Development of a high-throughput dual-stream liquid chromatography-tandem mass spectrometry method to screen for inhibitors of glutamate carboxypeptidase II.

RATIONALE: Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to yield glutamate (Glu) and N-acetylaspartate (NAA). Inhibition of GCPII has been shown to remediate the neurotoxicity of excess Glu in a variety of cell and animal disease models. A robust high-throughput liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was needed to quantify GCPII enzymatic activity in a biochemical high-throughput screening assay. METHODS: A dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2 × 30 mm) columns. Each LC channel was run independently, and the cycle time was 2 min per channel. Overall throughput was 1 min per sample for the dual-channel integrated system. Multiply injected acquisition files were split during data review, and batch metadata were automatically paired with raw data during the review process. RESULTS: Two LC sorbents, BEH-Amide and Penta-HILIC, were tested to separate the NAAG cleavage product Glu from isobaric interference and ion suppressants in the bioassay matrix. Early elution of NAAG and NAA on BEH-Amide allowed interfering species to be diverted to waste. The limit of quantification was 0.1 pmol for Glu. The Z-factor of this assay averaged 0.85. Over 36 000 compounds were screened using this method. CONCLUSIONS: A fast gradient dual-stream LC/MS/MS method for Glu quantification in GCPII biochemical screening assay samples was developed and validated. HILIC separation chemistry offers robust performance and unique selectivity for targeted positive mode quantification of Glu, NAA, and NAAG.

Computational investigations on the potential role of hygrophorones as quorum sensing inhibitors against LasR protein of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram negative, rod shape bacterium that infects people with compromised immune systems, such as those suffering from AIDS, organ transplantation and cancer. This bacterium is responsible for diseases like cystic fibrosis, chronic lung infection, and ulcerative keratitis. It is diagnosed in most of the patients who were on prolonged ventilation with long term critical care stay. P. aeruginosa develops rapid antimicrobial resistance that is challenging for the treatment and eventually it causes high mortality rate. Thus, the search for potential novel inhibitors that can inhibit the pathogenic activity of P. aeruginosa is of utmost importance. In P. aeruginosa, an important protein, LasR that participates in the gene regulations and expressions has been proposed to be a suitable drug target. Here, we identify a set of hygrophorone molecules as effective inhibitors for this LasR protein based on molecular docking and simulations studies. At first, large number of hygrophorone series of small molecules were screened against the LasR protein and their binding affinities were assessed based on the docking scores. Top scored molecules were selected for calculating various pharmacophore properties, and finally, their potential in inhibiting the LasR protein was delineated by atomistic molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area-based calculations. Both docking and simulations studies reveal that a subset of hygrophorone molecules have a good binding affinity for LasR protein and form stable LasR-inhibitor complexes. The present study illustrates that the hygrophorones can be effective inhibitors for the LasR protein and will spur further in vitro studies that would aid to the ongoing search for new antibiotics.Communicated by Ramaswamy H. Sarma.

Noscapine-Amino Acid Conjugates Suppress the Progression of Cancer Cells.

Lung cancer is the leading cause of cancer deaths globally; 1 in 16 people are diagnosed with lung cancer in their lifetime. Microtubules, a critical cytoskeletal assembly, have an essential role in cell division. Interference with the microtubule assembly leads to genetic instability during mitosis and cancer cell death. Currently, available antimitotic drugs such as vincas and taxanes are limited due to side effects such as alopecia, myelosuppression, and drug resistance. Noscapine, an opium alkaloid, is a tubulin-binding agent and can alter the microtubule assembly, causing cancer cell death. Amino acids are fundamental building blocks for protein synthesis, making them essential for the biosynthesis of cancer cells. However, the ability of amino acids in drug transportation has yet to be exploited in developing noscapine analogues as a potential drug candidate for cancer. Hence, in the present study, we have explored the ninth position of noscapine by introducing a hydroxymethylene group using the Blanc reaction and further coupled it with a series of amino acids to construct five target conjugates in good yields. The synthesized amino acid conjugate molecules were biologically evaluated against the A549 lung cancer cell line, among which the noscapine-tryptophan conjugate showed IC50 = 32 µM, as compared to noscapine alone (IC50 = 73 µM). Morphological changes in cancer cells, cell cycle arrest in the G1 phase, and ethidium bromide/acridine orange staining indicated promising anticancer properties. Molecular docking confirmed strong binding to tubulin, with a score of -41.47 kJ/mol with all 3D coordinates and significant involvement of molecular forces, including the hydrogen bonds and hydrophobic interactions. Molecular dynamics simulations demonstrated a stable binding of noscapine-tryptophan conjugate for a prolonged time (100 ns) with the involvement of free energy through the reaction coordinates analyses, solving the bioavailability of parent noscapine to the body.

