Since its early days in the 19th century, medicinal chemistry has concentrated its efforts on the treatment of diseases, using tools from areas such as chemistry, pharmacology, and molecular biology. The understanding of biological mechanisms and signaling pathways is crucial information for the development of potential agents for the treatment of diseases mainly because they are such complex processes. Given the limitations that the experimental approach presents, computational chemistry is a valuable alternative for the study of these systems and their behavior. Thus, classical molecular dynamics, based on Newton's laws, is considered a technique of great accuracy, when appropriated force fields are used, and provides satisfactory contributions to the scientific community. However, as many configurations are generated in a large MD simulation, methods such as Statistical Inefficiency and Optimal Wavelet Signal Compression Algorithm are great tools that can reduce the number of subsequent QM calculations. Accordingly, this review aims to briefly discuss the importance and relevance of medicinal chemistry allied to computational chemistry as well as to present a case study where, through a molecular dynamics simulation of AMPK protein (50 ns) and explicit solvent (TIP3P model), a minimum number of snapshots necessary to describe the oscillation profile of the protein behavior was proposed. For this purpose, the RMSD calculation, together with the sophisticated OWSCA method was used to propose the minimum number of snapshots.
- MeSH
- Algorithms MeSH
- Chemistry, Pharmaceutical MeSH
- Quantum Theory MeSH
- Humans MeSH
- AMP-Activated Protein Kinases metabolism chemistry MeSH
- Molecular Dynamics Simulation * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Pancreatic-β-cell-specifying transcription factor Nkx6.1, indispensable for embryonic development of the pancreatic epithelium and commitment to β-cell lineage, directly controls the expression of a glucose transporter (Glut2), pyruvate carboxylase (Pcx), and genes for insulin processing (endoplasmic reticulum oxidoreductase-1β, Ero1lb; zinc transporter-8, Slc30a8). The Nkx6.1 decline in aging diabetic Goto-Kakizaki rats contributes to β-cell trans-differentiation into δ-cells. Elucidating further Nkx6.1 roles, we studied Nkx6.1 ablation in rat INS-1E cells, prepared by CRISPR/Cas9 gene editing from single colonies. INS-1ENkx6.1-/- cells exhibited unchanged glucose-stimulated insulin secretion (GSIS), moderately decreased phosphorylating/non-phosphorylating respiration ratios at high glucose; unchanged but delayed ATP-elevation responses to glucose; delayed uptake of fluorescent glucose analog, but slightly improved cytosolic Ca2+-oscillations, induced by glucose; despite approximately halved Glut2, Pcx, Ero1lb, and Slc30a8 expression, and reduced nuclear receptors Nr4a1 and Nr4a3. Thus, ATP synthesis was time-compensated, despite the delayed GLUT2-mediated glucose uptake and crippled pyruvate-malate redox shuttle (owing to the PCX-deficiency) in INS-1ENkx6.1-/- cells. Nkx6.1 thus controls the expression of genes that are not essential for acute insulin secretion, the function of which can be compensated for. Considerations that Nkx6.1 deficiency is an ultimate determinant of β-cell pathology beyond cell trans-(de-)differentiation or β-cell identity are not supported by our results.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Insulin-Secreting Cells * metabolism MeSH
- Glucose metabolism MeSH
- Homeodomain Proteins * genetics metabolism MeSH
- Insulin * metabolism MeSH
- Rats MeSH
- Insulin Secretion MeSH
- Transcription Factors genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The pregnane X receptor (PXR) is a ligand-activated nuclear receptor controlling hepatocyte expression of numerous genes. Although expression changes in xenobiotic-metabolizing, lipogenic, gluconeogenic and bile acid synthetic genes have been described after PXR activation, the temporal dynamics of their expression is largely unknown. Recently, 3D spheroids of primary human hepatocytes (PHHs) have been characterized as the most phenotypically relevant hepatocyte model. We used 3D PHHs to assess time-dependent expression profiles of 12 prototypic PXR-controlled genes in the time course of 168 h of rifampicin treatment (1 or 10 μM). We observed a similar bell-shaped time-induction pattern for xenobiotic-handling genes (CYP3A4, CYP2C9, CYP2B6, and MDR1). However, we observed either biphasic profiles for genes involved in endogenous metabolism (FASN, GLUT2, G6PC, PCK1, and CYP7A1), a decrease for SHP or oscillation for PDK4 and PXR. The rifampicin concentration determined the expression profiles for some genes. Moreover, we calculated half-lives of CYP3A4 and CYP2C9 mRNA under induced or basal conditions and we used a mathematical model to describe PXR-mediated regulation of CYP3A4 expression employing 3D PHHs. The study shows the importance of long-term time-expression profiling of PXR target genes in phenotypically stable 3D PHHs and provides insight into PXR function in liver beyond our knowledge from conventional 2D in vitro models.
