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SUPPLEMENTAL TABLE 1. Mass Spectrometry on EGFRL858R/T790M Protein

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SUPPLEMENTAL TABLE 1. Mass Spectrometry on EGFRL858R/T790M Protein SUPPLEMENTAL TABLE 1. Mass spectrometry on EGFRL858R/T790M protein % Mass Modification on Compound EGFRL858R/T790M Protein 1 96% 2 95% 3 109% 4 108% SUPPLEMENTAL TABLE 2. EGFR modulation in A431, H1975 and HCC827 cells by compound 3. EC50 (nM) Cell Lines A431 H1975 HCC827 EGFR Genotype WT L858R/T790M DelE746-A750 pEGFR > 4331 58 ± 34 187 ± 88 pAKT > 4331 55 ± 12 80 ± 49 pERK > 5000 39 ± 25 65 ± 6 pS6RP > 4841 29 ± 22 74 ± 48 Occupancy > 4260 34 ± 9 nd n > 3; ave ± STD; nd = not determined SUPPLEMENTAL TABLE 3. Kinase selectivity profile of compound 3, afatinib and WZ4002 at 1 µM. Compound 3 WZ4002 Afatinib FLT3 94% TXK* 98% ERBB4/HER4* 99% EGFR (L858R/T790M)* 91% BTK* 90% EGFR (WT)* 99% JAK3* 87% EGFR (WT)* 89% ERBB2/HER2*§ 98% TXK* 84% EGFR (L858R, T790M)* 87% EGFR (L858R, T790M)* 93% Aurora A 82% FLT3 87% TXK* 87% EGFR (WT)* 81% JAK3* 86% BLK* 84% FAK/PTK2 77% ERBB4/HER4* 85% BTK* 79% BMX/ETK* 74% FMS 77% ITK* 63% CHK2 74% BLK* 74% YES/YES1 55% ERBB4/HER4* 66% FAK/PTK2 73% LYN 53% BTK* 64% BMX/ETK* 69% c-Src 54% TEC* 62% CHK2 64% ITK* 58% FGFR3 54% TEC* 53% §ERBB2/HER2 included in kinase panel Compounds were tested against a 62 kinase panel at Reaction Biology Corporation which includes representative kinases from each branch of the kinome tree. Kinases inhibited greater than 50% are indicated. Asterisks indicate kinases that share Cys 797 with EGFR. For afatinib, ERBB2/HER2 kinase was included in the 62 kinase panel. SUPPLEMENTAL TABLE 4. Multi-dose level PK/PD study in H1975 tumor-bearing mice Compound 3 Tumor Conc. 1h Tumor Conc. 6h Lung Conc. 1h Lung Conc. 6h Tumor pEGFR Tumor EGFR Lunggp pEGFR (mg/kg) (ng/g) (ng/g) (ng/g) (ng/g) inhibition (%) Occupancy (%) inhibition (%) 3 150 ± 14 not detected* 114 ± 19 138 9 ± 22 62 ± 8 nd 10 455 ± 27 75 ± 7 688 ± 35 not detected* 48 ± 13 69 ± 2 nd 30 1304 ± 178 200 ± 40 1903 ± 329 115 ± 20 79 ± 3 89 ± 1 nd 100 4065 ± 813 830 ± 106 5925 ± 601 498 ± 82 96 ± 1 94 ± 1 0 ± 12 n = 4; ave ± SEM; nd = not determined; *no compound was detected at indicated time points due to technical issues SUPPLEMENTAL FIGURE1 SUPPLEMENTAL Merge pS6RP Merge Merge EGFR Merge Total pS6RP Merge ERK Merge AKT Merge Total EGFR Total ERK Total AKT Tubulin pEGFR pS6RP pERK pAKT nM │DMSO │20 A431 (EGFR │78 │313 │1250 WT ) │5000 H1975 (EGFR │DMSO │20 │78 │313 L858R/T790M │1250 │5000 ) HCC827 (EGFR │DMSO │20 │78 │313 DelE746-A750 │1250 │5000 ) 5 SUPPLEMENTAL FIGURE 2 AB H1975 (EGFRL858R/T790M) HCC827 (EGFRDelE746-A750) DMSO 0h 2h 4h 8h 16h 24h DMSO 0h 2h 4h 8h 16h 24h │ │ │ │ │ │ │ │ │ │ │ │ │ │ pEGFR pEGFR Total EGFR Total EGFR Merge EGFR Merge EGFR pERK pERK Total ERK Total ERK Merge ERK Merge ERK pAKT pAKT Total AKT Total AKT Merge AKT Merge AKT Free EGFRL858R Tubulin Tubulin SUPPLEMENTAL FIGURE 3 AB H1975 (EGFRL858R/T790M) HCC827 (EGFRDelE746-A750) EGFR Myc EGFR Myc 140% 140% 120% 120% 100% 100% 80% 80% 60% 60% 40% % Inhibition % Inhibition 40% 20% 20% 0% 0% 0h 2h 4h 8h 16h 24h 0hr 1hr 2hr 4hr 8hr 16hr 24hr CHX CHX Need to add HCC827 (Del) here SUPPLEMENTAL FIGURE 4 125% 100% ion tt 75% 50% % Inhibi 25% 0% DMSO 0hr 2hr 4hr 8hr 16hr 24hr erlotinib (100 nM) SUPPLEMENTAL FIGURE 5 AB H1975 (EGFRL858R/T790M) HCC827 (EGFRDelE746-A750) 0 min 5 min 15 min 30 min 60 min 0 min 5 min 15 min 30 min 60 min │ │ │ │ │ │ │ │ │ │ pEGFR pEGFR Total EGFR Total EGFR Merge EGFR Merge EGFR pERK pERK Total ERK Total ERK Merge ERK Merge ERK pAKT pAKT Total AKT Total AKT Merge AKT Merge AKT Free EGFRL858R Tubulin Tubulin.
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