Investigation Of The Effects And Mechanisms Of A Next Generation TRK Inhibitor JND4135 In Overcoming Multiple Resistant Mutants In Vitro And In Vivo

TRK（tropomyosin receptor kinase）receptor tyrosine kinase family includes TRKA,TRKB and TRKC,which are encoded by NTRK1,NTRK2,and NTRK3genes,respectively.NTRK gene fusions are oncogenic drivers of various pediatric and adult cancers,TRK fusion proteins encoded by NTRK fusion genes can be constitutively activated without ligands to promote the proliferation and survival of tumor cells.Although the first-generation TRK inhibitors larotrectinib and entrectinib have a high response rate and good overall safety in the treatment of NTRK fusion-positive cancer patients,TRK mutation-mediated resistance inevitably occurs quickly,mainly including amino acid point mutations in solvent front regions,gatekeeper residues,x DFG motifs,etc.The second-generation TRK inhibitors which are in the clinical trials,LOXO-195 and TPX-0005,have improved the activity against wild-type TRKA/B/C kinases and can overcome the solvent front mutation resistance.However,neither of them can overcome the x DFG motif mutation resistance.In this study,23 kinds of NTRK gene fusion-driven Ba F3 model cells were constructed,including TRKA/B/C wild types and various drug-resistant mutations.Through compound library screening in our group,it was found that JND4135 has n M-level kinase inhibitory and affinity activity against wild-type TRKA/B/C.It showed good proliferation inhibitory activity against wild-type and multiple mutated Ba F3-TRK cells at a level of<20 n M,especially effectively against x DFG motif mutations,and it has comparable activity against solvent front mutations with that of LOXO-195.The kinase profile screening experiment on 468 kinases found that JND4135 is a potent and selective TRK inhibitor.The kinase inhibitory activity and affinity activity studies show that the Kd of JND4135 to wild-type TRKA/B/C is0.079 n M,0.29 n M,0.46 n M,respectively,IC₅₀ value is 6.966 n M,6.018 n M,3.006n M,respectively.Flow cytometry analysis showed that JND4135 can induce significant G0/G1 phase arrest and apoptosis in Ba F3-CD74-NTRK1-G667C cells at a concentration of 10 n M.WB studies have shown that JND4135 can dose-dependently inhibit the phosphorylation of TRK and its downstream signaling pathways in wild-type and multiple drug-resistant mutant TRK Ba F3 model cells.Molecular docking shows that JND4135 can stably bind to wild-type,solvent front and x DFG mutant TRKC protein the DFG-out conformation,and the mutated amino acids do not disturb the binding with the target protein.Molecular interaction experiments show that JND4135exhibits a slow binding and slow dissociation binding with TRKC and TRKC^G696Cproteins mode has similar affinities,but it is obviously different from other TRK inhibitors on the market or under development,and it.We also found that JND4135can recruit E3 ubiquitin ligase RNF123 to induce the degradation of EVT6-TRKC fusion protein through the proteasome pathway rapidly,but EVT6-TRKC will rapidly re-synthesize and compensate for the degradation caused by JND4135.In vivo studies have shown that the pharmacokinetic properties of JND4135 are poor,with low C_max,bioavailability,etc.Under the conditions of intraperitoneal administration,JND4135 can inhibit Ba F3-CD74-NTRK1-G667C transplantation tumor model at a dose of 20 mg/kg with 75%TGI,and the tumor can be completely eliminated at a dose of 40 mg/kg.Meanwhile,it was found that the above doses can significantly prolong the survival time of tumor-bearing mice.The WB study found that it can effectively inhibit the phosphorylation of TRKA and PLCγ1 in tumor tissues.During the in vivo experiment,JND4135 had little effect on mice’s body weight,and no other side effects were seen.In summary,we established 23 model cells expressing various TRK mutants based on Ba F3 cells,and identified that JND4135 can overcome the previously reported 1^st and 2^nd-generation TRK inhibitors-resistant x DFG and other mutations in vitro and in vivo for the first time.Its unique binding mode and the resultant degradation of TRKC were also discovered.While the PK properties of JND4135 are poor,and further structural modification is needed to improve its druggability.