Julian Engel

Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach.

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.

Hope and Disappointment: Covalent Inhibitors to Overcome Drug Resistance in Non-Small Cell Lung Cancer

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.

Insight into the Inhibition of Drug-Resistant Mutants of the Receptor Tyrosine Kinase EGFR.

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.

Identification of Type II and III DDR2 Inhibitors

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.

Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor

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.

Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically Resistant L858R/T790M/C797S EGFR Mutant: Targeting of Both Hydrophobic Regions and the Phosphate Binding Site

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.

Drugging the catalytically inactive state of RET kinase in RET-rearranged tumors

An examination of the activity profiles of RET inhibitors suggests potential treatment for RET-rearranged cancers. RET-ting out lung tumors Gene fusions and rearrangements serve as oncogenic drivers in a number of tumor types, and some of these can be targeted with existing drugs. RET rearrangements have been identified as drivers in some lung adenocarcinomas, but previous attempts to target RET have not been successful. Plenker et al. determined why the drugs previously proposed for inhibiting RET were not sufficiently potent and showed that successful inhibition of RET requires the ability to bind RET in its catalytically inactive conformation, known as the “DFG-out conformation,” thus locking it in an inactive state. The authors also identified drugs that bind RET in the desired conformation and demonstrated their efficacy in patient-derived xenograft models. 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.

Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34

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.

Identification and further development of potent TBK1 inhibitors.

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.

Structure-Guided Development of Covalent and Mutant-Selective Pyrazolopyrimidines to Target T790M Drug Resistance in Epidermal Growth Factor Receptor

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.