A reconstructed genome-scale metabolic model of Helicobacter pylori for predicting putative drug targets in clarithromycin and rifampicin resistance conditions.

BACKGROUND: Helicobacter pylori is considered a true human pathogen for which rising drug resistance constitutes a drastic concern globally. The present study aimed to reconstruct a genome-scale metabolic model (GSMM) to decipher the metabolic capability of H. pylori strains in response to clarithromycin and rifampicin along with identification of novel drug targets. MATERIALS AND METHODS: The iIT341 model of H. pylori was updated based on genome annotation data, and biochemical knowledge from literature and databases. Context-specific models were generated by integrating the transcriptomic data of clarithromycin and rifampicin resistance into the model. Flux balance analysis was employed for identifying essential genes in each strain, which were further prioritized upon being nonhomologs to humans, virulence factor analysis, druggability, and broad-spectrum analysis. Additionally, metabolic differences between sensitive and resistant strains were also investigated based on flux variability analysis and pathway enrichment analysis of transcriptomic data. RESULTS: The reconstructed GSMM was named as HpM485 model. Pathway enrichment and flux variability analyses demonstrated reduced activity in the ribosomal pathway in both clarithromycin- and rifampicin-resistant strains. Also, a significant decrease was detected in the activity of metabolic pathways of clarithromycin-resistant strain. Moreover, 23 and 16 essential genes were exclusively detected in clarithromycin- and rifampicin-resistant strains, respectively. Based on prioritization analysis, cyclopropane fatty acid synthase and phosphoenolpyruvate synthase were identified as putative drug targets in clarithromycin- and rifampicin-resistant strains, respectively. CONCLUSIONS: We present a robust and reliable metabolic model of H. pylori. This model can predict novel drug targets to combat drug resistance and explore the metabolic capability of H. pylori in various conditions.

Capsaicin-Sensitive Peptidergic Sensory Nerves Are Anti-Inflammatory Gatekeepers in the Hyperacute Phase of a Mouse Rheumatoid Arthritis Model.

Capsaicin-sensitive peptidergic sensory nerves play complex, mainly protective regulatory roles in the inflammatory cascade of the joints via neuropeptide mediators, but the mechanisms of the hyperacute arthritis phase has not been investigated. Therefore, we studied the involvement of these afferents in the early, "black box" period of a rheumatoid arthritis (RA) mouse model. Capsaicin-sensitive fibres were defunctionalized by pretreatment with the ultrapotent capsaicin analog resiniferatoxin and arthritis was induced by K/BxN arthritogenic serum. Disease severity was assessed by clinical scoring, reactive oxygen species (ROS) burst by chemiluminescent, vascular permeability by fluorescent in vivo imaging. Contrast-enhanced magnetic resonance imaging was used to correlate the functional and morphological changes. After sensory desensitization, both early phase ROS-burst and vascular leakage were significantly enhanced, which was later followed by the increased clinical severity scores. Furthermore, the early vascular leakage and ROS-burst were found to be good predictors of later arthritis severity. We conclude that the anti-inflammatory role of peptidergic afferents depends on their activity in the hyperacute phase, characterized by decreased cellular and vascular inflammatory components presumably via anti-inflammatory neuropeptide release. Therefore, these fibres might serve as important gatekeepers in RA.

Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair.

Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), an ancient disease which still today causes 1.4 million deaths worldwide per year. Long-term, multi-agent anti-tubercular regimens can lead to the anticipated non-compliance of the patient and increased drug toxicity, which in turn can contribute to the emergence of drug-resistant MTB strains that are not susceptible to first- and second-line available drugs. Hence, there is an urgent need for innovative antitubercular drugs and vaccines. A number of biochemical processes are required to maintain the correct homeostasis of DNA metabolism in all organisms. Here we focused on reviewing our current knowledge and understanding of biochemical and structural aspects of relevance for drug discovery, for some such processes in MTB, and particularly DNA synthesis, synthesis of its nucleotide precursors, and processes that guarantee DNA integrity and genome stability. Overall, the area of drug discovery in DNA metabolism appears very much alive, rich of investigations and promising with respect to new antitubercular drug candidates. However, the complexity of molecular events that occur in DNA metabolic processes requires an accurate characterization of mechanistic details in order to avoid major flaws, and therefore the failure, of drug discovery approaches targeting genome integrity.

An in silico strategy for identification of novel drug targets against Plasmodium falciparum.

