Irene Seipelt

Current View on the Mechanism of Action of Perifosine in Cancer

Perifosine treatment exhibits a complex molecular response including the inhibition of Akt or the induction of apoptosis via clustering of death receptors in lipid rafts. However, the molecular response can vary between different tumor entities and the contribution of each target pathway to the activity of Perifosine might be distinct depending on the tumor entity or the agent combined with Perifosine. In this review we discuss the current view on the mechanism of action of perifosine in cancer and the contribution of the molecular targets of Perifosine to its activity.

Discovery of 7‐Aryl‐Substituted (1,5‐Naphthyridin‐4‐yl)ureas as Aurora Kinase Inhibitors

As part of our research projects to identify new chemical entities of biological interest, we developed a synthetic approach and the biological evaluation of (7‐aryl‐1,5‐naphthyridin‐4‐yl)ureas as a novel class of Aurora kinase inhibitors for the treatment of malignant diseases based on pathological cell proliferation. 1,5‐Naphthyridine derivatives showed excellent inhibitory activities toward Aurora kinases A and B, and the most active compound, 1‐cyclopropyl‐3‐[7‐(1‐methyl‐1H‐pyrazol‐4‐yl)‐1,5‐naphthyridin‐4‐yl]urea (49), displayed IC50 values of 13 and 107 nM against Aurora kinases A and B, respectively. In addition, the selectivity toward a panel of seven cancer‐related protein kinases was highlighted. In vitro ADME properties were also determined in order to rationalize the difficulties in correlating antiproliferative activity with Aurora kinase inhibition. Finally, the good safety profile of these compounds imparts promising potential for their further development as anticancer agents.

Discovery of (7-aryl-1,5-naphthyridin-2-yl)ureas as dual inhibitors of ERK2 and Aurora B kinases with antiproliferative activity against cancer cells.

A novel series of (7-aryl-1,5-naphthyridin-2-yl)ureas was discovered as dual ERK2 and Aurora B kinases inhibitors. Several analogues were active at micromolar and submicromolar range against ERK2 and Aurora B, associated with very promising antiproliferative activity toward various cancer cell lines. Synthesis, structure activity relationship and docking study are reported. In vitro ADME properties and safety data are also discussed.

Abstract 871: Dual inhibition of PI3K and Erk1/2 shows synergy and efficacy in human tumor cells, either by using drug combinations or novel dual PI3K/Erk inhibitors

The Ras/Raf/Mek/Erk and the PI3K/Akt signaling pathways are frequently deregulated in cancer due to activation of upstream receptors, decreased expression of tumor suppressors like PTEN or activating mutations of Ras, B-Raf or PI3K. Several clinical studies with kinase inhibitors targeting single members of the Ras/Raf/Mek/Erk or the PI3K/Akt pathway are ongoing. However, preclinical and clinical trials indicated limited success by single pathway inhibition and different resistance mechanisms were defined. Further clinical trials with drug combinations of Ras/Raf/Mek/Erk and PI3K/Akt suppressors have been initiated, suggesting a more favorable outcome than targeting only one single pathway, Ras/Raf/Mek/Erk or PI3K/Akt. We performed in-vitro combination experiments with Raf, Mek or Erk inhibitors and PI3K or Akt inhibitors in several human tumor cell lines. Strong synergy was achieved with various combinations including the Mek inhibitor CI-1040 and the PI3K inhibitor GDC-0941 (CI = 0.17) or the Erk inhibitor AEZS-131 and the PI3K inhibitor D-117073 (CI = 0.23) in A549 cells. Also Raf inhibitors like Sorafenib and Zelboraf were combined with different PI3K inhibitors, resulting in synergistic anti-proliferative activity. Due to the attractiveness of parallel inhibition of the Ras/Raf/Mek/Erk and PI3K/Akt pathways, we developed AEZS-136 that concurrently inhibits Erk1/2 (IC50 ∼ 50nM) and PI3K (IC50 ∼ 100nM) by an ATP competitive mode of action. Derivatives of the dual PI3K/Erk inhibitor were co-crystallized with Erk2 and PI3Kα enabling an optimization process by SAR driven medicinal chemistry. The anti-proliferative efficacy of AEZS-136 was evaluated in more than 40 human tumor cell lines and physico-chemical as well as in-vitro ADMET properties were widely assessed. Furthermore, the in-vivo pharmacokinetics and anti-tumor efficacy was explored. AEZS-136 was well tolerated and showed dose dependent inhibition of human colon tumor growth of up to 74% in a Hct116 mouse model. Here we present the concept of dual targeting of the Ras/Raf/Mek/Erk and the PI3K/Akt pathways, either by using drug combinations or our novel dual PI3K/Erk inhibitor. AEZS-136 is a small molecule in preclinical development showing a uniquely advantageous kinase inhibition profile. Broad clinical anti-tumor activity is expected for AEZS-136 in tumors with deregulated Ras/Raf/Mek/Erk and PI3K-Akt signaling. 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 871. doi:1538-7445.AM2012-871

Efficient synthesis of novel disubstituted pyrido[3,4-b]pyrazines for the design of protein kinase inhibitors

A novel family of disubstituted pyrido[3,4-b]pyrazine-based compounds was discovered as valuable series for the design of promising protein kinase inhibitors. SAR study allowed the identification of 4-(piperidin-1-yl)aniline moiety as pharmacophoric group, in C-5 or C-8 positions of the pyrido[3,4-b]pyrazine ring, for binding to the selected therapeutic targets. Several analogues were active at low micromomolar IC50 values against a panel of seven cancer-related protein kinases providing an excellent starting point for further drug discovery optimization.

