The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.
L eishmaniasis is a prevalent disease that impacts 98 countries and territories, mainly in Africa, Asia, and South America. It can cause substantial illness and death, particularly in its visceral manifestation that can be specifically targeted in the development of medications to combat leishmaniasis. This study has found natural compounds with possible inhibitory activity against APX using a reliable and accurate QSAR model. Despite the severe side effects of current treatments and the absence of an effective vaccination, these compounds show promise as a potential treatment for the disease. Nine hit compounds were found, and subsequent molecular docking was performed. Estradiol cypionate showed the lowest binding energy (- 10.5 kcal/mol), thus showing the strongest binding, and also had the strongest binding affinity, with a ΔGTotal of - 26.31 ± 3.01 kcal/mol, second only to the control molecule. Additionally, three hits viz. cloxacillin-sodium (- 16.57 ± 2.89 kcal/mol), cinchonidine (- 16.04 ± 3.27 kcal/mol), and quinine hydrochloride dihydrate (13.38 ± 1.06 kcal/mol) also showed significant binding affinity. Multiple interactions between drugs and active site residues demonstrated a substantial binding affinity with the target protein. The identified compounds exhibited drug-like effects and were orally bioavailable based on their ADME-toxicology features. Overall, estradiol cypionate, cloxacillin sodium, cinchonidine, and quinine hydrochloride dihydrate all exhibited inhibitory effects on the APX enzyme of Leishmania donovani. These results suggest that further investigation is needed to explore the potential of developing novel anti-leishmaniasis drugs using these compounds.
- MeSH
- antiprotozoální látky * farmakologie chemie MeSH
- inhibitory enzymů * farmakologie chemie MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- leishmanióza * farmakoterapie MeSH
- lidé MeSH
- simulace molekulového dockingu MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The Takeda G protein-coupled receptor 5 (TGR5), also known as GPBAR1 (G protein-coupled bile acid receptor), is a membrane-type bile acid receptor that regulates blood glucose levels and energy expenditure. These essential functions make TGR5 a promising target for the treatment of type 2 diabetes and metabolic disorders. Currently, most research on developing TGR5 agonists focuses on modifying the structure of bile acids, which are the endogenous ligands of TGR5. However, TGR5 agonists with nonsteroidal structures have not been widely explored. This study aimed at discovering new TGR5 agonists using bile acid derivatives as a basis for a computational approach. We applied a combination of pharmacophore-based, molecular docking, and molecular dynamic (MD) simulation to identify potential compounds as new TGR5 agonists. Through pharmacophore screening and molecular docking, we identified 41 candidate compounds. From these, five candidates were selected based on criteria including pharmacophore features, a docking score of less than 9.2 kcal/mol, and similarity in essential interaction patterns with a reference ligand. Biological assays of the five hits confirmed that Hit-3 activates TGR5 similarly to the bile acid control. This was supported by MD simulation results, which indicated that a hydrogen bond interaction with Tyr240 is involved in TGR5 activation. Hit-3 (CSC089939231) represents a new nonsteroidal lead that can be further optimized to design potent TGR5 agonists.
- MeSH
- lidé MeSH
- ligandy MeSH
- molekulární struktura MeSH
- objevování léků MeSH
- receptory spřažené s G-proteiny * agonisté metabolismus MeSH
- simulace molekulární dynamiky * MeSH
- simulace molekulového dockingu * MeSH
- vztahy mezi strukturou a aktivitou MeSH
- žlučové kyseliny a soli chemie metabolismus farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mutations in CACNA1C, the gene encoding Cav1.2 voltage-gated calcium channels, are associated with a spectrum of disorders, including Timothy syndrome and other neurodevelopmental and cardiac conditions. In this study, we report a child with a de novo heterozygous missense variant (c.1973T > C; L658P) in CACNA1C, presenting with refractory epilepsy, global developmental delay, hypotonia, and multiple systemic abnormalities, but without overt cardiac dysfunction. Electrophysiological analysis of the recombinant Cav1.2 L658P variant revealed profound gating alterations, most notably a significant hyperpolarizing shift in the voltage dependence of activation and inactivation. Additionally, molecular modeling suggested that the L658P mutation disrupts interactions within the IIS5 transmembrane segment, reducing the energy barrier for state transitions and facilitating channel opening at more negative voltages. These findings establish L658P as a pathogenic CACNA1C variant primarily associated with severe neurological dysfunction and expands the phenotypic spectrum of CACNA1C-related disorders.
