TRI-611, a selective, brain-penetrant molecular glue degrader of ALK

a, Schematic of TR-FRET screen used to identify molecular glues of CRBN and ALK. Europium-labelled streptavidin binds to biotinylated ALK and Ulight-labelled anti-His antibody binds to CRBN. When ALK and CRBN are brought into proximity in a ternary complex by a molecular glue, a TR-FRET signal is generated. b, Scatter plot of TR-FRET screen and identification of compound 1 as an ALK:CRBN molecular glue hit. Data points represent individual compounds sorted on the x-axis by compound ID number. c, Key features of compound 1. P_app: apparent permeability coefficient; MDR1: multidrug resistance 1 (P-glycoprotein); A-B: apical-basolateral; efflux ratio: P_app (apical-basolateral)/P_app (basolateral-apical); CNS MPO: central nervous system multiparameter optimization score; MLM: mouse liver microsomes; HLM: human liver microsomes. d, Measurement of induced ALK:CRBN proximity measured by TR-FRET as a dose response of compound 1 in the presence and absence of 10 µM CC-885, a GSPT1 MGD that binds to CRBN but does not recruit ALK. Data from n = 2 replicate wells are plotted and experiment was conducted once. e, Measurement of ALK:compound 1:CRBN ternary complex formation by SPR with immobilized ALK kinase domain, fixed CRBN, and the indicated concentrations of compound 1. K_D value represents the mean and SD of 6 replicates run on two days, and traces are from one experiment. f and g, Measurement of induced cellular proximity (by luminescence relative to DMSO) between EML4-ALK v1 (f) or EML4-ALK v3 (g) and CRBN as a dose response of compound 1 or TRI-611 in 293 T cells by NanoBiT assay. Data points indicate mean and standard deviation from six replicate transfections. CI: confidence interval. h, Representative SPR single-cycle kinetics sensorgram of ternary complexes of ALK WT:TRI-611:CRBN measured by flowing a titration of CRBN/DDB1 and fixed TRI-611 over immobilized ALK kinase domain. K_D and residence time values represent mean and SD of 3 independent experiments, and traces are from one experiment. i, ALK kinase activity as measured with a PhosphoSens kinase enzyme assay in the presence or absence of an ALK:TRI-611:CRBN complex. Data points represent the mean and 95% CI of 4 replicate wells and experiment was conducted once. j, Representative SPR sensorgrams of CRBN and the indicated concentrations of TRI-611 to measure binary affinity. K_D value is represented as mean and SD of 4 experiments on two days, and traces are from one experiment. Source data can be found online.

