Unraveling the efficacy of verbascoside in thwarting MRSA pathogenicity by targeting sortase A.

In the fight against hospital-acquired infections, the challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel treatment methods. This study focused on undermining the virulence of S. aureus, especially by targeting surface proteins crucial for bacterial adherence and evasion of the immune system. A primary aspect of our approach involves inhibiting sortase A (SrtA), a vital enzyme for attaching microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to the bacterial cell wall, thereby reducing the pathogenicity of S. aureus. Verbascoside, a phenylethanoid glycoside, was found to be an effective SrtA inhibitor in our research. Advanced fluorescence quenching and molecular docking studies revealed a specific interaction between verbascoside and SrtA, pinpointing the critical active sites involved in this interaction. This molecular interaction significantly impedes the SrtA-mediated attachment of MSCRAMMs, resulting in a substantial reduction in bacterial adhesion, invasion, and biofilm formation. The effectiveness of verbascoside has also been demonstrated in vivo, as shown by its considerable protective effects on pneumonia and Galleria mellonella (wax moth) infection models. These findings underscore the potential of verbascoside as a promising component in new antivirulence therapies for S. aureus infections. By targeting crucial virulence factors such as SrtA, agents such as verbascoside constitute a strategic and potent approach for tackling antibiotic resistance worldwide. KEY POINTS: • Verbascoside inhibits SrtA, reducing S. aureus adhesion and biofilm formation. • In vivo studies demonstrated the efficacy of verbascoside against S. aureus infections. • Targeting virulence factors such as SrtA offers new avenues against antibiotic resistance.

Identification of 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids as promising DNMT1 inhibitors.

DNA methyltransferase 1 (DNMT1) is the primary enzyme responsible for maintaining DNA methylation patterns during cellular division, crucial for cancer development by suppressing tumor suppressor genes. In this study, we retained the phthalimide structure of N-phthaloyl-l-tryptophan (RG108) and substituted its indole ring with nitrogen-containing aromatic rings of varying sizes. We synthesized 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids and confirmed them as DNMT1 inhibitors through protein affinity testing, radiometric method using tritium labeled SAM, and MTT assay. Preliminary structure-activity relationship analysis revealed that introducing substituents on the carbazole ring could enhance inhibitory activity, with S-configuration isomers showing greater activity than R-configuration ones. Notably, S-3-(3,6-di-tert-butyl-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7r-S) and S-3-(1,3,6-trichloro-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7t-S) exhibited significant DNMT1 enzyme inhibition activity, with IC50 values of 8.147 µM and 0.777 µM, respectively (compared to RG108 with an IC50 above 250 µM). Moreover, they demonstrated potential anti-proliferative activity on various tumor cell lines including A2780, HeLa, K562, and SiHa. Transcriptome analysis and KEGG pathway enrichment of K562 cells treated with 7r-S and 7t-S identified differentially expressed genes (DEGs) related to apoptosis and cell cycle pathways. Flow cytometry assays further indicated that 7r-S and 7t-S induce apoptosis in K562 cells and arrest them in the G0/G1 phase in a concentration-dependent manner. Molecular docking revealed that 7t-S may bind to the methyl donor S-adenosyl-l-methionine (SAM) site in DNMT1 with an orientation opposite to RG108, suggesting potential for deeper penetration into the DNMT1 pocket and laying the groundwork for further modifications.

Exploring the therapeutic potential of rutin through investigating its inhibitory mechanism on lactate dehydrogenase: Multi-spectral methods and computer simulation.