The apicomplexan parasite Plasmodium falciparum is responsible for global malaria burden. With the reported resistance to artemisinin chemotherapy, there is an urgent need to maintain early phase drug discovery and identify novel drug targets for successful eradication of the pathogen from the host. In our previous work on comparative genomics study for identification of putative essential genes and therapeutic candidates in P. falciparum, we predicted 11 proteins as anti-malarial drug targets from PlasmoDB database. In this paper, we made an attempt for identification of novel drug targets in P. falciparum genome using a sequence of computational methods from Malaria Parasite Metabolic Pathway database. The study reported the identification of 71 proteins as potential drug targets for anti-malarial interventions. Furthermore, homology modeling and molecular dynamic simulation study of one of the potential drug targets, aminodeoxychorismate lyase, was carried to predict the 3D structure of the protein. Structure and ligand-based drug designing reported MMV019742 from Pathogen Box and TCAMS-141515 from GSK-TCAMS library as potential hits. The reliability of the binding mode of the inhibitors is confirmed by GROMACS for a simulation time of 20 ns in water environment. This will be helpful for experimental validation of the small-molecule inhibitor.

Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp).

Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.

An Insight into the Combat Strategies for the Treatment of Type 2 Diabetes Mellitus.

Diabetes is a chronic, and metabolic disorder that has gained epidemic proportions in the past few decades creating a threat throughout the globe. It is characterized by increased glucose levels that may be due to immune-mediated disorders (T1DM), insulin resistance or inability to produce sufficient insulin by ß-pancreatic cells (T2DM), gestational, or an increasingly sedentary lifestyle. The progression of the disease is marked by several pathological changes in the body like nephropathy, retinopathy, and various cardiovascular complications. Treatment options for T1DM are majorly focused on insulin replacement therapy. While T2DM is generally treated through oral hypoglycemics that include metformin, sulfonylureas, thiazolidinediones, meglitinides, incretins, SGLT-2 inhibitors, and amylin antagonists. Multidrug therapy is often recommended when patients are found incompliant with the first-line therapy. Despite the considerable therapeutic benefits of these oral hypoglycemics, there lie greater side effects (weight variation, upset stomach, skin rashes, and risk of hepatic disease), and limitations including short half-life, frequent dosing, and differential bioavailability which inspires the researchers to pursue novel drug targets and small molecules having promising clinical efficacy posing minimum side-effects. This review summarizes some of the current emerging novel approaches along with the conventional drug targets to treat type 2 diabetes.

Glucose Transporter and Sensor Mechanisms in Fungal Pathogens as Potential Drug Targets.

Fungal infections are emerging as major health challenges in recent years. The development of resistance against existing antifungal agents needs urgent attention and action. The limited classes of antifungal drugs available, their tendency to cause adverse effects, lack of effectiveness, etc., are the major limitations of current therapy. Thus, there is a pressing demand for new antifungal drug classes to cope with the present circumstances. Glucose is the key source of energy for all organisms, including fungi. Glucose plays a crucial role as a source of carbon and energy for processes like virulence, growth, invasion, biofilm formation, and resistance development. The glucose transport and sensing mechanisms are well developed in these organisms as an important strategy to sustain survival. Modulating these transport or sensor mechanisms may serve as an important strategy to inhibit fungal growth. Moreover, the structural difference between human and fungal glucose transporters makes them more appealing as drug targets. Limited literature is available for fungal glucose entry mechanisms. This review provides a comprehensive account of sugar transport mechanisms in common fungal pathogens.

The search for novel treatment strategies for Streptococcus pneumoniae infections.

This review provides an overview of the most important novel treatment strategies against Streptococcus pneumoniae infections published over the past 10 years. The pneumococcus causes the majority of community-acquired bacterial pneumonia cases, and it is one of the prime pathogens in bacterial meningitis. Over the last 10 years, extensive research has been conducted to prevent severe pneumococcal infections, with a major focus on (i) boosting the host immune system and (ii) discovering novel antibacterials. Boosting the immune system can be done in two ways, either by actively modulating host immunity, mostly through administration of selective antibodies, or by interfering with pneumococcal virulence factors, thereby supporting the host immune system to effectively overcome an infection. While several of such experimental therapies are promising, few have evolved to clinical trials. The discovery of novel antibacterials is hampered by the high research and development costs versus the relatively low revenues for the pharmaceutical industry. Nevertheless, novel enzymatic assays and target-based drug design, allow the identification of targets and the development of novel molecules to effectively treat this life-threatening pathogen.

