GoKawiila, Measurement of TRI-611 induced proximity of the ALK kinase domain and CRBN/DDB1 in the presence of DMSO or 10 µM of the indicated TKIs as measured by TR-FRET acceptor/donor ratio. Data from n = 2 replicate wells are plotted. Experiment was conducted once. b, Inhibition of kinase activity by alectinib and lorlatinib as measured with a PhosphoSens kinase enzyme assay in the presence or absence of ALK:TRI-611:CRBN ternary complex. Data points represent the mean and SD of 3 replicate wells. Experiment was conducted once c, Left, Relative proliferation of NCI-H3122 cells measured by CTG in response to a dose titration of alectinib in the presence of the indicated concentrations of TRI-611 for 4 d. Error bars represent SD of n = 4 replicate treatments. Right, Bliss synergy plot of the TRI-611/alectinib in vitro combination data. Darker red indicates increased synergy (greater than additivity). d, Body weight changes of Balb/c nude mice with oral once daily administration of vehicle or compounds at the indicated dose levels during the NCI-H3122 efficacy study in Figs. 5c and 5e. Data points represent mean and SEM of n = 8. e, Relative EML4-ALK protein (by AlphaLISA) and unbound plasma C max at 6 h following a second dose of the indicated treatments. Data points represent the mean and SEM of n = 4 animals (n = 3 for lorlatinib exposure in the TRI-611 0.3 mpk/lorlatinib 0.1 mpk due to concentration below the limit of detection). Values for individual animals are shown as open black circles (EML4-ALK protein), open red squares (TRI-611 concentration), or open blue triangles (lorlatinib concentration). P-values by unpaired, two-tailed student’s t-test. f, Percent change in tumour volume for individual animals bearing subcutaneous NCI-H3122 xenografts on day 28 relative to day 0 following the indicated treatments. g, Relative EML4-ALK protein (by AlphaLISA) and unbound plasma C max at 6 h following a second dose of the indicated treatments. Data points represent the mean and SEM of n = 4 animals (n = 3 for alectinib 5 mpk due to spurious AlphaLISA reading). Values for individual animals are shown as open black circles (EML4-ALK protein), open red squares (TRI-611 concentration), or open blue triangles (alectinib concentration). P-values by unpaired, two-tailed student’s t-test. h, Body weight changes of Balb/c nude mice with oral once daily administration of vehicle or compounds at the indicated dose levels during the NCI-H3122 efficacy study in f. Data points represent mean and SEM of n = 8. i, Unbound plasma concentrations at the indicated times following the day 21 dose in the intracranial efficacy study in Fig. 5d. Data points represent mean and SEM of n = 4 (different animals were sampled at 2 h and 4 h timepoints). Values for individual animals are shown. j, Plot: relative proliferation of NCI-H3122 WT or NCI-H3122MET (MET overexpressing) cells measured by CTG in response to a dose titration TRI-611 in the presence of the indicated concentrations of capmatinib for 4 d. Error bars represent SD of n = 4 replicate treatments. Western: levels of the indicated proteins following 14 h treatment of NCI-H3122 (WT) or NCI-H3122MET (MET) with 1 µM TRI-611. Image is representative of 2 Western blots with consistent results. k, Plot: relative proliferation of MGH915-4 patient-derived cells measured by CTG in response to a dose titration TRI-611 in the presence or absence of 100 nM capmatinib for 5 d. Error bars represent SD of n = 3 replicate treatments and curve is representative of 3 independent experiments. Western: levels of the indicated proteins following 14 h treatment of MGH915-4 with 0.0001, 0.001, 0.01, or 0.1 µM TRI-611, or 1 µM TRI-611 with and without 100 nM capmatinib. All lanes are from the same gel with intervening lanes cropped as indicated. Image is representative of 2 replicate Western blots with consistent results. Curve fitting parameters can be found in Supplementary Table 1, raw gel images can be found in Supplementary Fig. 1, and source data with statistical parameters can be found online.
Source data