Deepak K. Rajpal

Computational Drug Repositioning: From Data to Therapeutics

Traditionally, most drugs have been discovered using phenotypic or target‐based screens. Subsequently, their indications are often expanded on the basis of clinical observations, providing additional benefit to patients. This review highlights computational techniques for systematic analysis of transcriptomics (Connectivity Map, CMap), side effects, and genetics (genome‐wide association study, GWAS) data to generate new hypotheses for additional indications. We also discuss data domains such as electronic health records (EHRs) and phenotypic screening that we consider promising for novel computational repositioning methods.

Applications of Connectivity Map in drug discovery and development

Genome-wide expression profiling of gene transcripts has been successfully applied in biomedical discovery for over a decade. Based on the premises of this technology, Connectivity Map provides a data-driven and systematic approach for discovering associations among genes, chemicals and biological conditions such as diseases. Since its first introduction in 2006, the approach has shown emerging promises in uncovering avenues for drug discovery and development such as in identifying and suggesting new indications for existing drugs and elucidating mode of actions for novel chemicals in addition to potentially predicting side effects.

Novel Gut-Based Pharmacology of Metformin in Patients with Type 2 Diabetes Mellitus

Metformin, a biguanide derivate, has pleiotropic effects beyond glucose reduction, including improvement of lipid profiles and lowering microvascular and macrovascular complications associated with type 2 diabetes mellitus (T2DM). These effects have been ascribed to adenosine monophosphate-activated protein kinase (AMPK) activation in the liver and skeletal muscle. However, metformin effects are not attenuated when AMPK is knocked out and intravenous metformin is less effective than oral medication, raising the possibility of important gut pharmacology. We hypothesized that the pharmacology of metformin includes alteration of bile acid recirculation and gut microbiota resulting in enhanced enteroendocrine hormone secretion. In this study we evaluated T2DM subjects on and off metformin monotherapy to characterize the gut-based mechanisms of metformin. Subjects were studied at 4 time points: (i) at baseline on metformin, (ii) 7 days after stopping metformin, (iii) when fasting blood glucose (FBG) had risen by 25% after stopping metformin, and (iv) when FBG returned to baseline levels after restarting the metformin. At these timepoints we profiled glucose, insulin, gut hormones (glucagon-like peptide-1 (GLP-1), peptide tyrosine-tyrosine (PYY) and glucose-dependent insulinotropic peptide (GIP) and bile acids in blood, as well as duodenal and faecal bile acids and gut microbiota. We found that metformin withdrawal was associated with a reduction of active and total GLP-1 and elevation of serum bile acids, especially cholic acid and its conjugates. These effects reversed when metformin was restarted. Effects on circulating PYY were more modest, while GIP changes were negligible. Microbiota abundance of the phylum Firmicutes was positively correlated with changes in cholic acid and conjugates, while Bacteroidetes abundance was negatively correlated. Firmicutes and Bacteroidetes representation were also correlated with levels of serum PYY. Our study suggests that metformin has complex effects due to gut-based pharmacology which might provide insights into novel therapeutic approaches to treat T2DM and associated metabolic diseases. Trial Registration: www.ClinicalTrials.gov NCT01357876

Inhibition of apical sodium-dependent bile acid transporter as a novel treatment for diabetes.

Bile acids are recognized as metabolic modulators. The present study was aimed at evaluating the effects of a potent Asbt inhibitor (264W94), which blocks intestinal absorption of bile acids, on glucose homeostasis in Zucker Diabetic Fatty (ZDF) rats. Oral administration of 264W94 for two wk increased fecal bile acid concentrations and elevated non-fasting plasma total Glp-1. Treatment of 264W94 significantly decreased HbA1c and glucose, and prevented the drop of insulin levels typical of ZDF rats in a dose-dependent manner. An oral glucose tolerance test revealed up to two-fold increase in plasma total Glp-1 and three-fold increase in insulin in 264W94 treated ZDF rats at doses sufficient to achieve glycemic control. Tissue mRNA analysis indicated a decrease in farnesoid X receptor (Fxr) activation in small intestines and the liver but co-administration of a Fxr agonist (GW4064) did not attenuate 264W94 induced glucose lowering effects. In summary, our results demonstrate that inhibition of Asbt increases bile acids in the distal intestine, promotes Glp-1 release and may offer a new therapeutic strategy for type 2 diabetes mellitus.