Neurodegenerative Pathways in Alzheimer's Disease: A Review.

Alzheimer's disease (AD) is a complex neurodegenerative disease that leads to insidious deterioration of brain functions and is considered the sixth leading cause of death in the world. Alzheimer's patients suffer from memory loss, cognitive deficit and behavioral changes; thus, they eventually follow a low-quality life. AD is considered as a multifactorial disorder involving different neuropathological mechanisms. Recent research has identified more than 20 pathological factors that are promoting disease progression. Three significant hypotheses are said to be the root cause of disease pathology, which include acetylcholine deficit, the formation of amyloid-beta senile plaques and tau protein hyperphosphorylation. Apart from these crucial factors, pathological factors such as apolipoprotein E (APOE), glycogen synthase kinase 3ß, notch signaling pathway, Wnt signaling pathway, etc., are considered to play a role in the advancement of AD and therefore could be used as targets for drug discovery and development. As of today, there is no complete cure or effective disease altering therapies for AD. The current therapy is assuring only symptomatic relief from the disease, and progressive loss of efficacy for these symptomatic treatments warrants the discovery of newer drugs by exploring these novel drug targets. A comprehensive understanding of these therapeutic targets and their neuropathological role in AD is necessary to identify novel molecules for the treatment of AD rationally.

Subtractive proteomics to identify targets for vaccine development against vancomycin-resistant Enterococcus faecalis.

Aim: Due to the increased level of vancomycin resistance in Enterococci species, an aggressive treatment involving targeted antibiotics is required to manage this frequently occurring infection. Materials & methods: Here, subtractive proteomics and reverse vaccinology approaches were employed to identify potential target and for the prediction of B cell and T cell epitopes against vancomycin-resistant Enterococcus faecalis (VRE V583). Results: The results exhibited the presence of 73 out of 805 non-homologous protein sequences in the proteome which can be employed as unique targets to develop the novel drugs and vaccine to counter the deadly infections caused by this microbe. Conclusion: The identified novel target in VRE V583 will equip our knowledge to design effective vaccine against probable protease EEP proteins.

Novel targets for drug discovery in celiac disease.

Celiac disease is a lifelong, immunological disorder induced by dietary protein-gluten, in a genetically susceptible populations, resulting in different clinical manifestations, the release of antibodies, and damage to the intestinal mucosa. The only recommended therapy for the disease is to strictly follow a gluten-free diet (GFD), which is difficult to comply with. A GFD is found to be ineffective in some active Celiac disease cases. Therefore, there is an unmet need for an alternative nondietary therapeutic approach. The review focuses on the novel drug targets for Celiac disease.

Novel drug targets for ductus arteriosus manipulation: Looking beyond prostaglandins.

Forty years ago, non-steroidal anti-inflammatory drugs were first reported to decrease systemic prostaglandin levels and promote ductus arteriosus (DA) closure. And yet, prolonged patency of the DA (PDA) remains a significant clinical problem, complicated by imperfect therapies and wide variations in treatment strategy. There are few pharmacology-based tools available for treating PDA (indomethacin, ibuprofen, and acetaminophen), or for maintaining DA patency (PGE1) as is needed to facilitate corrective surgery for ductus-dependent congenital heart defects. Unfortunately, all of these treatments are inefficient and are associated with concerning adverse effects. This review highlights novel potential DA drug targets that may expand our therapeutic repertoire beyond the prostaglandin pathway.

Pharmacological Cyclophilin Inhibitors Prevent Intoxication of Mammalian Cells with Bordetella pertussis Toxin.

The Bordetella pertussis toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.

Impact of Genomics on Clarifying the Evolutionary Relationships amongst Mycobacteria: Identification of Molecular Signatures Specific for the Tuberculosis-Complex of Bacteria with Potential Applications for Novel Diagnostics and Therapeutics.

