Discovery of Next-Generation Tropomyosin Receptor Kinase Inhibitors for Combating Multiple Resistance Associated with Protein Mutation

Abstract

Tropomyosin receptor kinase (TRK) inhibition is an effective therapeutic approach for treatment of a variety of cancers. Despite the use of first-generation TRK inhibitor (TRKI) larotrectinib (1) resulting in significant therapeutic response in patients, acquired resistance develops invariably. The emergence of secondary mutations occurring at the solvent-front, xDFG, and gatekeeper regions of TRK represents a common mechanism for acquired resistance. However, xDFG mutations remain insensitive to second-generation macrocyclic TRKIs selitrectinib (3) and repotrectinib (4) designed to overcome the resistance mediated by solvent-front and gatekeeper mutations. Here, we report the structure-based drug design and discovery of a next-generation TRKI. The structure−activity relationship studies culminated in the identification of a promising drug candidate 8 that showed excellent in vitro potency on a panel of TRK mutants, especially TRKA G667C in the xDFG motif, and improved in vivo efficacy than 1 and 3 in TRK wild-type and mutant fusion-driven tumor xenograft models, respectively.

Structural Optimization and Structure–Activity Relationship Studies of 6,6-Dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one Derivatives as A New Class of Potent Inhibitors of Pan-Trk and Their Drug-Resistant Mutants

“…Due to the absence of crystal structures reported at present for TrkC mutants, the homology models of TrkC G623R and TrkC G696C were generated from TrkC wt (PDB: 6KZD). The TrkC G623R mutant is the substitution of the small glycine residue with an arginine-bearing additional bulky side chain, which causes the steric clash and directly interferes with larotrectinib and entrectinib binding to the protein. ,, Similarly, the TrkC G696C mutant is the substitution of the glycine residue G696 in the DFG motif with cysteine, which leads to steric hindrance between the thiol side chain of cysteine and the 2,5-difluorophenyl moiety of larotrectinib and compromises larotrectinib binding. , From the molecular docking, we found that the binding modes of TrkC wt and mutants with 11g were nearly identical. Most of the interactions observed are retained, and TrkC G623R or TrkC G696C mutants do not affect the binding modes (Figure C,D), which is consistent with the biological assay results that the type-I inhibitor larotrectinib lost potencies against TrkC G623R and TrkC G696C mutants when compared with 11g (Table ).…”

“…The TrkC G623R mutant is the substitution of the small glycine residue with an arginine-bearing additional bulky side chain, which causes the steric clash and directly interferes with larotrectinib and entrectinib binding to the protein. 69,78,80 Similarly, the TrkC G696C mutant is the substitution of the glycine residue G696 in the DFG motif with cysteine, which leads to steric hindrance between the thiol side chain of cysteine and the 2,5-difluorophenyl moiety of larotrectinib and compromises larotrectinib binding. 78,80 From the molecular docking, we found that the binding modes of TrkC wt and mutants with 11g were nearly identical.…”

“…69,78,80 Similarly, the TrkC G696C mutant is the substitution of the glycine residue G696 in the DFG motif with cysteine, which leads to steric hindrance between the thiol side chain of cysteine and the 2,5-difluorophenyl moiety of larotrectinib and compromises larotrectinib binding. 78,80 From the molecular docking, we found that the binding modes of TrkC wt and mutants with 11g were nearly identical. Most of the interactions observed are retained, and TrkC G623R or TrkC G696C mutants do not affect the binding modes (Figure 3C,D), which is consistent with the biological assay results that the type-I inhibitor larotrectinib lost potencies against TrkC G623R and TrkC G696C mutants when compared with 11g (Table 4).…”

NTRK Fusion in Non-Small Cell Lung Cancer: Diagnosis, Therapy, and TRK Inhibitor Resistance

“…Thus, resistance mediated by xDFG mutation remains a major challenge for next-generation TRK inhibitors. Though recent studies report that promising drug compounds designed to overcome multiple resistance possessed potent inhibitory activities to xDFG mutations as well as solvent-front and gatekeeper substitutions in vitro and in vivo ( 95 , 96 ), the exploration of new drugs to inhibit xDFG mutation is still facing unmet clinical needs.…”

Distribution- and Metabolism-Based Drug Discovery: A Potassium-Competitive Acid Blocker as a Proof of Concept

“…The in vitro metabolic stabilities of selected compounds were performed using human and rat liver microsomes in triplicate as described previously [ 50 ].…”