Source data

a-c, Relative change in protein abundance comparing cellular protein levels of NCI-H3122, SNU2535, or A549 cells treated in triplicate dishes with DMSO or 3 µM of TRI-611 for 3 h assessed on a proteome scale by mass spectrometry. Horizontal dashed line indicates adjusted p-value of 0.05; vertical dashed lines indicate 2-fold change relative to DMSO. Adjusted p-values were calculated by unpaired, 2-tailed student’s t-test with multiple hypothesis correction by the Benjamini-Hochberg method. d and e, Change in EML4-ALK protein by AlphaLISA following treatment of NCI-H3122 (d) or SNU2535 (e) cells with TRI-611. Each datapoint represents one well normalized to time 0. f, Levels of the indicated proteins following treatment of NCI-H3122 cells with 0.03, 0.15, or 2 µM TRI-611, or 0.2 µM lorlatinib for 6 h. Image is representative of 2 Western blots with consistent results. g, Proliferation relative to DMSO as measured by Cell Titer Glo following 4-day treatment with lorlatinib or CC-885. Data points represent mean and standard deviation of 3 replicate treatments. h, Proliferation relative to DMSO as measured by Cell Titer Glo following 4-day treatment with TRI-611 or lorlatinib in the indicated cell lines. Data are from the same experiments presented in Fig. 1i and above in panel g. Data points represent mean and standard deviation of 3 replicate treatments. i, plot: Levels of ALK protein by ELISA in Kelly cells treated with TRI-611 for 3 h. Data points represent the mean and SD of 3 replicate treatments. Western: Levels of ALK following treatment of Kelly cells with 0.1 µM TRI-611 for 5 h in the presence or absence of bortezomib at 0.1 µM. Experiment was conducted once but similar bortezomib rescue was observed at a second tested concentration of TRI-611. j, left, Levels of EML4-ALK protein by ELISA in NCI-H3122 WT or NCI-H3122-CRBN-OV (CRBN overexpressing) following treatment with TRI-611 for 6 h. middle, Relative proliferation by CTG in NCI-H3122 WT or NCI-H3122-CRBN-OV following treatment with TRI-611 for 4 days. For both, data points represent mean and SD of 3 replicate treatments. right, Levels of the indicated proteins following treatment of NCI-H3122-WT or NCI-H3122-CRBN OV with 0.03, 0.12, or 2 µM of TRI-611 for 12 h. Image is representative of 2 Western blots with consistent results. k, Levels of the indicated proteins following treatment with 1 µM TRI-611 for 14 h, or 0.3 µM CC-885 for 4 h. Images are representative of 2 Western blots with consistent results. l, Relative proliferation of KM12-luc cells following treatment with TRI-611, lorlatinib, or CC-885 for 4 days as measured by Cell Titer Glo. Data points represent the mean and SD of 3 replicate treatments. m, Cell cycle distribution as measured by BrdU/7-AAD staining following treatment of NCI-H3122 cells with TRI-611 at proliferation IC₅₀ (0.15 µM) or IC₉₀ (2 µM), or lorlatinib at proliferation IC₅₀ (0.003 µM) or IC₉₀ (0.1 µM). Error bars represent the SD of 3 replicate treatments. n, Induction of apoptosis as measured by Caspase-Glo assay following treatment of NCI-H3122 cells with TRI-611 or lorlatinib for 24 h. Data points represent the mean and SD of 3 replicate treatments. Curve fitting parameters can be found in Supplementary Table 1, raw gel images can be found in Supplementary Fig. 1, and source data can be found online.

Source data

a, ALK:TRI-611:CRBN ternary complex with key residues highlighted. ALK: blue; CRBN: purple. b, 2D representation of key ligand interactions. Residues are annotated with single letter codes and numbering for “C” (CRBN) or “A” (ALK). HIE: histidine with protonated epsilon nitrogen. Green line: pi-stack; pink line: h-bond. c, Composite cryo-EM density map for the entire sample assembly of ALK:TRI-611:CRBN:DDB1 (EMDB ID EMD-72685), protein models rigid-body fitted into the density. d, Cryo-EM data collection, refinement, and validation statistics for the structure of ALK:TRI-611:CRBN. e, Relative EML4-ALK protein by ELISA in NCI-H3122 WT or NCI-H3122^{EML4-ALK D1389W} following treatment with TRI-611 for 3 h. f, Relative proliferation by CTG in NCI-H3122 WT or NCI-H3122^{EML4-ALK D1389W} following treatment with TRI-611 (left) or lorlatinib (right) for 4 d. For all plots, data points represent mean and SD of 3 replicate treatments. Curve fitting parameters can be found in Supplementary Table 1 and source data can be found online.

