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1.
Commun Biol ; 5(1): 1206, 2022 11 09.
Article in English | MEDLINE | ID: mdl-36352263

ABSTRACT

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.


Subject(s)
Receptors, G-Protein-Coupled , Phosphorylation , Ligands , Receptors, G-Protein-Coupled/metabolism , Immunoassay
2.
Chem Sci ; 10(46): 10789-10801, 2019 Dec 14.
Article in English | MEDLINE | ID: mdl-31857889

ABSTRACT

Precision medicine has revolutionized the treatment of patients in EGFR driven non-small cell lung cancer (NSCLC). Targeted drugs show high response rates in genetically defined subsets of cancer patients and markedly increase their progression-free survival as compared to conventional chemotherapy. However, recurrent acquired drug resistance limits the success of targeted drugs in long-term treatment and requires the constant development of novel efficient inhibitors of drug resistant cancer subtypes. Herein, we present covalent inhibitors of the drug resistant gatekeeper mutant EGFR-L858R/T790M based on the pyrrolopyrimidine scaffold. Biochemical and cellular characterization, as well as kinase selectivity profiling and western blot analysis, substantiate our approach. Moreover, the developed compounds possess high activity against multi drug resistant EGFR-L858R/T790M/C797S in biochemical assays due to their highly reversible binding character, that was revealed by characterization of the binding kinetics. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR which provide detailed insight into their binding mode.

3.
J Med Chem ; 62(11): 5541-5546, 2019 06 13.
Article in English | MEDLINE | ID: mdl-31083997

ABSTRACT

Pyrazolopyrimidines are well-established as covalent inhibitors of protein kinases such as the epidermal growth factor receptor or Bruton's tyrosine kinase, and we recently described their potential in targeting mitogen-activated protein kinase kinase 7 (MKK7). Herein, we report the structure-activity relationship of pyrazolopyrimidine-based MKK7 inhibitors and solved several complex crystal structures to gain insights into their binding mode. In addition, we present two structures of apo-MKK7, exhibiting a DFG-out and an unprecedented DFG-in/Leu-in conformation.


Subject(s)
MAP Kinase Kinase 7/chemistry , MAP Kinase Kinase 7/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , Amino Acid Motifs , MAP Kinase Kinase 7/antagonists & inhibitors , Models, Molecular , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology
4.
J Med Chem ; 62(5): 2843-2848, 2019 03 14.
Article in English | MEDLINE | ID: mdl-30768270

ABSTRACT

The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold.


Subject(s)
JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , MAP Kinase Kinase 7/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Crystallography, X-Ray , Drug Design , Phosphorylation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology
5.
J Med Chem ; 60(18): 7725-7744, 2017 09 28.
Article in English | MEDLINE | ID: mdl-28853575

ABSTRACT

Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm , ErbB Receptors/chemistry , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Mice , Molecular Docking Simulation , Point Mutation , Protein Kinase Inhibitors/pharmacokinetics , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology
6.
J Med Chem ; 60(13): 5613-5637, 2017 07 13.
Article in English | MEDLINE | ID: mdl-28603991

ABSTRACT

Inhibition of the epidermal growth factor receptor represents one of the most promising strategies in the treatment of lung cancer. Acquired resistance compromises the clinical efficacy of EGFR inhibitors during long-term treatment. The recently discovered EGFR-C797S mutation causes resistance against third-generation EGFR inhibitors. Here we present a rational approach based on extending the inhibition profile of a p38 MAP kinase inhibitor toward mutant EGFR inhibition. We used a privileged scaffold with proven cellular potency as well as in vivo efficacy and low toxicity. Guided by molecular modeling, we synthesized and studied the structure-activity relationship of 40 compounds against clinically relevant EGFR mutants. We successfully improved the cellular EGFR inhibition down to the low nanomolar range with covalently binding inhibitors against a gefitinib resistant T790M mutant cell line. We identified additional noncovalent interactions, which allowed us to develop metabolically stable inhibitors with high activities against the osimertinib resistant L858R/T790M/C797S mutant.


Subject(s)
ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Imidazoles/chemistry , Imidazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm , ErbB Receptors/metabolism , Gefitinib , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Molecular Docking Simulation , Point Mutation , Quinazolines/pharmacology , Structure-Activity Relationship
7.
Sci Transl Med ; 9(394)2017 06 14.
Article in English | MEDLINE | ID: mdl-28615362

ABSTRACT

Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. We provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors, such as AD80 or ponatinib, that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells, we identify the CCDC6-RETI788N mutation and drug-induced mitogen-activated protein kinase pathway reactivation as possible mechanisms by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors.


Subject(s)
Adenocarcinoma/metabolism , Gene Rearrangement/genetics , Lung Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-ret/genetics , Adenocarcinoma of Lung , Animals , Cell Line, Tumor , Cytoskeletal Proteins/genetics , Drug Resistance, Neoplasm/genetics , Gene Rearrangement/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Imidazoles/pharmacology , Mice , Mutation , NIH 3T3 Cells , Pyridazines/pharmacology
8.
Angew Chem Int Ed Engl ; 56(28): 8153-8157, 2017 07 03.
Article in English | MEDLINE | ID: mdl-28544137

ABSTRACT

Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.


