Conformational diversity and protein-protein interfaces in drug repurposing in Ras signaling pathway.

We focus on drug repurposing in the Ras signaling pathway, considering structural similarities of protein-protein interfaces. The interfaces formed by physically interacting proteins are found from PDB if available and via PRISM (PRotein Interaction by Structural Matching) otherwise. The structural coverage of these interactions has been increased from 21 to 92% using PRISM. Multiple conformations of each protein are used to include protein dynamics and diversity. Next, we find FDA-approved drugs bound to structurally similar protein-protein interfaces. The results suggest that HIV protease inhibitors tipranavir, indinavir, and saquinavir may bind to EGFR and ERBB3/HER3 interface. Tipranavir and indinavir may also bind to EGFR and ERBB2/HER2 interface. Additionally, a drug used in Alzheimer's disease can bind to RAF1 and BRAF interface. Hence, we propose a methodology to find drugs to be potentially used for cancer using a dataset of structurally similar protein-protein interface clusters rather than pockets in a systematic way.

Reconstruction of the binding pathway of an anti-HIV drug, Indinavir, in complex with the HTLV-1 protease using unaggregated unbiased molecular dynamics simulation.

Retroviruses are a growing concern for the health of human beings, and one of the dangerous members of this family is the Human T-cell Leukemia Virus 1 (HTLV-1) virus. It has affected more than 20 million people so far, and since there are no registered treatments against it yet, urgent treatment solutions are needed. One of the most promising drug targets to fight this virus is the protease enzyme of the virus's protein machinery. In this study, by utilizing a computational method called Unaggregated Unbiased Molecular Dynamics (UUMD), we reconstructed the binding pathway of a HTLV-1 protease inhibitor, Indinavir, to find the details of the binding pathway, the influential residues, and also the stable states of the binding pathway. We achieved the native conformation of the inhibitor in 6 rounds, 360 replicas by performing over 4 micro-seconds of UMD simulations. We found 3 Intermediate states between the solvated state and the native conformation state in the binding pathway. We also discovered that aromatic residues such as Trp98 and Trp98', catalytic residues Asp32 and Asp32', and the flap region's residues have the most influential roles in the binding pathway and also have the most contribution to the total interaction energies. We believe that the details found in this study would be a great guide for developing new treatment solutions against the HTLV-1 virus by inhibiting the HTLV-1 protease.

Drug binding dynamics of the dimeric SARS-CoV-2 main protease, determined by molecular dynamics simulation.

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.

A search for medications to treat COVID-19 via in silico molecular docking models of the SARS-CoV-2 spike glycoprotein and 3CL protease.

BACKGROUND: The COVID-19 has now been declared a global pandemic by the World Health Organization. There is an emergent need to search for possible medications. METHOD: Utilization of the available sequence information, homology modeling, and in slico docking a number of available medications might prove to be effective in inhibiting the SARS-CoV-2 two main drug targets, the spike glycoprotein, and the 3CL protease. RESULTS: Several compounds were determined from the in silico docking models that might prove to be effective inhibitors for SARS-CoV-2. Several antiviral medications: Zanamivir, Indinavir, Saquinavir, and Remdesivir show potential as and 3CLPRO main proteinase inhibitors and as a treatment for COVID-19. CONCLUSION: Zanamivir, Indinavir, Saquinavir, and Remdesivir are among the exciting hits on the 3CLPRO main proteinase. It is also exciting to uncover that Flavin Adenine Dinucleotide (FAD) Adeflavin, B2 deficiency medicine, and Coenzyme A, a coenzyme, may also be potentially used for the treatment of SARS-CoV-2 infections. The use of these off-label medications may be beneficial in the treatment of the COVID-19.

Chemical composition and antioxidant potential of essential oil and methanol extract from Tunisian and French fennel (Foeniculum vulgare Mill.) seeds.