Source data

a, Measurement of induced proximity of ALK WT or ALK^{L1196M/G1202R} kinase domains and CRBN/DDB1 as measured by TR-FRET acceptor/donor ratio. Data from n = 2 replicate wells are plotted. Experiment with ALK MUT was repeated 15 times with consistent results. ALK WT data are the same as that shown in Fig. 1b, and data in the 2 graphs are from the same experiment. b, SPR single-cycle kinetics sensorgram of ternary complex of ALK MUT:TRI-611:CRBN measured by flowing a titration of CRBN/DDB1 and fixed TRI-611 over immobilized ALK kinase domain, where ALK MUT = ALK^{L1196M/G1202R}. Plot is representative of 3 independent experiments run in parallel with the ALK WT experiment shown in Extended Data Fig. 1h, and K_D and residence time represent the mean and SD of n = 3 (K_D) or n = 2 (t_1/2, value could not be determined in one experiment). c, TRI-611 mediated ubiquitin transfer to ALK^{L1196M/G1202R} (ALK MUT) kinase domain in the presence of E1, E2 and the E3 ligase complex containing CRBN as measured by Western blotting with an ALK-specific antibody following 1 h reaction at 30 °C in the presence of 0.01, 0.1, or 1 µM of TRI-611. Image is representative of 3 independent experiments. d, Degradation of EML4-ALK in stable Ba/F3^hCRBN cell lines was measured by Western blotting following treatment of cells with 0.1 µM TRI-611 for 2 h. Images are representative of at least 2 Westerns per cell line. e, Relative EML4-ALK protein by ELISA in the indicated Ba/F3^hCRBN cell lines following 3 h treatment with TRI-611. Data points represent mean and SD of 3 replicate treatments. f, Comparison of degradation pDC50 (-log₁₀DC₅₀) and proliferation pIC₅₀ (-log₁₀IC₅₀) among the indicated cell lines. Pearson r and 2-tailed p-value were calculated by GraphPad Prism. CI: 95% confidence interval. g, Expression of the indicated proteins in the indicated cell lines (extract from 1.2x10⁵ cells per lane). Relative CRBN expression levels were consistent across 2 replicate Westerns for ALK+ cell lines. h, Levels of the indicated proteins in NCI-H3122 cells with the indicated CRISPR engineered mutations treated with 1 µM TRI-611 for 14 h. Image is representative of at least 2 independent Westerns with consistent results, except for G1269A and I1171N, which were profiled once. i, Levels of the indicated proteins in patient-derived cell lines following treatment with 0.0001, 0.001, 0.01, 0.1, or 1 µM TRI-611 for 16 h. An equal amount of extract from NCI-H3122 cells was used as a comparison. Western blots from each cell line are from the same gel with intervening lanes cropped where indicated. Images are 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 can be found online.

Source data

a, Unbound plasma concentrations of TRI-611 (using measured mouse plasma protein binding of 98.6% bound) following once daily oral dosing on day 21 of the efficacy study in Fig. 4a. Dashed line indicates degradation DC₅₀ for in vitro treatment of NCI-H3122 with TRI-611, corrected for media protein binding (using measured media protein binding of 78% bound). Data points represent the mean and SEM of n = 4 per timepoint. b, Relative EML4-ALK protein by AlphaLISA in tumours normalized to vehicle at each timepoint (left y-axis) and unbound plasma TRI-611 concentration (right y-axis, calculated as in a) following a single oral dose with 1 mg/kg or 10 mg/kg TRI-611. Data points represent the mean and SEM of 4 animals. Dashed line indicates degradation DC₅₀ for in vitro treatment of NCI-H3122 with TRI-611, as in a. c, Body weight changes of Balb/c mice during the efficacy study in Fig. 4a. Data points represent mean and SEM of n = 8. d, Levels of pSTAT3 and STAT3 in individual NCI-H3122 tumours at 6 h following a second daily dose of TRI-611. Each lane represents an individual animal (same study as in Fig. 4b). Western analysis was performed once. e, Tumour volume of NCI-H2228 xenografts following once daily oral dosing with vehicle or TRI-611. Data points represent the mean and SEM of n = 8. f, Relative EML4-ALK protein as measured by AlphaLISA in NCI-H2228 xenografts 6 h after a second dose of TRI-611. Error bars represent SEM of n = 4 (n = 3 for TRI-611 0.1 mg/kg and 1 mg/kg dose due to spurious AlphaLISA counts for one animal in each cohort). Values for individual animals are shown. P-values by unpaired, 2-tailed student’s t-test. g, Body weight changes of CB-17 SCID mice during the efficacy study in e. Data points represent mean and SEM of n = 8. h, Levels of pSTAT3 and STAT3 in individual NCI-H2228 tumours at 6 h following a second daily dose of TRI-611. Each lane represents an individual animal (same study as in f). Western analysis was performed once. i, Body weight changes of mice during the efficacy study in Fig. 4c. Data points represent mean and SEM of n = 8. j, Maximum unbound plasma concentration of TRI-611 (calculated as described in a) or lorlatinib (calculated using published mouse plasma protein binding of 75% bound⁵⁶) in NCI-H3122 xenografts following a second dose of compound. Error bars represent the mean and SEM of n = 4. Values for individual animals are shown. Raw gel images can be found in Supplementary Fig. 1 and source data with statistical parameters and Western blotting band quantification can be found online.

