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To explore complex biological and chemical systems, pharmaceutical research has effectively included several molecular modeling tools into a range of drug development initiatives. Molecular docking methods are widely employed in current drug design to investigate ligand conformations within macromolecular targets' binding sites. This method also estimates the ligand-receptor binding free energy by assessing critical phenomena involved in the intermolecular recognition process. In an attempt, several natural products have been synthesized in our laboratory. All the synthesized compounds containing (6H-Dibenzo[b,d]pyran-6-one) framework were subjected to molecular docking studies for the inhibition of CYP1B1 and BCL2 proteins using Auto Dock Vina software and the interacting amino acid residues were visualized using Discovery Studio, to look into the binding modalities that might influence their anticancer properties. The in silico molecular docking study outcomes showed that all the synthesized compounds having optimum binding energy and have a decent affinity to the active pocket, thus, they may be considered as a respectable inhibitor of CYP1B1 and BCL2 proteins.

Heterogeneity in tumor expression as well as expression in normal tissues of various targets limit the usefulness of current ligand-based active targeting approaches. Incorporation of synthetic receptors, which can be recognized by delivery systems engineered to present specific functional groups on the surface, is a novel approach to improve tumor targeting. Alternatively, introduction of synthetic functionalities on cellular carriers can also enhance tumor targeting. We review various strategies that have been utilized for the introduction of synthetic targets in tumor tissues. The introduction of synthetic functional groups in the tumor through improved strategies is anticipated to result in improved target specificity and reduced heterogeneity in target expression.

Rationale: Obstructive sleep apnea is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep. The main goal of our study was to determine if designer receptors exclusively activated by designer drugs (DREADD) could be used to activate the genioglossus muscle as a potential novel treatment strategy for sleep apnea. We have previously shown that the prototypical DREADD ligand clozapine-N-oxide increased pharyngeal diameter in mice expressing DREADD in the hypoglossal nucleus. However, the need for direct brainstem viral injections and clozapine-N-oxide toxicity diminished translational potential of this approach, and breathing during sleep was not examined. Objectives: Here, we took advantage of our model of sleep-disordered breathing in diet-induced obese mice, retrograde properties of the adeno-associated virus serotype 9 (AAV9) viral vector, and the novel DREADD ligand J60. Methods: We administered AAV9-hSyn-hM3(Gq)-mCherry or control AAV9 into the genioglossus muscle of diet-induced obese mice and examined the effect of J60 on genioglossus activity, pharyngeal patency, and breathing during sleep. Measurements and Main Results: Compared with control, J60 increased genioglossus tonic activity by greater than sixfold and tongue uptake of 2-deoxy-2-[ 18 F]fluoro-d-glucose by 1.5-fold. J60 increased pharyngeal patency and relieved upper airway obstruction during non-REM sleep. Conclusions: We conclude that following intralingual administration of AAV9-DREADD, J60 can activate the genioglossus muscle and improve pharyngeal patency and breathing during sleep.

Background: Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown., Methods: We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice., Results: Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K + conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K + currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K + conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K + conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice., Conclusions: Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC., (Copyright © 2019 by the American Society of Nephrology.)

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of G q -mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that G q -mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple G q -coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the G q -coupled receptor 5-HT 2C R in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

As investigators, we use many methodologies to answer both practical and theoretical questions in our field. Occasionally, we must stop and collect the latest findings or trends and then look forward to where our ideas, findings, and hypotheses may take us. Similar to volumes that were published in previous years on drug discrimination (Glennon and Young, Drug discrimination applications to medicinal chemistry and drug studies. Wiley, Hoboken, 2011; Ho et al., Drug discrimination and state dependent learning. Academic Press, New York, 1978), this collection in Current Topics in Behavioral Neurosciences serves as a current analysis of the continued value of the drug discrimination procedure to the fields of pharmacology, neuroscience, and psychology and as a stepping stone to where drug discrimination methodology can be applied next, in both a practical and theoretical sense. This final chapter represents one investigator's perspective on the utility and possibilities for a methodology that she fell in love with over 30 years ago.

The identification of new molecular targets for diagnostic and therapeutic applications using in vitro methods is an important challenge in nuclear medicine. One such method is immunohistochemistry, increasingly popular because it is easy to perform. This review presents the case for conducting receptor immunohistochemistry to evaluate potential molecular targets in human tumor tissue sections. The focus is on the immunohistochemistry of G-protein-coupled receptors, one of the largest families of cell surface proteins, representing a major class of drug targets and thus playing an important role in nuclear medicine. This review identifies common pitfalls and challenges and provides guidelines on performing such immunohistochemical studies. An appropriate validation of the target is a prerequisite for developing robust and informative new molecular probes., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

To improve discovery of drugs for difficult targets, the opportunities of chemical space beyond the rule of 5 (bRo5) were examined by retrospective analysis of a comprehensive set of structures for complexes between drugs and clinical candidates and their targets. The analysis illustrates the potential of compounds far beyond rule of 5 space to modulate novel and difficult target classes that have large, flat, and groove-shaped binding sites. However, ligand efficiencies are significantly reduced for flat- and groove-shape binding sites, suggesting that adjustments of how to use such metrics are required. Ligands bRo5 appear to benefit from an appropriate balance between rigidity and flexibility to bind with sufficient affinity to their targets, with macrocycles and nonmacrocycles being found to have similar flexibility. However, macrocycles were more disk- and spherelike, which may contribute to their superior binding to flat sites, while rigidification of nonmacrocycles lead to rodlike ligands that bind well to groove-shaped binding sites. These insights should contribute to altering perceptions of what targets are considered "druggable" and provide support for drug design in beyond rule of 5 space.

