SUPPLEMENTARY MATERIAL
In vitro kinase activity assays
Recombinant enzyme for JAK1 was purchased from Invitrogen (Carlsbad, CA). Recombinant enzymes for FLT3, JAK2, JAK3, TYK2 were generated by Protein Biochemistry Group in S*BIO. The full kinase domain with a N-terminal GST-tag was expressed using the Bac-to-Bac Baculovirus Expression System (Invitrogen Life Technologies, #10359-016) and batch purified with Glutathione-Sepharose (Amersham Pharmacia, Uppsala, Sweden). All assays were carried out in 384-well white microtiter plates. Compounds were 4-fold serially diluted in 8 steps, starting from 10 µM. The reaction mixture consisted of 25 µl assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 5 mM β-glycerol phosphate). For FLT3 assays, the reaction contained 2.0 µg/mL FLT3 enzyme, 5 µM of poly(Glu,Tyr) substrate (Sigma, Cat # P0275) and 4 µM of ATP. For JAK1 assays, the reaction contained 2.5 µg/mL of JAK1 enzyme, 10 µM of poly(Glu,Ala,Tyr) substrate (Sigma, Cat # P3899) and 1.0 µM of ATP. For JAK2 assays, the reaction contained 0.35 µg/mL of JAK2 enzyme, 10 µM of poly (Glu,Ala,Tyr) substrate (Sigma, Cat # P3899) and 0.15 µM of ATP. For JAK3 assays, the reaction contained 3.5 µg/mL of JAK3 enzyme, 10 µM of poly (Glu,Ala,Tyr) substrate (Sigma, Cat # P3899) and 6.0 µM of ATP. For TYK2 assays, the reaction contained 2.5 µg/mL of TYK2 enzyme, 10 µM of poly (Glu,Ala,Tyr) substrate (Sigma, Cat # P3899) and 0.15 µM of ATP. The reaction was incubated at room temperature for 2 h prior to addition of 13 µL PKLight® detection reagent (Lonza, Rockford, ME). After 10 min incubation luminescent signals were read on a multi-label plate reader (Wallac 1420 Victor2™, Perkin-Elmer, Waltham, MA).
Other kinases tested at Millipore (c-Kit, KDR, PDGFRβ, Abl, ALK, AXL, BTK, EGFR, FGFR1, InsR, Lck, Met, RET, Src, Syk, Tie2, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, PKA, PKBα, PKCα, Aurora A, Aurora B, ASK1, MEK1, MKK6, CaMKVI, MAPKAPK2, PRAK, GSK3β, JNK1α1, p38αc-RAF, IRAK4, CK1δ, CK2α, IKKβ, TTK, Nek2) gave less than 30% inhibition at 100 nM.
lues were calculated using XLfit software (IDBS).
Pharmacokinetic analysis:
The Ba/F3-JAK2V617F xenograft model was established as described in the following section. Three days after cell injection, mice were allocated into 4 groups of 12 to receive oral doses of SB1518 at 15, 50, and 150 mg/kg q.d. for 12 days. On day 12, groups of 2 mice were sacrificed at pre-dose, 0.5, 1, 2, 4, 8 and 24 h post dose. Blood samples were collected by retro-orbital plexus puncture with 10 µL of 10% EDTA, centrifuged at 100Xg for 10 min and the plasma stored at -80°C.
50 µL of plasma (calibration standards or study sample) containing internal standard was extracted with 1.25 ml of methyl-tertiarybutylether for 30 minutes. Following extraction the samples were centrifuged at 13,000 g. for 10 minutes at 4°C in a microcentrifuge, and the supernatants were transferred to clean eppendorf tubes, and dried in a turbovap for 15 minutes. The residues were reconstituted with 100 µL of mobile phase and analyzed by LC/MS/MS. Analysis of murine plasma was performed using HPLC/MS on a Waters Alliance HT2795/QuattroMicro (Waters Corp., Milford, MA) using a C8 column (2 X 5 mm, 5 µm Zorbax, Quantum Analytics, Foster City, CA). Samples were resolved using an isocratic run with a mobile phase consisting of methanol: 0.1% formic acid in water (70:30v/v) at a flow rate of 0.3 mL/min. The tandem MS conditions for multiple reaction monitoring for SB1518 were transitions of mass to charge ratio (m/z) of 473 to m/z 96.8 and m/z 237 to 194 for the internal standard carbamazepine respectively, both in electrospray ionization-positive mode. The cone voltage (CV) was 40 V with collision energy (CE) of 35 eV for SB1518 and 20 V / 35 eV for carbamazepine. The assay was linear between 1 and 1000 ng/mL and the lower limit of quantitation was 1 ng/mL. Non-compartmental pharmacokinetic analysis was performed using WinNonlin (version 5.1, Pharsight, CA). The AUC was estimated by using the linear trapezoidal method. Dose proportionality was assessed by linear regression of day 12 AUC0-∞ and Cmax with dose using Prism 5 (GraphPad Software, La Jolla, CA).
Detection of JAK2V617F mutation
25 ng of extracted genomic DNA were used for Real Time PCR in the 7500 Real Time PCR system (Applied Biosystem, Foster City, CA), using a PCR Mastermix from Applied Biosystem, (Cat# 4324018) to determine the JAK2V617F mutation burden. The PCR cycle was as follows: (95C 10 min, 45 cycles at 95C 15 s, 62C 1 min). The TaqMan® probe used was AS-JAK2 (CTT GCT CAT CAT ACT TGC) and the forward primer used for JAK2 wild type/mutant determination was g-JAK2F (TTA TGG ACA ACA GTC AAA CAA CAA T). The reverse primers used for wild type/mutant JAK2 determination were G-gJAK2 (TTT ACT TAC TCT CGT CTC CAC AGt C) and T-gJAK2 (TTT ACT TAC TCT CGT CTC CAC AGt A), respectively. The mutation burden of the sample was calculated according to the following formula: JAK2 V617F%=[copy number of V617/(copy number of V617F+copy number of wild type)]*100%. All percentages above 0% indicate the presence of the JAK2V617F mutation.
RS11-0189R – Supplementary material Page 1