Neuroinflammatory Responses and Blood-Brain Barrier Injury in Chronic Alcohol Exposure: Role of Purinergic P2X7 Receptor Signaling.

Alcohol consumption leads to neuroinflammation and blood-brain barrier (BBB) damage, resulting in neurological impairment. We previously demonstrated that ethanol-induced disruption of barrier function in human brain endothelial cells was associated with mitochondrial injury, increased ATP and extracellular vesicle (EV) release, and purinergic receptor P2X7R activation. Therefore, we aimed to evaluate the effect of P2X7r blockade on peripheral and neuro-inflammation in EtOH-exposed mice. In a chronic intermittent ethanol (CIE)-exposed mouse model, P2X7R was inhibited by two different methods: Brilliant Blue G (BBG) or gene knockout. We assessed blood ethanol concentration (BEC), plasma P2X7R and P-gp, number of extra-cellular vesicles (EV), serum ATP and EV-ATP levels. Brain microvessel gene expression and EV mtDNA copy numbers were measured by RT2 PCR array and digital PCR, respectively. A RT2 PCR array of brain microvessels revealed significant upregulation of proinflammatory genes involved in apoptosis, vasodilation, and platelet activation in CIE-exposed animals, which were decreased 15-50-fold in BBG-treated CIE-exposed animals. Plasma P-gp levels and serum P2X7R shedding were significantly increased in CIE-exposed animals. Pharmacological or genetic suppression of P2X7R decreased P2X7R shedding to levels equivalent to those in control group. The increase in EV number and EV-ATP content in the CIE-exposed mice was significantly reduced by P2X7R inhibition. CIE mice showed augmented EV-mtDNA copy numbers which were reduced in EVs after P2X7R inhibition or receptor knockout. These observations suggested that P2X7R signaling plays a critical role in ethanol-induced brain injury. Increased eATP, EV-ATP, EV numbers, and EV-mtDNA copy numbers highlight a new mechanism of brain injury during alcohol exposure via P2X7R and biomarkers of such damage. In this study, for the first time, we report the in vivo involvement of P2X7R signaling in CIE-induced brain injury.

Michael Adduct of Sulfonamide Chalcone Targets Folate Metabolism in Brugia Malayi Parasite.

A series of Michael adducts of malononitrile and sulfonamide chalcones were synthesized, characterized, and evaluated for their antifilarial activity. Out of 14 compounds, N-(4-(4,4-dicyano-3-p-tolylbutanoyl)phenyl)benzenesulfonamide showed favorable drug-likeness properties with marked antifilarial effects at micro-molar dosages. Apoptosis in Brugia malayi microfilariae was confirmed by EB/AO staining, MTT assay, and cytoplasmic cytochrome c ELISA. Since chalcone and folate synthesis pathways share the same substrate, we hypothesize a structural analogy-based inhibition of folate metabolism by this compound. Molecular docking against a pre-validated BmDHFR protein showed more favorable thermodynamic parameters than a positive control, epicatechin-3-gallate. The compound significantly suppressed the DHFR activity in a parasite extract in vitro. Our hypothesis is also supported by a significant reversal of DHFR inhibition by folate addition, which indicated a plausible mechanism of competitive inhibition. These results demonstrate that targeting filarial folate metabolism through DHFR with consequent apoptosis induction might be rewarding for therapeutic intervention. This study reveals a novel rationale of the structural analogy-based competitive inhibition of DHFR by Michael adducts of sulfonamide chalcones.

Implications of Fragment-Based Drug Discovery in Tuberculosis and HIV.

Tuberculosis (TB) remains a global health problem and the emergence of HIV has further worsened it. Long chemotherapy and the emergence of drug-resistance strains of Mycobacterium tuberculosis as well as HIV has aggravated the problem. This demands urgent the need to develop new anti-tuberculosis and antiretrovirals to treat TB and HIV. The lack of diversity in drugs designed using traditional approaches is a major disadvantage and limits the treatment options. Therefore, new technologies and approaches are required to solve the current issues and enhance the production of drugs. Interestingly, fragment-based drug discovery (FBDD) has gained an advantage over high-throughput screenings as FBDD has enabled rapid and efficient progress to develop potent small molecule compounds that specifically bind to the target. Several potent inhibitor compounds of various targets have been developed using FBDD approach and some of them are under progression to clinical trials. In this review, we emphasize some of the important targets of mycobacteria and HIV. We also discussed about the target-based druggable molecules that are identified using the FBDD approach, use of these druggable molecules to identify novel binding sites on the target and assays used to evaluate inhibitory activities of these identified druggable molecules on the biological activity of the targets.

Fragment-Based Drug Discovery against Mycobacteria: The Success and Challenges.

The emergence of drug-resistant mycobacteria, including Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM), poses an increasing global threat that urgently demands the development of new potent anti-mycobacterial drugs. One of the approaches toward the identification of new drugs is fragment-based drug discovery (FBDD), which is the most ingenious among other drug discovery models, such as structure-based drug design (SBDD) and high-throughput screening. Specialized techniques, such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and many others, are part of the drug discovery approach to combat the Mtb and NTM global menaces. Moreover, the primary drawbacks of traditional methods, such as the limited measurement of biomolecular toxicity and uncertain bioavailability evaluation, are successfully overcome by the FBDD approach. The current review focuses on the recognition of fragment-based drug discovery as a popular approach using virtual, computational, and biophysical methods to identify potent fragment molecules. FBDD focuses on designing optimal inhibitors against potential therapeutic targets of NTM and Mtb (PurC, ArgB, MmpL3, and TrmD). Additionally, we have elaborated on the challenges associated with the FBDD approach in the identification and development of novel compounds. Insights into the applications and overcoming the challenges of FBDD approaches will aid in the identification of potential therapeutic compounds to treat drug-sensitive and drug-resistant NTMs and Mtb infections.

In vitro apoptotic effect on human lymphatic filarial parasite by piperidine derivatives and thymidine reversal study.

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.

SXP-RAL Family Filarial Protein, rWbL2, Prevents Development of DSS-Induced Acute Ulcerative Colitis.

Helminthic infections lead to the release of various molecules which play an important role in modulation of the host immune system. Such filarial proteins with immunomodulatory potential can be used for therapeutic purpose in inflammatory and immune mediated diseases. In the present study, we have explored the prophylactic effect of filarial SXP-RAL family protein of Wuchereria bancrofti i.e. rWbL2 protein in DSS induced inflammatory ulcerative colitis in a mouse model. Prior treatment of rWbL2, followed by induction of colitis, showed significantly reduced disease severity as indicated by the decreased disease manifestations and improved macroscopic and microscopic inflammation. This preventive effect was found to be associated with increased release of anti-inflammatory cytokine IL-10 and decreased release of proinflammatory cytokines IFN-γ, TNF-α, IL-6 and IL-17 by the splenocytes of treated mice. From this study, it can be envisaged that pretreatment with filarial protein, rWbL2, can prevent the establishment of ulcerative colitis in BALB/c mice. The underlying immunological mechanism may involve the up-regulation of Th2 immune response with down-regulation of Th1 response.