An alarming increase in tuberculosis (TB) caused by drug-resistant strains of Mycobacterium tuberculosis has created an urgent need for new antituberculosis drugs acting via novel mechanisms. Phylogenomic and comparative genomic analyses reviewed here reveal that the TB causing bacteria comprise a small group of organisms differing from all other mycobacteria in numerous regards. Comprehensive analyses of protein sequences from mycobacterial genomes have identified 63 conserved signature inserts and deletions (indels) (CSIs) in important proteins that are distinctive characteristics of the TB-complex of bacteria. The identified CSIs provide potential means for development of novel diagnostics as well as therapeutics for the TB-complex of bacteria based on four key observations: (i) The CSIs exhibit a high degree of exclusivity towards the TB-complex of bacteria; (ii) Earlier work on CSIs provide evidence that they play important/essential functions in the organisms for which they exhibit specificity; (iii) CSIs are located in surface-exposed loops of the proteins implicated in mediating novel interactions; (iv) Homologs of the CSIs containing proteins, or the CSIs in such homologs, are generally not found in humans. Based on these characteristics, it is hypothesized that the high-throughput virtual screening for compounds binding specifically to the CSIs (or CSI containing regions) and thereby inhibiting the cellular functions of the CSIs could lead to the discovery of a novel class of drugs specifically targeting the TB-complex of organisms.

AAA+ Machines of Protein Destruction in Mycobacteria.

The bacterial cytosol is a complex mixture of macromolecules (proteins, DNA, and RNA), which collectively are responsible for an enormous array of cellular tasks. Proteins are central to most, if not all, of these tasks and as such their maintenance (commonly referred to as protein homeostasis or proteostasis) is vital for cell survival during normal and stressful conditions. The two key aspects of protein homeostasis are, (i) the correct folding and assembly of proteins (coupled with their delivery to the correct cellular location) and (ii) the timely removal of unwanted or damaged proteins from the cell, which are performed by molecular chaperones and proteases, respectively. A major class of proteins that contribute to both of these tasks are the AAA+ (ATPases associated with a variety of cellular activities) protein superfamily. Although much is known about the structure of these machines and how they function in the model Gram-negative bacterium Escherichia coli, we are only just beginning to discover the molecular details of these machines and how they function in mycobacteria. Here we review the different AAA+ machines, that contribute to proteostasis in mycobacteria. Primarily we will focus on the recent advances in the structure and function of AAA+ proteases, the substrates they recognize and the cellular pathways they control. Finally, we will discuss the recent developments related to these machines as novel drug targets.

Drug Target Exploitable Structural Features of Adenylyl Cyclase Activity in Schistosoma mansoni.

The draft genome sequence of the parasitic flatworm Schistosoma mansoni (S. mansoni), a cause of schistosomiasis, encodes a predicted guanosine triphosphate (GTP) binding protein tagged Smp_059340.1. Smp_059340.1 is predicted to be a member of the G protein alpha-s subunit responsible for regulating adenylyl cyclase activity in S. mansoni and a possible drug target against the parasite. Our structural bioinformatics analyses identified key amino acid residues (Ser53, Thr188, Asp207 and Gly210) in the two molecular switches responsible for cycling the protein between active (GTP bound) and inactive (GDP bound) states. Residue Thr188 is located on Switch I region while Gly210 is located on Switch II region with Switch II longer than Switch I. The Asp207 is located on the G3 box motif and Ser53 is the binding residue for magnesium ion. These findings offer new insights into the dynamic and functional determinants of the Smp_059340.1 protein in regulating the S. mansoni life cycle. The binding interfaces and their residues could be used as starting points for selective modulations of interactions within the pathway using small molecules, peptides or mutagenesis.

In silico identification of common putative drug targets in Leptospira interrogans.

Infectious diseases are the leading causes of death worldwide. Hence, there is a need to develop new antimicrobial agents. Traditional method of drug discovery is time consuming and yields a few drug targets with little intracellular information for guiding target selection. Thus, focus in drug development has been shifted to computational comparative genomics for identifying novel drug targets. Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. Availability of L. interrogans serovars and human genome sequences facilitated to search for novel drug targets using bioinformatics tools. The genome sequence of L. interrogans serovar Copenhageni has 5,124 genes while that of serovar Lai has 4,727 genes. Through subtractive genomic approach 218 genes in serovar Copenhageni and 158 genes in serovar Lai have been identified as putative drug targets. Comparative genomic approach had revealed that 88 drug targets were common to both the serovars. Pathway analysis using the Kyoto Encyclopaedia of Genes and Genomes revealed that 66 targets are enzymes and 22 are non-enzymes. Sixty two common drug targets were predicted to be localized in cytoplasm and 16 were surface proteins. The identified potential drug targets form a platform for further investigation in discovery of novel therapeutic compounds against Leptospira.