- MeSH
- dítě MeSH
- gating iontového kanálu * MeSH
- lidé MeSH
- missense mutace genetika MeSH
- molekulární modely MeSH
- neurovývojové poruchy * genetika MeSH
- předškolní dítě MeSH
- sekvence aminokyselin MeSH
- vápníkové kanály - typ L * genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Investigation determines the beneficial effect of bergaptol against gestational diabetes (GD). Gestational diabetes was induced in female rats and treated them with bergaptol 20 and 40 mg/kg for eighteen days. Effect of bergaptol was assessed on blood glucose and insulin level in GD rat. Inflammatory mediators and oxidative stress parameters were also assessed in GD rats. Moreover, mRNA expression of INSR, NF-kappaB, Akt and GSK-3beta were assessed in the GD rats by qRT-PCR method. In silico network pharmacology study was performed, along with gene ontology and egg pathway to assessed the targets of bergaptol, molecular docking study was also performed for the confirmation of possible pathway involved in the management of GD. Blood glucose and insulin level was significantly reduces in the blood bergaptol treated group than GD group of rats. Treatment with bergaptol ameliorates the altered level of mediators of inflammation and oxidative stress parameters in GD rats. There was significant reduction in the mRNA expression of NF-kappaB and GSK-3beta and increase in expression of INSR and Akt in the tissue homogenate of bergaptol treated GD rats. Docking study shows effective binding strength of bergaptol individually with INSR, NF-kappaB, Akt and GSK-3beta-protein targets. In conclusion, data of investigation suggest that bergaptol improves the sensitivity of insulin receptor in GD, as it reduces parameters of oxidative stress and inflammatory mediators by regulating INSR/NF-kappaB/Akt/GSK-3beta pathway. Key words Gestational diabetes, Bergaptol, Insulin resistance, Inflammation, Oxidative stress.
- MeSH
- experimentální diabetes mellitus * farmakoterapie metabolismus MeSH
- gestační diabetes * farmakoterapie metabolismus MeSH
- inzulinová rezistence * fyziologie MeSH
- kinasa glykogensynthasy 3beta metabolismus MeSH
- krevní glukóza metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- oxidační stres účinky léků MeSH
- potkani Wistar MeSH
- receptor inzulinu metabolismus MeSH
- signální transdukce účinky léků MeSH
- simulace molekulového dockingu * MeSH
- těhotenství MeSH
- zánět farmakoterapie metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
AIM: This study aimed to investigate the phytochemical composition of Psychotria montana extract (PME) and evaluate its inhibitory effects on MCF7 breast cancer cells. METHODS: The chemical composition of PME was analyzed using UPLC-QToF-MS. The effects of PME on cell proliferation were evaluated using the MTT assay. Flow cytometry was used for cell cycle and apoptosis analysis. The effects of PME on the transcription of cell cycle control genes were assessed using real-time PCR. RESULTS: UPLC-QToF-MS analysis revealed major compounds of PME, including terpenoids and flavonoids, with the potential to inhibit proliferation, migration, and induce apoptosis in MCF7 cancer cells. PME effectively suppressed MCF7 cell proliferation under 2D culture, with a low IC50 value of 34.7 μg/ml. PME also hindered cell migration (p < 0.01) and reduced spheroid number (p < 0.001) and size (p < 0.001) in serum-free 3D culture. Apoptosis analysis via nuclear staining with DAPI and flow cytometry revealed an increase in the number of apoptotic cells after PME treatment (p < 0.001). Additionally, the PME induced cell cycle arrest at the G0/G1 phase (p < 0.05). PME altered the expression of cell cycle control genes (cyclins and CDKs) as well as cancer suppressor genes including p16, p27, and p53 at the transcriptional level (mRNA). The results of molecular docking suggest that the compounds present in PME exhibit a high binding affinity for CDK3, CDK4, CDK6, and CDK8 proteins, which are essential regulators of the cell cycle. CONCLUSION: Psychotria montana has the potential to inhibit cancer cells by inducing apoptosis and halting the cell cycle of MCF7 breast cancer cells.
