Augmenting a training dataset of the generative diffusion model for molecular docking with artificial binding pockets.

This study introduces the PocketCFDM generative diffusion model, aimed at improving the prediction of small molecule poses in the protein binding pockets. The model utilizes a novel data augmentation technique, involving the creation of numerous artificial binding pockets that mimic the statistical patterns of non-bond interactions found in actual protein-ligand complexes. An algorithmic method was developed to assess and replicate these interaction patterns in the artificial binding pockets built around small molecule conformers. It is shown that the integration of artificial binding pockets into the training process significantly enhanced the model's performance. Notably, PocketCFDM surpassed DiffDock in terms of non-bond interaction and steric clash numbers, and the inference speed. Future developments and optimizations of the model are discussed. The inference code and final model weights of PocketCFDM are accessible publicly via the GitHub repository: https://github.com/vtarasv/pocket-cfdm.git.

Computer-aided design of muscarinic acetylcholine receptor M3 inhibitors: Promising compounds among trifluoromethyl containing hexahydropyrimidinones/thiones.

The new high selective mAChRs M3 inhibitors with IC50 in nanomolecular ranges, which can be the prototypes for effective COPD and asthma treatment drugs, were discovered with computational approaches among trifluoromethyl containing hexahydropyrimidinones/thiones. Compounds [6-(4-ethoxy-3-methoxy-phenyl)-4-hydroxy-2-thioxo-4-(trifluoromethyl)hexahydropyrimidin-5-yl]-phenyl-methanone (THPT-1) and 5-benzoyl-6-(3,4-dimethoxyphenyl)-4-hydroxy-4-(trifluoromethyl)hexahydropyrimidin-2-one (THPO-4) have been proved to be a highly effective (with IC50 values of 1.62 ⋅ 10-7  M and 3.09 ⋅ 10-9  M, respectively) at the same concentrations significantly competitive inhibit the signal conduction through mAChR3 in comparison with ipratropium bromide, without significant effect on mAChR2, nicotinic cholinergic and adrenergic receptors.

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.

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.

Identification of Novel Antistaphylococcal Hit Compounds Targeting Sortase A.

Staphylococcus aureus (S. aureus) is a causative agent of many hospital- and community-acquired infections with the tendency to develop resistance to all known antibiotics. Therefore, the development of novel antistaphylococcal agents is of urgent need. Sortase A is considered a promising molecular target for the development of antistaphylococcal agents. The main aim of this study was to identify novel sortase A inhibitors. In order to find novel antistaphylococcal agents, we performed phenotypic screening of a library containing 15512 compounds against S. aureus ATCC43300. The molecular docking of hits was performed using the DOCK program and 10 compounds were selected for in vitro enzymatic activity inhibition assay. Two inhibitors were identified, N,N-diethyl-N'-(5-nitro-2-(quinazolin-2-yl)phenyl)propane-1,3-diamine (1) and acridin-9-yl-(1H-benzoimidazol-5-yl)-amine (2), which decrease sortase A activity with IC50 values of 160.3 µM and 207.01 µM, respectively. It was found that compounds 1 and 2 possess antibacterial activity toward 29 tested multidrug resistant S. aureus strains with MIC values ranging from 78.12 to 312.5 mg/L. These compounds can be used for further structural 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.

Dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-4-ones as a new class of CK2 inhibitors.

Identification of new small molecules inhibiting protein kinase CK2 is highly required for the study of this protein's functions in cell and for the further development of novel pharmaceuticals against a variety of disorders associated with CK2 activity. In this article, a virtual screening of a random small-molecule library was performed and 12 compounds were initially selected for biochemical tests toward CK2. Among them, the most active compound 1 ([Formula: see text]) belonged to dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-4-ones. The complex of this compound with CK2 was analyzed, and key ligand-enzyme interactions were determined. Then, a virtual screening of 231 dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-4-one derivatives was performed and 37 compounds were chosen for in vitro testing. It was found that 32 compounds inhibit CK2 with [Formula: see text] values from 2.5 to 7.5 [Formula: see text]. These results demonstrate that dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-4-one is a novel class of CK2 inhibitors.