Lactate dehydrogenase (LDH), a crucial enzyme in anaerobic glycolysis, plays a pivotal role in the energy metabolism of tumor cells, positioning it as a promising target for tumor treatment. Rutin, a plant-based flavonoid, offers benefits like antioxidant, antiapoptotic, and antineoplastic effects. This study employed diverse experiments to investigate the inhibitory mechanism of rutin on LDH through a binding perspective. The outcomes revealed that rutin underwent spontaneous binding within the coenzyme binding site of LDH, leading to the formation of a stable binary complex driven by hydrophobic forces, with hydrogen bonds also contributing significantly to sustaining the stability of the LDH-rutin complex. The binding constant (Ka) for the LDH-rutin system was 2.692 ± 0.015 × 104 M-1 at 298 K. Furthermore, rutin induced the alterations in the secondary structure conformation of LDH, characterized by a decrease in α-helix and an increase in antiparallel and parallel ß-sheet, and ß-turn. Rutin augmented the stability of coenzyme binding to LDH, which could potentially hinder the conversion process among coenzymes. Specifically, Arg98 in the active site loop of LDH provided essential binding energy contribution in the binding process. These outcomes might explain the dose-dependent inhibition of the catalytic activity of LDH by rutin. Interestingly, both the food additives ascorbic acid and tetrahydrocurcumin could reduce the binding stability of LDH and rutin. Meanwhile, these food additives did not produce positive synergism or antagonism on the rutin binding to LDH. Overall, this research could offer a unique insight into the therapeutic potential and medicinal worth of rutin.

Dual-targeted NAMPT inhibitors as a progressive strategy for cancer therapy.

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the nicotinamide adenine dinucleotide (NAD+) synthesis pathway catalyzing the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-pyrophosphate (PRPP) to produce nicotinamide mononucleotide (NMN). Given the pivotal role of NAD+ in a range of cellular functions, including DNA synthesis, redox reactions, cytokine generation, metabolism, and aging, NAMPT has become a promising target for many diseases, notably cancer. Therefore, various NAMPT inhibitors have been reported and classified as first and second-generation based on their chemical structures and design strategies, dual-targeted being one. However, most NAMPT inhibitors suffer from several limitations, such as dose-dependent toxicity and poor pharmacokinetic properties. Consequently, there is no clinically approved NAMPT inhibitor. Hence, research on discovering more effective and less toxic dual-targeted NAMPT inhibitors with desirable pharmacokinetic properties has drawn attention recently. This review summarizes the previously reported dual-targeted NAMPT inhibitors, focusing on their design strategies and advantages over the single-targeted therapies.

A consensus reverse docking approach for identification of a competitive inhibitor of acetyltransferase enhanced intracellular survival protein from Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which remains a significant global health challenge. The emergence of multidrug-resistant (MDR) Mtb strains imposes the development of new therapeutic strategies. This study focuses on the identification and evaluation of potential inhibitors against Mtb H37Ra through a comprehensive screening of an in-house chemolibrary. Subsequently, a promising pyrimidine derivative (LQM495) was identified as promising and then further investigated by experimental and in silico approaches. In this context, computational techniques were used to elucidate the potential molecular target underlying the inhibitory action of LQM495. Then, a consensus reverse docking (CRD) protocol was used to investigate the interactions between this compound and several Mtb targets. Out of 98 Mtb targets investigated, the enhanced intracellular survival (Eis) protein emerged as a target for LQM495. To gain insights into the stability of the LQM495-Eis complex, molecular dynamics (MD) simulations were conducted over a 400 ns trajectory. Further insights into its binding modes within the Eis binding site were obtained through a Quantum mechanics (QM) approach, using density functional theory (DFT), with B3LYP/D3 basis set. These calculations shed light on the electronic properties and reactivity of LQM495. Subsequently, inhibition assays and kinetic studies of the Eis activity were used to investigate the activity of LQM495. Then, an IC50 value of 11.0 ± 1.4 µM was found for LQM495 upon Eis protein. Additionally, its Vmax, Km, and Ki parameters indicated that it is a competitive inhibitor. Lastly, this study presents LQM495 as a promising inhibitor of Mtb Eis protein, which could be further explored for developing novel anti-TB drugs in the future.

Research progress on GPX4 targeted compounds.