- MeSH
- apoptóza * účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- fytogenní protinádorové látky farmakologie chemie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- nádory prsu * farmakoterapie patologie genetika metabolismus MeSH
- počítačová simulace MeSH
- pohyb buněk účinky léků MeSH
- proliferace buněk * účinky léků MeSH
- Psychotria * chemie MeSH
- rostlinné extrakty * farmakologie chemie MeSH
- simulace molekulového dockingu MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA * chemie metabolismus MeSH
- flavinmononukleotid * chemie metabolismus MeSH
- flaviny chemie metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- oxidace-redukce * MeSH
- simulace molekulární dynamiky MeSH
- světlo * MeSH
- Thermosynechococcus metabolismus MeSH
- transkripční faktory metabolismus chemie MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
AIM: This work explores the synthesis of new bi-heterocyclic hybrid compounds based on quinoline ring and investigates their potential as anticancer agents. MATERIALS & METHODS: The novel fused quinoline-thiazolo[3,2-a] benzimidazole-3(2 h)one hybrids were prepared by regioselective nucleophilic ring opening of the corresponding quinolinyl-oxiranes. In vitro cytotoxic activity was evaluated against human lung (A549) and gastric (AGS) cancer cell lines. RESULTS: Global results showed that all tested compounds have promising inhibitory properties. Compounds 17 and 18 bearing two methoxy groups on the quinoline ring have exhibited remarkable and interesting activities. The investigation of the cell death process showed that these compounds activated a caspase-dependent apoptosis pathway. Results were further supported by molecular docking studies. CONCLUSION: Both compounds exhibited good drug-like characteristics, which make them promising drug candidates.
- MeSH
- apoptóza * účinky léků MeSH
- benzimidazoly * farmakologie chemie chemická syntéza MeSH
- chinoliny * chemie farmakologie chemická syntéza MeSH
- kaspasy * metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- protinádorové látky * farmakologie chemie chemická syntéza MeSH
- screeningové testy protinádorových léčiv MeSH
- simulace molekulového dockingu * MeSH
- thiazoly chemie farmakologie chemická syntéza MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Interleukin-2-inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are two important members of the Tec family with crucial roles in immune system function. Deregulation in ITK and BTK activity is linked to several hematological malignancies, making them key targets for cancer immunotherapy. In this study, we synthesized a series of azaspirooxindolinone derivatives and evaluated their cytotoxic activity against ITK/BTK-negative and positive cancer cell lines, followed by enzymatic inhibition studies to assess the ITK/BTK kinase selectivity of two hit compounds. Several compounds demonstrated selective cytotoxicity against ITK- or BTK-expressing cells. Compound 3d exhibited high cytotoxicity in ITK-positive Jurkat (IC50 = 3.58 μM) and BTK-positive Ramos (IC50 = 3.06 μM) cells, while compound 3j showed strong cytotoxicity in Ramos (IC50 = 1.38 μM) and Jurkat (IC50 = 4.16 μM) cells. Compounds 3a and 3e were selectively cytotoxic in Jurkat cells (IC50 = 9.36 μM and 10.85 μM, respectively), while compounds 3f and 3g were highly cytotoxic in Ramos cells (IC50 = 1.82 μM and 1.42 μM, respectively). None of the active compounds exhibited cytotoxicity in non-cancer cell lines (IC50 > 50 μM), demonstrating their selectivity for malignant cells. Enzyme inhibition assay showed that 3d is a selective ITK inhibitor (IC50 = 0.91 μM) with no detectable BTK inhibition, aligning with its strong activity in ITK-positive cells. In contrast, compound 3j did not inhibit ITK or BTK enzymatically, suggesting an alternative mechanism of action. These findings highlight 3d as a promising ITK inhibitor and warrant further investigation to elucidate its mechanism of action.
- MeSH
- inhibitory proteinkinas * farmakologie chemická syntéza chemie MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- oxindoly farmakologie chemie chemická syntéza MeSH
- proliferace buněk účinky léků MeSH
- proteinkinasa BTK * antagonisté a inhibitory metabolismus MeSH
- protinádorové látky * farmakologie chemická syntéza chemie MeSH
- racionální návrh léčiv * MeSH
- screeningové testy protinádorových léčiv * MeSH
- simulace molekulového dockingu MeSH
- spirosloučeniny chemie farmakologie chemická syntéza MeSH
- tyrosinkinasy * antagonisté a inhibitory metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In this study, we investigated the stability of the fully activated conformation of the orexin receptor 2 (OX2R) embedded in a pure POPC bilayer using MD simulations. Various thermodynamic ensembles (i.e., NPT, NVT, NVE, NPAT, μVT, and NPγT) were employed to explore the dynamical heterogeneity of the system in a comprehensive way. In addition, informational similarity metrics (e.g., Jensen-Shannon divergence) as well as Markov state modeling approaches were utilized to elucidate the receptor kinetics. Special attention was paid to assessing surface tension within the simulation box, particularly under NPγT conditions, where 21 nominal surface tension constants were evaluated. Our findings suggest that traditional thermodynamic ensembles such as NPT may not adequately control physical properties of the POPC membrane, impacting the plausibility of the OX2R model. In general, the performed study underscores the importance of employing the NPγT ensemble for computational investigations of membrane-embedded receptors, as it effectively maintains zero surface tension in the simulated system. These results offer valuable insights for future research aimed at understanding receptor dynamics and designing targeted therapeutics.