Effect of Roux-en-Y Gastric Bypass Surgery on Bile Acid Metabolism in Normal and Obese Diabetic Rats

In addition to classic functions of facilitating hepatobiliary secretion and intestinal absorption of lipophilic nutrients, bile acids (BA) are also endocrine factors and regulate glucose and lipid metabolism. Recent data indicate that antiobesity bariatric procedures e.g. Roux-en-Y gastric bypass surgery (RYGB), which also remit diabetes, increase plasma BAs in humans, leading to the hypothesis that BAs may play a role in diabetes resolution following surgery. To investigate the effect of RYGB on BA physiology and its relationship with glucose homeostasis, we undertook RYGB and SHAM surgery in Zucker diabetic fatty (ZDF) and normoglycemic Sprague Dawley (SD) rats and measured plasma and fecal BA levels, as well as plasma glucose, insulin, Glucagon like peptide 1 (GLP-1) and Peptide YY (PYY), 2 days before and 3, 7, 14 and 28 days after surgery. RYGB decreased body weight and increased plasma GLP-1 in both SD and ZDF rats while decreasing plasma insulin and glucose in ZDF rats starting from the first week. Compared to SHAM groups, both SD-RYGB and ZDF-RYGB groups started to have increases in plasma total BAs in the second week, which might not contribute to early post-surgery metabolic changes. While there was no significant difference in fecal BA excretion between SD-RYGB and SD-SHAM groups, the ZDF-RYGB group had a transient 4.2-fold increase (P<0.001) in 24-hour fecal BA excretion on post-operative day 3 compared to ZDF-SHAM, which paralleled a significant increase in plasma PYY. Ratios of plasma and fecal cholic acid/chenodeoxycholic acid derived BAs were decreased in RYGB groups. In addition, tissue mRNA expression analysis suggested early intestinal BA reabsorption and potentially reduced hepatic cholic acid production in RYGB groups. In summary, we present novel data on RYGB-mediated changes in BA metabolism to further understand the role of BAs in RYGB-induced metabolic effects in humans.

The role of translational bioinformatics in drug discovery

The application of translational approaches (e.g. from bed to bench and back) is gaining momentum in the pharmaceutical industry. By utilizing the rapidly increasing volume of data at all phases of drug discovery, translational bioinformatics is poised to address some of the key challenges faced by the industry. Indeed, computational analysis of clinical data and patient records has informed decision-making in multiple aspects of drug discovery and development. Here, we review key examples of translational bioinformatics approaches to emphasize its potential to enhance the quality of drug discovery pipelines, reduce attrition rates and, ultimately, lead to more effective treatments.

Early Activation of Th2/Th22 Inflammatory and Pruritogenic Pathways in Acute Canine Atopic Dermatitis Skin Lesions

Determining inflammation and itch pathway activation in patients with atopic dermatitis (AD) is fraught with the inability to precisely assess the age of skin lesions, thus affecting the analysis of time-dependent mediators. To characterize inflammatory events occurring during early experimental acute AD lesions, biopsy samples were collected 6, 24, and 48 hours after epicutaneous application of Dermatophagoides farinae house dust mites to sensitized atopic dogs. The skin transcriptome was assessed using a dog-specific microarray and quantitative PCR. Acute canine AD skin lesions had a significant up-regulation of genes encoding T helper (Th) 2 (e.g., IL4, IL5, IL13, IL31, and IL33), Th9 (IL9), and Th22 (IL22) cytokines as well as Th2-promoting chemokines such as CCL5 and CCL17. Proinflammatory (e.g., IL6, LTB, and IL18) cytokines were also up-regulated. Other known pruritogenic pathways were also activated: there was significant up-regulation of genes encoding proteases cathepsin S (CTSS), mast cell chymase (CMA1), tryptase (TPS1) and mastin, neuromedin-B (NMB), nerve growth factor (NGF), and leukotriene-synthesis enzymes (ALOX5, ALOX5AP, and LTA4H). Experimental acute canine house dust mite-induced AD lesions exhibit an activation of innate and adaptive immune responses and pruritogenic pathways similar to those seen in humans with acute AD, thereby validating this model to test innovative therapeutics modalities for this disease.