Blocking the System Xc-_ GSH_GPX4 pathway to induce ferroptosis in tumor cells is a novel strategy for cancer treatment. GPX4 serves as the core of the System Xc-/GSH/GPX4 pathway and is a predominant target for inducing ferroptosis in tumor cells. This article summarizes compounds identified in current research that directly target the GPX4 protein, including inhibitors, activators, small molecule degraders, chimeric degraders, and the application of combination therapies with other drugs, aiming to promote further research on the target and related diseases.

Identification of sulfonylpyrimidines as novel selective aldosterone synthase (CYP11B2) inhibitors.

4-[(5-[2-Methyl-5-(methylsulfonyl)pentan-2-yl]sulfonylpyrimidin-4-yl)amino]benzonitrile 2 was identified as a novel potent aldosterone synthase inhibitor. Compound 2 was found to inhibit human CYP11B2 in the nanomolar range, and showed an aldosterone-lowering effect in a furosemide-treated cynomolgus monkey model. Although human CYP11B2 has the high homology sequence with human CYP11B1, compound 2 showed more than 80 times higher selectivity over human CYP11B1 in vitro.

Structure-function relationships in (poly)phenol-enzyme binding: Direct inhibition of human salivary and pancreatic α-amylases.

(Poly)phenols inhibit α-amylase by directly binding to the enzyme and/or by forming starch-polyphenol complexes. Conventional methods using starch as the substrate measure inhibition from both mechanisms, whereas the use of shorter oligosaccharides as substrates exclusively measures the direct interaction of (poly)phenols with the enzyme. In this study, using a chromatography-based method and a short oligosaccharide as the substrate, we investigated the detailed structural prerequisites for the direct inhibition of human salivary and pancreatic α-amylases by over 50 (poly)phenols from the (poly)phenol groups: flavonols, flavones, flavanones, flavan-3-ols, polymethoxyflavones, isoflavones, anthocyanidins and phenolic acids. Despite being structurally very similar (97% sequence homology), human salivary and pancreatic α-amylases were inhibited to different extents by the tested (poly)phenols. The most potent human salivary α-amylase inhibitors were luteolin and pelargonidin, while the methoxylated anthocyanidins, peonidin and petunidin, significantly blocked pancreatic enzyme activity. B-ring methoxylation of anthocyanidins increased inhibition against both human α-amylases while hydroxyl groups at C3 and B3' acted antagonistically in human salivary inhibition. C4 carbonyl reduction, or the positive charge on the flavonoid structure, was the key structural feature for human pancreatic inhibition. B-ring glycosylation did not affect salivary enzyme inhibition, but increased pancreatic enzyme inhibition when compared to its corresponding aglycone. Overall, our findings indicate that the efficacy of interaction with human α-amylase is mainly influenced by the type and placement of functional groups rather than the number of hydroxyl groups and molecular weight.

Watermelon: setup and validation of an in silico fragment-based approach.

We present a new computational approach, named Watermelon, designed for the development of pharmacophore models based on receptor structures. The methodology involves the sampling of potential hotspots for ligand interactions within a protein target's binding site, utilising molecular fragments as probes. By employing docking and molecular dynamics (MD) simulations, the most significant interactions formed by these probes within distinct regions of the binding site are identified. These interactions are subsequently transformed into pharmacophore features that delineates key anchoring sites for potential ligands. The reliability of the approach was experimentally validated using the monoacylglycerol lipase (MAGL) enzyme. The generated pharmacophore model captured features representing ligand-MAGL interactions observed in various X-ray co-crystal structures and was employed to screen a database of commercially available compounds, in combination with consensus docking and MD simulations. The screening successfully identified two new MAGL inhibitors with micromolar potency, thus confirming the reliability of the Watermelon approach.

Three neo-Clerodane Diterpenoids from Tinospora cordifolia Stems and Their Arginase Inhibitory Activities.

