Alcohol Pharmacology Education Partnership: Using Chemistry and Biology Concepts To Educate High School Students about Alcohol.

We developed the Alcohol Pharmacology Education Partnership (APEP), a set of modules designed to integrate a topic of interest (alcohol) with concepts in chemistry and biology for high school students. Chemistry and biology teachers (n = 156) were recruited nationally to field-test APEP in a controlled study. Teachers obtained professional development either at a conference-based workshop (NSTA or NCSTA) or via distance learning to learn how to incorporate the APEP modules into their teaching. They field-tested the modules in their classes during the following year. Teacher knowledge of chemistry and biology concepts increased significantly following professional development, and was maintained for at least a year. Their students (n = 14 014) demonstrated significantly higher scores when assessed for knowledge of both basic and advanced chemistry and biology concepts compared to students not using APEP modules in their classes the previous year. Higher scores were achieved as the number of modules used increased. These findings are consistent with our previous studies, demonstrating higher scores in chemistry and biology after students use modules that integrate topics interesting to them, such as drugs (the Pharmacology Education Partnership).

A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity.

Inhibiting protein prenylation is an attractive means to modulate cellular processes controlled by a variety of signaling proteins, including oncogenic proteins such as Ras and Rho GTPases. The largest class of prenylated proteins contain a so-called CaaX motif at their carboxyl termini and are subject to a maturation process initiated by the attachment of an isoprenoid lipid by either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I). Inhibitors of FTase, termed FTIs, have been the subject of intensive development in the past decade and have shown efficacy in clinical trials. Although GGTase-I inhibitors (GGTIs) have received less attention, accumulating evidence suggests GGTIs may augment therapies using FTIs and could be useful to treat a myriad of additional disease states. Here we describe the characterization of a selective, highly potent, and cell-active GGTase-I inhibitor, GGTI-DU40. Kinetic analysis revealed that inhibition by GGTI-DU40 is competitive with the protein substrate and uncompetitive with the isoprenoid substrate; the Ki for the inhibition is 0.8 nM. GGTI-DU40 is highly selective for GGTase-I both in vitro and in living cells. Studies indicate GGTI-DU40 blocks prenylation of a number of geranylgeranylated CaaX proteins. Treatment of MDA-MB-231 breast cancer cells with GGTI-DU40 inhibited thrombin-induced cell rounding via a process that involves inhibition of Rho proteins without significantly effecting parallel mobilization of calcium via Gbetagamma. These studies establish GGTI-DU40 as a prime tool for interrogating biologies associated with protein geranylgeranylation and define a novel structure for this emerging class of experimental therapeutics.

Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif.

Wrch-1 is a Rho family GTPase that shares strong sequence and functional similarity with Cdc42. Like Cdc42, Wrch-1 can promote anchorage-independent growth transformation. We determined that activated Wrch-1 also promoted anchorage-dependent growth transformation of NIH 3T3 fibroblasts. Wrch-1 contains a distinct carboxyl-terminal extension not found in Cdc42, suggesting potential differences in subcellular location and function. Consistent with this, we found that Wrch-1 associated extensively with plasma membrane and endosomes, rather than with cytosol and perinuclear membranes like Cdc42. Like Cdc42, Wrch-1 terminates in a CAAX tetrapeptide (where C is cysteine, A is aliphatic amino acid, and X is any amino acid) motif (CCFV), suggesting that Wrch-1 may be prenylated similarly to Cdc42. Most surprisingly, unlike Cdc42, Wrch-1 did not incorporate isoprenoid moieties, and Wrch-1 membrane localization was not altered by inhibitors of protein prenylation. Instead, we showed that Wrch-1 is modified by the fatty acid palmitate, and pharmacologic inhibition of protein palmitoylation caused mislocalization of Wrch-1. Most interestingly, mutation of the second cysteine of the CCFV motif (CCFV > CSFV), but not the first, abrogated both Wrch-1 membrane localization and transformation. These results suggest that Wrch-1 membrane association, subcellular localization, and biological activity are mediated by a novel membrane-targeting mechanism distinct from that of Cdc42 and other isoprenylated Rho family GTPases.

Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development.

Protein farnesyltransferase (FTase) is an enzyme responsible for posttranslational modification of proteins carrying a carboxy-terminal CaaX motif. Farnesylation allows substrates to interact with membranes and protein targets. Using gene-targeted mice, we report that FTase is essential for embryonic development, but dispensable for adult homeostasis. Six-month-old FTase-deficient mice display delayed wound healing and maturation defects in erythroid cells. Embryonic fibroblasts lacking FTase have a flat morphology and reduced motility and proliferation rates. Ablation of FTase in two ras oncogene-dependent tumor models has no significant consequences for tumor initiation. However, elimination of FTase during tumor progression had a limited but significant inhibitory effect. These results should help to better understand the role of protein farnesylation in normal tissues and in tumor development.

Improved loading and cleavage methods for solid-phase synthesis using chlorotrityl resins: synthesis and testing of a library of 144 discrete chemicals as potential farnesyltransferase inhibitors.

The use of chlorotrityl resins for the immobilization of amines is sometimes deterred by the lengthy process of loading the reactants on the resins and product decomposition caused by the reactive chlorotrityl group in the presence of 1% TFA as a cleavage agent. Here, we report improved methods developed for selective and efficient loading of aminobenzoic acid derivatives on chlorotrityl resins and for cleavage of aniline-containing products from the resins without decomposition. These methods led to the synthesis of a library of 144 discrete chemicals as potential farnesyltransferase inhibitors (FTIs) using IRORI's radio-frequency-encoded sorting technique and to the study of the applicability of the bivalence approach to the development of FTIs.

High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors.

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.

Prenylcysteine lyase deficiency in mice results in the accumulation of farnesylcysteine and geranylgeranylcysteine in brain and liver.

In in vitro experiments, prenylcysteine lyase (Pcly) cleaves the thioether bond of prenylcysteines to yield free cysteine and the aldehyde of the isoprenoid lipid. However, the importance of this enzyme has not yet been fully defined at the biochemical or physiologic level. In this study, we show that Pcly is expressed at high levels in mouse liver, kidney, heart, and brain. To test whether Pcly deficiency would cause prenylcysteines to accumulate in tissues and result in pathologic consequences, we produced Pcly-deficient cell lines and Pcly-deficient mice (Pcly-/-). Pcly activity levels were markedly reduced in Pcly-/- cells and tissues. Pcly-/- fibroblasts were more sensitive than wild-type fibroblasts to growth inhibition when prenylcysteines were added to the cell culture medium. To determine if the reduced Pcly enzyme activity levels led to an accumulation of prenylcysteines within cells, mass spectrometry was used to measure farnesylcysteine and geranylgeranylcysteine levels in the tissues of Pcly-/- mice and wild-type controls. These studies revealed a striking accumulation of both farnesylcysteine and geranylgeranylcysteine in the brain and liver of Pcly-/- mice. This accumulation did not appear to be accompanied by significant pathologic consequences. Pcly-/- mice were healthy and fertile, and surveys of more than 30 tissues did not uncover any abnormalities. We conclude that prenylcysteine lyase does play a physiologic role in cleaving prenylcysteines in mammals, but the absence of this activity does not lead to major pathologic consequences.