Showing papers by "Central Drug Research Institute published in 2019"

Metabolic Insight of Neutrophils in Health and Disease.

Abstract: Neutrophils are the most abundant, short lived, and terminally differentiated leukocytes with distinct tiers of arsenals to counter pathogens. Neutrophils were traditionally considered transcriptionally inactive cells, but recent researches in the field led to a paradigm shift in neutrophil biology and revealed subpopulation heterogeneity, and functions pivotal to immunity and inflammation. Furthermore, recent unfolding of metabolic plasticity in neutrophils has challenged the long-standing concept of their sole dependence on glycolytic pathway. Metabolic adaptations and distinct regulations have been identified which are critical for neutrophil differentiation and functions. The metabolic reprogramming of neutrophils by inflammatory mediators or during pathologies such as sepsis, diabetes, glucose-6-phosphate dehydrogenase deficiency, glycogen storage diseases (GSDs), systemic lupus erythematosus (SLE), rheumatoid arthritis, and cancer are now being explored. In this review, we discuss recent developments in understanding of the metabolic regulation, that may provide clues for better management and newer therapeutic opportunities for neutrophil centric immuno-deficiencies and inflammatory disorders.

Mitochondria Permeability Transition versus Necroptosis in Oxalate-Induced AKI.

Abstract: Background Serum oxalate levels suddenly increase with certain dietary exposures or ethylene glycol poisoning and are a well known cause of AKI. Established contributors to oxalate crystal–induced renal necroinflammation include the NACHT, LRR and PYD domains-containing protein-3 (NLRP3) inflammasome and mixed lineage kinase domain-like (MLKL) protein–dependent tubule necroptosis. These studies examined the role of a novel form of necrosis triggered by altered mitochondrial function. Methods To better understand the molecular pathophysiology of oxalate-induced AIK, we conducted in vitro studies in mouse and human kidney cells and in vivo studies in mice, including wild-type mice and knockout mice deficient in peptidylprolyl isomerase F (Ppif) or deficient in both Ppif and Mlkl. Results Crystals of calcium oxalate, monosodium urate, or calcium pyrophosphate dihydrate, as well as silica microparticles, triggered cell necrosis involving PPIF–dependent mitochondrial permeability transition. This process involves crystal phagocytosis, lysosomal cathepsin leakage, and increased release of reactive oxygen species. Mice with acute oxalosis displayed calcium oxalate crystals inside distal tubular epithelial cells associated with mitochondrial changes characteristic of mitochondrial permeability transition. Mice lacking Ppif or Mlkl or given an inhibitor of mitochondrial permeability transition displayed attenuated oxalate-induced AKI. Dual genetic deletion of Ppif and Mlkl or pharmaceutical inhibition of necroptosis was partially redundant, implying interlinked roles of these two pathways of regulated necrosis in acute oxalosis. Similarly, inhibition of mitochondrial permeability transition suppressed crystal-induced cell death in primary human tubular epithelial cells. PPIF and phosphorylated MLKL localized to injured tubules in diagnostic human kidney biopsies of oxalosis-related AKI. Conclusions Mitochondrial permeability transition–related regulated necrosis and necroptosis both contribute to oxalate-induced AKI, identifying PPIF as a potential molecular target for renoprotective intervention.

AT2R Activation Prevents Microglia Pro-inflammatory Activation in a NOX-Dependent Manner: Inhibition of PKC Activation and p47 phox Phosphorylation by PP2A