Significance: Mitochondria determine glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells by elevating ATP synthesis. As the metabolic and redox hub, mitochondria provide numerous links to the plasma membrane channels, insulin granule vesicles (IGVs), cell redox, NADH, NADPH, and Ca2+ homeostasis, all affecting insulin secretion. Recent Advances: Mitochondrial redox signaling was implicated in several modes of insulin secretion (branched-chain ketoacid [BCKA]-, fatty acid [FA]-stimulated). Mitochondrial Ca2+ influx was found to enhance GSIS, reflecting cytosolic Ca2+ oscillations induced by action potential spikes (intermittent opening of voltage-dependent Ca2+ and K+ channels) or the superimposed Ca2+ release from the endoplasmic reticulum (ER). The ATPase inhibitory factor 1 (IF1) was reported to tune the glucose sensitivity range for GSIS. Mitochondrial protein kinase A was implicated in preventing the IF1-mediated inhibition of the ATP synthase. Critical Issues: It is unknown how the redox signal spreads up to the plasma membrane and what its targets are, what the differences in metabolic, redox, NADH/NADPH, and Ca2+ signaling, and homeostasis are between the first and second GSIS phase, and whether mitochondria can replace ER in the amplification of IGV exocytosis. Future Directions: Metabolomics studies performed to distinguish between the mitochondrial matrix and cytosolic metabolites will elucidate further details. Identifying the targets of cell signaling into mitochondria and of mitochondrial retrograde metabolic and redox signals to the cell will uncover further molecular mechanisms for insulin secretion stimulated by glucose, BCKAs, and FAs, and the amplification of secretion by glucagon-like peptide (GLP-1) and metabotropic receptors. They will identify the distinction between the hub β-cells and their followers in intact and diabetic states. Antioxid. Redox Signal. 36, 920-952.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Insulin-Secreting Cells * metabolism MeSH
- Glucose metabolism MeSH
- Insulin metabolism MeSH
- Islets of Langerhans * metabolism MeSH
- Mitochondria metabolism MeSH
- NAD metabolism MeSH
- NADP metabolism MeSH
- Insulin Secretion MeSH
- Secretagogues metabolism MeSH
- Calcium metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- Phosphatidylcholines chemistry metabolism MeSH
- Ion Channel Gating genetics MeSH
- Genetic Vectors chemistry metabolism MeSH
- HEK293 Cells MeSH
- Protein Interaction Domains and Motifs MeSH
- Protein Conformation, alpha-Helical MeSH
- Protein Conformation, beta-Strand MeSH
- Humans MeSH
- Liposomes chemistry metabolism MeSH
- Luminescent Proteins genetics metabolism MeSH
- Patch-Clamp Techniques MeSH
- Mutation MeSH
- Neoplasm Proteins chemistry genetics metabolism MeSH
- ORAI1 Protein chemistry genetics metabolism MeSH
- Stromal Interaction Molecule 1 chemistry genetics metabolism MeSH
- Gene Expression Regulation MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Genes, Reporter MeSH
- Molecular Dynamics Simulation MeSH
- Amino Acid Substitution MeSH
- Calcium metabolism MeSH
- Calcium Signaling * MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The membrane cholesterol was found to bind and modulate the function of several G-protein coupled receptors including muscarinic acetylcholine receptors. We investigated the binding of 20 steroidal compounds including neurosteroids and steroid hormones to muscarinic receptors. Corticosterone, progesterone and some neurosteroids bound to muscarinic receptors with the affinity of 100 nM or greater. We established a structure-activity relationship for steroid-based allosteric modulators of muscarinic receptors. Further, we show that corticosterone and progesterone allosterically modulate the functional response of muscarinic receptors to acetylcholine at physiologically relevant concentrations. It can play a role in stress control or in pregnancy, conditions where levels of these hormones dramatically oscillate. Allosteric modulation of muscarinic receptors via the cholesterol-binding site represents a new pharmacological approach at diseases associated with altered cholinergic signalling.