Three neo-clerodane diterpenoids, including two new tinocordifoliols A (1) and B (2) and one known tinopanoid R (3), were isolated from the ethyl acetate-soluble fraction of the 70% ethanol extract of Tinospora cordifolia stems. The structures were elucidated by various spectroscopic methods, including one dimensional (1D) and 2D-NMR, high resolution-electrospray ionization (HR-ESI)-MS, and electronic circular dichroism (ECD) data. The T. cordifolia extract and all isolated compounds 1-3 possessed arginase I inhibitory activities. Among them, 3 exhibited moderate competitive inhibition of human arginase I (IC50 = 61.9 µM). Furthermore, docking studies revealed that the presence of a ß-substituted furan in 3 may play a key role in the arginase I inhibitory activities.

7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase.

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

Vitamin B6 is an important nutrient for optimal brain function, with deficiencies linked to impaired memory, learning and mood in various mental disorders. In older people, vitamin B6 deficiency is also associated with declining memory and dementia. Although this has been known for years, the precise role of vitamin B6 in these disorders and whether supplements can be used to treat or prevent them remained unclear. This is partly because vitamin B6 is actually an umbrella term for a small number of very similar and interchangeable molecules. Only one of these is 'bioactive', meaning it has a biological role in cells. However, therapeutic strategies aimed at increasing only the bioactive form of vitamin B6 are lacking. Previous work showed that disrupting the gene for an enzyme called pyridoxal phosphatase, which breaks down vitamin B6, improves memory and learning in mice. To investigate whether these effects could be mimicked by drug-like compounds, Brenner, Zink, Witzinger et al. used several biochemical and structural biology approaches to search for molecules that bind to and inhibit pyridoxal phosphatase. The experiments showed that a molecule called 7,8-dihydroxyflavone ­ which was previously found to improve memory and learning in laboratory animals with brain disorders ­ binds to pyridoxal phosphatase and inhibits its activity. This led to increased bioactive vitamin B6 levels in mouse brain cells involved in memory and learning. The findings of Brenner et al. suggest that inhibiting pyridoxal phosphatase to increase vitamin B6 levels in the brain could be used together with supplements. The identification of 7,8-dihydroxyflavone as a promising candidate drug is a first step in the discovery of more efficient pyridoxal phosphatase inhibitors. These will be useful experimental tools to directly study whether increasing the levels of bioactive vitamin B6 in the brain may help those with mental health conditions associated with impaired memory, learning and mood.

Prediction of the binding mechanism of a selective DNA methyltransferase 3A inhibitor by molecular simulation.

DNA methylation is an epigenetic mechanism that introduces a methyl group at the C5 position of cytosine. This reaction is catalyzed by DNA methyltransferases (DNMTs) and is essential for the regulation of gene transcription. The DNMT1 and DNMT3A or -3B family proteins are known targets for the inhibition of DNA hypermethylation in cancer cells. A selective non-nucleoside DNMT3A inhibitor was developed that mimics S-adenosyl-l-methionine and deoxycytidine; however, the mechanism of selectivity is unclear because the inhibitor-protein complex structure determination is absent. Therefore, we performed docking and molecular dynamics simulations to predict the structure of the complex formed by the association between DNMT3A and the selective inhibitor. Our simulations, binding free energy decomposition analysis, structural isoform comparison, and residue scanning showed that Arg688 of DNMT3A is involved in the interaction with this inhibitor, as evidenced by its significant contribution to the binding free energy. The presence of Asn1192 at the corresponding residues in DNMT1 results in a loss of affinity for the inhibitor, suggesting that the interactions mediated by Arg688 in DNMT3A are essential for selectivity. Our findings can be applied in the design of DNMT-selective inhibitors and methylation-specific drug optimization procedures.

