Discovery of 1-{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (PF-06459988), a Potent, WT Sparing, Irreversible Inhibitor of T790M-Containing EGFR Mutants

Abstract

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.

Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors

“…This competition profile contrasted with that observed for WT-EGFR, as measured in A431 cells, where probe labeling of a 170 kDa protein (in this case, using probe 7 that targets WT-EGFR (Lanning et al, 2014)) was competed by second-generation inhibitors such as afatinib, but less substantially by the third-generation T790M-EGFR inhibitors 1–3 and the recently described T790M-EGFR inhibitor PF-06459988 (Cheng et al, 2016) when tested at a concentration of 100 nM (below the IC 50 values of third-generation inhibitors for WT-EGFR) (Figure S3D). Additional protein targets beyond L858R/T790M-EGFR were identified for inhibitors 1 and 3 , which, for instance, blocked corresponding probe 4 and 6 labeling of 22 kDa (marked <1 and <1,3; Figure 1C) and 50 kDa (marked <3; Figure 1C) proteins, respectively, in a concentration-dependent manner (Figure S3E).…”

“…Analysis of probes and parent inhibitor pairs in Table S1 for inhibition of phosphorylated EGFR (pY1068) in H1975 cells and intrinsic reactivity in a conjugation assay with glutathione (GSH) were performed as described (Cheng et al, 2016). Briefly, the intrinsic chemical reactivity was accessed by evaluating the rate of electrophilic attack of the test compound to a strong nucleophile, glutathione (GSH), this was achieved by monitoring test compound loss during the reaction via LCMS/MS detection.…”

“…Generation of crystal structure of inhibitor 3 with L858R/T790M-EGFR was accomplished using methods as extensively described (Cheng et al, 2016, Planken et al, 2017). Briefly, EGFR kinase domain (spanning residues 696 – 1022 with L858R, T790M and V948R mutations) at 9 mg/mL was incubated with Inhibitor 3 (0.25 mM) for at least an hour and then set up for crystallization at 13°C in sitting drop configuration.…”

“…Combustion analyses were performed by Atlantic Microlab, Inc. (Norcross, Georgia). Inhibitors 1–3 , AZD5104, PF-06274484 and PF6459988 were synthesized as described previously (Planken et al, 2017, Cheng et al, 2016, Butterworth et al, 2013, Lee et al, 2015, Fakhoury et al, 2005). For specific synthetic details for Probes 4–6 , please see supplemental information (Methods S1).…”

Molecular Skin Surface-Based Transformation Visualization between Biological Macromolecules

“…The problem of Kruithof and Vegter's approach is that the triangles have completely no quality control and will be possibly distorted during the process of projecting. In 2004 and 2005, Cheng and Shi improved the triangulation quality of MSS by introducing the restricted Delaunay triangulation to the mesh representation [27, 28]. Instead of meshing sampled points on the skin surface and controlling the triangle quality by edge contraction and circumcenter insertion, they triangulate the sample points in 3 dimensions and take the surface triangle as the final mesh.…”