Highly selective MERTK inhibitors achieved by a single methyl group

Transcription

1 Highly selective MERTK inhibitors achieved by a single methyl group Jichen Zhao, ± Dehui Zhang, ±, Weihe Zhang, ± Michael A. Stashko, ± Deborah DeRyckere, Eleana Vasileiadi, Rebecca E. Parker, Debra Hunter, Qingyang Liu, ± Yuewei Zhang, ± Jacqueline Norris-Drouin, ± Bing Li, ± David Drewry, Dmitri Kireev, ± Douglas K. Graham, Henry Shelton Earp, Stephen V. Frye, ± Xiaodong Wang, ± * ± Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy Meryx, Inc., 45 West Dr., Chapel Hill, NC 27599, USA Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Aflac Cancer and Blood Disorders Center of Children s Healthcare of Atlanta and Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA 3322, USA Lineberger Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Supporting Information S1

2 Microfluidic Capillary Electrophoresis (MCE) Assay Activity assays were performed in a 384 well, polypropylene microplate in a final volume of 5 µl of 5 mm Hepes, Ph 7.4 containing 1 mm MgCl 2, 1. mm DTT,.1% Triton X-1,.1% Bovine Serum Albu (BSA), containing 1. µm fluorescent substrate (Table 5) and ATP at the Km for each enzyme (Table S1). All reactions were terated by addition of 2 µl of 7 mm EDTA. After a 18 incubation, phosphorylated and unphosphorylated substrate peptides (Table 5) were separated in buffer supplemented with 1 x CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Table S1. Assay conditions for MCE assays Kinase Peptide Substrate Kinase (nm) ATP (um) Mer 5-FAM-EFPIYDFLPAKKK-CONH Axl 5-FAM-KKKKEEIYFFF-CONH Tyro3 5-FAM-EFPIYDFLPAKKK-CONH Flt3 5-FAM-KKKKEEIYFFF-CONH Selectivity Profiling for 19 IC5 deteration by Carna Biosciences Compound preparation Test compound was dissolved in and diluted with dimethylsulfoxide (DMSO) to achieve 1-fold higher concentration as specified for the compound. The solution was further diluted 25-fold with assay buffer to make the final test compound solution. Reference compounds for assay control were prepared similarly. Assay reagents and procedures S2

3 Off-chip Mobility Shift Assay (MSA) 1) 5 ul of 4 compound solution, 5 ul of 4 Substrate/ATP/Metal solution, and 1 ul of 2 kinase solution were prepared with assay buffer (2 mm HEPES,.1% Triton X-1, 2 mm DTT, ph 7.5) and mixed and incubated in a polypropylene 384 well microplate for 1 or 5 h at room temperature, depending on kinase) 2) 6 ul of Teration Buffer (QuickScout Screening Assist MSA; Cama Biosciences) was added to the well. 3) The reaction mixture was applied to a LabChip3 system (Caliper Life Science), and the product and substrate peptide peaks were separated and quantitated. 4) The kinase reaction was evaluated by the product ratio calculated from peak heights of product (P) and substrate (S) peptides (P/(P+S)). Data Analysis The readout value of reaction control (complete reaction mixture) was set as % inhibition, and the readout value of background (Enzyme(-)) was set as 1% inhibition, then the percent inhibition of each test solution was calculated. Short PK Study A group of 4 male Swiss albino mice were dosed intravenously (i.v.) with solution formulation of the chosen compound in 1% DMSO, 1% Tween 8 in normal saline (.9% w/v NaCl solution) or normal saline only. From each mouse, three blood samples (6 µl/sample) were collected from retro orbital sinus such that samples were obtained at.5,.25, 1, 2, 4, & 8 h post dose. At each time point blood samples were collected from two mice in labeled micro centrifuge tube containing K2EDTA as anticoagulant. Plasma samples were separated by centrifugation of whole blood and stored below -7ºC until bioanalysis. All samples were processed for analysis by precipitation using acetonitrile (ACN) and analyzed with fit for purpose LC/MS/MS method (LLOQ was 2.47 S3

4 ng/ml for i.v. group and 9.87 ng/ml for p.o. group). Pharmacokinetic parameters were calculated using the Non-compartmental analysis tool of Phoenix WinNonlin (Version 6.3). Full PK Study A group of 9 male Swiss albino mice (group I) were dosed intravenously (iv) or intraperitoneally (ip) with solution formulation of 19 in normal saline (.9% w/v NaCl solution). Another group of 9 male Swiss albino mice (group II) were dosed orally (po) with formulation of 19 in normal saline. From each mouse, three blood samples (6 µl/sample) were collected from retro orbital plexus under light isoflurane anesthesia such that samples were obtained at pre-dose,.8,.25,.5, 1, 2, 4, 8 & 24 h (iv & ip) & pre-dose,.25,.5, 1, 2, 4, 6, 8 & 24 h (po) post dose. At each time point blood samples were collected from three mice in labeled micro centrifuge tube containing K2EDTA as anticoagulant. Plasma samples were separated by centrifugation of whole blood and stored below -7ºC until bioanalysis. All samples were processed for analysis by precipitation using acetonitrile (ACN) and analyzed with fit for purpose LC/MS/MS method (LLOQ was 2.47 ng/ml for iv group and 9.87 ng/ml for po group). Pharmacokinetic parameters were calculated using the Non-compartmental analysis tool of Phoenix WinNonlin (Version 6.3). S4

5 8/23/ AM Page 1 / 1 DZ23-16A Data Filename DZ23-16A.lcd Method Filename 5%-95%(22).lcm Batch Filename Vial # 1-1 Sample Type Unknown Injection Volume 1 ul Date Acquired 8/2/ PM Acquired by System Adistrator Date Processed 8/2/218 4 PM Processed by System Adistrator PDA Multi 1 254nm,4nm % C\LabSolutions\Data\li wang\dz23-16a.lcd

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7 f1 (ppm) BL1_7-2_PROTON_

8 BL1_7-4_CARBON_ f1 (ppm)

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