New natural protein tyrosine phosphatase 1B inhibitors from Gynostemma pentaphyllum.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease mainly caused by insulin resistance, which can lead to a series of complications such as cardiovascular disease, retinopathy, and its typical clinical symptom is hyperglycaemia. Glucosidase inhibitors, including Acarbose, Miglitol, are commonly used in the clinical treatment of hypoglycaemia. In addition, Protein tyrosine phosphatase 1B (PTP1B) is also an important promising target for the treatment of T2DM. Gynostemma pentaphyllum is a well-known oriental traditional medicinal herbal plant, and has many beneficial effects on glucose and lipid metabolism. In the present study, three new and nine known dammarane triterpenoids isolated from G. pentaphyllum, and their structures were elucidated by spectroscopic methods including HR-ESI-MS,1H and 13C NMR and X-ray crystallography. All these compounds were evaluated for inhibitory activity against α-glucosidase, α-amylase and PTP1B. The results suggested that compounds 7∼10 were potential antidiabetic agents with significantly inhibition activity against PTP1B in a dose-dependent manner.

Bacterial neuraminidase inhibitory chalcones from flowers of Coreopsis lanceolata, their kinetic characterization and antibiofilm effect.

BACKGROUND: Bacteria within biofilms are thousand times more resistant to antibiotics. Neuraminidase is a crucial enzyme for bacterial adhesion and biofilm formation, it hydrolyzes glycosidic residue of glycoproteins, glycolipids, and oligosaccharides. Coreopsis lanceolata L. flowers may have a significant potential of bacterial neuraminidase (BNA) inhibition because of high natural abundance of chalcones. PURPOSE: The investigation of bacterial biofilm inhibitors has emerged as a novel therapeutic strategy against antibiotic resistance. Therefore, individual chalcones were isolated from C. lanceolata and their capacity to inhibit BNA and formation of Escherichia coli biofilm were evaluated. METHODS: Different chromatographic techniques were used to isolate the compounds (1-12). Enzyme inhibition and detailed kinetic behavior of compounds was determined by estimation of kinetic parameters (Michaelis-Menten constants (Km), maximum velocity (Vmax), dissociation constant for binding with the free enzyme (KI) and enzyme-substate complex (KIS)). Binding affinities (KSV) and binding modes of inhibitors were elucidated by fluorescence quenching and molecular docking, respectively. The natural abundance of chalcones was established through UPLC-Q-TOF/MS. The most potent inhibitor (1) was tested for its ability to inhibit the formation of E. coli biofilm, which was examined by crystal violet assay, scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM). RESULTS: A series of eight chalcones (1-8) and four chalcone glucosides (9-12), inhibited BNA in a dose-dependent manner with IC50 of 8.3 ∼ 77.0 µM. The most potent chalcones were butein (1, IC50 = 8.3 µM) and its glucoside 9 (IC50 = 13.8 µM). The aglycones (1-8) showed non-competitive inhibition, while chalcone glucosides (9-12) displayed a mixed type I (KI < KIS). Inhibitory behaviors were doubly confirmed by KSV and matched with tendency of IC50. The functional group responsible for BNA inhibition were disclosed as 4'-hydroxyl group on B-ring by structure activity relationship (SAR) and molecular docking experiments. Butein (1) suppressed E. coli biofilm formation by > 50 % at 100 µM according to crystal violet assay, which was confirmed by SEM and CLSM imaging. CONCLUSION: The results showed that chalcones (1-8) and chalcone glucosides (9-12), metabolites isolated from the flowers of C. lanceolata, had BNA inhibitory and antibiofilm formation effect on E. coli.

Exploring Asphodelus microcarpus as a source of xanthine oxidase inhibitors: Insights from in silico and in vitro studies.

