Anti-cancer potential of natural products containing (6H-dibenzo[b,d]pyran-6-one) framework using docking tools.

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.

Synthetic Receptor-Based Targeting Strategies to Improve Tumor Drug Delivery.

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.

Designer Receptors Exclusively Activated by Designer Drugs Approach to Treatment of Sleep-disordered Breathing.

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-[18F]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.

In Vitro Evaluation of Molecular Tumor Targets in Nuclear Medicine: Immunohistochemistry Is One Option, but Under Which Conditions?

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.

Limits and possibilities experienced by nurses in the treatment of women with chronic venous ulcers / Límites y posibilidades vividas por enfermeras en el tratamiento de mujeres con úlcera venosa crónica / Limites e possibilidades vivenciados por enfermeiras no tratamento de mulheres com úlcera venosa crônica

Objective To understand the experiences and expectations of nurses in the treatment of women with chronic venous ulcers. Method Phenomenological research was based on Alfred Schütz, whose statements were obtained in January, 2012, through semi-structured interviews with seven nurses. Results The nurse reveals the difficulties presented by the woman in performing self-care, the perceived limitations in the treatment anchored in motivation, and the values and beliefs of women. It showed professional frustration because venous leg ulcer recurrence, lack of inputs, interdisciplinary work and training of nursing staff. There was an expected adherence to the treatment of women, and it emphasized the need for ongoing care, supported self-care and standard practices in treatment. Conclusion That treatment of chronic venous leg ulcers constitutes a challenge that requires collective investment, involving women, professionals, managers and health institutions. .

Objetivo Comprender las experiencias y expectativas de enfermeras en el tratamiento de mujeres con úlcera venosa crónica. Método Investigación fenomenológica fundamentada en Alfred Schutz, que buscó Se realizó entrevista semiestructurada con siete enfermeras, en enero del 2012. Resultados La enfermera revela dificultades presentadas por la mujer para realizar el autocuidado, percibe limitaciones en el tratamiento relacionadas con la desmotivación, los valores y las creencias de las mujeres. Refiere frustración profesional debido a la recidiva de la lesión, a la falta de insumos, al deficiente trabajo interdisciplinar y a la limitada capacitación del equipo de enfermeras. Espera la adhesión de la mujer al tratamiento y resalta la necesidad del cuidado continuo, del autocuidado apoyado y de estandarizar conductas de tratamiento. Conclusión El tratamiento de la úlcera venosa crónica es un desafío que requiere contribución colectiva, involucrando a las mujeres, a los profesionales, a los gestores y a las instituciones de salud. .

Objetivo Compreender as experiências e expectativas de enfermeiras no tratamento de mulheres com úlcera venosa crônica na Atenção Primária à Saúde. Método Pesquisa fundamentada na fenomenologia social de Alfred Schütz, com depoimentos obtidos em janeiro de 2012, por meio de entrevista semiestruturada com sete enfermeiras. Resultados As enfermeiras revelam dificuldades apresentadas pelas mulheres com úlcera venosa crônica para realizar o autocuidado, percebem limitações na terapêutica ancoradas na desmotivação e nos valores e crenças das mulheres. Referem frustração profissional em razão da recidiva da lesão, falta de insumos e tecnologia, de trabalho interdisciplinar e da capacitação da equipe de enfermagem. Esperam a adesão das mulheres ao tratamento e ressaltam a necessidade do cuidado contínuo, do autocuidado apoiado e da padronização de condutas no tratamento. Conclusão O tratamento da úlcera venosa crônica constitui-se em um desafio que requer investimento coletivo, envolvendo a mulher, os profissionais, os gestores e as instituições de saúde. .

Novel thiazole derivatives: a patent review (2008 - 2012. Part 2).

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.

Label-free cell-based dynamic mass redistribution assays.

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.

Sulfonylurea receptor 1 in central nervous system injury: a focused review.

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.

Treating diabetes today: a matter of selectivity of sulphonylureas.

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 ß-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 ß-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 ß-cell K(ATP) channel and does not activate Epac2.

The personalised medicine. A paradigm of evidence-based medicine.

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.

Human oocytes express ATP-sensitive K(+) channels.

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.

Renin-angiotensin gene polymorphisms and neurohormonal inhibition.

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.

Attenuation of cadmium-induced necrotic cell death by necrostatin-1: potential necrostatin-1 acting sites.

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.

Levcromakalim and MgGDP activate small conductance ATP-sensitive K+ channels of K+ channel pore 6.1/sulfonylurea receptor 2A in pig detrusor smooth muscle cells: uncoupling of cAMP signal pathways.

Pharmacological studies have suggested the existence of ATP-sensitive K(+) (K(ATP)) channel as a therapeutic target in urinary bladders; however, electrical properties have not yet been shown. Patch-clamp techniques were applied to investigate the properties of K(ATP) channels in pig detrusor cells. In whole-cell configuration, levcromakalim, a K(ATP) channel opener, induced a long-lasting outward current in a concentration-dependent manner. The current-voltage curve of the levcromakalim-induced membrane current intersected at approximately -80 mV. This current was abolished by glibenclamide. Intracellular application of 0.1 mM GDP significantly enhanced the levcromakalim-induced membrane current, whereas cAMP did not. Furthermore, neurotransmitters related to cAMP signaling, such as calcitonin gene-related peptide, vasointestinal peptide, adenosine, and somatostatin, had little effect on the membrane current. In cell-attached configuration, levcromakalim activated K(+) channels with a unitary conductance of approximately 12 pS. When the patch configuration was changed to inside-out mode, the K(+) channel activity ran down. Subsequent application of 1 mM GDP reactivated the channels. The openings of the approximately 12 pS K(+) channels in the presence of 1 mM GDP was suppressed by ATP and glibenclamide. In reverse transcription-polymerase chain reaction, K(+) channel pore 6.1 and sulfonylurea receptor (SUR)2A were predominant in pig detrusor cells. The 12 pS K(+) channel activated by levcromakalim in pig detrusor smooth muscle cells is a K(ATP) channel. The predominant expression of SUR2A can account for the lack of effect of neurotransmitters related to cAMP.

