A major focus of tuberculosis drug discovery is aimed at the development of novel antibiotics with activity against drug-resistant strains of Mycobacterium tuberculosis. Results: We have synthesized ten isoniazid derivatives and investigated for antibacterial activity toward M. tuberculosis H37Rv and isoniazid-resistant strain SRI 1369. It was revealed that only one compound, isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide (1), is active toward isoniazid-resistant strain with minimum inhibitory concentration value of 0.14 µM. This compound is not cytotoxic toward human liver cells (HepG2; IC50 >100 µM), demonstrates good permeability in Caco-2 cells. Accordingly to the results of plasma protein binding assay, unbound fraction of compound 1, which potentially exhibits pharmacologic effects, is 57.9%. Conclusion: Therefore, isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide is a promising compound for further preclinical studies.

Dual-targeted hit identification using pharmacophore screening.

Mycobacterium tuberculosis infection remains a major cause of global morbidity and mortality due to the increase of antibiotics resistance. Dual/multi-target drug discovery is a promising approach to overcome bacterial resistance. In this study, we built ligand-based pharmacophore models and performed pharmacophore screening in order to identify hit compounds targeting simultaneously two enzymes-M. tuberculosis leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS). In vitro aminoacylation assay revealed five compounds from different chemical classes inhibiting both enzymes. Among them the most active compound-3-(3-chloro-4-methoxy-phenyl)-5-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-3H-[1,2,3]triazol-4-ylamine (1) inhibits mycobacterial LeuRS and MetRS with IC50 values of 13 µM and 13.8 µM, respectively. Molecular modeling study indicated that compound 1 has similar binding mode with the active sites of both aminoacyl-tRNA synthetases and can be valuable compound for further chemical optimization in order to find promising antituberculosis agents.

Benzaldehyde thiosemicarbazone derivatives against replicating and nonreplicating Mycobacterium tuberculosis.

In this article, we report a series of benzaldehyde thiosemicarbazone derivatives possessing high activity toward actively replicating Mycobacterium tuberculosis strain with minimum inhibitory concentration (MIC) values in the range from 0.14 to 2.2 µM. Among them, two compounds-2-(4-phenethoxybenzylidene)hydrazine-1-carbothioamide (13) and 2-(3-isopropoxybenzylidene)hydrazine-1-carbothioamide (20) also demonstrate submicromolar antimycobacterial activity against M. tuberculosis under hypoxia with MIC values of 0.68 and 0.74 µM, respectively. The activity of compounds 13 and 20 toward five investigated isoniazid-, rifampicin-, and fluoroquinolone-resistant M. tuberculosis isolates is similar to commercially available antituberculosis drugs. The compounds 13 and 20 possess good ADME properties and have low cytotoxicity toward human liver cells (HepG2). Therefore, 2-(4-phenethoxybenzylidene)hydrazine-1-carbothioamide (13) and 2-(3-isopropoxybenzylidene)hydrazine-1-carbothioamide (20) are valuable candidates for further preclinical studies.

Dual-target inhibitors of mycobacterial aminoacyl-tRNA synthetases among N-benzylidene-N'-thiazol-2-yl-hydrazines.

Effective treatment of tuberculosis is challenged by the rapid development of Mycobacterium tuberculosis (Mtb) multidrug resistance that presumably could be overcome with novel multi-target drugs. Aminoacyl-tRNA synthetases (AARSs) are an essential part of protein biosynthesis machinery and attractive targets for drug discovery. Here, we experimentally verify a hypothesis of simultaneous targeting of structurally related AARSs by a single inhibitor. We previously identified a new class of mycobacterial leucyl-tRNA synthetase inhibitors, N-benzylidene-N'-thiazol-2-yl-hydrazines. Molecular docking of a library of novel N-benzylidene-N'-thiazol-2-yl-hydrazine derivatives into active sites of M. tuberculosis LeuRS (MtbLeuRS) and MetRS (MtbMetRS) resulted in a panel of the best ranking compounds, which were then evaluated for enzymatic potency. Screening data revealed 11 compounds active against MtbLeuRS and 28 compounds active against MtbMetRS. The hit compounds display dual inhibitory potency as demonstrated by IC50 values for both enzymes. Compound 3 is active against Mtb H37Rv cells in in vitro bioassays.

Design, synthesis and evaluation of 3-quinoline carboxylic acids as new inhibitors of protein kinase CK2.

In this article, the derivatives of 3-quinoline carboxylic acid were studied as inhibitors of protein kinase CK2. Forty-three new compounds were synthesized. Among them 22 compounds inhibiting CK2 with IC50 in the range from 0.65 to 18.2 µM were identified. The most active inhibitors were found among tetrazolo-quinoline-4-carboxylic acid and 2-aminoquinoline-3-carboxylic acid derivatives.

Identification of Mycobacterium tuberculosis leucyl-tRNA synthetase (LeuRS) inhibitors among the derivatives of 5-phenylamino-2H-[1,2,4]triazin-3-one.

