Title: Analysis of suppressive effects of pesticide triazophos on leucocyte immune responses in a teleost, Channa Punctatus Bhardwaj AK, Chandra RK, Tripathi MK Ref: Drug & Chemical Toxicology, :1, 2021 : PubMed
Triazophos is a commonly used organophosphate insecticide, which inhibits the acetylcholinesterase enzyme and causes paralysis and death of insects. Impact of the pesticides on immunity has scarcely been investigated, especially in fishes. The present study was designed to analyze the immunotoxic role of in vitro triazophos exposure to the leucocytes in freshwater teleost, Channa punctatus. Triazophos, at in vitro concentrations of 0.1, 0.5, and 1 microg ml(-1), was used to study leucocyte phagocytosis, superoxide production, nitrite release, and lymphocyte proliferation. Dose-dependent suppression of various immune responses was observed. Nitrite release and superoxide production by leucocytes were reduced in cultures incubated with triazophos. Mitogen-induced lymphocyte proliferation was significantly reduced at 0.5 and 1 microg ml(-1) but not at 0.1 microg ml(-1) concentration of pesticide. The biphasic suppressive effect was also discovered while evaluating phagocytic response. These investigations describe the effects of pesticide on immune responses in C. punctatus, which are helpful in understanding the immunotoxicity in fish. Substantially more researches are required to help design the measures to combat ecotoxicity in freshwater bodies.
Title: Design, synthesis, and evaluation of N-benzylpyrrolidine and 1,3,4-oxadiazole as multitargeted hybrids for the treatment of Alzheimer's disease Choubey PK, Tripathi A, Tripathi MK, Seth A, Shrivastava SK Ref: Bioorg Chem, 111:104922, 2021 : PubMed
Novel N-Benzylpyrrolidine hybrids were designed, synthesized, and tested against multiple in-vitro and in-vivo parameters. Among all the synthesized molecules, 8f and 12f showed extensive inhibition against beta-secretase-1 (hBACE-1), human acetylcholinesterase (hAChE) & human butyrylcholinesterase (hBuChE). These molecules are also endowed with significant AChE-peripheral anionic site (PAS) binding capability, blood-brain barrier permeability, potential disassembly of Abeta aggregates along with neuroprotection ability on SHSY-5Y cell lines. Results of the Y-Maze and Morris water maze test concluded that compounds 8f and 12f ameliorated cognitive dysfunction induced by scopolamine and Abeta. The ex-vivo activity was executed on rat's brain homogenate indicating a reduction in AChE level and oxidative stress. The pharmacokinetic investigation ascertained considerable oral absorption profile of the lead 12f. The results of the in silico docking studies and molecular dynamics simulations demonstrated stable interactions of compounds 8f and 12f with the target residues of hAChE, hBuChE and hBACE-1.
Title: Cholinesterase as a Target for Drug Development in Alzheimer's Disease Sharma P, Tripathi MK, Shrivastava SK Ref: Methods Mol Biol, 2089:257, 2020 : PubMed
Alzheimer's disease (AD) is an enormous healthcare challenge, and 50 million people are currently suffering from it. There are several pathophysiological mechanisms involved, but cholinesterase inhibitors remained the major target from the last 2-3 decades. Among four available therapeutics (donepezil, rivastigmine, galantamine, and memantine), three of them are cholinesterase inhibitors. Herein, we describe the role of acetylcholine sterase (AChE) and related hypothesis in AD along with the pharmacological and chemical aspects of the available cholinesterase inhibitors. This chapter discusses the development of several congeners and hybrids of available cholinesterase inhibitors along with their binding patterns in enzyme active sites.