This study was designed to examine the effect of provenance on the phytochemical and antioxidant properties of essential oil and methanolic extract from Tunisian and French fennel seeds (FFS). Analysis of the essential oil showed that although the same main compounds were found in Tunisian and FFS cultivars, some differences were present in their proportions allowing to classify them in two chemotypes. The first class was composed by trans-anethole (63.41%-78.26%) for Tunisian cultivars and the second one by estragole (44.72%-88.92%) for French cultivars. The phenolic composition of all fennel seed extracts was characterized by its richness in quinic acid, 4-O-caffeoylquinic acid, p-coumaric acid, and 4-O-caffeoylquinic acid. All fennel seed extracts showed a better antioxidant potential than their essential oils depending on the origin. Principal component analysis showed a dispersion of the cultivars on three groups depending on the chemotype diversity. PRACTICAL APPLICATIONS: Recently, much attention has been focused on fennel due to the nutritional and health-protective value of their seeds. Several studies have highlighted the importance of fennel seed extracts and essential oils as key ingredients rich in bioactive compounds serving in formulation of new functional food products. This investigation designed to examine the effect of provenance on phytochemical and antioxidant potentials of Tunisian and French fennel seed extracts and essential oils.

Core-Shell Palladium Telluride Quantum Dot-Hemethiolate Cytochrome Based Biosensor for Detecting Indinavir Drug.

Indinavir is a first-generation HIV protease inhibitor anti-retroviral (ARV) drug. Due to interindividual differences in the rate of indinavir metabolism, clinicians and pharmacologists have expressed urgent need for sensor devices that will enable real time determination of appropriate dosage. In this study, an indinavir biosensor was developed by the functionalization of a cysteamine-modified gold (Cyst|Au) electrode with biocompatible core-shell 3-mercaptopropionic acid (3-MPA)-capped palladium telluride quantum dot (PdTeQD) and the heme-thiolate cytochrome P450-3A4 (CYP3A4) enzyme. The PdTeQD was capped with 3-mercaptopropionic acid (3-MPA) to improve its reactivity, biocompatibility and thermal stability. Small angle X-ray scattering (SAXS) studies revealed that the 3-MPA-PdTeQD particles formed core-shells with diameters of 4.7 nm. Fourier transformed infrared spectroscopy (FTIR) experiments confirmed the formation of 3-MPA-PdTeQD by the presence of specific COOH and CH2 FTIR signature bands. Ultraviolet-visible (UV-Vis) spectrophotometric analysis of the quantum dot, exhibited a broad characteristic band at ~320 nm, corresponding to a band gap energy (Eg) value of 3.87 eV, indicating that the QD is a semiconducting material. Cyclic voltammetry (CV) responses of the biosensor (i.e., CYP3A4|3-MPA-PdTeQD|Cyst|Au) indicated that 0.26 V was the suitable potential for measuring indinavir metabolism. The biosensor has a sensitivity, dynamic linear range (DLR) and limit of detection (LOD) values of 0.0218 µA/nM, 0.0004-0.01 nM (i.e., 3×10-7 -7×10-6 mg L-1) and 0.023×10-7 mg L-1, respectively, for indinavir. The LOD value was lower than the maximum plasma concentration (Cmax) value (0.13-8.6 mg L-1) of indinavir which is normally measure 8 h after intake. The low DLR value makes the biosensor suitable for application at point-of-care, where indinavir concentration is expected to be in ng L-1 level in physiological samples within a few minutes of the drug administration.

The mechanism for differential effect of nelfinavir and indinavir on collagen metabolism in human skin fibroblasts.

The mechanism for differential effects of human immune deficiency virus protease inhibitors (HIVPIs), nelfinavir (NEL) and indinavir (IND) on collagen metabolism disturbances was studied in human skin fibroblasts. It has been considered that HIVPIs-dependent deregulation of collagen biosynthesis involves prolidase (an enzyme providing proline for collagen biosynthesis), glutamine (Gln) (a substrate for proline biosynthesis), nuclear factor-κB (NF-κB) (a transcription factor that inhibit expression of type I collagen genes), ß1 integrin receptor and Akt signalling. It was found that NEL impaired collagen biosynthesis and the process was more pronounced in the presence of Gln, while IND stimulated collagen biosynthesis. NEL-dependent inhibition of collagen biosynthesis was accompanied by massive intracellular accumulation of type I collagen, while IND slightly induced this process. This effect of NEL was reversed by ascorbic acid but not N-acetylcysteine. The mechanism for the NEL-dependent defect in collagen metabolism was found at the level of prolidase activity, ß1 integrin signalling and NF-κB. NEL inhibited expression of ß1 integrin receptor, Akt and ERK1/2 and increased expression of p65 NF-κB. However, inhibitors of p65 NF-κB did not prevent NEL-dependent inhibition of collagen biosynthesis suggesting that this transcription factor is not involved in studied mechanism. Using PI3K inhibitor wortmannin that prevent phosphorylation of Akt revealed that NEL-dependent inhibition of Akt results in inhibition of collagen biosynthesis. The data suggest that differential effect of NEL and IND on collagen metabolism involves NEL-dependent down-regulation of Akt signalling and proline availability for collagen biosynthesis.

