1 generation EGF receptor tyrosine kinase inhibitors (EGFR TKIs) provide significant clinical benefit in patients with advanced EGFR mutant (EGFRm+) PRT062607 HCL non-small cell lung cancer (NSCLC). potential activity is afatinib plus the anti-EGFR antibody cetuximab which induced a 32% unconfirmed response rate in a phase IB trial for patients with EGFRm+ lung cancer and acquired resistance to erlotinib (33). However this combination has substantial skin toxicity with 18% of patients reporting CTCAE grade 3 or higher rash (33). Therefore there remains a significant unmet need for an EGFR TKI agent that can more effectively target T790M tumors while sparing the activity of wild-type EGFR. This has led to the development of “third generation” EGFR TKIs that are designed to target T790M and EGFR TKI-sensitizing mutations more selectively than wild-type EGFR. WZ4002 was the first such agent to be published (34) although it has not progressed to clinical trials. A second agent closely related to the WZ4002 series CO-1686 has been recently reported (35) and is currently in early Phase II clinical trials. HM61713 is another “third generation” agent that is currently in early Phase I trials. Here we describe identification characterization and early clinical development of AZD9291 a novel irreversible EGFR TKI with selectivity against mutant versus wild-type forms of EGFR. AZD9291 is a mono-anilino-pyrimidine compound that is structurally and pharmacologically specific from all the TKIs PRT062607 HCL including CO-1686 and WZ4002. Results AZD9291 is a mutant-selective irreversible inhibitor of EGFR kinase activity AstraZeneca developed a novel series of irreversible small-molecule inhibitors to target the sensitizing and T790M resistant mutant forms of the EGFR tyrosine kinase with selectivity over the wild-type form of the receptor. These compounds bind to the EGFR kinase irreversibly by targeting the cysteine-797 residue in the ATP binding site via covalent bond formation (36) as depicted in the modeling structure for AZD9291 (Fig. 1A). Further work on this chemotype allowed additional structure activity relationships (SAR) to be discerned that enabled target potency to be increased without driving increased lipophilicity thus maintaining favorable drug-like properties. Continued medicinal chemistry efforts achieved further improvements including increased kinase selectivity ultimately arriving at the mono-anilino-pyrimidine AZD9291 (Fig. 1B). Mass spectrometry of chymotrypsin digests confirmed that AZD9291 can covalently PRT062607 HCL modify recombinant EGFR (L858R/T790M) at the target cysteine 797 amino acidity (Supplementary Fig. S1 A&B). Shape 1 AZD9291 binding framework and setting. A Structural model displaying the covalent setting of binding of AZD9291 to EGFR T790M via Cys-797. Displays pyrimidine core developing two hydrogen bonds towards the hinge area (Met-793) orientation from the indole group adjacent … AZD9291 includes a specific chemical structure through the additional third-generation TKIs WZ4002 (34) and PRT062607 HCL CO-1686 (35). Whilst the previous two substances share a few common structural features (e.g. placing from the electrophilic features that undergoes response having a conserved cysteine residue within EGFR (Cys 797) heteroatom-linked pyrimidine 4-substituents and existence of the pyrimidine 5-substituent) AZD9291 can be architecturally exclusive. Amongst other variations the electrophilic features resides for the pyrimidine C-2 substituent band the pyrimidine 4-substituent can be Mouse monoclonal antibody to Pyruvate Dehydrogenase. The pyruvate dehydrogenase (PDH) complex is a nuclear-encoded mitochondrial multienzymecomplex that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO(2), andprovides the primary link between glycolysis and the tricarboxylic acid (TCA) cycle. The PDHcomplex is composed of multiple copies of three enzymatic components: pyruvatedehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and lipoamide dehydrogenase(E3). The E1 enzyme is a heterotetramer of two alpha and two beta subunits. This gene encodesthe E1 alpha 1 subunit containing the E1 active site, and plays a key role in the function of thePDH complex. Mutations in this gene are associated with pyruvate dehydrogenase E1-alphadeficiency and X-linked Leigh syndrome. Alternatively spliced transcript variants encodingdifferent isoforms have been found for this gene. C-linked and heterocyclic as well as the pyrimidine 5-placement is without substitution. In EGFR recombinant enzyme assays (Millipore) AZD9291 demonstrated an obvious IC50 of 12 nM against L858R and 1 nM against L858R/T790M; they are known as apparent because the quantity of energetic enzyme changes as time passes and therefore IC50 can be time-dependent for irreversible real estate agents. The medication exhibited almost 200 times higher strength against L858R/T790M than wild-type EGFR PRT062607 HCL (Supplementary Desk S1A) in keeping with the design objective of the mutant EGFR selective agent compared to early era TKIs. Following murine studies exposed that AZD9291 was metabolized to create at least two circulating metabolite varieties AZ5104 and AZ7550. In biochemical assays AZ7550 got a comparable strength and selectivity profile towards the mother or father (Supplementary Desk S1A). On the other hand although AZ5104 exhibited the same overall profile it PRT062607 HCL was more potent against mutant and wild-type EGFR forms thus.