Structural Hypervariability of the Two Human Protein Kinase CK2 Catalytic Subunit Paralogs Revealed by Complex Structures with a Flavonol- and a Thieno[2,3-d]pyrimidine-Based Inhibitor.

Protein kinase CK2 is associated with a number of human diseases, among them cancer, and is therefore a target for inhibitor development in industry and academia. Six crystal structures of either CK2α, the catalytic subunit of human protein kinase CK2, or its paralog CK2α' in complex with two ATP-competitive inhibitors-based on either a flavonol or a thieno[2,3-d]pyrimidine framework-are presented. The structures show examples for extreme structural deformations of the ATP-binding loop and its neighbourhood and of the hinge/helix αD region, i.e., of two zones of the broader ATP site environment. Thus, they supplement our picture of the conformational space available for CK2α and CK2α'. Further, they document the potential of synthetic ligands to trap unusual conformations of the enzymes and allow to envision a new generation of inhibitors that stabilize such conformations.

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.

Design and synthesis of novel protein kinase CK2 inhibitors on the base of 4-aminothieno[2,3-d]pyrimidines.

An extension of our previous research work has resulted in a number of new ATP-competitive CK2 inhibitors that have been identified among 4-aminothieno[2,3-d]pyrimidine derivatives. The most active compounds obtained in the course of the research are 3-(5-p-tolyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5e (NHTP23, IC50 = 0.01 µM), 3-(5-phenyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5g (NHTP25, IC50 = 0.065 µM) and 3-(6-methyl-5-phenyl-thieno[2,3-d]pyrimidin-4-ylamino)-benzoic acid, 5n (NHTP33, IC50 = 0.008 µM). Structure-activity relationships of the tested 4-aminothieno[2,3-d]pyrimidine derivatives have been studied and their binding mode with ATP-acceptor site of CK2 has been proposed. A negative effect of intramolecular hydrogen bonding in the compounds' structure is discussed.

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.

A Note of Caution on the Role of Halogen Bonds for Protein Kinase/Inhibitor Recognition Suggested by High- And Low-Salt CK2α Complex Structures.

CK2 is a Ser/Thr kinase recruited by tumor cells to avoid cell death. 4'-Carboxy-6,8-dibromo-flavonol (FLC26) is a nanomolar CK2 inhibitor reducing the physiological phosphorylation of CK2 biomarkers and inducing cell death. Its binding mode to the ATP site was predicted to depend primarily on noncovalent interactions not comprising halogen bonds. We confirm this by two independent cocrystal structures which additionally show that FLC26 is selective for an open, protein kinase-untypical conformation of the hinge/helix αD region. The structures suggest how the bromo substituents, found previously in lead optimization studies, contribute to the inhibitory efficacy. In this context, one of the complex structures, obtained by crystallization with the kosmotropic salt NaCl, revealed an unconventional π-halogen bond between the 8-bromo substituent of FLC26 and an aromatic side chain which is absent under low-salt conditions. The kosmotropic salt sensitivity of π-halogen bonds is a novel feature which requires attention in structural comparisons and halogen-bond-based explanations.

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.

Design, synthesis and biological evaluation of naphthostyril derivatives as novel protein kinase FGFR1 inhibitors.

New class of FGFR1 kinase inhibitors with naphthostyril heterocycle has been identified. A series of N-phenylnaphthostyril-1-sulfonamides has been synthesized and tested in vitro. It was revealed that the most active compound N-(4-hydroxyphenyl)naphthostyril-1-sulfonamide inhibited FGFR1 with IC50 of 2 µM. In our preliminary studies, N-phenylnaphthostyril-1-sulfonamides demonstrated selectivity of FGFR1 inhibition and antiproliferative activity on cancer cell line. N-phenylnaphthostyril-1-sulfonamides have a good potential for further development as anticancer agents.