Thomas A. Engler

Orally bioavailable GSK-3α/β dual inhibitor increases markers of cellular differentiation in vitro and bone mass in vivo

GSK‐3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK‐3 inhibitor was evaluated in pre‐osteoblasts and in osteopenic rats. GSK‐3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats.

Novel, potent and selective cyclin D1/CDK4 inhibitors: indolo[6,7-a]pyrrolo[3,4-c]carbazoles.

The synthesis and CDK inhibitory properties of a series of indolo[6,7-a]pyrrolo[3,4-c]carbazoles is reported. In addition to their potent CDK activity, the compounds display antiproliferative activity against two human cancer cell lines. These inhibitors also effect strong G1 arrest in these cell lines and inhibit Rb phosphorylation at Ser780 consistent with inhibition of cyclin D1/CDK4.

Changes in Osteoblast, Chondrocyte, and Adipocyte Lineages Mediate the Bone Anabolic Actions of PTH and Small Molecule GSK‐3 Inhibitor

Parathyroid hormone (PTH) and glycogen synthase kinase‐3 (GSK‐3) inhibitor 603281‐31‐8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281‐31‐8 in rat osteopenia model. Female 6‐month‐old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1–38) at 10 µg/kg/day s.c. or 603281‐31‐8 at 3 mg/kg/day p.o. for 60 days. Twenty‐four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (Pu2009<u20090.05) were subjected to pathway analysis to delineate relevant bio‐processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK‐3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three‐fold increase in marrow adiposity. PTH and GSK‐3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK‐3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU‐fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK‐3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes. J. Cell. Biochem. 102: 1504–1518, 2007.

A robust high-content imaging approach for probing the mechanism of action and phenotypic outcomes of cell cycle modulators

High-content screening is increasingly used to elucidate changes in cellular biology arising from treatment with small molecules and biological probes. We describe a cell classifier for automated analysis of multiparametric data from immunofluorescence microscopy and characterize the phenotypes of 41 cell-cycle modulators, including several protein kinase inhibitors in preclinical and clinical development. This method produces a consistent assessment of treatment-induced phenotypes across experiments done by different biologists and highlights the prevalence of nonuniform and concentration-dependent cellular response to treatment. Contrasting cell phenotypes from high-content screening to kinase selectivity profiles from cell-free assays highlights the limited utility of enzyme potency ratios in understanding the mechanism of action for cell-cycle kinase inhibitors. Our cell-level approach for assessing phenotypic outcomes is reliable, reproducible and capable of supporting medium throughput analyses of a wide range of cellular perturbations. Mol Cancer Ther; 10(2); 242–54. ©2011 AACR.

Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma – Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3

It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

An improved method for direct conversion of heteroaryl-aldehydes to heteroaryl-acetonitriles

Abstract Treatment of heteroaryl-aldehydes with diethyl cyanophosphonate in the presence of a catalytic amount of LiCN affords phosphorylated cyanohydrins which are reduced in situ with SmI 2 to give heteroaryl-acetonitriles in generally good overall yields (50–100%). The generality of the process is demonstrated.

A new regioselective synthesis of pterocarpans

Pterocarpans are formed directly and efficiently via titanium(IV) catalysed reactions of 2H-chromenes and 2-alkoxy-1,4-benzoquinones.

Chemical Proteomic Characterization of a Covalent KRASG12C Inhibitor

The KRASG12C protein product is an attractive, yet challenging, target for small molecule inhibition. One option for therapeutic intervention is to design small molecule ligands capable of binding to and inactivating KRASG12C via formation of a covalent bond to the sulfhydryl group of cysteine 12. In order to better understand the cellular off-target interactions of Compound 1, a covalent KRASG12C inhibitor, we have completed a series of complementary chemical proteomics experiments in H358 cells. A new thiol reactive probe (TRP) was designed and used to construct a cellular target occupancy assay for KRASG12C. In addition, the thiol reactive probes allowed us to profile potential off-target interactions of Compound 1 with over 3200 cysteine residues. In order to complement the TRP data we designed Compound 2, an alkyne containing version of Compound 1, to serve as bait in competitive chemical proteomics experiments. Herein, we describe and compare data from both the TRP and the click chemistry probe pull down experiments.

Abstract 5374: LY-GSK-3i, a novel glycogen synthase kinase-3 inhibitor, potentiates the apoptotic activity of cytotoxic agents in combination chemotherapy protocols in in vitro and in vivo tumor models

