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A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase.

ABSTRACT: An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.

SUBMITTER: Irague R

PROVIDER: S-EPMC5819781 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase.

Irague Romain R Topham Christopher M CM Martineau Nelly N Baylac Audrey A Auriol Clément C Walther Thomas T François Jean-Marie JM André Isabelle I Remaud-Siméon Magali M

PloS one 20180220 2

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. Th ...[more]

PMID: 29462203

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Project description:Quantitative high throughput screening (qHTS) assays use cells or tissues to screen thousands of compounds in a short period of time. Data generated from qHTS assays are then evaluated using nonlinear regression models, such as the Hill model, and decisions regarding toxicity are made using the estimates of the parameters of the model. For any given compound, the variability in the observed response may either be constant across dose groups (homoscedasticity) or vary with dose (heteroscedasticity). Since thousands of compounds are simultaneously evaluated in a qHTS assay, it is not practically feasible for an investigator to perform residual analysis to determine the variance structure before performing statistical inferences on each compound. Since it is well-known that the variance structure plays an important role in the analysis of linear and nonlinear regression models it is therefore important to have practically useful and easy to interpret methodology which is robust to the variance structure. Furthermore, given the number of chemicals that are investigated in the qHTS assay, outliers and influential observations are not uncommon. In this article we describe preliminary test estimation (PTE) based methodology which is robust to the variance structure as well as any potential outliers and influential observations. Performance of the proposed methodology is evaluated in terms of false discovery rate (FDR) and power using a simulation study mimicking a real qHTS data. Of the two methods currently in use, our simulations studies suggest that one is extremely conservative with very small power in comparison to the proposed PTE based method whereas the other method is very liberal. In contrast, the proposed PTE based methodology achieves a better control of FDR while maintaining good power. The proposed methodology is illustrated using a data set obtained from the National Toxicology Program (NTP). Additional information, simulation results, data and computer code are available online as supplementary materials.

Dataset Information

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase.

Publications

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase.

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