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1. Introduction 6. Summary 4. Wee1 Selectivity Enhances Development of Selective WEE1 inhibitors A. Scott1*, A. Watson1, R. Bawn2, A. Churn2, C. Doe1, P. Doyle2, S. Hallworth3, A. Huxley2, M. Isherwood2, B. Leslie2, I. Morrison3, J. Morton2, G. Nelson3, C. Rigby2, M. Shelbourne2, M. Bingham2, T. Pesnot2 [1] Department of Discovery Biology, Concept Life Sciences, Block 35S, Alderley Park, Cheshire, SK10 4TG; [2] Department of Medicinal Chemistry, Concept Life Sciences, Block 35S, Alderley Park, Cheshire, SK10 4TG; [3] Department of ADMET services, Concept Life Sciences, Block 35S, Alderley Park, Cheshire, SK10 4TG 1. INTRODUCTION 4. WEE1 SELECTIVITY ENHANCES CELL VIABILITY WEE1 regulates the G2/M cell cycle checkpoint via phosphorylation ► Multiple CLS analogues are < 10 nM inhibitors of WEE1 kinase and of CDK1 (aka Cdc2) at Tyr15, which inhibits CDK1/cyclin B kinase display a measured logD between 1 and 3 (Fig. 4). activity (Fig. 1; Matheson et al, 2016). Inhibition of WEE1 overrides ► A range of structurally diverse analogues are >100-fold selective for DNA damage-induced cell cycle arrest in cells with a dysfunctional WEE1 over PLK1. G1 checkpoint and drives TP53 mutant cancer cells into mitotic ► None of the tested WEE1 literature references are > 50-fold selective catastrophe (Fig. 2; Duda et al, 2016). It is therefore an attractive (AZD1775 is 22-fold selective). Figure 1: Schematic representation of the role pf WEE1 in the G2/M target for enhancing the effects of chemotherapeutic DNA- checkpoint. Taken from Matheson et al, 2016 ► A number of CLS analogues show comparable potency to AZD1775 in the cellular mechanistic assay (pCDK1 levels; Fig. 5A); compound damaging therapies. Figure 4: WEE1 biochemical IC (Eu-LanthaScreen, Thermo) vs ranking was further confirmed by Western blotting (Fig. 5B). 50 PLK1 biochemical IC (ADP-Glo, Promega). Colours correspond to The potent WEE1 inhibitor AZD1775 (aka MK1775) has advanced 50 AZD1775 measured logD, with analogues highlighted in the presentation 150 in brighter colouring. (Spheres are CLS analogues, squares are to clinical trials in combination with DNA-damaging therapies in PLK1i 1 literature examples.) several cancers (https://clinicaltrials.gov). However, recent reports 1 100 2 K D 3 C Figure : CLS compound ( - ) effects on cellular WEE activity show that AZD1775 has single agent antiproliferative activity p 5 1 6 1 4 % 50 compared to AZD1775 and selective PLK1 inhibitor (PLK1i) after 6 (Matheson et al, 2016), which is counter-intuitive considering its 5 6 h treatment in non-synchronised cells by (A) ELISA (mean ± sd, n= ) and Western blotting ( , , and , nM doses). postulated mode of action. Other studies suggest AZD1775 exerts 0 2 (B) 100 1 000 10 000 1 10 100 1000 10000 100000 Con = vehicle control. poor kinase selectivity, and inhibits polo-like kinase 1 (PLK1) with Compound (nM) similar potency as WEE1 (Wright et al, 2017). PLK1 is also known to ► WEE1 active and selective compounds show reduced single agent cytotoxicity in MDA-MB-231 (Fig. 6A), HEK293 (non-cancer) directly regulate WEE1 activity by phosphorylation of Ser53, which and DAOY (data not shown) cells. leads to ubiquitination and subsequent proteasomal degradation ► There is a strong correlation between cancer (MDA-MB-231) and non-cancer (HEK293) viability IC50 data, suggesting that single of WEE1 (Kousholt et al, 2012). These findings suggest that some agent cytotoxicity is not dependent on TP53 status (Fig. 6B). of the observed effects of AZD1775 may not be solely due to WEE1 ► Compound ranking was further confirmed by real-time kinetic measurement of anti-proliferative effects (RealTime-Glo inhibition. Highly selective (in vitro and in vivo) tool compounds are (Promega); Fig. 6C and IncuCyte (Essen); data not shown) in MDA-MB-231 cells. Figure 6: (A) Compound critical for biological research and further elucidation of the role 150 AZD1775 100000 30000 effects on MDA-MB-231 cell 1 AZD1775 2 2 10000 R =0.953 1 viability (CellTiter-Glo assay, of WEE1. We present here our work at Concept Life Sciences (CLS) ) 0 h y t 5 t 100 Promega, mean ± sd, n=2). (B) i 3 20000 3 C l w I i 1000 o 3 b Figure : Proposed mechanism by which WEE inhibition may lead to r 2 1 9 6 towards the identification and development of a selective WEE1 a 4 Correlation of MDA-MB-231 g i 2 ( V K 100 U cell death by MUS81-SLX4-dependent chromosome breakage (mitotic 5 E and HEK293 viability IC values. % L 50 H 10000 50 tool compound for these studies. 6 R catastrophe). Taken from Duda et al, 2016. 