Subject(s)
Autophagy/drug effects , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Autophagosomes/drug effects , Drug Discovery , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , Pyrimidines/chemistry , Sirolimus/pharmacology , Structure-Activity Relationship
9.
J Med Chem ; 60(6): 2361-2372, 2017 03 23.
Article in English | MEDLINE | ID: mdl-28225269

ABSTRACT

The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacokinetics , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/genetics , Humans , Indazoles , Lung/drug effects , Lung/metabolism , Lung Neoplasms/genetics , Mice , Molecular Docking Simulation , Mutation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology
10.
ACS Omega ; 2(4): 1563-1575, 2017 Apr 30.
Article in English | MEDLINE | ID: mdl-29963651

ABSTRACT

Within the spectrum of kinase inhibitors, covalent-reversible inhibitors (CRIs) provide a valuable alternative approach to classical covalent inhibitors. This special class of inhibitors can be optimized for an extended drug-target residence time. For CRIs, it was shown that the fast addition of thiols to electron-deficient olefins leads to a covalent bond that can break reversibly under proteolytic conditions. Research groups are just beginning to include CRIs in their arsenal of compound classes, and, with that, the understanding of this interesting set of chemical warheads is growing. However, systems to assess both characteristics of the covalent-reversible bond in a simple experimental setting are sparse. Here, we have developed an efficient methodology to characterize the covalent and reversible properties of CRIs and to investigate their potential in targeting clinically relevant variants of the receptor tyrosine kinase EGFR.

11.
Angew Chem Int Ed Engl ; 55(36): 10909-12, 2016 08 26.
Article in English | MEDLINE | ID: mdl-27496389

ABSTRACT

Targeting acquired drug resistance represents the major challenge in the treatment of EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we describe the structure-based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR-mutant drug-resistant cells. Protein X-ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR-T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR-C797S.


Subject(s)
ErbB Receptors/metabolism , Protein Kinase Inhibitors/metabolism , Binding Sites , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Humans , Kinetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Molecular Docking Simulation , Phosphorylation , Point Mutation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyrazoles/pharmacology , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacology
12.
ACS Med Chem Lett ; 7(1): 2-5, 2016 Jan 14.
Article in English | MEDLINE | ID: mdl-26819655

ABSTRACT

In the last five years, the detailed understanding of how to overcome T790M drug resistance in non-small cell lung cancer (NSCLC) has culminated in the development of a third-generation of covalent EGFR inhibitors with excellent clinical outcomes. However, the emergence of a newly discovered acquired drug resistance challenges the concept of small molecule targeted cancer therapy in NSCLC.

13.
J Med Chem ; 58(17): 6844-63, 2015 Sep 10.
Article in English | MEDLINE | ID: mdl-26275028

ABSTRACT

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.


Subject(s)
Antineoplastic Agents/chemistry , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Cell Membrane Permeability , Crystallography, X-Ray , Databases, Chemical , Drug Design , ErbB Receptors/genetics , Humans , Kinetics , Lung Neoplasms , Models, Molecular , Molecular Conformation , Mutation , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Quinazolines/chemistry , Quinazolines/pharmacology , Small Molecule Libraries , Solubility , Structure-Activity Relationship , src-Family Kinases/chemistry , src-Family Kinases/genetics
14.
ACS Chem Biol ; 10(1): 289-98, 2015 Jan 16.
Article in English | MEDLINE | ID: mdl-25540906

ABSTRACT

The cytosolic Ser/Thr kinase TBK1 was discovered to be an essential element in the mediation of signals that lead to tumor migration and progression. These findings meet the need for the identification of novel tool compounds and potential therapeutics to gain deeper insights into TBK1 related signaling and its relevance in tumor progression. Herein, we undertake the activity-based screening for unique inhibitors of TBK1 and their subsequent optimization. Initial screening approaches identified a selection of TBK1 inhibitors that were optimized using methods of medicinal chemistry. Variations of the structural characteristics of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or basic contributions to drug design approaches are anticipated for our improved small molecules.


Subject(s)
Drug Design , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Cell Line , Humans , Macrophages/drug effects , Macrophages/metabolism , Mice , Molecular Docking Simulation , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/chemistry , Small Molecule Libraries/chemistry , Structure-Activity Relationship
15.
J Med Chem ; 57(10): 4252-62, 2014 May 22.
Article in English | MEDLINE | ID: mdl-24754677

ABSTRACT

Discoidin domain-containing receptors (DDRs) exhibit a unique mechanism of action among the receptor tyrosine kinases (RTKs) because their catalytic activity is induced by extracellular collagen binding. Moreover, they are essential components in the assimilation of extracellular signals. Recently, DDRs were reported to be significantly linked to tumor progression in breast cancer by facilitating the processes of invasion, migration, and metastasis. Here, we report the successful development of a fluorescence-based, direct binding assay for the detection of type II and III DFG-out binders for DDR2. Using sequence alignments and homology modeling, we designed a DDR2 construct appropriate for fluorescent labeling. Successful assay development was validated by sensitive detection of a reference DFG-out binder. Subsequent downscaling led to convenient application to high-throughput screening formats. Screening of a representative compound library identified high-affinity DDR2 ligands validated by orthogonal activity-based assays, and a subset of identified compounds was further investigated with respect to DDR1 inhibition.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Mitogen/antagonists & inhibitors , Discoidin Domain Receptors , Drug Design , Fluorescence , High-Throughput Screening Assays , Ligands , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Structure, Tertiary , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Mitogen/chemistry , Receptors, Mitogen/metabolism , Structure-Activity Relationship
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