Investigation of pharmacokinetic properties of CK2 Inhibitors with an - PowerPoint PPT Presentation

Investigation of pharmacokinetic properties of CK2 Inhibitors with an Indeno[1,2- b ]indole scaffold Robin Birus 1, *, Marc Le Borgne 2 , and Joachim Jose 1 1 Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Corrensstrasse 48, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany 2 ISPB-Faculte de Pharmacie, Universite Claude Bernard Lyon 1, EA 4446 Bioactive Molecules and Medicinal Chemistry, 8 avenue Rockefeller, 69373 Lyon cedex 08,France. * Corresponding author: robin.birus@uni-muenster.de 1

Investigation of pharmacokinetic properties of CK2 Inhibitors with an Indeno[1,2- b ]indole scaffold Graphical Abstract 100 80 confluence [%] 60 40 20 0 0 12 24 36 48 t [h] Uptake Influence on tumor Induction of Metabolism analysis cell growth apoptosis studies 2

Abstract: The highly pleiotropic and constitutively active protein kinase CK2 plays an important role in several cellular mechanisms. Due to its overexpression and elevated activity in tumor cells, CK2 became an important target in tumor therapy nowadays. It was shown, that the kinase causes antiapoptotic and proliferation enhancing effects in neoplastic tissues [1,2]. Moreover, the reduction of CK2 activity in tumor cells leads to apoptosis while normal cells stay unaffected [3]. Indeno[1,2- b ]indoles are ATP competitive CK2 inhibitors with IC 50 values in the nanomolar range of concentration. NA16 was described as one of the most potent indeno[1,2- b ]indoles with an IC 50 value of 25 nM [4]. Therefore, the pharmacokinetic properties of NA16 were further analyzed during this study. It could be shown that NA16 reduces the growth of different tumor cell lines and induces cancer cell apoptosis. Furthermore, its effect on HUVEC cell growth was comparable with the effect of CX-4945, a CK2 inhibitor in clinical trials [5], which was used as control. Intracellular concentrations of NA16 were higher than concentrations of CX-4945 at different time points. Metabolism studies showed that NA16 is moderate metabolic stable and is not glucuronidated. These results underline the potential of NA16 as an antitumor drug. References: [1] Ahmed, K et al. : Trends Cell Biol 2002, 12, 226-230. [2] Meggio, F. and Pinna, L.A.: FASEB J. 2003, 17, 349-368. [3] Slaton, J. W. et al. : Mol Cancer Res 2004, 2, 172. [4] Gozzi, G. J. et al. : J Med Chem 2015, 58, 265-277. [5] Siddiqui-Jain, A. et al. : Cancer Res 2010, 70, 24. Keywords: Protein kinase CK2; ATP-competitive inhibitor; cell culture; pharmacokinetic 3

Introduction • CK2: • Heterotetrameric holoenzyme • Ubiquitous Ser/Thr kinase • Constitutively active; highly pleiotropic • Overexpression and higher activity in tumor cells • Important target in tumor therapy • Indeno[1,2- b ]indoles: • Potent, ATP competitive CK2 inhibitors • Low IC 50 values: <1 µM • NA16: One of the most potent Indeno[1,2- b ]indole derivatives ➢ IC 50 = 25 nM NA16

Results and discussion 1. Influence of NA16 on tumor cell growth A431 A549 LNCaP 100 100 100 30 µM 30 µM 40 µM 15 µM 15 µM 35 µM 80 80 80 12.5 µM 12.5 µM 30 µM confluence [%] confluence [%] confluence [%] 10 µM 10 µM 25 µM 60 60 60 7.5 µM 7.5 µM 20 µM 3.75 µM 3.75 µM 15 µM 40 40 40 1.88 µM 1.88 µM 10 µM 0.94 µM 0.94 µM 5 µM 20 20 20 0.47 µM 0.47 µM 1 µM DMSO 1% DMSO 1% DMSO 1% 0 0 0 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 t [h] t [h] t [h] • Investigation of tumor cell growth via Live Cell Imaging • Analyzing the effect of NA16 on growth of different cancer cell lines: A431 (epidermal), A549 (lung) and LNCaP (prostate) • NA16 reduces growth of all tested tumor cell line dose-dependently 5

Results and discussion 1. Influence of NA16 on tumor and normal cell growth • EC 50 : Inhibitor concentration which causes a reduction of cell 30 growth by 50% compared to A431 cells treated with 1% DMSO A549 • HUVEC: Human umbilical vein LNCaP 20 endothelial cell HUVEC EC 50 [µM] ➢ Non-cancer cell line • 10 NA16 reduces tumor cell growth in the same dimension as CX-4945 • 0 EC 50 of NA16 on HUVEC cells CX-4945 NA16 comparable to EC 50 of CX-4945 6

Results and discussion 2. Induction of apoptosis by NA16 DMSO 1% NA16 20 µM 25000 Green Object Count (1/Well) 20000 0 h CX-4945 1 µM 15000 CX-4945 10 µM CX-4945 20 µM 10000 NA16 1 µM NA16 10 µM NA16 20 µM 5000 DMSO 1% 48 h 0 0 12 24 36 48 t [h] • Apoptosis analysis of A431 cells via Live Cell Imaging • Fluorescence labeling of apoptotic cells after 24 h treatment with CK2 inhibitors ➢ Induction of apoptosis detectable as well after treatment with CX-4945 as with NA16 7

Results and discussion 3. Cellular uptake • Uptake analysis of CK2 inhibitors into A431 cells Intracellular concentrations • Extracellular concentration: 1 µM 1 h 6000 • Different incubation times: 1 h, 5 h, 12 h 5 h • 4000 12 h Cell lysis and sample purification • Quantification of intracellular inhibitor 2000 c [nM] concentration via HPLC-MS/MS 200 • c (NA16) > c (CX-4945) 100 • NA16: Decrease of intracellular concentration 0 • CX-4945 NA16 CX-4945: Increase of intracellular concentration 8

Results and discussion 4. Metabolism studies • Investigation of metabolic stability of NA16: Amount of intact parent [%] 80 • Quantification of intact NA16 after incubation with phase I metabolism enzymes via HPLC-MS (Börgel et al . 60 2019) • Comparison with imipramine (control 40 for metabolic labile substance) • Amount of intact NA16 higher than amount 20 of imipramine ➢ NA16 has a moderate metabolic 0 stability Imipramine NA16 9

Results and discussion 4. Metabolism studies • Metabolite identification: • NA16 + phase I metabolism enzymes vs. NA16 + phase I & II metabolism enzymes (glucuronidation was investigated representative for phase II metabolism) • Analysis of metabolites via HPLC-MS (Börgel et al. 2019) • Metabolites in both samples were equivalent • No glucuronidation products of NA16 detectable 10

Conclusions • Effects of NA16: • Reduction of growth of A431, A549 and LNCaP cells • Influence on HUVEC growth comparable with influence of CX-4945 • Induction of apoptosis weaker compared to treatment with CX-4945 • Intracellular concentration higher than concentration of CX-4945; time-dependent decrease Amount of intact parent [%] 80 60 • NA16 is moderate metabolic stable; no 40 20 0 glucuronidation products detectable e 6 n 1 m i A N a r p i m I ➢ NA16 is worth analyzing further effects concerning its anti-tumor activity 11

Acknowledgements Thanks to Prof. Jose and all members of the working group. 12