Selective Spectrum Antibiotic Modulation of the Gut Microbiome in Obesity and Diabetes Rodent Models

The gastrointestinal tract microbiome has been suggested as a potential therapeutic target for metabolic diseases such as obesity and Type 2 diabetes mellitus (T2DM). However, the relationship between changes in microbial communities and metabolic disease-phenotypes are still poorly understood. In this study, we used antibiotics with markedly different antibacterial spectra to modulate the gut microbiome in a diet-induced obesity mouse model and then measured relevant biochemical, hormonal and phenotypic biomarkers of obesity and T2DM. Mice fed a high-fat diet were treated with either ceftazidime (a primarily anti-Gram negative bacteria antibiotic) or vancomycin (mainly anti-Gram positive bacteria activity) in an escalating three-dose regimen. We also dosed animals with a well-known prebiotic weight-loss supplement, 10% oligofructose saccharide (10% OFS). Vancomycin treated mice showed little weight change and no improvement in glycemic control while ceftazidime and 10% OFS treatments induced significant weight loss. However, only ceftazidime showed significant, dose dependent improvement in key metabolic variables including glucose, insulin, protein tyrosine tyrosine (PYY) and glucagon-like peptide-1 (GLP-1). Subsequently, we confirmed the positive hyperglycemic control effects of ceftazidime in the Zucker diabetic fatty (ZDF) rat model. Metagenomic DNA sequencing of bacterial 16S rRNA gene regions V1-V3 showed that the microbiomes of ceftazidime dosed mice and rats were enriched for the phylum Firmicutes while 10% OFS treated mice had a greater abundance of Bacteroidetes. We show that specific changes in microbial community composition are associated with obesity and glycemic control phenotypes. More broadly, our study suggests that in vivo modulation of the microbiome warrants further investigation as a potential therapeutic strategy for metabolic diseases.

The epigenetic signature of systemic insulin resistance in obese women

Aims/hypothesisInsulin resistance (IR) links obesity to type 2 diabetes. The aim of this study was to explore whether white adipose tissue (WAT) epigenetic dysregulation is associated with systemic IR by genome-wide CG dinucleotide (CpG) methylation and gene expression profiling in WAT from insulin-resistant and insulin-sensitive women. A secondary aim was to determine whether the DNA methylation signature in peripheral blood mononuclear cells (PBMCs) reflects WAT methylation and, if so, can be used as a marker for systemic IR.MethodsFrom 220 obese women, we selected a total of 80 individuals from either of the extreme ends of the distribution curve of HOMA-IR, an indirect measure of systemic insulin sensitivity. Genome-wide transcriptome and DNA CpG methylation profiling by array was performed on subcutaneous (SAT) and visceral (omental) adipose tissue (VAT). CpG methylation in PBMCs was assayed in the same cohort.ResultsThere were 647 differentially expressed genes (false discovery rate [FDR] 10%) in SAT, all of which displayed directionally consistent associations in VAT. This suggests that IR is associated with dysregulated expression of a common set of genes in SAT and VAT. The average degree of DNA methylation did not differ between the insulin-resistant and insulin-sensitive group in any of the analysed tissues/cells. There were 223 IR-associated genes in SAT containing a total of 336 nominally significant differentially methylated sites (DMS). The 223 IR-associated genes were over-represented in pathways related to integrin cell surface interactions and insulin signalling and included COL5A1, GAB1, IRS2, PFKFB3 and PTPRJ. In VAT there were a total of 51 differentially expressed genes (FDR 10%); 18 IR-associated genes contained a total of 29 DMS.Conclusions/interpretationIn individuals discordant for insulin sensitivity, the average DNA CpG methylation in SAT and VAT is similar, although specific genes, particularly in SAT, display significantly altered expression and DMS in IR, possibly indicating that epigenetic regulation of these genes influences metabolism.

Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.