High throughput screening via mass spectrometry: a case study using acetylcholinesterase.
ABSTRACT Mass spectrometry-based screening can be applied to a wide range of targets, including those intractable targets that use substrates such as lipids, fatty acids, phospholipids, steroids, prostaglandins, and other compounds not generally amenable to conventional screening techniques. The major limitation to this approach is throughput, making HTS via mass spectrometry impractical. We present a mass spectrometry-based technique and hardware for lead discovery applications. Mass spectrometry enables the design of label-free assays using biologically native substrates for a wide range of enzymatic targets. This system can be used for the direct quantification of analytes in complex reaction mixtures with typical throughputs of 4-5 s per sample. A mass spectrometry-based assay was developed to identify inhibitors of acetylcholinesterase, an enzyme with clinical importance in Alzheimer's disease. The system was used to screen a small chemical library. Several potent inhibitors were identified, and the IC(50) values of the inhibitors were determined.
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ABSTRACT: A derivatization method in mass spectrometry for small molecular analysis was developed to solve the problems of volatility of many analytes, difficult ionization of analytes, and undiscriminating isobaric analytes. This derivatization method, oximation of -ketoacid, in the transaminase reaction leads to change of mass difference between amine reactants and ketoacid products. In addition, regardless of the kinds of ketoacid, the linear relationship between the peak intensity and its concentration (from 1 mM to 10 mM) shows that quantitative analysis of the conversion of the reaction can be executed by the analysis of peak intensity of the corresponding oximated ketoacids. Furthermore, this method can be used for identifying transaminase as well as determining its substrate specificity01/2004; 19(5).
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ABSTRACT: Helicobacter pylori is motile by means of polar flagella and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an anti-virulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This study describes screening the five Pse biosynthetic enzymes for small molecule inhibitors using both high throughput (HTS) and in silico (VS) approaches. Using a 100,000 compound library, 1773 hits were identified by HTS that exhibited a 40% threshold inhibition at 10 μM concentration. In addition, VS efforts using a 1.6 million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at 10 μM concentration in vitro. Further secondary screening was performed which identified 320 unique molecular structures or validated hits. Following kinetic studies and SAR of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with IC50 values of approximately 14 μM, and which belonged to a distinct chemical cluster, were able to penetrate the Gram negative cell membrane and prevent flagella formation.Antimicrobial Agents and Chemotherapy 09/2014; 58(12). DOI:10.1128/AAC.03858-14 · 4.45 Impact Factor
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ABSTRACT: In our previous findings, we have demonstrated that aspirin/acetyl salicylic acid (ASA) might induce sirtuins via aryl hydrocarbon receptor (Ah receptor). Induction effects included an increase in cellular paraoxonase 1 (PON1) activity and apolipoprotein A1 (ApoA1) gene expression. As predicted, ASA and salicylic acid (SA) treatment resulted in generation of H2O2, which is known to be an inducer of mitochondrial gene Sirt4 and other downstream target genes of Sirt1. Our current mass spectroscopic studies further confirm the metabolism of the drugs ASA and SA. Our studies show that HepG2 cells readily converted ASA to SA, which was then metabolized to 2,3-DHBA. HepG2 cells transfected with aryl hydrocarbon receptor siRNA upon treatment with SA showed the absence of a DHBA peak as measured by LC-MS/MS. MS studies for Sirt1 action also showed a peak at 180.9 m/z for the deacetylated and chlorinated product formed from N-acetyl lε-lysine. Thus an increase in Sirt4, Nrf2, Tfam, UCP1, eNOS, HO1 and STAT3 genes could profoundly affect mitochondrial function, cholesterol homeostasis, and fatty acid oxidation, suggesting that ASA could be beneficial beyond simply its ability to inhibit cyclooxygenase. Copyright © 2015. Published by Elsevier B.V.European Journal of Pharmacology 12/2014; 749. DOI:10.1016/j.ejphar.2014.12.020 · 2.68 Impact Factor