The cholinesterases are essential targets implicated in the pathogenesis of Alzheimer's disease (AD). In the present study, virtual screening and molecular docking are performed to identify the potential hits. Docking-post processing (DPP) and pose filtration protocols against AChE and BChE resulted in three hits (AW00308, HTS04089, and JFD03947). Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) and molecular dynamics simulation analysis affirmed the stability and binding pattern of the docked complex JFD03947, which was further synthesized and evaluated for in vitro cholinesterase inhibition (AChE, IC50 = 0.062 microM; BChE, IC50 = 1.482 microM) activity. The enzyme kinetics study of the JFD03947 against hAChE and hBChE suggested a mixed type of inhibition. The results of thioflavin T-assay also elicited anti-Abeta aggregation activity by JFD03947. Further, biological evaluation of identified compound JFD03947 also showed neuroprotective ability against the SH-SY5Y neuroblastoma cell lines.
The multitarget-directed strategy offers an effective and promising paradigm to treat the complex neurodegenerative disorder, such as Alzheimer's disease (AD). Herein, a series of N-benzylpiperidine analogs (17-31 and 32-46) were designed and synthesized as multi-functional inhibitors of acetylcholinesterase (AChE) and beta-secretase-1 (BACE-1) with moderate to excellent inhibitory activities. Among the tested inhibitors, 25, 26, 40, and 41 presented the most significant and balanced inhibition against both the targets. Compounds 40 and 41 exhibited high brain permeability in the PAMPA-BBB assay, significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE, and were devoid of neurotoxicity towards SH-SY5Y neuroblastoma cell lines up to the maximum tested concentration of 80muM. Meanwhile, both these compounds inhibited self- and AChE-induced Abeta aggregation in thioflavin T assay, which was also re-affirmed by morphological characterization of Abeta aggregates using atomic force microscopy (AFM). Moreover, 40 and 41 ameliorated the scopolamine-induced cognitive impairment in elevated plus and Y-maze experiments. Ex vivo and biochemical analysis established the brain AChE inhibitory potential and antioxidant properties of these compounds. Further, improvement in Abeta1-42-induced cognitive impairment was also observed by compound 41 in the Morris water maze experiment with significant oral absorption characteristics ascertained by the pharmacokinetic studies.
The diverse nature of Alzheimer's disease (AD) has prompted researchers to develop multi-functional agents. Herein, we have designed and synthesized molecular hybrids of 2-pyridylpiperazine and 5-phenyl-1,3,4-oxadiazoles. Biological activities of synthesized compounds suggested significant and balanced inhibitory potential against target enzymes. In particular, compound 49 containing 2,4-difluoro substitution at terminal phenyl ring considered as most potential lead with inhibition of acetylcholinesterase (hAChE, IC50=0.054muM), butyrylcholinesterase (hBChE, IC50=0.787muM) and beta-secretase-1 (hBACE-1, IC50=0.098muM). The enzyme kinetics study of 49 against hAChE suggested a mixed type of inhibition (Ki=0.030muM). Also, 48 and 49 showed significant displacement of propidium iodide from the peripheral anionic site (PAS) of hAChE, excellent blood-brain barrier (BBB) permeability in parallel artificial membrane permeation assay (PAMPA), and neuroprotective ability against SH-SY5Y neuroblastoma cell lines. Further, 49 also exhibited anti-Abeta aggregation activity in self- and AChE-induced thioflavin T assay, which was ascertained by morphological characterization by atomic force microscopy (AFM). Moreover, in vivo behavioral studies signified learning and memory improvement by compound 49 in scopolamine- and Abeta-induced cognitive dysfunctions performed on Y-maze and Morris water maze. The ex vivo studies suggested decreased AChE activity and antioxidant potential of compound 49, with good oral absorption characteristics ascertained by pharmacokinetic studies.