Various thiosemicarbazones (TSCs) and their heterocyclic thiadiazolines (TDZ) possess important biological effects. In addition, chromenyl derivatives exhibit a wide range of pharmacological activities. Based on these findings and as a continuation of our research on nitrogen and sulfur containing compounds, we investigated a series of previously reported chromenyl-TSCs (1a-j) and chromenyl-TDZs (2a-j) for their in vitro antimicrobial activities against two bacterial and four fungal strains. MIC and MBC/MFC (µg/mL) values of these compounds were evaluated and compared to those of Spectinomycin, Moxifloxacin and Fluconazole, used as reference drugs. For a better understanding of the drug-receptor interactions, all the compounds were further subjected to molecular docking against four targets that were chosen based on the specific mechanism of action of the reference drugs used in the antimicrobial screening. All compounds tested showed equal or higher antibacterial/antifungal activities relative to the used reference drugs. In silico studies (molecular docking) revealed that all the investigated compounds showed good binding energies towards four receptor protein targets and supported their antimicrobial properties.

We investigated the susceptibility of 10 enterovirus D68 (EV-D68) isolates (belonging to clusters A, B, and C) to (entero)virus inhibitors with different mechanisms of action. The 3C-protease inhibitors proved to be more efficient than enviroxime and pleconaril, which in turn were more effective than vapendavir and pirodavir. Favipiravir proved to be a weak inhibitor. Resistance to pleconaril maps to V69A in the VP1 protein, and resistance to rupintrivir maps to V104I in the 3C protease. A structural explanation of why both substitutions may cause resistance is provided., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Receptor-based pharmacophore modeling is an efficient computer-aided drug design technique that uses the structure of the target protein to identify novel leads. However, most methods consider protein flexibility and desolvation effects in a very approximate way, which may limit their use in practice. The Site-Identification by Ligand Competitive Saturation (SILCS) assisted pharmacophore modeling protocol (SILCS-Pharm) was introduced recently to address these issues, as SILCS naturally takes both protein flexibility and desolvation effects into account by using full molecular dynamics simulations to determine 3D maps of the functional group-affinity patterns on a target receptor. In the present work, the SILCS-Pharm protocol is extended to use a wider range of probe molecules including benzene, propane, methanol, formamide, acetaldehyde, methylammonium, acetate and water. This approach removes the previous ambiguity brought by using water as both the hydrogen-bond donor and acceptor probe molecule. The new SILCS-Pharm protocol is shown to yield improved screening results, as compared to the previous approach based on three target proteins. Further validation of the new protocol using five additional protein targets showed improved screening compared to those using common docking methods, further indicating improvements brought by the explicit inclusion of additional feature types associated with the wider collection of probe molecules in the SILCS simulations. The advantage of using complementary features and volume constraints, based on exclusion maps of the protein defined from the SILCS simulations, is presented. In addition, reranking using SILCS-based ligand grid free energies is shown to enhance the diversity of identified ligands for the majority of targets. These results suggest that the SILCS-Pharm protocol will be of utility in rational drug design.

Alzheimer's disease is an age related disorder and is defined to be progressive, irreversible neurodegenerative disease. The potential targets which are associated with the Alzheimer's disease are cholinesterases, N-methyl-D-aspartate receptor, Beta secretase 1, Pregnane X receptor (PXR) and P-glycoprotein (Pgp). P-glycoprotein is a member of the ATP binding cassette (ABC) transporter family, which is an important integral of the blood-brain, blood-cerebrospinal fluid and the blood-testis barrier. Reports from the literature provide evidences that the up-regulation of the efflux pump is liable for a decrease in β -amyloid intracellular accumulation and is an important hallmark in Alzheimer's disease (AD). Thus, targeting β-amyloid clearance by stimulating Pgp could be a useful strategy to prevent Alzheimer's advancement. Currently available drugs provide limited effectiveness and do not assure to cure Alzheimer's disease completely. On the other hand, the current research is now directed towards the development of synthetic or natural based therapeutics which can delay the onset or progression of Alzheimer's disease. Since ancient time medicinal plants such as Withania somnifera, Bacopa monieri, Nerium indicum have been used to prevent neurological disorders including Alzheimer's disease. Till today around 125 Indian medicinal plants have been screened on the basis of ethnopharmacology for their activity against neurological disorders. In this paper, we report bioactives from natural sources which show binding affinity towards the Pgp receptor using ligand based pharmacophore development, virtual screening, molecular docking and molecular dynamics simulation studies for the bioactives possessing acceptable ADME properties. These bioactives can thus be useful to treat Alzheimer's disease.

