Supplementary Figures 1-4 - PDF file 294K, Supplementary Figure 1: PKM2 activation by screening hit XC-409; Supplementary Figure 2: Phosphorylation state of PKM2 at Tyr105 in cancer cell lines; supplementary Figure 3: Viability of a selection of lung adenocarcinoma cells in media with and without serine; Supplementary Figure 4: Sensitivity of a panel of adherent cancer cell lines to serine deprivation and treatment with PKM2 activator SGI-10067

Pim-1 has emerged as an attractive target for developing therapeutic agents for treating disorders involving abnormal cell growth, especially cancers. Herein we present lead optimization, chemical synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine compounds as potent and selective inhibitors of Pim-1 starting from a hit from virtual screening. These pyrazolo[1,5-a]pyrimidine compounds strongly inhibited Pim-1 and Flt-3 kinases. Selected compounds suppressed both the phosphorylation of BAD protein in a cell-based assay and 2-dimensional colony formation in a clonogenic cell survival assay at submicromolar potency, suggesting that cellular activity was mediated through inhibition of Pim-1. Moreover, these Pim-1 inhibitors did not show significant hERG inhibition at 30 μM concentration. The lead compound proved to be highly selective against a panel of 119 oncogenic kinases, indicating it had an improved safety profile compared with the first generation Pim-1 inhibitor SGI-1776.

The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

Activators of the pyruvate kinase M2 (PKM2) are currently attracting significant interest as potential anticancer therapies. They may achieve a novel antiproliferation response in cancer cells through modulation of the classic 'Warburg effect' characteristic of aberrant metabolism. In this Letter, we describe the optimization of a weakly active screening hit to a structurally novel series of small molecule 3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as potent PKM2 activators.

We present the discovery and optimization of a novel series of inhibitors of bacterial UDP-N-acetylglucosamine 2-epimerase (called 2-epimerase in this paper). Starting from virtual screening hits, the activity of various inhibitory molecules was optimized using a combination of structure-based and rational design approaches. We successfully designed and identified a 2-epimerase inhibitor (compound 12-ES-Na, that we named Epimerox) which blocked the growth of methicillin-resistant Staphylococcus aureus (MRSA) at 3.9 μM MIC (minimum inhibitory concentration) and showed potent broad-range activity against all Gram-positive bacteria that were tested. Additionally a microplate coupled assay was performed to further confirm that the 2-epimerase inhibition of Epimerox was through a target-specific mechanism. Furthermore, Epimerox demonstrated in vivo efficacy and had a pharmacokinetic profile that is consonant with it being developed into a promising new antibiotic agent for treatment of infections caused by Gram-positive bacteria.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11) frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

2-Arylamino-4-aryl-pyrimidines were found to be potent inhibitors of PAK1 kinase. The synthesis and SAR are described. The incorporation of a bromide at the 5-position of the pyrimidine core and in combination with a 1,2-dimethylpiperazine pendant domain yielded a lead compound with potent PAK1 inhibition and anti-proliferative activity in various colon cancer cell lines.

Rationale: 5-diphosphoinositol pentakisphosphate (IP7), formed by a family of inositol hexakis phosphate kinases (IP6Ks), has been demonstrated to be a physiologic inhibitor of Akt. IP6K inhibition may increase Akt activation in mesenchymal stem cells (MSCs), resulting in enhanced cardiac protective effect after transplantation. Objective: To investigate the role of IP6Ks for improving MSCs’ functional survival and cardiac protective effect after transplantation into infarcted mice hearts. Methods and results: Bone marrow-derived mesenchymal stem cells (BMMSCs), isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein positive (Fluc + -eGFP + ) transgenic mice, were preconditioned with IP6Ks inhibitor TNP (0.5μmol/L, 1μmol/L, 5μmol/L, and 10μmol/L) for 2 h followed by 6 hours of hypoxia and serum deprivation (H/SD) injury. TNP dose dependently significantly decreased IP7 production with increased Akt phosphorylation. Moreover,TNP at 10μmol/L significantly improved the viability and enhanced the paracrine effect of MSCs after H/SD. Furthermore, MSCs were transplanted into infarcted hearts with or without selective IP6Ks inhibition. Longitudinal in vivo bioluminescence imaging (BLI) and immunofluorescent staining revealed that TNP pretreatment enhanced the survival of engrafted MSCs, which promoted the anti-apoptotic and pro-angiogenic efficacy of MSCs in vivo . Furthermore, MSCs therapy with IP6Ks inhibition significantly decreased fibrosis and preserved heart function. Conclusion: Inhibition of IP6Ks promotes MSCs engraftment and paracrine effect in infarcted hearts at least in part by down-regulating IP7 production and enhancing Akt activation, which might contribute to the preservation of myocardial function after MI.

Abstract Inactivation of the M2 form of pyruvate kinase (PKM2) in cancer cells is associated with increased tumorigenicity. To test the hypothesis that tumor growth may be inhibited through the PKM2 pathway, we generated a series of small-molecule PKM2 activators. The compounds exhibited low nanomolar activity in both biochemical and cell-based PKM2 activity assays. These compounds did not affect the growth of cancer cell lines under normal conditions in vitro, but strongly inhibited the proliferation of multiple lung cancer cell lines when serine was absent from the cell culture media. In addition, PKM2 activators inhibited the growth of an aggressive lung adenocarcinoma xenograft. These findings show that PKM2 activation by small molecules influences the growth of cancer cells in vitro and in vivo, and suggest that such compounds may augment cancer therapies. Mol Cancer Ther; 12(8); 1453–60. ©2013 AACR.

