Gupta RD

References (7)

Title : Chlorpyrifos and parathion regulate oxidative stress differentially through the expression of paraoxonase 2 in human neuroblastoma cell - Parween_2022_Neurotoxicol__
Author(s) : Parween F , Sarker A , Gupta RD
Ref : Neurotoxicology , : , 2022
Abstract : Organophosphate (OP) compounds are frequently linked to both chronic and acute forms of nervous system disorders. Chlorpyrifos (CPF) and parathion (PA) are two of the most widely used OP insecticides throughout the world. These compounds are acetylcholinesterase inhibitors and cause a cholinergic crisis. However, there are other non-cholinergic effects of the OP compounds as well. The role of Paraoxonase 1 (PON1) in the metabolism of OP compounds is well established owing to its significant organophosphatase activity. Since PON2 has no paraoxonase activity and the level of its expression is 20-40 fold higher in the brain, in this article the role of PON2 in response to CPF and PA exposure concerning both cholinergic and non-cholinergic effects are explored. The effect of these OPs on cell viability, reactive oxygen species (ROS), PON2 gene expression, and function was studied. Glutathione level, esterase activity, and paraoxonase activity were also measured in CPF- and PA-treated IMR-32 cells. At these levels, both CPF and PA showed different impacts on IMR-32 cells. PA at higher concentrations (50-200microM) proved to be less toxic than CPF. Interestingly, induction of ROS was also lower in the case of PA-treated cells as compared to the CPF. However, PON2 protein expression was increased with the increasing concentration of PA and decreased with the increasing concentration of CPF. To explore the possible mechanism of the differential regulation of PON2 gene expression by CPF and PA, we investigated the possible binding and signaling through the human M2 muscarinic acetylcholine receptor (M2AChR). Since M2AChRs are similar to G-protein coupled receptors and function through cAMP signalling, we measured the cAMP level after CPF and PA treatment CPF- and PA-treated IMR-32 cells can be used as a model to study the mechanism by which PON2 acts as a ROS scavenger in response to xenobiotics stimulation in the brain.
ESTHER : Parween_2022_Neurotoxicol__
PubMedSearch : Parween_2022_Neurotoxicol__
PubMedID: 36058312

Title : Association between human paraoxonase 2 protein and efficacy of acetylcholinesterase inhibiting drugs used against Alzheimer's disease - Parween_2021_PLoS.One_16_e0258879
Author(s) : Parween F , Hossain MS , Singh KP , Gupta RD
Ref : PLoS ONE , 16 :e0258879 , 2021
Abstract : Serum Paraoxonase 2 (PON2) level is a potential biomarker owing to its association with a number of pathophysiological conditions such as atherosclerosis and cardiovascular disease. Since cholinergic deficiency is closely linked with Alzheimer's disease (AD) progression, acetylcholinesterase inhibitors (AChEIs) are the treatment of choice for patients with AD. However, there is a heterogenous response to these drugs and mostly the subjects do not respond to the treatment. Gene polymorphism, the simultaneous occurrence of two or more discontinuous alleles in a population, could be one of the important factors for this. Hence, we hypothesized that PON2 and its polymorphic forms may be hydrolyzing the AChEIs differently, and thus, different patients respond differently. To investigate this, two AChEIs, donepezil hydrochloride (DHC) and pyridostigmine bromide (PB), were selected. Human PON2 wildtype gene and four mutants, two catalytic sites, and two polymorphic sites were cloned, recombinantly expressed, and purified for in vitro analysis. Enzyme activity and AChE activity were measured to quantitate the amount of DHC and PB hydrolyzed by the wildtype and the mutant proteins. Herein, PON2 esterase activity and AChE inhibitor efficiency were found to be inversely related. A significant difference in enzyme activity of the catalytic site mutants was observed as compared to the wildtype, and subsequent AChE activity showed that esterase activity of PON2 is responsible for the hydrolysis of DHC and PB. Interestingly, PON2 polymorphic site mutants showed increased esterase activity; therefore, this could be the reason for the ineffectiveness of the drugs. Thus, our data suggested that the esterase activity of PON2 was mainly responsible for the hydrolysis of AChEI, DHC, and PB, and that might be responsible for the variation in individual response to AChEI therapy.
ESTHER : Parween_2021_PLoS.One_16_e0258879
PubMedSearch : Parween_2021_PLoS.One_16_e0258879
PubMedID: 34714861