Background: The mechanisms by which general anesthetics, including xenon and nitrous oxide, act are only beginning to be discovered. However, structural approaches revealed weak but specific protein-gas interactions., Methods: To improve knowledge, we performed x-ray crystallography studies under xenon and nitrous oxide pressure in a series of 10 binding sites within four proteins., Results: Whatever the pressure, we show (1) hydrophobicity of the gas binding sites has a screening effect on xenon and nitrous oxide binding, with a threshold value of 83% beyond which and below which xenon and nitrous oxide, respectively, binds to their sites preferentially compared to each other; (2) xenon and nitrous oxide occupancies are significantly correlated respectively to the product and the ratio of hydrophobicity by volume, indicating that hydrophobicity and volume are binding parameters that complement and oppose each other's effects; and (3) the ratio of occupancy of xenon to nitrous oxide is significantly correlated to hydrophobicity of their binding sites., Conclusions: These data demonstrate that xenon and nitrous oxide obey different binding mechanisms, a finding that argues against all unitary hypotheses of narcosis and anesthesia, and indicate that the Meyer-Overton rule of a high correlation between anesthetic potency and solubility in lipids of general anesthetics is often overinterpreted. This study provides evidence that the mechanisms of gas binding to proteins and therefore of general anesthesia should be considered as the result of a fully reversible interaction between a drug ligand and a receptor as this occurs in classical pharmacology.

Allosteric cooperativity, which nature uses to improve the sensitivity with which biomolecular receptors respond to small changes in ligand concentration, could likewise be of use in improving the responsiveness of artificial biosystems. Thus motivated, we demonstrate here the rational design of cooperative molecular beacons, a widely employed DNA sensor, using a generalizable population-shift approach in which we engineer receptors that equilibrate between a low-affinity state and a high-affinity state exposing two binding sites. Doing so we achieve cooperativity within error of ideal behavior, greatly steepening the beacon's binding curve relative to that of the parent receptor. The ability to rationally engineer cooperativity should prove useful in applications such as biosensors, synthetic biology and "smart" biomaterials, in which improved responsiveness is of value., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Thiazole is a well-known five-membered heterocyclic compound. Various methods have been worked out for its synthesis. In the last few decades, a lot of work has been done on the thiazole ring to find new drugs with antioxidant, analgesic, anti-inflammatory, antimicrobial, antifungal, antiviral, diuretic, anticonvulsant, neuroprotective and antitumor or cytotoxic properties and fewer side effects. This review presents the up-to-date development of different thiazole derivatives., Areas Covered: The present review gives an account of the recent therapeutic patent literature (2008 - 2012) describing the applications of thiazole and its derivatives on selected activities. In this review, many relevant biological properties and therapeutic applications of thiazole derivatives reported in international patents from all companies have been discussed; an overview of the chemical matter has also been given. Because of the huge amount of patents registered in this period relative to thiazole derivatives, attention has been focused on thiazole derivatives having pharmacological activity toward receptors., Expert Opinion: Based on the large variety of possible therapeutic applications proposed in patents for thiazole derivatives having pharmacological activity toward receptors, it is possible to point out the unpredictability of pharmacological activity consequent to structural modification, more or less simple, of a prototype drug molecule. In any case, the thiazole scaffold continues to have great potential in chemical pharmaceutical research.

Model-based analysis of routinely generated pharmacokinetic and pharmacodynamic (PK-PD) data is a key component of preclinical drug discovery. The work process of such analyses can be automated by properly designed computer programs that reduce the number of manual steps, resulting in time saving and significantly fewer errors. Critical decisions can still be made by modelers. Using concrete animal data examples this paper illustrates when, and demonstrates how, automated PK-PD approaches can be used and what benefits they offer to the modeling and simulation community. Specifically, we describe two compound optimization case studies from drug discovery projects, and also demonstrate how a subsequent optimization step to predict the human dose can be coupled to an automated approach.

Label-free cell-based assays offer a powerful approach to drug discovery and compound profiling for endogenously expressed receptors in a variety of cell types, including primary and stem cells. Dynamic mass redistribution (DMR) responses in whole cells following receptor stimulation provide phenotypic activity profiles that are readily amenable to evaluation of compound pharmacology. Protocols are provided in this unit to obtain DMR response profiles in adherent and suspension cells, and then to use known tool compounds to delineate the biology of the underlying signaling pathways from the information-rich kinetic traces that are recorded., (Copyright © 2014 John Wiley & Sons, Inc.)

The sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channel is a nonselective cation channel that is regulated by intracellular calcium and adenosine triphosphate. The channel is not constitutively expressed, but is transcriptionally upregulated de novo in all cells of the neurovascular unit, in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). The channel is linked to microvascular dysfunction that manifests as edema formation and delayed secondary hemorrhage. Also implicated in oncotic cell swelling and oncotic (necrotic) cell death, the channel is a major molecular mechanism of 'accidental necrotic cell death' in the CNS. In animal models of SCI, pharmacological inhibition of Sur1 by glibenclamide, as well as gene suppression of Abcc8, prevents delayed capillary fragmentation and tissue necrosis. In models of stroke and TBI, glibenclamide ameliorates edema, secondary hemorrhage, and tissue damage. In a model of SAH, glibenclamide attenuates the inflammatory response due to extravasated blood. Clinical trials of an intravenous formulation of glibenclamide in TBI and stroke underscore the importance of recent advances in understanding the role of the Sur1-regulated NC(Ca-ATP) channel in acute ischemic, traumatic, and inflammatory injury to the CNS.

Aim: Insulin secretagogues promote insulin release by binding to sulfonylurea receptors on pancreatic β-cells (SUR1). However, these drugs also bind to receptor isoforms on cardiac myocytes (SUR2A) and vascular smooth muscle (SUR2B). Binding to SUR2A/SUR2B may inhibit ischaemic preconditioning, an endogenous protective mechanism enabling cardiac tissue to survive periods of ischaemia. This study was designed to identify insulin secretagogues that selectively bind to SUR1 when given at therapeutic doses., Methods: Using accepted systematic review methods, three electronic databases were searched from inception to 13 June 2011. Original studies measuring the half-maximal inhibitory concentration (IC(50)) for an insulin secretagogue on K(ATP) channels using standard electrophysiological techniques were included. Steady-state concentrations (C(SS)) were estimated from the usual oral dose and clearance values for each drug., Results: Data were extracted from 27 studies meeting all inclusion criteria. IC(50) values for SUR1 were below those for SUR2A/SUR2B for all insulin secretagogues and addition of C(SS) values identified three distinct patterns. The C(SS) for gliclazide, glipizide, mitiglinide and nateglinide lie between IC(50) values for SUR1 and SUR2A/SUR2B, suggesting that these drugs bind selectively to pancreatic receptors. The C(SS) for glimepiride and glyburide (glibenclamide) was above IC(50) values for all three isoforms, suggesting these drugs are non-selective. Tolbutamide and repaglinide may have partial pancreatic receptor selectivity because IC(50) values for SUR1 and SUR2A/SUR2B overlapped somewhat, with the C(SS) in the midst of these values., Conclusions: Insulin secretagogues display different tissue selectivity characteristics at therapeutic doses. This may translate into different levels of cardiovascular risk., (© 2011 Blackwell Publishing Ltd.)

It is well known that sulphonylureas (SUs), commonly used in the treatment of type 2 diabetes mellitus, stimulate insulin secretion by closing ATP-sensitive K(+) (K(ATP) ) channels in pancreatic β-cells by binding to the SU receptor SUR1. SUs are now known also to activate cAMP sensor Epac2 (cAMP-GEFII) to Rap1 signalling, which promotes insulin granule exocytosis. For SUs to exert their full effects in insulin secretion, they are required to activate Epac2 as well as to inhibit the β-cell K(ATP) channels. As Epac2 is also necessary for potentiation of glucose-induced insulin secretion by cAMP-increasing agents, such as incretin, Epac2 is a target of both cAMP and SUs. The distinct effects of various SUs appear to be because of their different actions on Epac2/Rap1 signalling as well as K(ATP) channels. Differently from other SUs, gliclazide is unique in that it is specific for β-cell K(ATP) channel and does not activate Epac2., (© 2011 Blackwell Publishing Ltd.)

Type 2 diabetes is making up to 90% of the all diabetic cases. In addition to insulin resistance, insufficient B-cell function also plays an important role in the pathogenesis of the disease. The insufficient production and secretion of insulin can be increased by secretagogue drugs, like sulfonylureas and incretin mimetics/enhancers. In recent years growing number of genetic failures of the B-cells has been detected. These genetic variants can influence the efficacy of secretagogue drugs. Some of these gene polymorphisms were identified in the genes encoding the KATP channel (KCNJ11 and ABCC8). These mutations are able either to reduce or increase the insulin secretion and can modify the insulin response to sulfonylurea treatment. Other polymorphisms were found on genes encoding enzymes or transcription factors. In recent years, the genetic variants of TCF7L2 and its clinical importance have been intensely studied. Authors give a summary of the above gene polymorphisms and their role in insulin secretion.

There is a huge void in the current pharmacological treatment options for obesity. This gap is surprising given the high prevalence and associated costs of obesity. Many factors have prevented active drug development, including the poor safety and efficacy of earlier antiobesity drugs. However, there are now several compelling targets on the horizon. The new generation of antiobesity drugs offers hope for the management of obesity, but no single agent is likely to be a panacea. Rather, obesity will need to be managed like many other chronic diseases, with combination therapies and long-term treatment in order to achieve sustained success. New targets have arisen as more research has been performed to understand the complex circuitry that controls energy homeostasis. The goal of this review is to discuss the latest pharmacological agents and strategies that are under development and that may eventually be used for the treatment of obesity.

The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono- and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.