Combination chemotherapy regimens for the treatment of cancer arise from the need to either enhance single agent activity, circumvent drug resistance or reduce toxicity by allowing lower dosing of cytotoxic agents. Glycogen synthase kinase 3 (GSK-3) is a constitutively active protein kinase with two highly homologous isoforms, GSK-3α and GSK-3β. Elevated GSK-3 activity has been described in a variety of tumors types and pharmacological or genetic inhibition of this kinase has been shown to reduce the survival of these tumors. In order to explore the potential for GSK-3 inhibitors in cancer therapy we developed LY-GSK-3i, which potently inhibits the enzymatic activity of GSK-3α and GSK-3β with IC50s of 1.5 nM and 0.9nM respectively and reduces the phosphorylation (Ser-396) of its substrate protein, Tau, with an EC50 of 1.4 nM. Here we report the novel and potent chemo-potentiating activity of LY-GSK-3i in a broad range of tumor models in combination with cytotoxic agents having different mechanisms of action. In in vitro caspase 3 activation assays, LY-GSK-3i enhances the apoptotic activity of pemetrexed, a multi-targeted anti-folate, in the NCI-H460 NSCLC cell line. The LY-GSK-3i/ pemetrexed combination enhances apoptotic activity 4-fold over the additive effect of each individual chemotherapeutic agent. DNA damaging agents such as platinum containing cytotoxics are also subject to chemopotentiation by LY-GSK-3i. In the NCI-H460 model, LY-GSK-3i potentiates the action of carboplatin 2 fold. Similarly, in the FaDu and SiHa squamous carcinoma cell lines, a 2-3 fold increase in apoptotic activity is observed with the combination treatment of LY-GSK-3i and cisplatin relative to treatment with either agent alone. Camptothecin is a DNA topoisomerase I inhibitor with modest apoptotic activity in the A2780, ovarian tumor cell line. The combination of camptothecin with LY-GSK-3i resulted in significant elevation in caspase 3 activation. Interestingly, commercially available GSK3/CDK inhibitors, such as SB216763, LiCl, alsterpaullone and purvalanol A have modest chemo-potentiating effect on Camptothecin toxicity compared to LY-GSK-3i. In vivo xenograft growth studies indicated that administration of LY-GSK-3i significantly improved the antitumor efficacy of cisplatin against Colo-205 colorectal tumors. Furthermore, LY-GSK-3i also potentiated in vivo the antitumor activity of carboplatin, or a combination of carboplatin/pemetrexed in NCI-H460 xenografts. Taken together, these data provide evidence for the potential therapeutic utility of LY-GSK-3i, in combination with cytotoxic agents, for the treatment of cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5374.

Abstract C211: Therapeutic utility of GSK‐3 inhibitors for the treatment of cancer

Glycogen synthase kinase‐3 (GSK‐3) is a constitutively active protein kinase that plays a role in diverse signaling pathways. Initially described as a regulator of glycogen synthesis and metabolism, GSK‐3 is now known to play a central role in a variety of cellular functions ranging from maintenance of stem cell function to regulation of cell cycle and apoptosis. In mammals, there are two highly homologous isoforms of GSK‐3 (GSK‐3α and GSK‐3β) which share substrate specificity and functional properties. GSK3β is one of the key components of the β‐catenin destruction complex and phosphorylation of β‐catenin by GSK‐3 targets the substrate protein for ubiquitylation and proteosomal destruction ‐ conversely, inhibition of GSK‐3β activity results in loss of phosphorylation sites and stabilization of β‐catenin in the cellular cytoplasm and nucleus. GSK‐3 expression and/or activity have been reported to be elevated in multiple tumor types, including pancreatic, gastric and colorectal carcinomas. Equally important, inhibition of GSK‐3 expression in colorectal tumor cell lines via specific siRNAs induced apoptosis and attenuated the proliferation of tumor cells. Together, these findings suggest that GSK‐3 inhibitors may represent a new class of therapeutic agents against colorectal cancer. LY‐GSK‐3i is a novel, potent and selective ATP competitive inhibitor of both isoforms of GSK‐3. In vitro, LY potently inhibits the enzymatic activity of GSK‐3α and GSK‐3β with IC50s of 1.5 nM and 0.9 nM, respectively. Pharmacologic activity of other GSK‐3 inhibitors in the literature against cancer is not clear. LY‐GSK‐3i did not evidence useful antitumor activity in its own right against colorectal, ovarian or non‐small cell lung (NSCL) tumor lines. However, when combined with different chemotherapeutic agents, LY was found to potentiate their pro‐apoptotic activity. LY‐GSK‐3i was shown to significantly potentiate caspase‐3 activation when combined cisplatin in HCT‐116 and Colo‐205 colorectal carcinoma lines. Enhancement of caspase‐3 activity was also observed when LY‐GSK‐3i was combined with 5‐fluorouracyl (5‐FU) in HCT‐116 cells. In order to explore the potential of LY‐GSK‐3i in other tumor types, we assessed the pro‐apoptotic activity of the LY/cisplatin combination against ovarian (A2780) and lung (A549 and NCI‐H460) cell lines. In all cases, LY‐GSK‐3i was shown to significantly elevate caspase‐3 activity when compared to cisplatin alone. In vivo, administration of a single dose of LY‐GSK‐3i to xenograft‐bearing mice lead to a time‐and dose‐dependent accumulation of the substrate protein ‐catenin in tumor tissues. In vivo xenograft growth studies revealed that administration of 5 mg/kg LY‐GSK‐3i in combination with cisplatin, given every 7 or 14 days, was sufficient to significantly improve the efficacy as compared to cisplatin alone given at the same intervals. Together, our findings provide evidence for the potential therapeutic utility of GSK‐3 inhibitors for the treatment of cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C211.