10 (C) Compound effects on cell 0 1 0 proliferation (RealTime-Glo 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 assay, mean ± sd, n=2). Compound (nM) MDA-MB-231 IC50 Compound (nM) Figure 7 (A) Correlation of MDA- 100000 100000 No correlation 10000 MB-231 and HEK293 viability 10000 10000 IC50 values. (B) Correlation . DISCOVERY CASCADE 0 2 0 1000 0 5 5 5 C No Correlation C C I I I 1000 1000 of MDA-MB-231 viability data y y y t t t i 2 i i l l l 100 i i R =0.73 i b b b with WEE1 enzyme IC (C) a 100 a 100 a i i i 50 V V V 10 Correlation of MDA-MB-231 10 10 viability data with PLK1 enzyme MedChem Design 1 1 1 1 10 100 1000 10000 1 10 100 1000 10000 100000 IC50 Data derived from mean of Chemical synthethis 100 1000 10000 WEE1 Enzyme IC50 WEE1 Cell IC50 PLK1 Enzyme IC50 2 independent measurements. DMPK SAR On target SAR ► Single agent toxicity in MDA-MB-231 cells does not correlate with WEE1 enzyme (Fig. 7A) or cellular (Fig. 7B) activity. In vitro DMPK Biochemical Assays ► We observe a weak correlation of cytotoxicity with PLK1 enzyme activity (Fig. 7C); the significance of this finding is not yet known. LogD, solubility, PPB, microsomal/ WLEE1 hepatocyte stability PLK1 Low Clint (Mics & Heps) High solubility WEE1 IC50 < 100 nM & > 100-fold selectivity against PLK1 Cellular POM Assays Viability . TOWARDS A WEE SELECTIVE TOOL COMPOUND PK cassette MDA-MB-231 (breast cancer) 5 1 WEE1 (pCDK1 – ELISA/Western) IV dosing – serial sampling DAOY (Medulloblastoma) PLK1 (pTCTP – Western) HEK293 (non cancer, kidney) WEE1 IC < 1 µM & > 10-fold ► i.v./i.v. correlation 50 Viability > 1 μM CLS analogue 13 shows similar WEE1 activity (both biochemical, Fig. 8A and cellular Fig. 8C), increased selectivity versus PLK1 selectivity against PLK1 (biochemical, Fig. 8B), and reduced cytotoxicity (Fig. 8D) compared to AZD1775. PK analysis Kinase panel Cytotoxicity Margin 150 150 150 150 ) IV dosing – serial sampling MDA-MB-231 (cancer) vs HEK293 (non- AZD1775 ) % % ( ( ) 468 kinases, including WEE1/WEE2/PLK1 131 ) y y % t t 100 100 % 100 100 i PO dosing – tissue collection cancer) in combination with e.g. olaparib ( i ( v v i y i 1 t t t i c l c K i A D A b a 1 C 1 No Kinase < 10-fold WEE1 IC Margin > 10-fold i 50 50 p 50 50 50 E K V E L P Sufficient free W concentration to cover In vitro WEE1 selective 0 0 0 0 WEE IC (pCDK1) for 12 h 0.1 1 10 100 1000 10000 100000 0.1 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 Selective in vivo tool Selective in vitro tool inhibition enhances cellular toxicity margins (Y/N) Compound (nM) Compound (nM) Compound (nM) Compound (nM) Figure 8: Profiling CLS analogue 13 versus AZD1775. Compound effects on (A) WEE1 biochemical inhibition, (B) PLK1 biochemical inhibition, (C) WEE1 cellular activity (pCDK1 in MDA-MB-231), (D) cellular viability (MDA-MB-231). E cacy study In vitro WEE1 selective inhibition is MDA-MB-231 xenograft efficacious and safe (Y/N) ► Cross screening of a panel of 468 kinases confirmed WEE1 activity and selectivity against PLK1. Follow up IC50s against the top hits identified in the screen FLT3(D835V), MAP3K2/15, OSR1, TAOK1/2, TYK2(JH2domain-pseudokinase) is ongoing. ► Profiling of 13 shows selectivity over PLK1 can be achieved whilst retaining comparable in vitro DMPK properties to AZD1775 (Table 1) 1 2 3 4 5 CLS ID WEE1 PLK1 PLK1/WEE1 pCDK1 Cell viability logD7.4 Kinetic Solubility PPB Hepatocytes IC50 IC50 IC50 IC50 Human Mouse Human Mouse Fold N.A. μM nM nM selectivity μM μM %Fu Clint (μL/min/106 cells) 3. STRUCTURE BASED DESIGN OF WEE1 VS PLK1 SELECTIVITY AZD1775 4 86 22 0.21 0.25 2.4 8.6 25 28 3 123 13 4 1,562 391 0.86 10.2 2.9 7 12 40 17 148 ► Human WEE1 and human PLK1 kinases have 44% sequence similarity (BLAST alignment, kinase domain). Table 1: In vitro biological and DMPK characterisation of AZD1775 and 13. 1Eu-LanthaScreenTM (assay wall = 5 nM), 2ADP-GloTM, 3WEE1 cellular activity (MDA- MB-231), 4MDA-MB-231 cells, 5Plasma protein binding. ► Analysis of WEE1 (Zhu et al, 2017) and PLK1 (Kothe et al, 2007) Xray crystallographic data highlights key residue differences 376 130 378 132 303 47 (e.g. Asn /Leu , Tyr /Leu , Glu /Arg ), cavities and clashes that could be exploited within the active site to enhance kinase ► i.v. bolus PK cassette (0.4 mg/kg) administration of 4 CLS analogues (including 13) and Compound AZD1775 13 selectivity (Fig. 3A). AZD1775 to CD1 male mice (3 animals using micro-sampling) carried out (Table 2) In vivo PK parameters Dose (mg/kg) 0.4 0.4 ► Our Hit identification strategy aims to identify novel compounds with high WEE1 selectivity over PLK1 by exploiting these active ► No i.v./i.v.
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