Title : Organophosphorus Nerve Agents: Types, Toxicity, and Treatments - Mukherjee_2020_J.Toxicol_2020_3007984
Author(s) : Mukherjee S , Gupta RD
Ref : J Toxicol , 2020 :3007984 , 2020
Abstract : Organophosphorus compounds are extensively used worldwide as pesticides which cause great hazards to human health. Nerve agents, a subcategory of the organophosphorus compounds, have been produced and used during wars, and they have also been used in terrorist activities. These compounds possess physiological threats by interacting and inhibiting acetylcholinesterase enzyme which leads to the cholinergic crisis. After a general introduction, this review elucidates the mechanisms underlying cholinergic and noncholinergic effects of organophosphorus compounds. The conceivable treatment strategies for organophosphate poisoning are different types of bioscavengers which include stoichiometric, catalytic, and pseudocatalytic. The current research on the promising treatments specifically the catalytic bioscavengers including several wild-type organophosphate hydrolases such as paraoxonase and phosphotriesterase, phosphotriesterase-like lactonase, methyl parathion hydrolase, organophosphate acid anhydrolase, diisopropyl fluorophosphatase, human triphosphate nucleotidohydrolase, and senescence marker protein has been widely discussed. Organophosphorus compounds are reported to be the nonphysiological substrate for many mammalian organophosphate hydrolysing enzymes; therefore, the efficiency of these enzymes toward these compounds is inadequate. Hence, studies have been conducted to create mutants with an enhanced rate of hydrolysis and high specificity. Several mutants have been created by applying directed molecular evolution and/or targeted mutagenesis, and catalytic efficiency has been characterized. Generally, organophosphorus compounds are chiral in nature. The development of mutant enzymes for providing superior stereoselective degradation of toxic organophosphorus compounds has also been widely accounted for in this review. Existing enzymes have shown limited efficiency; hence, more effective treatment strategies have also been critically analyzed.
ESTHER : Mukherjee_2020_J.Toxicol_2020_3007984
PubMedSearch : Mukherjee_2020_J.Toxicol_2020_3007984
PubMedID: 33029136

Title : Directed evolution of hydrolases for prevention of G-type nerve agent intoxication - Gupta_2011_Nat.Chem.Biol_7_120
Author(s) : Gupta RD , Goldsmith M , Ashani Y , Simo Y , Mullokandov G , Bar H , Ben-David M , Leader H , Margalit R , Silman I , Sussman JL , Tawfik DS
Ref : Nat Chemical Biology , 7 :120 , 2011
Abstract : Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by approximately 10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) approximately 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
ESTHER : Gupta_2011_Nat.Chem.Biol_7_120
PubMedSearch : Gupta_2011_Nat.Chem.Biol_7_120
PubMedID: 21217689

Title : Genome sequence of Rheinheimera sp. strain A13L, isolated from Pangong Lake, India - Gupta_2011_J.Bacteriol_193_5873
Author(s) : Gupta HK , Gupta RD , Singh A , Chauhan NS , Sharma R
Ref : Journal of Bacteriology , 193 :5873 , 2011
Abstract : Rheinheimera sp. strain A13L, which has antimicrobial activity, was isolated from alkaline brackish water of the high-altitude Pangong Lake of Ladakh, India. Here we report the draft genome sequence of Rhienheimera sp. strain A13L (4,523,491 bp with a G+C content of 46.23%). The genome is predicted to contain genes for marinocine and colicin V production, which may be responsible for the antimicrobial activity of the strain.
ESTHER : Gupta_2011_J.Bacteriol_193_5873
PubMedSearch : Gupta_2011_J.Bacteriol_193_5873
PubMedID: 21742876
Gene_locus related to this paper: 9gamm-f7p184 , 9gamm-f7nsp4 , 9gamm-f7nsu1

Title : Stereo-specific synthesis of analogs of nerve agents and their utilization for selection and characterization of paraoxonase (PON1) catalytic scavengers - Ashani_2010_Chem.Biol.Interact_187_362
Author(s) : Ashani Y , Gupta RD , Goldsmith M , Silman I , Sussman JL , Tawfik DS , Leader H
Ref : Chemico-Biological Interactions , 187 :362 , 2010
Abstract : Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentially the less-toxic R(P) enantiomers of nerve agents and of their fluorogenic surrogates containing the fluorescent leaving group, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC). To increase the sensitivity and reliability of the screening protocol so as to directly select rePON1 clones displaying stereo-preference towards the toxic S(P) enantiomer, and to determine accurately K(m) and k(cat) values for the individual isomers, two approaches were used to obtain the corresponding S(P) and R(P) isomers: (a) stereo-specific synthesis of the O-ethyl, O-n-propyl, and O-i-propyl analogs and (b) enzymic resolution of a racemic mixture of O-cyclohexyl methylphosphonylated CHMC. The configurational assignments of the S(P) and R(P) isomers, as well as their optical purity, were established by X-ray diffraction, reaction with sodium fluoride, hydrolysis by selected rePON1 variants, and inhibition of AChE. The S(P) configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with S(P)/R(P) inhibition ratios of 10-100, whereas the R(P) isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the S(P) counterpart. Wt rePON1-induced R(P)/S(P) hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be >>1000. The various S(P) enantiomers of O-alkyl-methylphosphonyl esters of CHMC provide suitable ligands for screening rePON1 libraries, and can expedite identification of variants with enhanced catalytic proficiency towards the toxic nerve agents.
ESTHER : Ashani_2010_Chem.Biol.Interact_187_362
PubMedSearch : Ashani_2010_Chem.Biol.Interact_187_362
PubMedID: 20303930

Title : Directed enzyme evolution via small and effective neutral drift libraries - Gupta_2008_Nat.Methods_5_939
Author(s) : Gupta RD , Tawfik DS
Ref : Nat Methods , 5 :939 , 2008
Abstract : Small libraries for directed evolution can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants. We describe methods for preparing such libraries, using serum paraoxonase (PON1). An optimized GFP variant fused to PON1 reported levels of soluble, functional enzyme, enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities toward several substrates, including toxic organophosphates.
ESTHER : Gupta_2008_Nat.Methods_5_939
PubMedSearch : Gupta_2008_Nat.Methods_5_939
PubMedID: 18931667