The crucial event in the pharmacological action of drugs is the interaction between a drug molecule and its receptor. Molecular recognition and binding affinity of a drug is driven by a flexible steric fit and a molecular field match between two complementary molecular surfaces of the receptor and the approaching ligand. Rational drug design methods, which attempt to predict the free energy change resulting from ligand binding, rely on a detailed knowledge of the physical forces that govern the drug-receptor interactions in several populated configurations of the intermolecular complex. The overall strength of the interaction is determined by the fine balance between the forces contributed by the individual chemical function groups of the two entities and the physiological medium. This paper recounts some basic information about the description of molecular structures and highlights the methods and approximations commonly used to calculate the binding affinity of a drug to its receptor.

The practice of "evidence-based medicine" aims at the conscientious, explicit and judicious use of the current best evidence in making decisions about the individualised patient care. The clinical genetics evolved from translational genetics research and contributes to the clinical care of patients and families through evidence-based health care in managing inherited disorders through accurate diagnosis, molecular pathology and assessing phenotypic correlations. Translational genetics and genomics research has led to the development of powerful tools for clinical diagnosis, assessing individual's genomic profile for disease prediction/prevention, high-throughput genome-wide screening for predisposition and/or protection to complex medical conditions, and discovery and development of new drugs and vaccines. Gene mapping and deciphering pathogenic mutations have helped in unravelling the basic biological mechanisms leading to new drug discovery and development. Targeted pharmacotherapy is now possible for managing the highly penetrant multi-system dominantly inherited conditions. Notable examples include rapamycin (sirolimus) in suppressing the mTOR pathway associated hamartomas in dominantly inherited cancer family syndromes and angiotensin converting enzyme receptor blockers (ACE-RB) in preventing aortic dilatation in Marfan syndrome and related familial arteriopathies. The translational genomic research is the essential prerequisite for developing sound evidence-based diagnostic, therapeutic and prognostic clinical protocols for the practice of personalised clinical medicine.

Glucokinase Glucokinase (GK GK ; EC 2.7.1.1.) phosphorylates and regulates glucose metabolism in insulin-producing pancreatic beta-cells, hepatocytes, and certain cells of the endocrine and nervous systems allowing it to play a central role in glucose homeostasis glucose homeostasis . Most importantly, it serves as glucose sensor glucose sensor in pancreatic beta-cells mediating glucose-stimulated insulin biosynthesis and release and it governs the capacity of the liver to convert glucose to glycogen. Activating and inactivating mutations of the glucokinase gene cause autosomal dominant hyperinsulinemic hypoglycemia and hypoinsulinemic hyperglycemia in humans, respectively, illustrating the preeminent role of glucokinase in the regulation of blood glucose and also identifying the enzyme as a potential target for developing antidiabetic drugs antidiabetic drugs . Small molecules called glucokinase activators (GKAs) glucokinase activators (GKAs) which bind to an allosteric activator allosteric activator site of the enzyme have indeed been discovered and hold great promise as new antidiabetic agents. GKAs increase the enzyme's affinity for glucose and also its maximal catalytic rate. Consequently, they stimulate insulin biosynthesis and secretion, enhance hepatic glucose uptake, and augment glucose metabolism and related processes in other glucokinase-expressing cells. Manifestations of these effects, most prominently a lowering of blood glucose, are observed in normal laboratory animals and man but also in animal models of diabetes and patients with type 2 diabetes mellitus (T2DM T2DM ) type 2 diabetes mellitus (T2DM) . These compelling concepts and results sustain a strong R&D effort by many pharmaceutical companies to generate GKAs with characteristics allowing for a novel drug treatment of T2DM.

Background: ATP-sensitive K(+) (K(ATP)) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The K(ATP) channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express K(ATP) channels., Methods: Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for K(ATP) channel subunits was determined using real-time RT-PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional K(ATP) channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used., Results: Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate K(ATP) channels, increased whole cell K(+) current. On the other hand, K(+) current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of K(ATP) channels., Conclusions: In conclusion, mammalian oocytes express K(ATP) channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception.

Objective: To incorporate structural biology, enzyme kinetics, and visualization of protein structures in a medicinal chemistry course to teach fundamental concepts of drug design and principles of drug action., Design: Pedagogy for active learning was incorporated via hands-on experience with visualization software for drug-receptor interactions and concurrent laboratory sessions. Learning methods included use of clicker technology, in-class assignments, and analogies., Assessment: Quizzes and tests that included multiple-choice and open-ended items based on Bloom's taxonomy were used to assess learning. Student feedback, classroom exercises, and tests were used to assess teaching methods and effectiveness in meeting learning outcomes., Conclusion: The addition of active-learning activities increased students' understanding of fundamental medicinal chemistry concepts such as ionization state of molecules, enzyme kinetics, and the significance of protein structure in drug design.