Risk factors for kidney disease among HIV-1 positive persons in the methadone program.

BACKGROUND: Kidney injury is a serious comorbidity among HIV-infected patients. Intravenous drug use is listed as one of the risk factors for impaired renal function; however, this group is rarely assessed for specific renal-related risks. METHODS: Patients attending methadone program from 1994 to 2015 were included in the study. Data collected included demographic data, laboratory tests, antiretroviral treatment history, methadone dosing and drug abstinence. Patients' drug abstinence was checked monthly on personnel demand. We have evaluated two study outcomes: (1) having at least one or (2) three eGFR < 60 ml/min (MDRD formula). RESULTS: In total, 267 persons, with 2593 person-years of follow-up were included into analyses. At the time of analyses, 251 (94%) were on antiretroviral therapy (ARV). Fifty-two (19.5%) patients had 1eGFR and 20 (7.5%) 3eGFR < 60. In univariate analysis, factors significantly increasing the odds of impaired renal function were: female gender, detectable HIV RNA on ART, age at registration per 5 years older, atazanavir use and time on antiretroviral treatment per 1 year longer. In the multivariate model, only female gender (OR 4.7; p = 0.002), time on cART (OR 1.11; p = 0.01) and baseline eGFR (OR 0.71; p = 0.001) were statistically significant. CONCLUSIONS: We have demonstrated a high rate of kidney function impairment among HIV-1 positive patients in the methadone program. All risk factors for decreased eGFR in this subpopulation of patients were similar to those described for general HIV population with very high prevalence in women. These findings imply the need for more frequent kidney function monitoring in this subgroup of patients.

Indinavir Plus Methylprednisolone Ameliorates Experimental Acute Lung Injury In Vitro and In Vivo.

BACKGROUND: An abnormal HMGB1 activation plays a key role in the pathogenesis of ALI. METHODS: In this study, the effects of Indinavir plus methylprednisolone on the LPS-mediated activation in human pulmonary microvascular endothelial cells (HPMECs), on the injury of AT I in vitro, and on rats with LPS-induced two-hit model with or without methylprednisolone were investigated. RESULTS: Indinavir treatment resulted in a reduction of HMGB1, its receptor TLR-4, and HMGB1's downstream p-NF-κB, attenuating a decrease of VE-cadherin in LPS-stimulated HPMECs. Apoptosis of AT I was attenuated with an increase of RAGE and aquaporin 5. Compared to methylprednisolone alone, methylprednisolone plus Indinavir attenuated the decrease of GRα and IκB-α in cytoplasm and avoid GRα deficiency in LPS-stimulated HPMECs for 96 h, attenuated the increase of p-NF-κB in nucleus. Indinavir ameliorated histopathological changes of two-hit ALI model of rats with reductions in microvascular permeability, lower HMGB1, TLR4, p-NF-κB, and MPO expression, whereas higher RAGE, aquaporin 5, and VE-cadherin in LPS-instilled lungs. Compared to methylprednisolone alone, methylprednisolone plus Indinavir attenuated the decrease of GRα and IκB-α in cytoplasm, decreased p-NF-κB in nucleus of lung tissue of two-hit ALI rats, and enhanced the anti-inflammatory effect of methylprednisolone for avoiding GRα deficiency. CONCLUSION: It demonstrated that Indinavir prevented experimental ALI model of rats by modulating the HMGB1/TLR-4 pathway to resolve systemic inflammation response in a greater degree with methylprednisolone, reduced the use time and dose of methylprednisolone, and avoided GRα deficiency in ALI and ARDS.

Differences in the Prediction of Area Under the Curve for a Protease Inhibitor Using Trough Versus Peak Concentration: Assessment Using Published Pharmacokinetic Data for Indinavir.