Abstract 3563: A highly selective Erk1/2 Inhibitor with in-vivo anti tumor potency

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Mutations in receptor tyrosine kinases, Ras or B-Raf frequently manifest constitutive activation of the Ras/Raf/Mek/Erk pathway in a variety of human cancers. The mitogen activated protein kinase Erk1/2 is an important regulator of cell proliferation and was shown to be over-activated by these dysregulated or mutated upstream signals. AEZS-131 selectively inhibits Erk1/2 with an IC50 of 4 nM, blocks cellular Rsk-1 phosphorylation, modulates downstream cellular substrate activation, arrests tumor cells in G1 and inhibits the growth of multiple human tumor cell lines in the nanomolar range. We have studied the response of human cancer cells (Hct116) in in-vivo mouse xenograft experiments. AEZS-131 significantly inhibited tumor growth at daily doses of 30mg/kg. Currently, AEZS-131 is undergoing in-vivo combination experiments with PI3K inhibitors. Since pharmacological inhibition of Erk1/2 reverses Ras and Raf activation also in cells demonstrating resistancy to common Raf inhibitors like GDC-0879 and PLX4720, it appears likely to put more attention on the downstream kinase Erk1/2 as therapeutic target. Furthermore, in instances of coincident activation of the Raf and PI3K pathways, combinations of AEZS-131 and PI3K inhibitors may prove efficacious. Here we present the in-vitro and in-vivo characterisation of the first in-class Erk1/2 inhibitor showing in-vivo efficacy as single therapy agent. Early development of the Erk1/2 inhibitor AEZS-131 by AEterna Zentaris is integral part of our in-house kinase research program comprising the investigation of different compounds for single Erk1/2 inhibition, single PI3K inhibition and dual kinase inhibitions. All compounds are exclusively synthesized by our Drug Discovery Department. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2011-3563

P3-18-06: AEZS-131 – A Highly Selective ERK-Inhibitor: Characterization and Preclinical Testing in Triple Negative Breast Cancer (TNBC).

Introduction: Overexpression of MAPK has been detected in 34 % of TNBC and has been found to be associated with anthracycline resistance (1). AEZS-131 is a highly selective orally active ERK1/2-inhibitor, which has shown in vivo activity (first in class) in colon cancer. The following study explores mode of action and efficacy in models of TNBC. Study design: AEZS-131 was tested in a kinase panel of 36 STK, 26 TK and 7 PIK to check for selectivity. Inhbition of Rsk-phosphorylation (cellular substrate of ERK) was evaluated by western blot analysis. Mode of action was explored by cell cycle FACS-analysis. Cleavage of PARP was explored by Western blot. Cytotoxic efficacy was evaluated in 5 TNBC, of which 2 had mutations in the MAPK signal transduction pathway lines, by MTT assay. Additionally it was explored if inhibition of classical apoptosis could abrogate the effect of AEZS-131. Results: AEZS-131 selectively inhibited ERK with an IC50 Conclusions: AZS-131 was shown to selectively inhibit ERK at low nM concentrations and to induce G1-arrest. Accordingly, the cytotoxic effect most pronounced in TNBC cell lines with mutations in the MAPK pathway. If classical apoptosis is inhibited, cytotoxic effects remain unchanged, suggesting that AEZS-131 can also induce nonclassical forms of programmed cell death. AEZS-131 should be further explored in TNBCs with overexpression of MAPK or mutations in the MAPK-pathway. *AEterna Zentaris GmbH, Frankfurt/M, Germany. (1): MAPK overexpression is associated with anthracycline resistance and increased risk for recurrence in patients with triple-negative breast cancer. Eralp Y, Derin D, Ozluk Y, Yavuz E, Guney N, Saip P, Muslumanoglu M, Igci A, Kucucuk S, Dincer M, Aydiner A, Topuz E. Ann Oncol. 2008 Apr;19(4):669–74. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-06.

Abstract 4474: Dual inhibitors for PI3K and Erk induce growth inhibition of tumor cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The Ras/Raf/Mek/Erk and the PI3K-Akt signaling pathways are prime targets for drug discovery in proliferative diseases such as cancer. The results of research to date indicate that both the MAPK and the PI3K signaling pathways represent therapeutic intervention points for the clinical treatment of malignant tumors. Our multi-parameter optimization program for kinase inhibitor selectivity, cellular efficacy, physico-chemical and in-vitro ADMET properties has led to the identification of a small molecular compound class with an uniquely advantageous dual kinase inhibition profile. These ATP competitive compounds inhibit Erk and PI3K in the nanomolar range and exert high selectivity against other serine threonine and tyrosine kinases. The anti-tumor efficacy of these dual kinase inhibitors was evaluated in diverse human tumor cell lines like HCT116, A549, MDA-MB 468, PC-3 and others. Physicochemical and in-vitro ADMET and safety parameters have been widely assessed. Furthermore in-vivo pharmacokinetic experiments showed plasma profiles expected to result in beneficial in-vivo anti-tumor efficacy. Here we present the key characteristics of the compound class that led to the selection of AEZS-132 for in-vivo experiments with tumor bearing nude mice. The optimization of ADME and physicochemical properties such as solubility, permeability and metabolic stability by medicinal chemistry is ongoing. 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 4474.