Gamma-hydroxybutyrate is found both naturally in the brain and self-administered as a drug of abuse. It has been reported to act at endogenous γ-hydroxybutyrate (GHB) receptors and GABA(B) receptors [GABA(B)R], and may also be metabolized to GABA. Here, the metabolic fingerprints of a range of concentrations of GHB were measured in brain cortical tissue slices and compared with those of ligands active at GHB and GABA-R using principal components analysis (PCA) to identify sites of GHB activity. Low concentrations of GHB (1.0 μM) produced fingerprints similar to those of ligands active at GHB receptors and α4-containing GABA(A)R. A total of 10 μM GHB clustered proximate to mainstream GABAergic synapse ligands, such as 1.0 μM baclofen, a GABA(B)R agonist. Higher concentrations of GHB (30 μM) clustered with GABA(C)R agonists and the metabolic responses induced by blockade of the GABA transporter-1 (GAT1). The metabolic responses induced by 60 and 100 μM GHB were mimicked by simultaneous blockade of GAT1 and GAT3, addition of low concentrations of GABA(C)R antagonists, or increasing cytoplasmic GABA concentrations by incubation with the GABA transaminase inhibitor vigabatrin. These data suggest that at concentrations > 30 μM, GHB may be active via metabolism to GABA, which is then acting upon an unidentified GABAergic master switch receptor (possibly a high-affinity extrasynaptic receptor), or GHB may itself be acting directly on an extrasynaptic GABA-R, capable of turning off large numbers of cells. These results offer an explanation for the steep dose-response curve of GHB seen in vivo, and suggest potential target receptors for further investigation., (© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.)

The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the distal convoluted tubule. Recently, we described a novel pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl-releasing protein 1 (RasGRP1), triggering a cascade ultimately activating ERK1/2 MAPK and decreasing NCC cell surface expression (Ko B, Joshi LM, Cooke LL, Vazquez N, Musch MW, Hebert SC, Gamba G, Hoover RS. Proc Natl Acad Sci USA 104: 20120-20125, 2007). Little is known about the mechanisms which underlie these effects on NCC activity. Regulation of NCC via changes in NCC surface expression has been reported, but endocytosis of NCC has not been demonstrated. In this study, utilizing biotinylation, internalization assays, and a dynamin dominant-negative construct, we demonstrate that the regulation of NCC by PE occurs via an enhancement in internalization of NCC and is dynamin dependent. In addition, immunoprecipitation of NCC and subsequent immunoblotting for ubiquitin showed increased ubiquitination of NCC with phorbol ester treatment. MEK1/2 inhibitors and gene silencing of RasGRP1 indicated that this effect was dependent on RasGRP1 and ERK1/2 activation. Inhibition of ubiquitination prevents any PE-mediated decrease in NCC surface expression as measured by biotinylation or NCC activity as measured by radiotracer uptake. These findings confirmed that the PE effect on NCC is mediated by endocytosis of NCC. Furthermore, ubiquitination of NCC is essential for this process and this ubiquitination is dependent upon RasGRP1-mediated ERK1/2 activation.

Systems biology provides a platform for integrating multiple components and interactions underlying cell, organ, and organism processes in health and disease. Beyond traditional approaches focused on individual molecules or pathways, bioinformatic network analysis of high-throughput data sets offers an opportunity for integration of biological complexity and multilevel connectivity. Emerging applications in rational drug discovery range from targeting and modeling disease-corrupted networks to screening chemical or ligand libraries to identification/validation of drug-target interactions for improved efficacy and safety.

Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na+-Cl- cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11beta-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V(2) receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT.

Introduction: It has become apparent in recent years that there is a need for a substantial improvement in the processes used to clarify the inter-relationships between a compound's pharmacokinetic (PK) characteristics, its target actions and its safety profile. This is particularly challenging because most of the techniques currently used to obtain meaningful data involve low efficiency animal studies. To maximize the value of such in vivo studies, time and resources need to be better utilized. One way of making major advances is to adopt an integrative approach to designing and analysing in vivo pharmacodynamic (PD) studies. In this paper we address experimental design issues related to maximizing the information content about target engagement. Refined integration of PK and PD (known as 'quantitative pharmacology' or a PKPD approach) is advocated, enabling clarification of the interdependence of a drug's pharmacological properties on its target physiological system(s) and its systemic exposure characteristics., Methods: Several in vivo pharmacological Case Studies are presented which are used to illustrate the discussion of issues of experimental design. The impact of rate, extent and mode of administration is discussed from a pharmacological viewpoint, and we examine the consequences of temporal differences between concentration and response from the perspective of experimental design. We also consider the importance of differences in plasma protein binding (PPB) on our ability to assess a pharmacodynamic property or safety margin, and examine the usefulness of measuring ex vivo PPB. Finally, we outline the value of implementing a clearly pre-determined, integrated work flow to generate, validate and maximize in vivo PKPD or preclinical disease model data, and summarize the key issues of experimental design., Results and Discussion: Quantitative pharmacological reasoning focuses on concentration-response and response-time relationships with special emphasis on the impact of drugs on disease (Levy, 1993) and this perspective was written to raise awareness among in vivo scientists as to why PKPD integration is essential in their studies., (Copyright 2010 Elsevier Inc. All rights reserved.)