In the present day antiretroviral therapy, Ctrough is a key tool for efficacy assessment. The present work explored the feasibility of using Ctrough or Cmax in the area under the concentration-time curve (AUC) prediction of indinavir. A simple unweighted linear regression model was developed to describe the relationship between Cmax versus AUC (r = 0.8101, P < 0.001) and Ctrough versus AUC (r = 0.8127, P < 0.001) for indinavir. The regression lines were used to predict the AUC values from literature Cmax or Ctrough data of indinavir in HIV and healthy subjects. The fold difference, defined as the quotient of the observed and predicted AUC values, was evaluated along with statistical comparison, including root mean square error (RMSE) prediction for the 2 models. The correlation between Cmax versus AUC and Ctrough versus AUC was established. Majority of the predicted values for Cmax versus AUC were within 0.75- to 1.5-fold differences. However, the Ctrough versus AUC model showed larger variability with approximately one-third of the predictions within 0.75- to 1.5-fold differences. The r value and %RMSE for observed versus predicted AUC for Ctrough (r = 0.5925, n = 65, P < 0.001, and RMSE: 67%) were inferior to the Cmax (r = 0.8773, n = 86, P < 0.001, and RMSE: 46%). In conclusion, Cmax versus AUC and Ctrough versus AUC relationships were established for indinavir showing the utility of a single concentration time point for therapeutic drug monitoring purpose. The Cmax model for indinavir may be more relevant for AUC prediction as determined by the statistical criteria.

Brain GLUT4 Knockout Mice Have Impaired Glucose Tolerance, Decreased Insulin Sensitivity, and Impaired Hypoglycemic Counterregulation.

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose.

Hyperglycemia exacerbates antiretroviral drug combination induced blood-brain barrier endothelial toxicity.

In this study, we sought to investigate how concomitant hyperglycemia influences the impact of combination antiretroviral therapy on blood-brain barrier (BBB) endothelial function. Immortalized human brain microvascular endothelial cell line (hCMEC/D3) was exposed to azidothymidine (AZT; a nucleoside reverse transcriptase inhibitor) and/or indinavir (IND; protease inhibitor) in normal glycemic (5.5mM) or hyperglycemic (HG; 25mM) media containing D-glucose for 24-72h. Cellular reactive oxygen species (ROS) and mitochondria-specific superoxide levels were assayed in addition to membrane potential to determine the extent of mitochondrial dysfunction. Nrf2 expression was analyzed by immunofluorescence. Our results indicated a significant increase in BBB endothelial toxicity (decreased ATP) by HG and AZT+IND with progression of time (24-72h). Concurrent HG and antiviral drug combination synergistically elevated BBB endothelial ROS induced by either condition alone. Further, HG and AZT+IND mutually interact to elicit a pronounced increase in mitochondrial superoxide levels post 24h (vs. either condition alone or controls). In addition, HG and AZT+IND complemented each other to induce potential loss of mitochondrial membrane potential. While HG or AZT+IND alone for 24h increased Nrf2 nuclear distribution, co-exposure conditions induced a potential loss of Nrf2 expression/nuclear translocation in BBB endothelium. In summary, our data strongly suggest that antiretroviral drug combination potentially interacts with concomitant HG and triggers exacerbated mitochondrial dysfunction and BBB endothelial toxicity, possibly through dysregulation of Nrf2 signaling. Thus, this study warrants the critical need for safety evaluation and monitoring of neurovascular complications of HAART regimens in HIV-infected diabetic patient cohort.

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

BACKGROUND: HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. RESULTS: A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. CONCLUSIONS: This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

CD26/DPPIV inhibition alters the expression of immune response-related genes in the thymi of NOD mice.

The transmembrane glycoprotein CD26 or dipeptidyl peptidase IV (DPPIV) is a multifunctional protein. In immune system, CD26 plays a role in T-cell function and is also involved in thymic maturation and emigration patterns. In preclinical studies, treatment with DPPIV inhibitors reduces insulitis and delays or even reverses the new -onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the specific mechanisms involved in these effects remain unknown. The aim of the present study was to investigate how DPPIV inhibition modifies the expression of genes in the thymus of NOD mice by microarray analysis. Changes in the gene expression of ß-cell autoantigens and Aire in thymic epithelial cells (TECs) were also evaluated by using qRT-PCR. A DPPIV inhibitor, MK626, was orally administered in the diet for 4 and 6 weeks starting at 6-8 weeks of age. Thymic glands from treated and control mice were obtained for each study checkpoint. Thymus transcriptome analysis revealed that 58 genes were significantly over-expressed in MK626-treated mice after 6 weeks of treatment. Changes in gene expression in the thymus were confined mainly to the immune system, including innate immunity, chemotaxis, antigen presentation and immunoregulation. Most of the genes are implicated in central tolerance mechanisms through several pathways. No differences were observed in the expression of Aire and ß-cell autoantigens in TECs. In the current study, we demonstrate that treatment with the DPPIV inhibitor MK626 in NOD mice alters the expression of the immune response-related genes in the thymus, especially those related to immunological central tolerance, and may contribute to the prevention of T1D.