Repurposing of FDA Approved Drugs Against SARS-CoV-2 Papain-Like Protease: Computational, Biochemical, and in vitro Studies.

The pandemic caused by SARS-CoV-2 (SCoV-2) has impacted the world in many ways and the virus continues to evolve and produce novel variants with the ability to cause frequent global outbreaks. Although the advent of the vaccines abated the global burden, they were not effective against all the variants of SCoV-2. This trend warrants shifting the focus on the development of small molecules targeting the crucial proteins of the viral replication machinery as effective therapeutic solutions. The PLpro is a crucial enzyme having multiple roles during the viral life cycle and is a well-established drug target. In this study, we identified 12 potential inhibitors of PLpro through virtual screening of the FDA-approved drug library. Docking and molecular dynamics simulation studies suggested that these molecules bind to the PLpro through multiple interactions. Further, IC50 values obtained from enzyme-inhibition assays affirm the stronger affinities of the identified molecules for the PLpro. Also, we demonstrated high structural conservation in the catalytic site of PLpro between SCoV-2 and Human Coronavirus 229E (HCoV-229E) through molecular modelling studies. Based on these similarities in PLpro structures and the resemblance in various signalling pathways for the two viruses, we propose that HCoV-229E is a suitable surrogate for SCoV-2 in drug-discovery studies. Validating our hypothesis, Mefloquine, which was effective against HCoV-229E, was found to be effective against SCoV-2 as well in cell-based assays. Overall, the present study demonstrated Mefloquine as a potential inhibitor of SCoV-2 PLpro and its antiviral activity against SCoV-2. Corroborating our findings, based on the in vitro virus inhibition assays, a recent study reported a prophylactic role for Mefloquine against SCoV-2. Accordingly, Mefloquine may further be investigated for its potential as a drug candidate for the treatment of COVID.

Stereoselective total synthesis of (-)-hygrophorone A12 and 4-epi-2,3-dihydrohygrophorone H12.

Stereoselective total synthesis of anti-fungal cyclopentenone (-)-hygrophorone A12 and cyclopentanone 4-epi-2,3-dihydrohygrophorone H12 were achieved in high overall yields from d-ribose. An aqueous KOH mediated diastereoselective formation of ß-hydroxy ketone with three contiguous chiral centres served as a key step in this synthesis.

Stereoselective total syntheses of (-)-hygrophorone A12, 4-O-acetyl-hygrophorone A12 and (+)-hygrophorone B12.

Total syntheses of anti-fungal cyclopentenones (-)-hygrophorone A12, 4-O-acetyl-hygrophorone A12 and (+)-hygrophorone B12 were achieved in high overall yields from d-(-)-tartaric acid. The key feature of these syntheses is the aqueous KOH-mediated diastereoselective intramolecular aldol reaction to form ß-hydroxy ketone with three contiguous chiral centres, which was further elaborated to (-)-hygrophorone A12 and (+)-hygrophorone B12. The synthetic route reported here is operationally simple and highly diastereoselective and is amenable for the synthesis of several analogues of hygrophorones.

Identification and analysis of organic explosives from post-blast debris by nuclear magnetic resonance.

The growing threat of terrorism has triggered an urgent need to find effective ways to improve the analysis of explosives. This will aid forensic scientists in analysing the post-blast debris, which in turn helps the law enforcement agencies to frame suitable regulations. Analysis of post-blast debris is challenging as it hosts a massive amount of complexity. There are various techniques reported till date such as mass spectrometry, gas chromatography, high-performance liquid chromatography, Fourier transform infrared spectroscopy, and Raman spectroscopy for the analysis of post-blast residues. However, none of them has been able to identify the structural composition of the explosives. The current research study focuses on identifying the structural composition of the explosives from the post-blast debris using the nuclear magnetic resonance (NMR) technology. The post-blast analytes were extracted from soil samples, cleaned by the solid phase extraction (SPE) method and were rapidly analysed by the nuclear magnetic resonance spectrometer. This paper reports the identification of nitro organic explosives such as pentaerythritol tetranitrate (PETN), trinitrotoluene (TNT) and 2,4,6-trinitrophenylmethylnitramine (tetryl) in post-blast debris by 400 MHz nuclear magnetic resonance spectrometer.