Neuropeptides play essential roles in many physiological systems in vertebrates and invertebrates. Peptides per se are difficult to use as therapeutic agents, as they are generally very unstable in biological fluid environments and cross biological membranes poorly. Recognition that nonpeptide ligands for peptide receptors have clinical utility came from the discovery that opiates (such as morphine) act by binding to G protein-coupled receptors (GPCRs) for which the endogenous ligands are a family of neuropeptides (enkephalins and endorphins). Basic research has revealed a very large number of distinct neuropeptides that influence virtually every aspect of mammalian physiology and considerable effort has been expended in the pursuit of new drugs that act through peptidergic signaling systems. Although useful drugs have been found to affect various aspects ofneuropeptide biology, most work has been devoted to the discovery of nonpeptide ligands that act as agonists or antagonists at peptidergic GPCRs. Similar opportunities are apparent for the discovery of nonpeptide ligands that act on invertebrate GPCRs. A consideration of the knowledge gained from the process as conducted for mammalian peptidergic systems can inform and illuminate promising strategies for the discovery of new drugs for the treatment and control of pests and parasites.

Analyses of polymorphisms in the renin-angiotensin system may never completely predict responsiveness to pharmacotherapy that blocks angiotensin-converting enzyme or the angiotensin receptor type 1. However, the pharmacogenetic studies that have been conducted to date are intriguing, and they illustrate the potential benefit of designing larger genome-wide clinical studies. Such studies will continue to define the role of renin-angiotensin pharmacogenetics in patients with heart failure due to underlying cardiac dysfunction.

Insect kinin neuropeptides share a common C-terminal pentapeptide sequence Phe1-Xaa1(2)-Xaa2(3)-Trp4-Gly5-NH2 (Xaa1(2) = His, Asn, Phe, Ser or Tyr; Xaa2(3) = Pro, Ser or Ala) and have been isolated from a number of insects, including species of Dictyoptera, Orthoptera and Lepidoptera. They have been associated with the regulation of such diverse processes as hindgut contraction, diuresis and the release of digestive enzymes. In this chapter, the chemical, conformational and stereochemical aspects of the activity ofthe insect kinins with expressed receptors and/or biological assays are reviewed. With this information, biostable analogs are designed that protect peptidase-susceptible sites in the insect kinin sequence and demonstrate significant retention of activity on both receptor and biological assays. The identification of the most critical residue of the insect kinins for receptor interaction is used to select a scaffold for a recombinant library that leads to identification ofa nonpeptide mimetic analog. C-terminal aldehyde insect kinin analogs modify the activity of the insect kinins leading to inhibition of weight gain and mortality in corn earworm larvae and selective inhibition ofdiuresis in the housefly. Strategies for the modification of insect neuropeptide structures for the enhancement ofthe topical and oral bioavailability of insect neuropeptides and the promotion of time-release from the cuticle and/or foregut are reviewed. Promising mimetic analog leads for the development of selective agents capable of disrupting insect kinin regulated processes are identified that may provide interesting tools for arthropod endocrinologists and new pest insect management strategies in the future.

Drug discovery is an iterative process with high risks and low chance of success. New genomics technologies allow veterinary medicine and agrochemical companies to validate and functionally screen new receptor-based targets, including neuropeptide G-protein coupled receptors, which were previously not amenable to high throughput screening. However this is just the first step in a long process to translate a mechanistic assay hit into a drug on the market. In addition to effectively eradicating pests on crops and parasites on their host, the molecules must also be safe, cheap to synthesise, formulatable and patentable. This is a costly process in which early attrition of unsuitable molecules is key to any successful program. Although first principle discovery is risky the ultimate benefits are considerable and future genomics resources will help to generate higher quality hits to strengthen the discovery pipeline.

There is no one ligand for visualising "the epileptogenic zone". Several PET ligands, however, can help by noninvasively or minimally invasively refining hypotheses regarding its location. Their relative merits depend not only on local availability and expertise, but also on epilepsy syndrome and overall diagnostic category. FDG, flumazenil (FMZ), alpha-methyl-tryptophan (AMT), the 5-HT1A ligands, and diprenorphine are discussed.

Target-mediated drug disposition (TMDD) is frequently reported for therapeutic monoclonal antibodies and is linked to the high affinity and high specificity of antibody molecules for their target. Understanding TMDD of a monoclonal antibody should go beyond the empirical description of its non-linear PK since valuable insights on the antibody-target interaction itself can be gained. This makes its mechanistic understanding precious for the drug development process, in particular for the optimization of new antibody molecules, for the design and interpretation of pharmacokinetic studies, and possibly even for the evaluation of efficacy and dose selection of drug candidates. Using the observation that the molecular (microscopic) processes are usually much more rapid than the pharmacokinetic (macroscopic) processes, a series of quasi-steady-state conditions on the microscopic level is proposed to bridge the gap between simple empirical and complex mechanistic descriptions of TMDD. These considerations show the impact of parameters such as target turnover, target expression, and target accessibility on the pharmacokinetics and pharmacodynamics of monoclonal antibodies.

