Structural analysis of the macrocyclic inhibitor BI-4020 binding to EGFR kinase

Abstract

A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants. Structures show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure- inspired approaches to developing targeted therapies in medicinal oncology.

Structural elements that enable specificity for mutant EGFR kinase domains with next-generation small-molecule inhibitors

“…One example is the phase 1 metabolite of AZD9291 (AZD5104), which is known to have similar potency against WT EGFR and the demethylation of the indole nitrogen leads to a H-bond with D855 (PDB ID 7JXL). (Tyler S. Beyett, To, et al, 2022) Other inhibitors that show Hbonding interactions with K745 include compound 43 (1) (related to EGF816)(Gérald Lelais et al, 2016) through a -CF3 group (Figure 2C). Moreover, CO-1686 (Yan, Zhu, Liang, Zhao, Geun Choi, et al, 2017) shows a dipole-dipole interaction through a -CF3 group with K745.…”

“…At Boehringer Ingelheim, structure-guided design of a macrocyclic benzimidazole inhibitor (BI-4020), related to the third-generation EGF816, shows tight binding to C797S-containing EGFR and interacts with K745 in both active and inactive conformations in through a novel pyrazole moiety (Figure 3C). (Tyler S. Beyett, Rana, et al, 2022;Engelhardt et al, 2019) In another case, an extensive drug development campaign by Blueprint Therapeutics led to the development of Compound 24 (3) the analogue of BLU-945 based on an isoquinoline scaffold, which exhibits selectivity for C797S (Figure 3E) (Eno et al, 2022). Like other inhibitors for C797S variants, the crystal structures of analogs of Compound 24 (3) show that a critical alcohol is positioned toward the K745 residues in the inactive conformation.…”

“…(Jia et al, 2016) Inhibitors that bind this pocket are intrinsically mutant selective as the L858R point mutation allows for access to this site via permanent unfolding of the A-loop. (Tyler S. Beyett, To, et al, 2022;Jia et al, 2016) Cocrystal structures containing the L858R mutation, which are underrepresented due to being relatively difficult to crystallize, show H-bonding interactions between the mutant Arg and the 2-isoindolinone of JBJ-09-063 potentially also contributing to L858R selectivity (Figure 3C). (Tyler S. Beyett, To, et al, 2022) Additionally, selectivity for T790M is enabled by thioether-aromatic ring interactions and otherwise insensitive to the C797S mutation due to the relatively long distance from the allosteric pocket to this amino acid side chain.…”

“…(To, Beyett, Jang, Feng, Bahcall, Haikala, Shin, Heppner, Rana, Leeper, et al, 2022) Recent cocrystal structures of allosteric inhibitors simultaneously bound with ATP-competitive third generation inhibitors, such as AZD9291 (Figure 3D), show a "swing" of K745 from the ATP side and over to the allosteric inhibitor now H-bonding with the 2-isoindolinone ketone (Figure 4D-E). (Tyler S. Beyett, To, et al, 2022;Niggenaber et al, 2020) Additionally, structural rearrangement of the P-loop F723 into a pi-stack with certain phenyl-containing allosteric inhibitors (e.g., JBJ-04-125-02, JBJ-09-063) are the source of positive cooperativity of ATP-competitive and allosteric inhibitors,(Tyler S. Beyett, To, et al, 2022) which is an important insight given that these inhibitors synergize with TKIs in vivo. (To, Beyett, Jang, Feng, Bahcall, Haikala, Shin, Heppner, Rana, Leeper, et al, 2022; Both the structural rearrangements of F723 and K745 toward the allosteric pocket indicate that displacement of ATP can lead to binding differences of amino acid side chains toward the allosteric site, which likely impact pharmacological efficacy of these next-generation inhibitors.…”