Source data

a, Tumour volume of NCI-H3122^unedited xenografts following once daily oral dosing with vehicle, TRI-611, or lorlatinib. Data points represent the mean and SEM of n = 8 (n = 7 for TRI-611 1 mg/kg and TRI-611 3 mg/kg). b, 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^unedited (left) or NCI-H3122^{EML4-ALK L1196M/G1202R} (right) efficacy study in Fig. 4d. Data points represent mean and SEM of n = 8 (left: 1 animal euthanized on day 14 in each of TRI-611 1 mg/kg and TRI-611 3 mg/kg dose groups; right: 1 mouse died in vehicle group on day 12). c, Relative EML4-ALK protein as measured by AlphaLISA in NCI-H3122^unedited (left) or NCI-H3122^{EML4-ALK L1196M/G1202R} (right) xenografts 6 h after a second dose of compounds. Error bars represent SEM of n = 4 (left: n = 3 for TRI-611 10 mg/kg and lorlatinib 3 mg/kg dose due to insufficient tumour volume for animals 4-3 and 5-1; right: n = 3 for TRI-611 10 mg/kg due to exclusion of spurious AlphaLISA signal in animal 4-2). Values for individual animals are shown. P-values by unpaired, 2-tailed student’s t-test. d, Levels of pEML4-ALK (pTyr1282/1283) and EML4-ALK in individual tumours in NCI-H3122^unedited (top) or NCI-H3122^{EML4-ALK L1196M/G1202R} (bottom) tumours at 6 h following a second dose of compounds. Each lane represents an individual animal (same study as in c). Western analysis was performed once. e, Maximum unbound plasma concentration of TRI-611 (calculated as in Extended Data Fig. 6a) or lorlatinib (using published mouse plasma protein binding of 75% bound⁵⁶) in the indicated xenografts following a second dose of compound. Error bars represent the mean and SEM of n = 4. Values for individual animals are shown. f, Body weight changes of NSG mice with oral once daily administration of vehicle or compounds at the indicated dose levels in the DFCI-669 study in Fig. 4e. Data points represent mean and SEM of n = 8 (n = 7 for vehicle cohort after day 14 and n = 4 for lorlatinib cohort). g, Maximum unbound plasma concentration of TRI-611 or lorlatinib (calculated as described in e) in the DFCI-669 study following a second dose of compound. Error bars represent the mean and SEM of n = 4. Values for individual animals are shown. h, Levels of the indicated proteins in individual DFCI-669 tumours at 6 h after a second dose of compounds as measured by Western blotting. Each lane represents an individual animal (same study as in g). Image is representative of 2 Western blots with consistent results. i, Body weight changes of NSG mice with oral once daily administration of vehicle or compounds at the indicated dose levels in the MGH953-7 study in Fig. 4f. Error bars represent the mean and SEM of n = 8. j, Unbound plasma concentration of TRI-611 or lorlatinib (calculated as described in e) in the MGH953-7 study at 2 h following a second dose of compound. Error bars represent the mean and SEM of n = 4. Values for individual animals are shown. k, Levels of the indicated proteins in individual MGH953-7 tumours at 2 h after a second dose of compounds as measured by Western blotting. Red arrow indicates sample with low tumour cell fraction due to presence of subcutaneous fat. Asterisk in EML4-ALK pY1282/1283 blot indicates non-specific background bands. Bands corresponding to EML4-ALK pY1282/1283 are indicated with a black arrowhead. Each lane represents an individual animal (same study as in j). Image is representative of 2 Western blots with consistent results. Raw gel images can be found in Supplementary Fig. 1 and source data with statistical parameters can be found online.