Abstract: Microglia-induced reactive oxygen species (ROS) production and inflammation play an imperative role in neurodegenerative diseases like Alzheimer's disease (AD) and Parkinson's disease (PD). It has been established that angiotensin II type-2 receptor (AT2R) activation is neuroprotective in central nervous system diseases like stroke and AD. However, the involvement of AT2R in NADPH oxidase (NOX)-mediated microglia activation is still elusive. Therefore, the present study investigated the role of AT2R in angiotensin II (Ang II) or Phorbol 12-myristate 13-acetate (PMA)-induced microglia activation in BV2 cells, primary microglia, p47phox knockout (p47KO) microglia, and in vivo. Treatment of microglia with Ang II or PMA induced a significant ROS generation and promoted pro-inflammatory microglia in a NOX-dependent manner. In contrast, AT2R activation by CGP42112A (CGP) inhibited NOX activation, ROS production, and pro-inflammatory microglia activation, while promoting the immunoregulatory microglia. This inhibitory effect of AT2R on NOX and pro-inflammatory activation was attenuated by AT2R antagonist, PD123319. Essentially, NOX inhibition (by DPI) or scavenging cellular ROS (by NAC) or p47KO microglia were immune to Ang II- or PMA-induced pro-inflammatory microglia activation. Mechanistically, AT2R, via activation of protein phosphatase-2A (PP2A), prevented the Ang II- or PMA-induced protein kinase C (PKC) activation and phosphorylation of p47phox, an effect that was reversed by the addition of PP2A inhibitor, Okadaic acid (OA). Importantly, PKC inhibitor, Rottlerin, inhibited the Ang II- or PMA-induced p47phox phosphorylation and ROS generation to the similar extent as AT2R activation. In addition, AT2R activation or p47KO prevented ROS production, pro-inflammatory microglial activation, and sickness behavior in mice model of neuroinflammation. Therefore, the present findings suggested that AT2R, via PP2A-mediated inhibition of PKC, prevents the NOX activation, ROS generation, and subsequent pro-inflammatory activation of microglia.

Inducible nitric oxide synthase: An asset to neutrophils.

Abstract: Neutrophils play a key role in innate immune responses against foreign intrusion and influence the subsequent instigation of adaptive immune response. Nitric oxide (NO) synthesized by neutrophil nitric oxide synthase (NOS) profoundly modulates their diverse physiological responsibilities furthermore encompassing pathological implications. Neutrophils are the active participants in diverse inflammatory and cardiovascular disorders but neutrophil nitric oxide synthase (NOS) remains enigmatic on various aspects. This review focuses on inducible NOS (iNOS) and makes an attempt to address its potential impact in neutrophil pathophysiology, their differentiation, functionality, and survival. We described the scenario from its expressional modulation, by pro- and anti-inflammatory cytokines governing the extent and duration of neutrophil immune response, to iNOS catalysis, the intracellular compartmentalization, and protein-protein interactions determining its microenvironment, activity and its contribution as a potential signaling protein apart from its role as signal transducer. Further, the relevance of investigating the unexplored facets of iNOS biology in neutrophils and possible prototypes of iNOS regulation is also exemplified in related cellular systems.

Mycobacterial tuberculosis Enzyme Targets and their Inhibitors

Abstract: Tuberculosis (TB) still continues to be a major killer disease worldwide. Unlike other bacteria Mycobacterium tuberculosis (Mtb) has the ability to become dormant within the host and to develop resistance. Hence efforts are being made to overcome these problems by searching for new antitubercular agents which may be useful in the treatment of multidrug-(MDR) and extensively drugresistant (XDR) M. tuberculosis and shortening the treatment time. The recent introduction of bedaquiline to treat MDR-TB and XDR-TB may improve the status of TB treatment. The target enzymes in anti-TB drug discovery programs play a key role, hence efforts have been made to review the work on molecules including antiTB drugs acting on different enzyme targets including ATP synthase, the target for bedaquiline. Literature searches have been carried out to find the different chemical molecules including drugs and their molecular targets responsible for their antitubercular activities in recent years. This review provides an overview of the chemical structures with their antitubercular activities and enzyme targets like InhA, ATP synthase, Lip Y, transmembrane transport protein large (MmpL3), and decaprenylphospho-β-D-ribofuranose 2-oxidase, (DprE1). The major focus has been on the new target ATP synthase. Such an attempt may be useful in designing new chemical entities (NCEs) for specific and multi-drug targeting against Mtb.

Triacylglycerols: Fuelling the Hibernating Mycobacterium tuberculosis.

