Characterisation of the PTEN inhibitor VO-OHpic
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Characterisation of the PTEN inhibitor VO-OHpic Lok Hang Mak & Ramón Vilar & Rudiger Woscholski
Received: 9 April 2010 / Accepted: 17 May 2010 / Published online: 4 June 2010 # Springer-Verlag 2010
Abstract PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases and has subsequently been implied as a valuable drug target for diabetes and cancer. Recently, we demonstrated that VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo. Given the importance of this inhibitor for future drug design and development, its mode of action needed to be elucidated. It was discovered that inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both Km and Vmax are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants Kic and Kiu were determined to be 27±6 and 45±11 nM, respectively. Using the artificial phosphatase substrate 3-O-methylfluorescein phosphate (OMFP) or the physiological substrate phosphatidylinositol 3,4,5-triphosphate (PIP3) comparable parameters were obtained suggesting that OMFP is a suitable substrate for PTEN inhibition studies and PTEN drug screening. Keywords PTEN . VO-OHpic . OMFP . PIP3
L. H. Mak (*) : R. Woscholski Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, UK e-mail: [email protected] R. Vilar Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK
Introduction PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a well-known tumour suppressor [1, 2], which loss in function through mutation, deletion, or transcriptional silencing is found in a wide spectrum of advanced human cancers [3]. It belongs to the family of the CX5R phosphatases and was initially believed to be a tyrosine phosphatase until Maehama et al. (1998) reported that PTEN exhibits a much greater affinity towards phosphoinositides than tyrosine phosphates [4]. PTEN hydrolyses the phosphate group in the 3′ position from phosphatidylinositol 3,4,5-triphosphate (PIP3) to form phosphatidylinositol 4,5-biphosphate and, in doing so, is counteracting phosphoinositide 3-kinases (PI3K). The balance of PTEN and PI3K activities determines the cellular PIP3 levels, which in turn is recognised by other signalling components such as Akt [5]. Increased activity of Akt as a result of increased PIP3 levels due to loss in PTEN function has been shown to play a major role in PTENmediated tumourigenesis [6]. However, there are diseases that could benefit from elevated levels of PIP3, which could be achieved by inhibiting PTEN. In particular, wound healing [7], asthma [8], neuroprotection and regeneration [9], and indeed some cancers [10] have been linked with PTEN as a drug target. Vanadium complexes are capable to mimic a variety of insulin-like effects, which were linked to the inhibition of protein tyrosine phosphatases (PTPs) [11–13]. M
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