Overcoming EGFR T790M and C797S resistance with mutant-selective allosteric inhibitors

EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors

NMR structural studies of interactions of a small, nonpeptidyl Tpo mimic with the thrombopoietin receptor extracellular juxtamembrane and transmembrane domains.

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation

Design, synthesis, and biological evaluation of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase

A series of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues lacking the benzoylglutamate subunit were prepared and examined as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase)

Identification and characterization of small molecule modulators of the EBI2 receptor

Oxysterols such as 7a, 25-dihydroxycholesterol (7a,25-OHC) have recently been identified as natural ligands for a G protein-coupled receptor called Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) 1, 2. EBI2 is highly expressed in immune cells and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes 3. In order to further delineate the physiological role of the oxysterol/EBI2 pathway in health and disease, here we describe the isolation and characterization of a potent small molecule antagonist for the EBI2 receptor. Prior to the isolation of the natural ligand, we first identified a surrogate small molecule agonist NIBR51 (NVP-AIU198) 1, which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (NVP-AQV967) 2 was used as a starting point for a medicinal chemistry campaign which yielded NIBR189 (NVP-CHY976) 4m. This compound was extensively characterized in binding and various functional signaling assays to show that it is a potent and selective antagonist for the EBI2 receptor. Furthermore we have used NIBR189 (NVP-CHY976) 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells

Optimisation of a 5-[3-phenyl-(2-cyclic-ether)-methyl-ether]-4-aminopyrrolopyrimidine series of IGF-1R inhibitors

Taking the pyrrolopyrimidine derived IGF-1R inhibitor NVP-AEW541 as the starting point, the benzyl ether back-pocket binding moiety was replaced with a series of 2-cyclic ether methyl ethers leading to the identification of novel achiral [2.2.1]-bicyclic ether methyl ether containing analogues with improved IGF-1R activities and kinase selectivities. Further exploration of the series, including a fluorine scan of the 5-phenyl substituent, and optimisation of the sugar-pocket binding moiety identified compound 41 containing (S)-2-tetrahydrofuran methyl ether 6-fluorophenyl ether back-pocket, and cis-N-Ac-Pip sugar-pocket binding groups. Compound 41 showed improved selectivity and pharmacokinetics compared to NVP-AEW541, and produced comparable in vivo efficacy to linsitinib in inhibiting the growth of an IGF-1R dependent tumor xenograft model in the mouse

Design and synthesis of novel selective anaplastic lymphoma kinase inhibitors

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described

Identification and Characterization of Small Molecule Modulators of the Epstein–Barr Virus-Induced Gene 2 (EBI2) Receptor

Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) (Nature 2011, 475, 524; 519). EBI2 is highly expressed in immune cells (J. Biol. Chem. 2006, 281, 13199), and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes (Nature 2010, 467, 460). Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First, we identified a small molecule agonist NIBR51 (<b>1</b>), which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (<b>2</b>) was used as a starting point for a medicinal chemistry campaign, which yielded NIBR189 (<b>4m</b>). This compound was extensively characterized in binding and various functional signaling assays. Furthermore, we have used <b>4m</b> to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells