Tyrosine Kinase Inhibitors
Gefitinib (IressaTM) and erlotinib (TarcevaTM) were kindly provided by Dr. Nisana Namwat (Khon Kaen University, Khon Kaen, Thailand). Imatinib mesylate (GleevecTM) was synthesized by American Custom Chemicals Corporation (San Diego, CA) and supplied by the Ludwig Institute for Cancer Research. Sunitinib malate (SutentTM) and sorafenib p-toluenesulfonate (NexavarTM) were purchased from LC laboratories (Woburn, MA). MET inhibitor (SU11274), FGFR inhibitors (SU4984, SU5402 and PD173074) and PDGFR tyrosine kinase inhibitor III were purchased from Calbiochem (La Jolla, CA). Inhibitors were dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 10 mM or 100 mM and stored at 220uC until used.intracranially implanted in 32 F344 female rats weighing 150?200 gms as described previously [24].demonstrate increased inhibition in 020913 and 060919 the cell growth compared to other combinations. (TIF)Figure S3 RTK combination treatment. 020913 cells were treated with FDA approved and unapproved RTK inhibitors at 25% of their IC50 concentrations.
Animal Drug Treatment
All the animals were treated with drug doses equivalent to FDA approved human doses. A weekly human dose was calculated for gefitinib and sunitinib and was converted to a mouse or a rat dose using the formula prescribed by Reagan-Shaw et al [25]. The weekly animal dose was then equally divided into three installments to be delivered on Monday, Wednesday and Friday and the treatment continued until the animals were dead or sacked due to tumor burden.
ment. 020913 cells were treated with vandetanib in combination with other FDA approved drugs such as imatinib (ima), sunitinib (sun) and sorafenib (sor). Vandetanib is an EGFR and VEGFR inhibitor. (TIF)
Figure S5 Inhibition of phosphorylation in 020913 cells when treated with a combination of Gefitinib and Sunitinib. (TIF) Table S1 IC50 values of RTK inhibitors in GBM oncosphere and adherent cell lines. (DOCX) Data S1 Calculation of FDA equivalent dose of RTK inhibitors for the animal studies. (XLS)Statistical and graphical analyses were performed using GraphPad Prism 5 (GraphPad, LaJolla, CA). The Student T-test was used to compare the growth inhibition in various groups. Animal survival was analyzed using Kaplan-Meier survival and log rank test.
Abstract
Cyclohexyl ketone substrate analogue inhibitors (AcSer-Y[C = OCH]-Pipryptamine) of Pin1, the cell cycle regulatory peptidyl-prolyl isomerase (PPIase), were designed and synthesized as potential electrophilic acceptors for the Pin1 active site Cys113 nucleophile to test a proposed nucleophilic addition-isomerization mechanism. Because they were weak inhibitors, models of all three stereoisomers were docked into the active site of Pin1. Each isomer consistently minimized to a trans-diaxial cyclohexane conformation. From this, we hypothesize that Pin1 stretches substrates into a trans-pyrrolidine conformation to lower the barrier to isomerization. Our reduced amide inhibitor of Pin1 adopted a similar trans-pyrrolidine conformation in the crystal structure. The molecular model of 1, which mimics the L-Ser-L-Pro stereochemistry, in the Pin1 ?active site showed a distance of 4.4 A, and an angle of 31u between Cys113-S and the ketone carbon. The computational models suggest that the mechanism of Pin1 PPIase is not likely to proceed through nucleophilic addition.
Citation: Xu GG, Slebodnick C, Etzkorn FA (2012) Cyclohexyl Ketone Inhibitors of Pin1 Dock in a Trans-Diaxial Cyclohexane Conformation. PLoS ONE 7(9): e44226. doi:10.1371/journal.pone.0044226 Editor: Joseph J. Barchi, National Cancer Institute at Frederick, United States of America Received May 23, 2012; Accepted August 3, 2012; Published September 19, 2012 Copyright: ?2012 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: We thank National Institutes of Health (NIH) (http://www.nih.gov/) grant R01 CA110940, and NIH grant S10 RR16658 for the LC-MSMS, and Oxford Instruments for the use of the diffractometer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Pin1 (peptidyl-prolyl isomerase (PPIase) interacting with neverin-mitosis A kinase-1) was discovered in 1996 as a PPIase enzyme that regulates mitosis [1]. The two domains of Pin1, a WW and a PPIase domain, are connected by a flexible linker that serves as a communication conduit between the domains [2]. Both of these domains recognize the phospho-Ser/Thr-Pro bonds present in mitotic phosphoproteins [3]. Pin1 is distinct from two other PPIase families, cyclophilin and FK506 binding protein (FKBP) [4], since Pin1 only has PPIase activity for phosphorylated substrates [3]. Pin1 catalyzes prolyl cis-trans isomerization to function as a molecular timer regulating the cell cycle, cell signaling, gene expression, immune response, and neuronal function [5]. Pin1 is overexpressed in many cancer lines, and plays an important role in oncogenesis [6]. Because of its significant role in cell cycle regulation by a unique mechanism, Pin1 represents an intriguing diagnostic and therapeutic target for cancer [7,8]. Several promising classes of Pin1 inhibitors have been synthesized as potential lead compounds [7], including designed inhibitors [9,10,11,12,13,14], and natural products [15,16]. The mechanisms of the PPIases, cyclophilins and FKBPs, were shown to go through a twisted amide transition state. Evidence included secondary deuterium isotope effects, molecular modeling, mutagenesis, and bound inhibitor structure [17,18,19,20,21,22, 23,24]. There are two proposed mechanisms for Pin1 catalysis: (1) the twisted-amide mechanism [25], and (2) the nucleophilicaddition mechanism (Figure 1) [26].