A series of compounds based on a 4-phenyl-2-phenylaminopyridine scaffold that are potent and selective inhibitors of Traf2- and Nck-interacting kinase (TNIK) activity are described. These compounds were used as tools to test the importance of TNIK kinase activity in signaling and proliferation in Wnt-activated colorectal cancer cells. The results indicate that pharmacological inhibition of TNIK kinase activity has minimal effects on either Wnt/TCF4/β-catenin-driven transcription or viability. The findings suggest that the kinase activity of TNIK may be less important to Wnt signaling than other aspects of TNIK function, such as its putative role in stabilizing the TCF4/β-catenin transcriptional complex.

Tumor cells primarily utilize aerobic glycolysis, rather than oxidative phosphorylation, to metabolize glucose (the Warburg effect). The M2 splice form of pyruvate kinase (PKM2), the enzyme catalyzing the rate-limiting final step of glycolysis, is highly upregulated in tumors. Unlike the M1 splice form (PKM1), a constitutively active tetramer found predominantly in non-cancerous tissues, PKM2 is an inactive dimer under normal physiological conditions. Tetramerization of PKM2 requires binding of the allosteric activator fructose-1,6-bisphosphate (FBP), an upstream glycolytic intermediate, resulting in a fully active enzyme. Inactivation of PKM2 by cancer cells may allow glycolytic intermediates to be diverted into other biosynthetic pathways necessary for biomass production. The finding that PKM2 rather than PKM1 expression enhances tumorigenicity suggests that activators of PKM2 may have anti-tumor properties. We have identified and developed a series of small molecule PKM2 activators that exhibit low nM activation activity in biochemical and cell-based assays that measure pyruvate and ATP production. The extent of activation of these compounds is equal to or greater than that of FBP in biochemical assays. In addition, preliminary studies show that PKM2 activators inhibit the growth of lung cancer cell lines in vitro. The current lead compound was tested in established subcutaneously implanted A549 lung adenocarcinoma xenografts, where we observed a statistically significant 54% decrease in tumor growth, with no observable toxicity. These data suggest that this class of PKM2 activators is effective as tumor cell metabolic regulators with anti-tumor activity for lung cancer and potentially other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B143.

Tumor cells primarily utilize aerobic glycolysis, rather than oxidative phosphorylation, to metabolize glucose (the Warburg effect). The M2 splice form of pyruvate kinase (PKM2), the enzyme catalyzing the rate-limiting final step of glycolysis, is highly upregulated in tumors. Unlike the M1 splice form (PKM1), a constitutively active tetramer found predominantly in non-cancerous tissues, PKM2 is an inactive dimer under normal physiological conditions. Tetramerization of PKM2 requires binding of the allosteric activator fructose-1,6-bisphosphate (FBP), an upstream glycolytic intermediate, resulting in a fully active enzyme. Inactivation of PKM2 by cancer cells may allow glycolytic intermediates to be diverted into other biosynthetic pathways necessary for biomass production. The finding that PKM2 rather than PKM1 expression enhances tumorigenicity suggests that activators of PKM2 may have anti-tumor properties. We have identified and developed a series of small molecule PKM2 activators that exhibit low nM activation activity in biochemical and cell-based assays that measure pyruvate and ATP production. The extent of activation of these compounds is equal to or greater than that of FBP in biochemical assays. In addition, preliminary studies show that PKM2 activators inhibit the growth of lung cancer cell lines in vitro. The current lead compound was tested in established subcutaneously implanted A549 lung adenocarcinoma xenografts, where we observed a statistically significant decrease in tumor growth, with no observable toxicity. These data suggest that this class of PKM2 activators is effective as tumor cell metabolic regulators with anti-tumor activity for lung cancer and potentially other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3226. doi:1538-7445.AM2012-3226

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) includes constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphomas, leukemias, multiple myeloma, prostate and bladder cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, and promoting tumor cell survival and proliferation. In the HEK-293T cell line, enhanced PIM kinase substrate BAD phosphorylation (pBAD) was observed following PIM and BAD overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, and more effectively by second generation PIM inhibitors exhibiting 4-10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-µM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the UM-UC-3 human bladder cancer cell line. PIM1 and PIM2 overexpression models were established in the human prostate cancer cell line 22RV-1 and the non-tumorigenic mouse NIH-3T3 cell background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In vivo xenograft studies using both PIM overexpression models and a clinically relevant solid tumor model facilitated identification of a lead candidate with demonstrated efficacy and favorable toxicity. IND-enabling studies with a lead candidate are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3762. doi:1538-7445.AM2012-3762

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) comprises constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, prostate, bladder, gastric and head & neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, promoting tumor cell survival and proliferation. PIM-1 and PIM-2 overexpression models were obtained in the human prostate cancer cell lines PC-3M and 22RV-1 and the non-tumorigenic mouse NIH-3T3 background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In the PC-3M cell line, enhanced phosphorylation of the PIM kinase substrate BAD (pBAD) was observed following PIM overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, as well as next generation PIM inhibitors exhibiting 4–10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-μM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the PC-3M prostate cancer cell line, the UM-UC-3 bladder cancer cell line, and the HSC3 head & neck cancer cell line. The second generation PIM inhibitors possess favorable hERG and CYP inhibition profiles compared with SGI-1776, and demonstrate excellent oral bioavailability. In vivo xenograft studies using both PIM overexpression models and clinically relevant solid tumor models will facilitate identification of a clinical candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C200.