02147nas a2200193 4500008004100000245010200041210006900143260000900212300001200221490000700233520143900240100002701679700002301706700002401729700002501753700002701778700002501805856012301830 2020 eng d00aDevelopment of multiple reaction monitoring assay for quantification of carnosine in human plasma0 aDevelopment of multiple reaction monitoring assay for quantifica cJAN  a763-7690 v103 a

Carnosine, a histidine containing dipeptide, exerts beneficial effects by scavenging reactive carbonyl compounds (RCCs) that are implicated in pathogenesis of diabetes. However, the reduced carnosine levels may aggravate the severity of diabetes. The precise quantification of carnosine levels may serve as an indicator of pathophysiological state of diabetes. Therefore, we have developed a highly sensitive targeted multiple reaction monitoring (MRM) method for quantification of carnosine in human plasma samples. Various mass spectrometry parameters such as ionization of precursor, fragment abundance and stability, collision energy, tube lens offset voltage were optimized to develop a sensitive and robust assay. Using the optimized MRM assay, the lower limit of detection (LOD) and limit of quantification (LOQ) for carnosine were found to be 0.4 nM and 1.0 nM respectively. Standard curves were constructed ranging from 1.0 nM to 15.0 mu M and the levels of carnosine in mice and human plasma were determined. Further, the MRM assay was extended to study carnosine hydrolyzing activity of human carnosinases, the serum carnosinase (CN1) and the cytosolic carnosinase (CN2). CN1 showed three folds higher activity than CN2. The MRM assay developed in this study is highly sensitive and can be used for basal plasma carnosine quantification, which can be developed as a novel marker for scavenging of RCCs in diabetes.

1 aPandya, Vaibhav, Kumar1 aSonwane, Babasaheb1 aRathore, Rajeshwari1 aUnnikrishnan, A., G.1 aKumaran, Sangaralingam1 aKulkarni, Mahesh, J. uhttp://library.ncl.res.in/content/development-multiple-reaction-monitoring-assay-quantification-carnosine-human-plasma02931nas a2200313 4500008004100000022001400041245013200055210006900187260000800256520189100264653001602155653001302171653001102184653000902195653003302204653002302237653000902260653002102269100002202290700002502312700002302337700001902360700002602379700002702405700001702432700002002449700002502469856012302494 2020 eng d a0739-110200aDiscovery of potential multi-target-directed ligands by targeting host-specific SARS-CoV-2 structurally conserved main protease0 aDiscovery of potential multitargetdirected ligands by targeting  cMAY3 a

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in the current COVID-19 pandemic. Worldwide this disease has infected over 2.5 million individuals with a mortality rate ranging from 5 to 10%. There are several efforts going on in the drug discovery to control the SARS-CoV-2 viral infection. The main protease (M-Pro) plays a critical role in viral replication and maturation, thus can serve as the primary drug target. To understand the structural evolution of M-Pro, we have performed phylogenetic and Sequence Similarity Network analysis, that depicted divergence of Coronaviridae M-Pro in five clusters specific to viral hosts. This clustering was corroborated with the comparison of M-Pro structures. Furthermore, it has been observed that backbone and binding site conformations are conserved despite variation in some of the residues. These attributes can be exploited to repurpose available viral protease inhibitors against SARS-CoV-2 M-Pro. In agreement with this, we performed screening of similar to 7100 molecules including active ingredients present in the Ayurvedic anti-tussive medicines, anti-viral phytochemicals and synthetic anti-virals against SARS-CoV-2 M-Pro as the primary target. We identified several natural molecules like delta-viniferin, myricitrin, taiwanhomoflavone A, lactucopicrin 15-oxalate, nympholide A, afzelin, biorobin, hesperidin and phyllaemblicin B that strongly binds to SARS-CoV-2 M-Pro. Intrestingly, these molecules also showed strong binding with other potential targets of SARS-CoV-2 infection like viral receptor human angiotensin-converting enzyme 2 (hACE-2) and RNA dependent RNA polymerase (RdRp). We anticipate that our approach for identification of multi-target-directed ligand will provide new avenues for drug discovery against SARS-CoV-2 infection. Communicated by Ramaswamy H. Sarma

10aCoronavirus10aCOVID-1910ahACE-210aMPro10amulti-target-directed ligand10aprotease inhibitor10aRdRp10aSARS-CoV-2 virus1 aJoshi, Rakesh, S.1 aJagdale, Shounak, S.1 aBansode, Sneha, B.1 aShankar, Shiva1 aTellis, Meenakshi, B.1 aPandya, Vaibhav, Kumar1 aChugh, Anita1 aGiri, Ashok, P.1 aKulkarni, Mahesh, J. uhttp://library.ncl.res.in/content/discovery-potential-multi-target-directed-ligands-targeting-host-specific-sars-cov-202456nas a2200289 4500008004100000022001400041245008200055210006900137260000800206490000900214520156800223653001301791653001601804653001401820653001301834653001001847653000701857653003901864653001601903100002701919700001901946700001501965700002401980700002702004700002502031856011002056 2023 eng d a0304-416500aMechanistic insights on anserine hydrolyzing activities of human carnosinases0 aMechanistic insights on anserine hydrolyzing activities of human cMAR0 v18673 a

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (Vmax/[E]t) was 21.6 s-1 and 2.8 s-1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (Km) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LCMS/MS approach.

10aAnserine10aCarnosinase10aCarnosine10aDiabetes10aLC-MS10aMS10aMultiple reaction monitoring (MRM)10aPromiscuity1 aPandya, Vaibhav, Kumar1 aShankar, Shiva1 aRajesh, S.1 aRathore, Rajeshwari1 aKumaran, Sangaralingam1 aKulkarni, Mahesh, J. uhttp://library.ncl.res.in/content/mechanistic-insights-anserine-hydrolyzing-activities-human-carnosinases