Elsevier

Biochemical Pharmacology

Volume 68, Issue 9, 1 November 2004, Pages 1807-1814
Biochemical Pharmacology

Using yeast to screen for inhibitors of protein tyrosine phosphatase 1B

https://doi.org/10.1016/j.bcp.2004.06.024Get rights and content

Abstract

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been proposed as a novel therapy to treat type 2 diabetes and obesity. In order to identify novel PTP1B inhibitors, we have developed a robust screen in Saccharomyces cerevisiae where growth is dependent on PTP1B catalytic activity. This was based on the observation that overexpression of v-Src, a tyrosine kinase, in yeast leads to lethality through mitotic dysfunction and this lethality can be reversed by co-expression of PTP1B. The expression levels of v-Src and PTP1B were optimized to obtain a balance between robust growth and sensitivity to inhibitors. Screening was carried out in 96-well plates and growth of the liquid culture measured by absorbance at 600 nm. Initial characterization was performed using vanadate as well as some novel PTP1B inhibitors. Vanadate specifically inhibited PTP1B-dependent growth in a dose dependent manner with an EC50 of 0.92 ± 0.07 mM. This simple yeast growth interference assay has the potential for use as a high throughput screen for PTP1B inhibitors in sample collections or crude mixtures.

Section snippets

Yeast strain

YPH499: Mata ura 3-52 lys 2-801amber ade 2-101ocher trp 1-Δ63 his3-Δ200 leu2Δ1 (Stratagene).

Plasmids constructs

EcoRI and SalI were used to cut out the catalytic domain (amino acids 1-320) of human PTP1B previously cloned in a pFLAG vector [18]. The resulting fragment was gel purified and ligated to p416GAL1 (American type culture collection (ATCC) 87332) previously digested with EcoRI and SalI and de-phosphorylated. The plasmid p416GAL1-PTP1B was sequenced to confirm the correct construction. PTP1B mutants C215S

Optimization of the rescue of yeast from v-Src lethality by PTP1B

It was previously shown that expression of v-Src in yeast results in the arrest of cell growth and this cell arrest can be rescued by the co-expression of PTP1B [16]. To reproduce these results and to establish the optimal balance between this lethality and the rescue mediated by PTP1B, the expression levels of v-Src in yeast were varied by using mutated GAL promoters [19]. In fact, v-Src expression driven by any of the GAL promoters, GAL1 (wild type) or the attenuated promoters GALL or GALS

Discussion

A major issue with PTP1B drug screening programs is the lack of a robust cell-based functional assay. Typically, increased tyrosine phosphorylation of the IR or glucose uptake has been used as functional readouts for PTP1B inhibition. However, the effect on IR phosphorylation due to either PTP1B mRNA knockdown or inhibition, as measured by quantitation of Western blots, results in at most a two to three-fold increase in phosphorylation over unstimulated controls [9], [10]. This small window and

Acknowledgements

We are grateful to Ernest Asante-Appiah for helpful discussion during the development of this assay and to Kathryn Skorey for assistance with the DiFMUP assay. We would like to thank Rick Friesen, Yongxin Han, C.K. Lau and Yves Leblanc for the inhibitors used in this study. We also want to give special thanks to Ann M. English and her laboratory at Concordia University for support and critical insight. J.M. is supported by Canadian NSERC Industrial Postgraduate Scholarship, FCAR-MRST Bourse de

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