- MeSH
- Acetylcholine metabolism MeSH
- Allosteric Regulation MeSH
- Adrenal Cortex Hormones metabolism MeSH
- Corticosterone metabolism MeSH
- Cricetinae MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Neurosteroids metabolism MeSH
- Gonadal Steroid Hormones metabolism MeSH
- Progesterone metabolism MeSH
- Receptors, Muscarinic metabolism MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Lysosomal sequestration of weak base drugs has been identified as one of the stress-related mechanisms that trigger in vitro lysosomal biogenesis controlled by transcription factor EB (TFEB). Whether such mechanism can induce lysosomal biogenesis in vivo is unknown. In this study, we addressed the question whether prolonged treatment with sunitinib (SUN) in patients with advanced renal cell carcinoma (n = 22) and with imatinib (IM) in those with gastrointestinal stromal tumor (n = 6) could induce lysosomal biogenesis in leukocytes. Lysosomal biogenesis was monitored using immunoblotting of three lysosomal membrane proteins: lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) and vacuolar H+-ATPase, B2 subunit (ATP6V1B2). Present results indicate that prolonged treatment with SUN affects LAMP1 and LAMP2 expression only marginally in most patients. In contrast, changes in ATP6V1B2 expression were marked and resembled irregular oscillations. Very similar changes in the expression of lysosomal membrane proteins were also found in IM-treated patients. Conclusion: prolonged treatment of cancer patients with SUN and IM did not induce leucocyte lysosomal biogenesis but dramatically affected expression of ATP6V1B2.
- MeSH
- Gastrointestinal Stromal Tumors drug therapy metabolism MeSH
- Imatinib Mesylate therapeutic use MeSH
- Protein Kinase Inhibitors therapeutic use MeSH
- Carcinoma, Renal Cell drug therapy metabolism MeSH
- Leukocytes metabolism MeSH
- Humans MeSH
- Lysosomes metabolism MeSH
- Lysosomal Membrane Proteins metabolism MeSH
- Sunitinib therapeutic use MeSH
- Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Although deep brain stimulation of the subthalamic nucleus (STN-DBS) in Parkinson's disease (PD) is generally a successful therapy, adverse events and insufficient clinical effect can complicate the treatment in some patients. We studied clinical parameters and cortical oscillations related to STN-DBS to identify patients with suboptimal responses. High-density EEG was recorded during a visual oddball three-stimuli paradigm in DBS "off" and "on" conditions in 32 PD patients with STN-DBS. Pre-processed data were reconstructed into the source space and the time-frequency analysis was evaluated. We identified a subgroup of six patients with longer reaction times (RT) during the DBS "on" state than in the DBS "off" state after target stimuli. These subjects had lower motor responsiveness to DBS and decreased memory test results compared to the other subjects. Moreover, the alpha and beta power decrease (event-related desynchronizations, ERD), known as an activation correlate linked to motor and cognitive processing, was also reduced in the DBS "on" condition in these patients. A subgroup of PD patients with a suboptimal response to STN-DBS was identified. Evaluation of RT could potentially serve as a biomarker for responsiveness to STN-DBS.
- MeSH
- Deep Brain Stimulation * MeSH
- Cognition MeSH
- Humans MeSH
- Subthalamic Nucleus * MeSH
- Parkinson Disease * therapy MeSH
- Reaction Time MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of β-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, β-cell-specific knockout mice (NOX4βKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4βKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause β-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.
- MeSH
- Potassium Channels physiology MeSH
- Glucose pharmacology MeSH
- Insulin Resistance MeSH
- Cells, Cultured MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- NADPH Oxidase 4 physiology MeSH
- Hydrogen Peroxide metabolism MeSH
- Insulin Secretion * MeSH
- Signal Transduction physiology MeSH
- Calcium metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Clinical symptoms of Parkinson's disease (PD) are accompanied by pathological phenomena detected locally in the basal ganglia (BG) as changes in local field potentials (LFPs) and also in cortical regions by electroencephalography (EEG). The literature published mainly between 2000 and 2017 was reviewed with an emphasis on approaches emerging after 2000, in particular on oscillatory dynamics, connectivity studies, and deep brain stimulation. Eighty-five articles were reviewed. The main observations were a general slowing of background activity, excessive synchronization of beta activity, and disturbed movement-related gamma oscillations in the BG and in the cortico-subcortical and cortico-cortical motor loops, suppressible by dopaminergic medication as well as by high-frequency deep brain stimulation (DBS). Non-motor symptoms are related mainly to changes in the alpha frequency range. EEG parameters can be useful in defining the risk of dementia in PD. Further progress was reported recently using advanced analytical technologies and high-performance computing (graph theory). Detailed knowledge of LFPs in PD enabled progress particularly in DBS therapy, which requires optimizing the clinical effect and minimizing adverse side effects. The neurocognitive networks and their dysfunction in PD and DBS therapy are promising targets for future research.
- MeSH
- Electroencephalography methods MeSH
- Deep Brain Stimulation methods MeSH
- Humans MeSH
- Brain physiopathology MeSH
- Nerve Net physiopathology MeSH
- Parkinson Disease diagnosis physiopathology therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