Herb-Drug Pharmacokinetic Interactions: Transport and Metabolism of Indinavir in the Presence of Selected Herbal Products.

Patients receiving anti-retroviral drug treatment are sometimes simultaneously taking herbal remedies, which may result in pharmacokinetic herb-drug interactions. This study aimed to determine if pharmacokinetic interactions exist between selected commercially available herbal products (i.e., Linctagon Forte(®), Viral Choice(®) and Canova(®)) and indinavir in terms of in vitro transport and metabolism. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in the presence and absence of the selected herbal products and verapamil (positive control). Metabolism of indinavir was determined in LS180 cells in the presence and absence of the selected herbal products as well as ketoconazole (positive control). The secretory transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte(®) and Viral Choice(®) when compared to that of indinavir alone. Canova(®) only slightly affected the efflux of indinavir compared to that of the control group. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir's bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made.

Tamizaje virtual de compuestosinhibidores de la SA P-2de Candida albicans / Virtual screening of inhibitory compounds SEP-2 Candida albicans

El incremento de la resistencia a antifúngicos en Candida spp. una levadurade carácter oportunista asociada a múltiples infecciones superficiales y sistémicas,plantea la necesidad de buscar diferentes estrategias para hallar nuevas opciones terapéuticas más selectivas y específicas. Una alternativa es el tamizaje basado en el acoplamiento virtual. En el presente trabajo, se analizaron 13418 compuestos con estructuras similares a compuestos reconocidos como inhibidores de la proteína SAP-2,seleccionada como diana antifúngica para el análisis virtual. Materiales y métodos: el estudio se realizó utilizando el programa SYBYL 8, al tiempo que se evaluó la afinidadde estas estructuras con los sitios activos de la enzima seleccionada mediante el programa FlexX integrado en SYBYL 8 y se realizó un consenso comparando los resultados con el programa Autodock Vina. Resultados: los resultados obtenidos mostraron una mayor afinidad por móleculas con estructura similar a los antirretrovirales,inhibidores de proteasas; amprenavir, saquinavir e indinavir. Conclusiones: los resultados obtenidos sugieren que estos lnhibidores de proteasas pueden ser propuestos como modelos para la búsqueda de nuevos antifúngicos que sean más selectivos contra Candida spp...

Development of an enantioselective assay for simultaneous separation of venlafaxine and O-desmethylvenlafaxine by micellar electrokinetic chromatography-tandem mass spectrometry: Application to the analysis of drug-drug interaction.

To-date, there has been no effective chiral capillary electrophoresis-mass spectrometry (CE-MS) method reported for the simultaneous enantioseparation of the antidepressant drug, venlafaxine (VX) and its structurally-similar major metabolite, O-desmethylvenlafaxine (O-DVX). This is mainly due to the difficulty of identifying MS compatible chiral selector, which could provide both high enantioselectivity and sensitive MS detection. In this work, poly-sodium N-undecenoyl-L,L-leucylalaninate (poly-L,L-SULA) was employed as a chiral selector after screening several dipeptide polymeric chiral surfactants. Baseline separation of both O-DVX and VX enantiomers was achieved in 15 min after optimizing the buffer pH, poly-L,L-SULA concentration, nebulizer pressure and separation voltage. Calibration curves in spiked plasma (recoveries higher than 80%) were linear over the concentration range 150-5000 ng/mL for both VX and O-DVX. The limit of detection (LOD) was found to be as low as 30 ng/mL and 21 ng/mL for O-DVX and VX, respectively. This method was successfully applied to measure the plasma concentrations of human volunteers receiving VX or O-DVX orally when co-administered without and with indinivar therapy. The results suggest that micellar electrokinetic chromatography electrospray ionization-tandem mass spectrometry (MEKC-ESI-MS/MS) is an effective low cost alternative technique for the pharmacokinetics and pharmacodynamics studies of both O-DVX and VX enantiomers. The technique has potential to identify drug-drug interaction involving VX and O-DVX enantiomers while administering indinivar therapy.

