ABSTRACT
The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The 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. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), 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.
Subject(s)
Antineoplastic Agents/pharmacology , Benzeneacetamides/pharmacology , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Mutant Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Thiazoles/pharmacology , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cetuximab/pharmacology , Disease Models, Animal , Drug Resistance, Multiple/drug effects , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/drug effects , Drug Synergism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Conformation/drug effects , Protein Multimerization/drug effectsABSTRACT
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 33 containing (S)-2-tetrahydrofuran methyl ether 6-fluorophenyl ether back-pocket, and cis-N-Ac-Pip sugar-pocket binding groups. Compound 33 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 tumour xenograft model in the mouse.
Subject(s)
Antineoplastic Agents/pharmacology , Imidazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Mice , Mice, Nude , Molecular Structure , NIH 3T3 Cells , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazines/chemical synthesis , Pyrazines/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrroles/chemical synthesis , Pyrroles/chemistry , Receptor, IGF Type 1/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemical synthesis , Drug Design , Protein Kinase Inhibitors/chemical synthesis , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , 4-Aminopyridine/analogs & derivatives , 4-Aminopyridine/chemistry , Anaplastic Lymphoma Kinase , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Half-Life , Humans , Lymphoma, Large-Cell, Anaplastic/drug therapy , Mice , Mice, SCID , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary , Rats , Receptor Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.
Subject(s)
ATP-Dependent Proteases/antagonists & inhibitors , Drug Design , Mitochondrial Proteins/antagonists & inhibitors , Protease Inhibitors/chemistry , ATP-Dependent Proteases/metabolism , Binding Sites , Boronic Acids/chemistry , Boronic Acids/metabolism , Boronic Acids/pharmacology , Bortezomib/chemistry , Bortezomib/metabolism , Cell Line , Cell Survival/drug effects , Humans , Mitochondrial Proteins/metabolism , Molecular Docking Simulation , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Subunits/antagonists & inhibitors , Protein Subunits/metabolism , Structure-Activity RelationshipABSTRACT
The lead optimization of a novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. The chemical instability of the dihydro-benzo[a]carbazole lead 2 was successfully addressed in the design and evaluation of compounds which also demonstrated improved potency compared to 2. Members of the scaffold have been identified which are full agonists that demonstrate cellular functional potency <50 nM. Analog 21 demonstrates equivalent efficacy in the human megakaryocyte differentiation (CFU-mega) assay compared to Eltrombopag.
Subject(s)
Benzene Derivatives/chemical synthesis , Benzene Derivatives/pharmacology , Carbazoles/chemical synthesis , Carbazoles/pharmacology , Receptors, Thrombopoietin/agonists , Thrombopoietin , Benzene Derivatives/chemistry , Carbazoles/chemistry , Combinatorial Chemistry Techniques , Drug Design , Humans , Inhibitory Concentration 50 , Megakaryocytes/metabolism , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Receptors, Thrombopoietin/chemistry , Structure-Activity Relationship , Thrombopoietin/chemistry , Thrombopoietin/metabolismABSTRACT
A novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. Starting from a 3.4 microM high throughput screen hit, members of this series have been identified which are full agonists with functional potency <50 nM and oral bioavailability in mice.
Subject(s)
Benzene Derivatives/chemical synthesis , Benzene Derivatives/pharmacology , Carbazoles/chemical synthesis , Carbazoles/pharmacology , Receptors, Thrombopoietin/agonists , Thrombopoietin , Administration, Oral , Animals , Benzene Derivatives/chemistry , Carbazoles/chemistry , Combinatorial Chemistry Techniques , Drug Design , Humans , Mice , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Receptors, Thrombopoietin/chemistry , Structure-Activity Relationship , Thrombopoietin/chemistry , Thrombopoietin/metabolismABSTRACT
Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.
Subject(s)
Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Discovery , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Nicotine/analogs & derivatives , Protein Kinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Molecular Conformation , Mutation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Nicotine/chemical synthesis , Nicotine/chemistry , Nicotine/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Rats , Rats, Wistar , Structure-Activity RelationshipABSTRACT
The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.
Subject(s)
Neoplasms/drug therapy , Protein Kinase Inhibitors/chemical synthesis , Pyrimidines/chemical synthesis , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Sulfones/chemical synthesis , Anaplastic Lymphoma Kinase , Animals , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Dogs , Humans , Macaca fascicularis , Male , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Rats , Structure-Activity Relationship , Sulfones/pharmacokinetics , Sulfones/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
The reaction between a novel butadienyl di-indium reagent and aldehydes showed remarkable 1,6-diastereoselectivity to give acetylenic diol products as a single diastereomer. A bicyclic transition state formed through intramolecular chelation control was proposed that might account for the high 1,6-diastereoselection result.