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.

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.

Structural characterization of the RLCK family member BSK8: a pseudokinase with an unprecedented architecture.

Brassinosteroid signaling kinases (BSKs) are plant-specific receptor-like cytoplasmic protein kinases involved in the brassinosteroid signaling pathway. Unlike common protein kinases, they possess a naturally occurring alanine residue at the "gatekeeper" position, as well as other sequence variations. How BSKs activate downstream proteins such as BSU1, as well as the structural consequences of their unusual sequential features, was unclear. We crystallized the catalytic domain of BSK8 and solved its structure by multiple-wavelength anomalous dispersion phasing methods to a resolution of 1.5Å. In addition, a co-crystal structure of BSK8 with 5-adenylyl imidodiphosphate (AMP-PNP) revealed unusual conformational arrangements of the nucleotide phosphate groups and catalytic key motifs, typically not observed for active protein kinases. Sequential analysis and comparisons with known pseudokinase structures suggest that BSKs represent constitutively inactive protein kinases that regulate brassinosteroid signal transfer through an allosteric mechanism.

Targeting gain of function and resistance mutations in Abl and KIT by hybrid compound design.

Mutations in the catalytic domain at the gatekeeper position represent the most prominent drug-resistant variants of kinases and significantly impair the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular and atomic levels will aid in the design and development of inhibitors that have the potential to overcome these resistance mutations. Herein, by introducing adaptive elements into the inhibitor core structure, we undertake the structure-based development of type II hybrid inhibitors to overcome gatekeeper drug-resistant mutations in cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-T670I and Abl-T315I variants, as essential targets in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies.

Selective detection of allosteric phosphatase inhibitors.

Normal cellular function, such as signal transduction, is largely controlled by the reversible phosphorylation of cellular proteins catalyzed by two major classes of enzymes, kinases and phosphatases. A misbalance in this complex and dynamic interplay leads to a variety of severe diseases, such as cancer, inflammation, or autoimmune diseases. This makes kinases as well as phosphatases equally attractive targets for therapeutic manipulation by small molecules. While the development of kinase inhibitors has resulted in several blockbuster drugs, such as imatinib, with remarkable success in the clinic and sales of many billions of U.S. dollars per year, not a single phosphatase inhibitor has yet been approved for clinical use. Similar to the kinase world, substrate-competitive phosphatase inhibitors have been developed but were not suitable for further development into clinical candidates due to their charge and limited selectivity. Research efforts, therefore, have shifted to the exploitation of allosteric sites that can regulate phosphatase activity and may enable the discovery of novel modulators of phosphatase activity with much improved pharmacological properties. However, assay systems, which enable the straightforward discovery of these inhibitor types, are missing. Here, we present a novel binding assay capable of detecting ligands of an allosteric pocket of the protein tyrosine phosphatase 1B. This assay is suitable for high-throughput screening and selectively detects ligands which bind to this unique site with a clear discrimination from substrate-competitive ligands.

Dibenzosuberones as p38 mitogen-activated protein kinase inhibitors with low ATP competitiveness and outstanding whole blood activity.

p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a "deep pocket" moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy.

Strategies for the selective regulation of kinases with allosteric modulators: exploiting exclusive structural features.

The modulation of kinase function has become an important goal in modern drug discovery and chemical biology research. In cancer-targeted therapies, kinase inhibitors have been experiencing an upsurge, which can be measured by the increasing number of kinase inhibitors approved by the FDA in recent years. However, lack of efficacy, limited selectivity, and the emergence of acquired drug resistance still represent major bottlenecks in the clinic and challenge inhibitor development. Most known kinase inhibitors target the active kinase and are ATP competitive. A second class of small organic molecules, which address remote sites of the kinase and stabilize enzymatically inactive conformations, is rapidly moving to the forefront of kinase inhibitor research. Such allosteric modulators bind to sites that are less conserved across the kinome and only accessible upon conformational changes. These molecules are therefore thought to provide various advantages such as higher selectivity and extended drug target residence times. This review highlights various strategies that have been developed to utilizing exclusive structural features of kinases and thereby modulating their activity allosterically.

Natural-product-derived fragments for fragment-based ligand discovery.