Accurate and efficient quantum chemistry calculations for noncovalent interactions in many-body systems: the XSAPT family of methods.

We present an overview of "XSAPT", a family of quantum chemistry methods for noncovalent interactions. These methods combine an efficient, iterative, monomer-based approach to computing many-body polarization interactions with a two-body version of symmetry-adapted perturbation theory (SAPT). The result is an efficient method for computing accurate intermolecular interaction energies in large noncovalent assemblies such as molecular and ionic clusters, molecular crystals, clathrates, or protein-ligand complexes. As in traditional SAPT, the XSAPT energy is decomposable into physically meaningful components. Dispersion interactions are problematic in traditional low-order SAPT, and two new approaches are introduced here in an attempt to improve this situation: (1) third-generation empirical atom-atom dispersion potentials, and (2) an empirically scaled version of second-order SAPT dispersion. Comparison to high-level ab initio benchmarks for dimers, water clusters, halide-water clusters, a methane clathrate hydrate, and a DNA intercalation complex illustrate both the accuracy of XSAPT-based methods as well as their limitations. The computational cost of XSAPT scales as O(N(3))-O(N(5)) with respect to monomer size, N, depending upon the particular version that is employed, but the accuracy is typically superior to alternative ab initio methods with similar scaling. Moreover, the monomer-based nature of XSAPT calculations makes them trivially parallelizable, such that wall times scale linearly with respect to the number of monomer units. XSAPT-based methods thus open the door to both qualitative and quantitative studies of noncovalent interactions in clusters, biomolecules, and condensed-phase systems.

Promiscuity and the conformational rearrangement of drug-like molecules: insight from the protein data bank.

Selectivity is a central aspect of lead optimization in the drug discovery process. Medicinal chemists often try to decrease molecular flexibility to improve selectivity, given the common belief that the two are interdependent. To investigate the relationship between polypharmacology and conformational flexibility, we mined the Protein Data Bank and constructed a dataset of pharmaceutically relevant ligands that crystallized in more than one protein target while binding to each co-crystallized receptor with similar in vitro affinities. After analyzing the molecular conformations of these 100 ligands, we found that 59 ligands bound to different protein targets without significantly changing conformation, suggesting that there is no distinct correlation between conformational flexibility and polypharmacology within our dataset. Ligands crystallized in similar proteins and highly ligand-efficient compounds with five or fewer rotatable bonds were less likely to adjust conformation when binding.

cAMP-dependent insulin modulation of synaptic inhibition in neurons of the dorsal motor nucleus of the vagus is altered in diabetic mice.

Pathologies in which insulin is dysregulated, including diabetes, can disrupt central vagal circuitry, leading to gastrointestinal and other autonomic dysfunction. Insulin affects whole body metabolism through central mechanisms and is transported into the brain stem dorsal motor nucleus of the vagus (DMV) and nucleus tractus solitarius (NTS), which mediate parasympathetic visceral regulation. The NTS receives viscerosensory vagal input and projects heavily to the DMV, which supplies parasympathetic vagal motor output. Normally, insulin inhibits synaptic excitation of DMV neurons, with no effect on synaptic inhibition. Modulation of synaptic inhibition in DMV, however, is often sensitive to cAMP-dependent mechanisms. We hypothesized that an effect of insulin on GABAergic synaptic transmission may be uncovered by elevating resting cAMP levels in GABAergic terminals. We used whole cell patch-clamp recordings in brain stem slices from control and diabetic mice to identify insulin effects on inhibitory neurotransmission in the DMV in the presence of forskolin to elevate cAMP levels. In the presence of forskolin, insulin decreased the frequency of inhibitory postsynaptic currents (IPSCs) and the paired-pulse ratio of evoked IPSCs in DMV neurons from control mice. This effect was blocked by brefeldin-A, a Golgi-disrupting agent, or indinavir, a GLUT4 blocker, indicating that protein trafficking and glucose transport were involved. In streptozotocin-treated, diabetic mice, insulin did not affect IPSCs in DMV neurons in the presence of forskolin. Results suggest an impairment of cAMP-induced insulin effects on GABA release in the DMV, which likely involves disrupted protein trafficking in diabetic mice. These findings provide insight into mechanisms underlying vagal dysregulation associated with diabetes.