Xanthine oxidase (XO) plays a critical role in purine catabolism, catalyzing the conversion of hypoxanthine to xanthine and xanthine to uric acid, contributing to superoxide anion production. This process is implicated in various human diseases, particularly gout. Traditional XO inhibitors, such as allopurinol and febuxostat, while effective, may present side effects. Our study focuses on Asphodelus microcarpus, a plant renowned for traditional anti-inflammatory uses. Recent investigations into its phenolic-rich flowers, notably abundant in luteolin derivatives, reveal its potential as a natural source of XO inhibitors. In the present research, XO inhibition by an ethanolic flowers extract from A. microcarpus is reported. In silico docking studies have highlighted luteolin derivatives as potential XO inhibitors, and molecular dynamics support that luteolin 7-O-glucoside has the highest binding stability compared to other compounds and controls. In vitro studies confirm that luteolin 7-O-glucoside inhibits XO more effectively than the standard inhibitor allopurinol, with an IC50 value of 4.8 µg/mL compared to 11.5 µg/mL, respectively. These findings underscore the potential therapeutic significance of A. microcarpus in managing conditions related to XO activity. The research contributes valuable insights into the health-promoting properties of A. microcarpus and its potential application in natural medicine, presenting a promising avenue for further exploration in disease management.

Glucoconjugated monoterpene indole alkaloids with xanthine oxidase inhibitory activity from Ophiorrhiza japonica.

Continued interest in the bioactive alkaloids led to the isolation of five undescribed alkaloids (1-5), ophiorglucidines A-E, and seven known analogues (6-12) from the water-soluble fraction of Ophiorrhiza japonica. The structures were elucidated based on spectroscopic data and quantum calculations as well as X-ray crystallographic analysis. The structure of 1 was characterized as a hexacyclic skeleton including a double bridge linking the indole and the monoterpene moieties, which is the first report of a single crystal with this type of structure. Moreover, the inhibitory effect of zwitterionic indole alkaloid glycosides on xanthine oxidase was found for the first time. The alkaloids 2 and 3, both of which have a pentacyclic zwitterionic system, were more active than the reference inhibitor, allopurinol (IC50 = 11.1 µM) with IC50 values of 1.0 µM, and 2.5 µM, respectively. Structure-activity relationships analyses confirmed that the carbonyl group at C-14 was a key functional group responsible for the inhibitory effects of these alkaloids.

Identification of DprE1 inhibitors for tuberculosis through integrated in-silico approaches.

Decaprenylphosphoryl-ß-D-ribose-2'-epimerase (DprE1), a crucial enzyme in the process of arabinogalactan and lipoarabinomannan biosynthesis, has become the target of choice for anti-TB drug discovery in the recent past. The current study aims to find the potential DprE1 inhibitors through in-silico approaches. Here, we built the pharmacophore and 3D-QSAR model using the reported 40 azaindole derivatives of DprE1 inhibitors. The best pharmacophore hypothesis (ADRRR_1) was employed for the virtual screening of the chEMBL database. To identify prospective hits, molecules with good phase scores (> 2.000) were further evaluated by molecular docking studies for their ability to bind to the DprE1 enzyme (PDB: 4KW5). Based on their binding affinities (< - 9.0 kcal/mole), the best hits were subjected to the calculation of free-binding energies (Prime/MM-GBSA), pharmacokinetic, and druglikeness evaluations. The top 10 hits retrieved from these results were selected to predict their inhibitory activities via the developed 3D-QSAR model with a regression coefficient (R2) value of 0.9608 and predictive coefficient (Q2) value of 0.7313. The induced fit docking (IFD) studies and in-silico prediction of anti-TB sensitivity for these top 10 hits were also implemented. Molecular dynamics simulations (MDS) were performed for the top 5 hit molecules for 200 ns to check the stability of the hits with DprE1. Based on their conformational stability throughout the 200 ns simulation, hit 2 (chEMBL_SDF:357100) was identified as the best hit against DprE1 with an accepted safety profile. The MD results were also in accordance with the docking score, MM-GBSA value, and 3D-QSAR predicted activity. The hit 2 molecule, (N-(3-((2-(((1r,4r)-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)acrylamide) could serve as a lead for the discovery of a novel DprE1 inhibiting anti-TB drug.

Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism. Here, we selected two evolutionary related glutamine amidotransferase complexes, aminodeoxychorismate synthase and anthranilate synthase, that are required for the biosynthesis of folate and tryptophan in most prokaryotic organisms. Both enzymes rely on the interplay of a glutaminase and a synthase subunit that is conferred by a highly conserved subunit interface. Consequently, inhibiting subunit association in both enzymes by one competing bispecific inhibitor has the potential to suppress bacterial proliferation. We comprehensively verified two conserved interface hot-spot residues as potential inhibitor-binding sites in vitro by demonstrating their crucial role in subunit association and enzymatic activity. For in vivo target validation, we generated genomically modified Escherichia coli strains in which subunit association was disrupted by modifying these central interface residues. The growth of such strains was drastically retarded on liquid and solid minimal medium due to a lack of folate and tryptophan. Remarkably, the bacteriostatic effect was observed even in the presence of heat-inactivated human plasma, demonstrating that accessible host metabolite concentrations do not compensate for the lack of folate and tryptophan within the tested bacterial cells. We conclude that a potential inhibitor targeting both enzyme complexes will be effective against a broad spectrum of pathogens and offer increased resilience against antibiotic resistance. IMPORTANCE: Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.

Elucidating the structural basis of vitamin B12 derivatives as novel potent inhibitors of PTP1B: Insights from inhibitory mechanisms using Gaussian accelerated molecular dynamics (GaMD) and in vitro study.

Vitamin B12 is a group of biologically active cobalamin compounds. In this study, we investigated the inhibitory effects of methylcobalamin (MeCbl) and hydroxocobalamin acetate (OHCbl Acetate) on protein tyrosine phosphatase 1B (PTP1B). MeCbl and OHCbl Acetate exhibited an IC50 of approximately 58.390 ± 2.811 µM and 8.998 ± 0.587 µM, respectively. The Ki values of MeCbl and OHCbl Acetate were 25.01 µM and 4.04 µM respectively. To elucidate the inhibition mechanism, we conducted a 500 ns Gaussian accelerated molecular dynamics (GaMD) simulation. Utilizing PCA and tICA, we constructed Markov state models (MSM) to examine secondary structure changes during motion. Our findings revealed that the α-helix at residues 37-42 remained the most stable in the PTP1B-OHCbl Acetate system. Furthermore, upon binding of OHCbl Acetate or MeCbl, the WPD loop of PTP1B moved inward to the active pocket, forming a closed conformation and potentially obstructs substrate entry. Protein-ligand interaction analysis and MM-PBSA showed that OHCbl Acetate exhibited lower binding free energy and engaged in more residue interactions with PTP1B. In summary, our study confirmed the substantial inhibitory activity of OHCbl Acetate against PTP1B, with its inhibitory potency notably surpassing that of MeCbl. We demonstrated potential molecular mechanisms of OHCbl Acetate inhibiting PTP1B.

Neuraminidase inhibition promotes the collective migration of neurons and recovery of brain function.

In the injured brain, new neurons produced from endogenous neural stem cells form chains and migrate to injured areas and contribute to the regeneration of lost neurons. However, this endogenous regenerative capacity of the brain has not yet been leveraged for the treatment of brain injury. Here, we show that in healthy brain chains of migrating new neurons maintain unexpectedly large non-adherent areas between neighboring cells, allowing for efficient migration. In instances of brain injury, neuraminidase reduces polysialic acid levels, which negatively regulates adhesion, leading to increased cell-cell adhesion and reduced migration efficiency. The administration of zanamivir, a neuraminidase inhibitor used for influenza treatment, promotes neuronal migration toward damaged regions, fosters neuronal regeneration, and facilitates functional recovery. Together, these findings shed light on a new mechanism governing efficient neuronal migration in the adult brain under physiological conditions, pinpoint the disruption of this mechanism during brain injury, and propose a promising therapeutic avenue for brain injury through drug repositioning.