Abstract: Mycobacterium tuberculosis (Mtb) has the remarkable ability to persist with a modified metabolic status and phenotypic drug tolerance for long periods in the host without producing symptoms of active tuberculosis. These persisters may reactivate to cause active disease when the immune system becomes disrupted or compromised. Thus, the infected hosts with the persisters serve as natural reservoir of the deadly pathogen. Understanding the host and bacterial factors contributing to Mtb persistence is important to devise strategies to tackle the Mtb persisters. Host lipids act as the major source of carbon and energy for Mtb. Fatty acids derived from the host cells are converted to triacylglycerols (triglycerides or TAG) and stored in the bacterial cytoplasm. TAG serves as a dependable, long-term energy source of lesser molecular mass than other storage molecules like glycogen. TAG are found in substantial amounts in the mycobacterial cell wall. This review discusses the production, accumulation and possible roles of TAG in mycobacteria, pointing out the aspects that remain to be explored. Finally, the essentiality of TAG synthesis for Mtb is discussed with implications for identification of intervention strategies.

Polyphenolic flavonoid (Myricetin) upregulated proteasomal degradation mechanisms: Eliminates neurodegenerative proteins aggregation.

Abstract: Major neurodegenerative disorders are characterized by the formation of misfolded proteins aggregates inside or outside the neuronal cells. Previous studies suggest that aberrant proteins aggregates play a critical role in protein homeostasis imbalance and failure of protein quality control (PQC) mechanism, leading to disease conditions. However, we still do not understand the precise mechanisms of PQC failure and cellular dysfunctions associated with neurodegenerative diseases caused by the accumulation of protein aggregates. Here, we show that Myricetin, a flavonoid, can eliminate various abnormal proteins from the cellular environment via modulating endogenous levels of Hsp70 chaperone and quality control (QC)-E3 ubiquitin ligase E6-AP. We have observed that Myricetin treatment suppresses the aggregation of different aberrant proteins. Myricetin also enhances the elimination of various toxic neurodegenerative diseases associated proteins from the cells, which could be reversed by the addition of putative proteasome inhibitor (MG132). Remarkably, Myricetin can also stabilize E6-AP and reduce the misfolded proteins inclusions, which further alleviates cytotoxicity. Taken together these findings suggested that new mechanistic and therapeutic insights based on small molecules mediated regulation of disturbed protein quality control mechanism, which may result in the maintenance of the state of proteostasis.

Statins Promote Interleukin-1β-Dependent Adipocyte Insulin Resistance Through Lower Prenylation, Not Cholesterol.

Abstract: Statins lower cholesterol and adverse cardiovascular outcomes, but this drug class increases diabetes risk. Statins are generally anti-inflammatory. However, statins can promote inflammasome-mediated adipose tissue inflammation and insulin resistance through an unidentified immune effector. Statins lower mevalonate pathway intermediates beyond cholesterol, but it is unknown whether lower cholesterol underpins statin-mediated insulin resistance. We sought to define the mevalonate pathway metabolites and immune effectors that propagate statin-induced adipose insulin resistance. We found that LDL cholesterol lowering was dispensable, but statin-induced lowering of isoprenoids required for protein prenylation triggered NLRP3/caspase-1 inflammasome activation and interleukin-1β (IL-1β)-dependent insulin resistance in adipose tissue. Multiple statins impaired insulin action at the level of Akt/protein kinase B signaling in mouse adipose tissue. Providing geranylgeranyl isoprenoids or inhibiting caspase-1 prevented statin-induced defects in insulin signaling. Atorvastatin (Lipitor) impaired insulin signaling in adipose tissue from wild-type and IL-18-/- mice, but not IL-1β-/- mice. Atorvastatin decreased cell-autonomous insulin-stimulated lipogenesis but did not alter lipolysis or glucose uptake in 3T3-L1 adipocytes. Our results show that statin lowering of prenylation isoprenoids activates caspase-1/IL-1β inflammasome responses that impair endocrine control of adipocyte lipogenesis. This may allow the targeting of cholesterol-independent statin side effects on adipose lipid handling without compromising the blood lipid/cholesterol-lowering effects of statins.

