Assay strategies for the discovery and validation of therapeutics targeting <i>Brugia pahangi</i> Hsp90

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target

Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and β) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/β)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/β) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/β)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90β/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in 18F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/β)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients

ASSOCIATION OF HYPERTENSION AND DIABETES WITH OBESITY

Introduction: Hypertension, diabetes and obesity are one of the top risk factors for cardiovascular complications in the world. Indians have high burden of obesity compared to western population. Hypertension and type 2 diabetes mellitus are major non communicable diseases and it is important to examine modifiable risk factors such as obesity, physical inactivity , tobacco use and alcohol consumption. Metabolic syndrome (MS) is defines as presence of type 2 diabetes or impaired glucose tolerance with any two of the following characteristics: obesity, low levels of high-density lipoprotein, high levels of triglycerides and hypertension by World Health Organization (WHO).&#x0D; Materials and Methods: 1485 samples were selected from the patients attending OPD of General Medicine Department of D.Y. Patil University School of Medicine, Mumbai. They were assessed for height, weight and body mass index (BMI) measurement. Hypertension was defined according to the guidelines given in Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure. Abdominal obesity is defined as an abdominal circumference &gt; 102cm in males and &gt;88cm in females for NCEP criteria and &gt; 90cm in males and &gt; 80cm in females for IDF criteria for South Asians. Blood glucose concentration was measured, the diagnosis of diabetes was defines by either casual plasma glucose ≥ 200mg/dl associated with symptoms of diabetes and on fasting samples- plasma glucose ≥ 126mg/dl.&#x0D; Results: Among all 1485 patients, 764(51.44%) were males and 721(48.56%) were females. It was observed that 32(21.6%) cases were obese, hypertension was observed in 289(19.5%) cases and diabetes in 168(11.3%). Among 289 hypertensive subjects, 214(74%) were obese and 75(36%) were non-obese. Out of 168 diabetic cases 96(57.1%) cases were obese and 72(42.9%) were non-obese. A statistically significant association was observed between diabetes, hypertension and obesity. Conclusion: There is significant association between obesity, hypertension and diabetes. BMI can provide useful information to classify the presence of hypertension and diabetes in population.&#x0D; Keywords: Obesity, Hypertension, Diabetes, BMI.</jats:p

Bilateral breast in brothers - abreast

Gynecomastia is a common occurrence in pubertal age group, and is physiological in up to 65 percent of cases. When occurs in the family it should be investigated in order not to miss on a treatable etiology. Two brothers within the same family, presenting with bilateral gynecomastia of different causes and requiring different treatment are presented

An unusual cause of flash pulmonary oedema

We report a case of young woman who presented with acute cardiogenic pulmonary oedema and respiratory failure. She underwent emergent endotracheal intubation and was transferred to the intensive care unit. She responded to intravenous diuretics and positive pressure ventilation. Subsequent workup revealed that she had Graves’ disease and was in thyrotoxic crisis. Therapy with propranolol and propylthiouracil was instituted to which she showed remarkable improvement