The increase of antibiotic resistance amongst Mycobacterium tuberculosis strains has become one of the most pressing problems of modern medicine. Therefore, the search of antibiotics against M. tuberculosis with novel mechanisms of action is very important. We have identified inhibitors of M. tuberculosis leucyl-tRNA synthetase (LeuRS) among the derivatives of 5-phenylamino-2H-[1,2,4]triazin-3-one. The most active compounds 5-(5-chloro-2-hydroxy-phenylamino)-6-methyl-2H-[1,2,4]triazin-3-one and 5-(5-chloro-2-hydroxy-phenylamino)-2H-[1,2,4]triazin-3-one inhibit M. tuberculosis LeuRS with IC50 of 7.6 µÐœ and 7.2 µÐœ, respectively. It was established that the inhibitory activity of compounds against pathogenic LeuRS is 10-fold better, than for human enzyme.

Production of recombinant human apoptosis signal-regulating kinase 1 (ASK1) in Escherichia coli.

Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies.

Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase.

Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol (1) inhibits LeuRS with IC50 of 6µM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC50 of 2.27µM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50=10.01µM and IC90=13.53µM.

Non-enzymatic activation of prothrombin induced by interaction with fibrin ß26-42 region.

We have discovered that addition of monomeric desAB fibrin to prothrombin leads to appearance of the thrombin-like activity of prothrombin towards S2238 chromogenic substrate. DesA and desABß(15-42)2 fibrin forms did not cause any activation of prothrombin. From this observation we could suggested that amino acid residues of the 15-42 fragment of BßN-domain presented in desAB fibrin, cleaved in desABß(15-42)2 fibrin and protected in desA fibrin, play a crucial role in the non-enzymatic activation of prothrombin. To identify the Bß amino acid residues involved in the fibrin-prothrombin binding we used monoclonal antibodies 1-5G and 2d2a with epitopes in Bß26-42 and Bß12-26 fibrin fragments respectively. The thrombin-like activity in the mixture of prothrombin and desAB fibrin was monitored in the presence of each of these monoclonal antibodies. It was found that anti-Bß12-26 antibody does not exhibit any inhibitory effect on the thombin-like activity of the mixture. In contrast, adding of Bß26-42 antibody into the mixture of desAB fibrin with prothrombin diminished the thrombin-like activity by 70%. Recombinant dimeric peptides Bß(15-44)2 and Bß(15-66)2 that mimic amino acid residues in fibrin were also tested for their ability to activate prothrombin. It was found that both peptides were able to induce non-enzymatic activation of prothrombin. The activation was more evident in the case of Bß(15-44)2 peptide. From the data obtained we can conclude that desAB fibrin binds to prothrombin through the Bß26-42 amino acid residues and the formation of such a complex caused a non-enzymatic activation of prothrombin.

Identification of apoptosis signal-regulating kinase 1 (ASK1) inhibitors among the derivatives of benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one.

Apoptosis signal-regulating kinase 1 (ASK1) plays important roles in the pathogenesis of type 1 and type 2 diabetes, autoimmune disorders, cancer and neurodegenerative diseases suggesting that small compounds inhibiting ASK1 could be used for the treatment of these pathologies. We have identified novel chemical class of ASK1 inhibitors, namely benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one, using molecular modeling techniques. It was found that the most active compound 1-(6-fluoro-benzothiazol-2-yl)-3-hydroxy-5-[3-(3-methyl-butoxy)-phenyl]-4-(2-methyl-2,3-dihydro-benzofuran-5-carbonyl)-1,5-dihydro-pyrrol-2-one (BPyO-34) inhibits ASK1 with IC50 of 0.52µM in vitro in kinase assay. The structure-activity relationships of 34 derivatives of benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one have been studied and binding mode of this chemical class has been proposed.

ASK1 pharmacophore model derived from diverse classes of inhibitors.

The three-dimensional pharmacophore model of apoptosis signal-regulating kinase 1 (ASK1) inhibitors has been developed with PharmaGist program. The positions of pharmacophore features in the model correspond to conformations of ASK1 highly active inhibitors in which they interact with ATP-binding site of ASK1. The generated pharmacophore model allows accurately predict active and inactive compounds and can be of great use for virtual screening aimed at discovering novel ASK1 inhibitors.

Rational design of apoptosis signal-regulating kinase 1 inhibitors: discovering novel structural scaffold.

Increased activity of apoptosis signal-regulating kinase 1 (ASK1) is associated with a number of human disorders and the inhibitors of ASK1 may become important compounds for pharmaceutical application. Here we report novel ASK1 inhibitor scaffold, namely 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one, that has been identified using virtual screening and biochemical tests. A series of derivatives has been synthesized and evaluated in vitro towards human protein kinase ASK1. It was revealed that the most active compounds 4-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid and 6-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)hexanoic acid inhibit ASK1 with IC50 of 0.2 µM. Structure-activity relationships of 33 derivatives of 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one have been studied and binding mode of this chemical class has been predicted.

Identification of 3H-naphtho[1,2,3-de]quinoline-2,7-diones as inhibitors of apoptosis signal-regulating kinase 1 (ASK1).

Apoptosis signal-regulating kinase 1 (ASK1) has recently emerged as an attractive therapeutic target for the treatment of cardiac and neurodegenerative disorders. The selective inhibitors of ASK1 may become important compounds for the development of clinical agents. We have identified the ASK1 inhibitor among 3H-naphtho[1,2,3-de]quinoline-2,7-diones using receptor-based virtual screening. In vitro kinase assay revealed that ethyl 2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinoline-1-carboxylate (NQDI-1) inhibited ASK1 with a K(i) of 500 nM. The competitive character of inhibition is demonstrated in Lineweaver-Burk plots. In our preliminary selectivity study this compound exhibited strong specific inhibitory activity toward ASK1.