Dendrimer-Based Nanocarriers in Lung Cancer Therapy

Abstract: The development of lung cancer treatment has urged the delivery of chemotherapeutics and diagnostic agents to the lungs and their adjacent lymph nodes. The unavailability of a proper tool in current chemotherapy or diagnostic procedures leads to a lack of diagnosis at the early stage of lung cancer, which ultimately leads to it becoming the leading cause of cancer-related deaths worldwide. The introduction of dendrimers through the extension of nanotechnological research has brought a multifunctional nanotherapeutic or diagnostic system to the formulation scientists. Dendrimers contribute to encapsulation and solubilization of chemotherapeutics, active targeting via tunable surface properties, and passive targeting which leads to the development of suitable therapeutic and diagnostic tools in cancer treatment. This chapter provides a broad perspective of dendrimer-based tools in the treatment of lung cancer. Overall, the utility and potential of this nano-delivery tool are predicted to make it a prospective tool in the improvement of the quality of life of lung cancer patients.

Quorum sensing pathways in Gram-positive and -negative bacteria: potential of their interruption in abating drug resistance.

Abstract: Quorum sensing (QS) is an inter-cell communication between bacterial populations through release of tiny diffusible compounds as signalling agents, called auto-inducers, abetting bacteria to track population density. QS allows bacterial population to perform collectively in coordination to wide phenotypes like alterations in expression of virulence genes to achieve advancement over their competitors, drug resistance and biofilm formation. Several classes of autoinducers have been described that are involved in bacterial virulence. This review gives an insight into the multitudinous QS systems in Gram-positive and Gram-negative bacteria to explore their role in microbial physiology and pathogenesis. Bacterial resistance to antibiotics has clinically become a super challenge. Strategies to interrupt QS pathways by natural and synthetic QS inhibitors or quorum quenchers or analogs provide a potential treatment. We highlight the advancements in discovery of promising new targets for development of next generation antimicrobials to control infections caused by multidrug resistant bacterial pathogens.

Repurposing disulfiram to target infections caused by non-tuberculous mycobacteria.

Abstract: BACKGROUND Non-tuberculous mycobacteria are emerging pathogens of significant worldwide interest because they have inherent drug resistance to a wide variety of FDA-approved drugs and cause a broad range of serious infections. In order to identify new drugs active against non-tuberculous mycobacteria, we identified disulfiram, utilized for treatment of alcohol dependence, as exhibiting potent growth-inhibitory activity against non-tuberculous mycobacteria. METHODS Whole-cell growth inhibition assays were used to screen and identify novel inhibitors. The hit compounds were tested against Vero cells to determine the selectivity index, and this was followed by determining time-kill kinetics against Mycobacterium fortuitum and Mycobacterium abscessus. Disulfiram's ability to synergize with several approved drugs utilized for the treatment of M. fortuitum and M. abscessus was determined using fractional inhibitory concentration indexes followed by determining its ability to reduce mycobacterial infections ex vivo. Finally, disulfiram's in vivo potential was determined in a neutropenic murine model mimicking mycobacterial infection. RESULTS We identified disulfiram as possessing potent antimicrobial activity against non-tuberculous mycobacteria. Disulfiram exhibited concentration- and time-dependent bactericidal activity against M. fortuitum as well as against M. abscessus and synergized with all drugs utilized for their treatment. Additionally, disulfiram reduced bacterial load in macrophages in an intracellular killing assay better than amikacin. When tested in a murine neutropenic M. fortuitum infection model, disulfiram caused significant reduction in bacterial load in kidneys. CONCLUSIONS Disulfiram exhibits all properties required for it to be repositioned as a novel anti-mycobacterial therapy and possesses a potentially new mechanism of action. Thus, it can be considered as a potent structural lead for the treatment of non-tuberculous mycobacterial infections.