Costunolide-induced apoptosis is caused by receptor-mediated pathway and inhibition of telomerase activity in NALM-6 cells.

Costunolide, isolated from the stem bark of Magnolia sieboldii, is a sesquiterpene lactone that exhibits various biological and immunological actions. We investigated the induction mechanism of apoptosis by costunolide in a human B cell leukemia NALM-6 cell culture system. Costunolide (10 microM)-induced apoptosis time-dependently increased, estimated by nuclear damage observation and flow cytometric analysis. Costunolide did not change Fas-associated factor 1 (FAF1), but the phosphorylation of Fas-associated death domain (FADD) at serine 194 increased from early treatment. The activation of caspase-8 and -9 and degradation of poly-(ADP-ribose) polymerase (PARP) was time-dependently detected by incubation with costunolide. Pretreatment of cells with caspase-3, -8 and broad spectrum caspase inhibitors significantly blocked costunolide-induced apoptosis, but caspase-9 inhibitor failed to block apoptosis. Telomerase activity was significantly suppressed after treatment with costunolide, and human telomerase reverse transcriptase (hTERT), a critical determinant of the enzyme activity of telomerase, decreased the expression of both mRNA and protein levels by costunolide. Costunolide-induced repression of telomerase was prevented by pretreatment of cells with caspase-3, -8 and broad spectrum caspase inhibitors, but caspase-9 inhibitor was no effect. These data suggest that one of the costunolide-induced apoptotic mechanisms is that the receptor-mediated pathway precedes the mitochondria-dependent pathway, caused by the inhibition of telomerase activity via suppression of hTERT in NALM-6 cells.

Bombesin marine toxin conjugates inhibit the growth of lung cancer cells.

Hemiasterlin (Hem) and dolastatin (Dol) are marine natural products which are cytotoxic for cancer cells. Hem, a tripeptide, and Dol, a hexapeptide, were conjugated with linkers (L) to the universal BB agonist DPhe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2(BA1) and the effects of the Hem-BB and Dol-BB conjugates investigated on NCI-H1299 lung cancer cells. Hem-LA-BA1 and Hem-LB-BA1 inhibited specific (125I-Tyr4)BB binding to NCI-H1299 cells, which have BB2 receptors (R), with IC50 values of 15 and 25 nM, respectively. Addition of Hem-LA-BA1 and Hem-LB-BA1 to Fura-2 AM loaded cells containing BB2R, caused elevated cytosolic Ca2+. In a growth assay, Hem-LA-BA1 and Hem-LB-BA1 inhibited the proliferation of NCI-H1299 cells. Dol-succinamide (Dols)-LD-BA1 and Dols-LE-BA1 bound with high affinity to NCI-H1299 cells and elevated cytosolic Ca2+, but did not inhibit the proliferation of NCI-H1299 cells. Also, Hem-LA-BA1 inhibited 125I-DTyr-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 (BA2) binding to Balb/3T3 cells transfected with BB1R or BB2R as well as with BRS-3 with IC50 values of 130, 8, and 540 nM, respectively. These results show that Hem-BB conjugates are cytotoxic for cancer cells containing BB2R.

Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans.

The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.

K(ATP) channel pharmacogenomics: from bench to bedside.

Inheritance plays a significant role in defining drug response and toxicity. Advances in molecular pharmacology and modern genomics emphasize genetic variation in dictating inter-individual pharmacokinetics and pharmacodynamics. A case in point is the homeostatic ATP-sensitive potassium (K(ATP)) channel, an established drug target that adjusts membrane excitability to match cellular energetic demand. There is an increased recognition that genetic variability of the K(ATP) channel impacts therapeutic decision-making in human disease.

alpha-Latrotoxin and its receptors.

alpha-Latrotoxin (alpha-LTX) from black widow spider venom induces exhaustive release of neurotransmitters from vertebrate nerve terminals and endocrine cells. This 130-kDa protein has been employed for many years as a molecular tool to study exocytosis. However, its action is complex: in neurons, alpha-LTX induces massive secretion both in the presence of extracellular Ca(2+) (Ca(2+) (e)) and in its absence; in endocrine cells, it usually requires Ca(2+) (e). To use this toxin for further dissection of secretory mechanisms, one needs an in-depth understanding of its functions. One such function that explains some alpha-LTX effects is its ability to form cation-permeable channels in artificial lipid bilayers. The mechanism of alpha-LTX pore formation, revealed by cryo-electron microscopy, involves toxin assembly into homotetrameric complexes which harbor a central channel and can insert into lipid membranes. However, in biological membranes, alpha-LTX cannot exert its actions without binding to specific receptors of the plasma membrane. Three proteins with distinct structures have been found to bind alpha-LTX: neurexin Ialpha, latrophilin 1, and receptor-like protein tyrosine phosphatase sigma. Upon binding a receptor, alpha-LTX forms channels permeable to cations and small molecules; the toxin may also activate the receptor. To distinguish between the pore- and receptor-mediated effects, and to study structure-function relationships in the toxin, alpha-LTX mutants have been used.