During the process of drug discovery, INPHARMA can be used to derive the structure of receptor/lead compound complexes binding to each other with a K(d) in the microM to mM range. To be successful, the methodology needs adjustment of various parameters that depend on the physical constants of the binding event and on the receptor size. Here we present a thorough theoretical analysis of the INPHARMA interligand NOE effect in dependence of experimental parameters and physical constants. This analysis helps the experimentalist to choose the correct experimental parameters and consequentially to achieve optimal performance of the methodology.

Cadmium (Cd) induces necrotic death in Chinese hamster ovary (CHO) K1 cells and we have established the responsible signaling pathway. Reportedly, necrostatin-1 (Nec-1) rescues cells from necrotic death by mediating through the death domain receptor (DR) signaling pathway. We show here that Nec-1 also effectively attenuates necrotic death triggered by Cd. Two other treatments that cause necrotic cell death, one can (z-VAD-fmk/TNF-alpha on U937 cells) and the other cannot (etherynic acid (EA) on DLD-1 cells) be rescued by Nec-1, were also studied in parallel for comparison. Results show that Nec-1 is ineffectual in modulating intracellular calcium contents, calpain activity (a downstream protease), or reactive oxygen species production. It can counteract the reduction in mitochondrial membrane potential (MMP) caused by treating CHO K1 or U937 cells with necrosis-inducing agent. However, this effect was not found in EA-treated DLD-1 cells. Notably, Nec-1 elevates NF-kappaB activity in the presence or absence of necrosis-inducing agents. Our study shows that, in addition to DR-mediated necrosis, Nec-1 is effective in attenuating Cd-induced necrosis. It rescues cells with reduced MMP implying that mitochondrion is its major acting site.

Activation of the large-conductance Ca(2+)-activated K(+) channel (BK) in the cardiac inner mitochondrial membrane has been suggested to protect the heart against ischemic injury. However, these findings are limited by the low selectivity profile and potency of the BK channel activator (NS1619) used. In the present study, we address the cardioprotective role of BK channels using a novel, potent, selective, and chemically unrelated BK channel activator, NS11021. Using electrophysiological recordings of heterologously expressed channels, NS11021 was found to activate BK alpha + beta1 channel complexes, while producing no effect on cardiac K(ATP) channels. The cardioprotective effects of NS11021-induced BK channel activation were studied in isolated, perfused rat hearts subjected to 35 min of global ischemia followed by 120 min of reperfusion. 3 microM NS11021 applied prior to ischemia or at the onset of reperfusion significantly reduced the infarct size [control: 44.6 +/- 2.0%; NS11021: 11.4 +/- 2.0%; NS11021 at reperfusion: 19.8 +/- 3.3% (p < 0.001 for both treatments compared to control)] and promoted recovery of myocardial performance. Co-administration of the BK-channel inhibitor paxilline (3 microM) antagonized the protective effect. These findings suggest that tissue damage induced by ischemia and reperfusion can be reduced by activation of cardiac BK channels.

K(ATP) channels are generally cardioprotective under conditions of metabolic impairment, consisting of pore-forming (Kir6.1 and/or Kir6.2) and sulphonylurea-binding, modulatory subunits [sulfonylurea receptor (SUR) 1, 2A, or 2B]. Cardiovascular K(ATP) channels are generally thought to consist of Kir6.2/SUR2A subunits (in the case of heart muscle) or Kir6.1/SUR2B subunits (smooth muscle), whereas SUR1-containing channels have well-documented roles in pancreatic insulin release. Recent data, however, demonstrated the presence of SUR1 subunits in mouse cardiac tissue (particularly in atria) and a surprising protection from myocardial ischemia/reperfusion in SUR1-null mice. Here, we review some of the extra-pancreatic roles assigned to SUR1 subunits and consider whether these might be involved in the sequelae of ischemia/reperfusion.

Although stimulants as the treatment of choice are widely prescribed in ADHD, little is known about their long-term neurobiological effects. Hence, for the first time the present study examined the long-term effects of chronic methylphenidate (MPH) administration on striatal 5-hydroxytryptamine transporter (5-HTT) densities in an animal model of ADHD. First, it compared the normal development of striatal 5-HTT densities of spontaneously hypertensive rats (SHR) as an animal model of ADHD and Wistar Kyoto (WKY) rats as controls; binding of the highly selective ligand of 5-HTT [(3)H]paroxetine was determined on membrane preparations of the striatum of SHR and WKY rats on postnatal days 25, 50, and 90, i.e. from the time of weaning until adulthood. Second, the long-term effect of chronic administration of 2 mg/kg per day MPH at two different developmental stages (days 25-39 or 50-64) on the striatal 5-HTT density was examined in both rat strains at day 90. Long-term effects of MPH treatment on striatal 5-HTT density in adulthood could be ruled out in both healthy (WKY) and "ADHD" rats (SHR). But a higher striatal 5-HTT density in older SHR versus WKY rats might indicate ADHD specific changes in the 5-HT system that needs further investigation not only in animals.