Source data

a, K_{pu,u brain} for the indicated compounds after a single oral dose in naïve Balb/c mice at the indicated timepoints. Animals were perfused prior to brain collection. Error bars represent the SD of n = 3. Values for individual animals are shown. P-values were calculated by unpaired, 2-tailed student’s t-test. TRI-611 mouse plasma protein binding was described in Extended Data Fig. 6a; mouse brain protein binding was measured at 98% bound. Lorlatinib mouse plasma and brain protein binding were measured at 77% and 92% bound, respectively. Neladalkib mouse plasma and brain protein binding were measured at 99.7% and 99.8% bound, respectively. b, Luminescence (left) or body weight changes (right) in Balb/c nude mice during the NCI-H2228-luc intracranial efficacy study. Data points represent the mean and SEM of n = 8 (n = 7 for vehicle at 16 d, n = 4 for vehicle at 20 d). c, Images of luminescent intracranial xenografts of NCI-H2228-luc during the efficacy study. d, Waterfall plot showing percent change in luminescence from day 0 to day 16 for individual animals with the indicated treatments in the NCI-H2228-luc efficacy study in c. e, Maximum unbound plasma concentration of TRI-611 (calculated as described in Extended Data Fig. 6a) or lorlatinib (calculated as described in Extended Data Fig. 6j) in the NCI-H2228-luc intracranial study in Fig. 5c following a second dose of compound. Error bars represent the mean and SEM of n = 4. Values for individual animals are shown. f, K_{pu,u brain} for TRI-611 and lorlatinib on day 21 of the efficacy study at 6 h post the last dose. Animals were perfused prior to brain collection. Error bars represent the SEM of n = 4 (n = 3 for TRI-611 3 mg/kg cohort due to animal euthanasia on day 20). Values for individual animals are shown. Protein binding parameters are described in a. Differences were not statistically significant (p > 0.05 by unpaired, 2-tailed student’s t-test). g, Unbound brain concentrations of TRI-611 at 6 h following the day 21 dose in the efficacy study. Animals were perfused prior to brain collection. Protein binding parameters are described in a. Error bars represent the SEM of n = 3 (TRI-611 3 mg/kg) or n = 4 (TRI-611 10 mg/kg). Values for individual animals are shown. Dashed line indicates NCI-H2228 in vitro DC₅₀ corrected for media protein binding (measured at 78% bound). Unbound brain exposure in the TRI-611 1 mg/kg cohort was below the limit of detection of 1.6x10⁻⁹ M. h, ALK protein expression (brown) by IHC in brain sections of mice inoculated intracranially with NCI-H2228-luc cells and treated for two days with vehicle or TRI-611 at the indicated doses. Tumours are from different animals in the same study as in Fig. 4h. Tumour cells are outlined in the grey boxes. Study was performed once after antibody validation with control tumour sections. i, Luminescence (top) or body weight changes (bottom) during the NCI-H2228-luc intracranial efficacy study in Fig. 5d. Error bars represent SEM of n = 8. j, Images of luminescent intracranial xenografts of NCI-H2228-luc during the efficacy study in Fig. 5d. Source data with statistical parameters can be found online.

Source data

a, Luminescence images of intracranial tumours for the efficacy study in Fig. 4i. b, Mean luminescence of NCI-H3122^{EML4-ALK L1196M/G1202}-luc intracranial tumours in animals treated with TRI-611 or lorlatinib. Data points represent the mean and SEM of n = 8. c, Body weight change of mice during the NCI-H3122^{EML4-ALK L1196M/G1202}-luc intracranial study. Data points represent the mean and SEM of n = 8. d, Waterfall plot showing percent change in luminescence from day 0 to day 20 for individual animals with the indicated treatments in the NCI-H3122^{EML4-ALK L1196M/G1202R}-luc efficacy study. e, Unbound brain concentrations of TRI-611 at 6 h following the second dose in the NCI-H3122^{EML4-ALK L1196M/G1202}-luc study. Animals were perfused prior to brain collection. Protein binding parameters are described in Extended Data Fig. 8a. Dashed line indicates NCI-H3122 in vitro DC₅₀ corrected for media protein binding (measured at 78% bound). Error bars represent the mean and SEM of n = 4. Values for individual animals are shown. f, Maximum unbound plasma concentration of TRI-611 or lorlatinib (calculated as in Extended Data Figs. 6a and 7e) following a second dose of compound in the NCI-H3122^{EML4-ALK L1196M/G1202}-luc efficacy study. Error bars represent SEM of n = 4. Values for individual animals are shown. Source data can be found online.

Source data

a, 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-H3122^MET (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-H3122^MET (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