Based on the quantitative structure-activity relationship (QSAR), some novel p-aminobenzoic acid derivatives as promising cholinesterase enzyme inhibitors were designed, synthesized, characterized and evaluated to enhance learning and memory. The in vitro enzyme kinetic study of the synthesized compounds revealed the type of inhibition on the respective acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The in vivo studies of the synthesized compounds exhibited significant reversal of cognitive deficits in the animal models of amnesia as compared to standard drug donepezil. Further, the ex vivo studies in the specific brain regions like the hippocampus, hypothalamus, and prefrontal cortex regions also exhibited AChE inhibition comparable to standard donepezil. The in silico molecular docking and dynamics simulations studies of the most potent compound 22 revealed the consensual interactions at the active site pocket of the AChE.
A series of novel piperazine tethered biphenyl-3-oxo-1,2,4-triazine derivatives were designed, and synthesized. Amongst the synthesized analogs, compound 6g showed significant non-competitive inhibitory potential against acetylcholinesterase (AChE, IC50; 0.2+/-0.01muM) compared to standard donepezil (AChE, IC50: 0.1+/-0.002muM). Compound 6g also exhibited significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE (22.22+/-1.11%) and showed good CNS permeability in PAMPA-BBB assay (Pe(exp), 6.93+/-0.46). The in vivo behavioral studies of compound 6g indicated significant improvement in cognitive dysfunctions against scopolamine-induced amnesia mouse models. Further, ex vivo studies showed a significant AChE inhibition and reversal of the scopolamine-induced oxidative stress by compound 6g. Moreover, molecular docking and dynamics simulations of compound 6g showed a consensual binding affinity and active site interactions with the PAS and active catalytic site (CAS) residues of AChE.
Title: Cloning, sequencing and structural features of a novel Streptococcus lipase Tripathi MK, Roy U, Jinwal UK, Jain SK, Roy PK Ref: Enzyme Microb Technol, 34:437, 2004 : PubMed
An expression library generated from genomic DNA of Streptococcus sp. N1 resulted in C1N1 Escherichia coli recombinants forming haloes on lipase indicator tributyrin/Tween-80 agar plates. The excised insert in the recombinant C1N1 was of 3,489 kb size. Analysis of the nucleotide sequence of the insert showed two significant operons of 1032 bp (ORF1) and 729 bp (ORF3). ORF1 on the (+)-strand is the structural lipase gene codes for a protein of 343 amino acid residues with a theoretical mass of 38.6 kDa, which is close to the value of the lipase expressed in E. coli (39.36 kDa). The primary structure of the lipase deduced from the nucleotide sequence shows a consensus sequence containing the active serine [VAGHSIGG], a conserved H-G dipeptide in the N-terminal part of the enzyme and a potential site for N-linked glycosylation at amino acid residues [129-131]. The sequence, AAGGA at -6 upstream of the start codon ATG seems to be putative ribosomal binding site (rbs) and is similar to the rbs of lip3 of Moraxella sp. TA 144. The putative-10[TTTAT] and -35[AATG] promoter sequences showed similarity to lip3 of Moraxella sp. TA 144. The lipase shared no significant homology with the members of different lipases families except with group V comprising of Psychrobacter immobilis B-10 [28% identical and 56% positive similarity] and Moraxella sp. TA 144, lip3 [28% identical and 54% similarities]. The 729bp operon present on (-) strand of 3.489 kb C1N1 insert was essential for the secretion of the enzyme. The putative protein on BLAST/FASTA search showed 57% identities and 72% positives with ATP-binding component of ABC-transporter. The recombinant lipase was resistant to organic solvents, non-ionic surfactants, metal ions and showed alkaline pH optima (pH 8.4).
The stereocontrolled reduction of 3-aryl-5-acetylisoxazolines (1) to the corresponding alcohols (2 and 3) in the presence of four different yeast strains, recognized as Baker's yeast (commercial), Candida krusei (ATCC 14243), Pichia farinosa (NRRL Y110) and Sacchromyces sp. (soil isolate) have been attempted. The C. krusei was found to be diastereoselective for the (R)-1 while the Sacchromyces sp. led to complete reduction to yield the RS- and SS-alcohol in 1:1 ratio at 10 g/L scale.