Fragment-based ligand and drug discovery predominantly employs sp(2)-rich compounds covering well-explored regions of chemical space. Despite the ease with which such fragments can be coupled, this focus on flat compounds is widely cited as contributing to the attrition rate of the drug discovery process. In contrast, biologically validated natural products are rich in stereogenic centres and populate areas of chemical space not occupied by average synthetic molecules. Here, we have analysed more than 180,000 natural product structures to arrive at 2,000 clusters of natural-product-derived fragments with high structural diversity, which resemble natural scaffolds and are rich in sp(3)-configured centres. The structures of the cluster centres differ from previously explored fragment libraries, but for nearly half of the clusters representative members are commercially available. We validate their usefulness for the discovery of novel ligand and inhibitor types by means of protein X-ray crystallography and the identification of novel stabilizers of inactive conformations of p38α MAP kinase and of inhibitors of several phosphatases.

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer.

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Fluorophore labeled kinase detects ligands that bind within the MAPK insert of p38α kinase.

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway.

Design, synthesis, and biological evaluation of novel disubstituted dibenzosuberones as highly potent and selective inhibitors of p38 mitogen activated protein kinase.

Synthesis, biological testing, structure-activity relationships (SARs), and selectivity of novel disubstituted dibenzosuberone derivatives as p38 MAP kinase inhibitors are described. Hydrophilic moieties were introduced at the 7-, 8-, and 9-position of the 2-phenylamino-dibenzosuberones, improving physicochemical properties as well as potency. Extremely potent inhibitors were obtained, with half-maximal inhibitory concentration (IC(50)) values in the low nM range in a whole blood assay measuring the inhibition of cytokine release. The high potency of the target compounds together with the outstanding selectivity of this novel class of compounds toward p38 mitogen activated protein (MAP) kinase as compared to other kinases indicate them to be most applicable as tools in pharmacological research and eventually they may foster a new generation of anti-inflammatory drugs.

Targeting GSK3 from Ustilago maydis: type-II kinase inhibitors as potential antifungals.

Protein kinases are key enzymes in the complex regulation of cellular processes in almost all living organisms. For this reason, protein kinases represent attractive targets to stop the growth of eukaryotic pathogens such as protozoa and fungi. However, using kinase inhibitors to fight against these organisms bears several challenges since most of them are unselective and will also affect crucial host kinases. Here we present the X-ray structure of glycogen synthase kinase 3 from the fungal plant pathogen Ustilago maydis (UmGSK3) and its inhibition by type-II kinase inhibitors. Despite the high sequence homology between the human and the fungal variant of this vital kinase, we found substantial differences in the conformational plasticity of their active sites. Compounds that induced such conformational changes could be used to selectively inhibit the fungal kinase. This study serves as an example of how species-specific selectivity of inhibitors can be achieved by identifying and addressing the inactive state of a protein kinase. In addition to this, our study gives interesting insights into the molecular plasticity of UmGSK3 by revealing a previously unknown inactive conformation of this important kinase family.

Structure-based design, synthesis and biological evaluation of N-pyrazole, N'-thiazole urea inhibitors of MAP kinase p38α.

In this paper, we present the structure-based design, synthesis and biological activity of N-pyrazole, N'-thiazole-ureas as potent inhibitors of p38α mitogen-activated protein kinase (p38α MAPK). Guided by complex crystal structures, we employed the initially identified N-aryl, N'-thiazole urea scaffold and introduced key structural elements that allowed the formation of novel hydrogen bonding interactions within the allosteric site of p38α, resulting in potent type III inhibitors. [4-(3-tert-Butyl-5-{[(1,3-thiazol-2-ylamino)carbonyl]amino}-1H-pyrazol-1-yl)-phenyl]acetic acid 18c was found to be the most potent compound within this series and inhibited p38α activity with an IC(50) of 135 ± 21 nM. Its closest analog, ethyl [4-(3-tert-butyl-5-{[(1,3-thiazol-2-ylamino)carbonyl]amino}-1H-pyrazol-1-yl)phenyl]acetate 18b, effectively inhibited p38α mediated phosphorylation of the mitogen activated protein kinase activated protein kinase 2 (MK2) in HeLa cells.

Skepinone-L is a selective p38 mitogen-activated protein kinase inhibitor.

Until now, a lack of inhibitors with high potency and selectivity in vivo has hampered investigation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We describe the design of skepinone-L, which is, to our knowledge, the first ATP-competitive p38 MAPK inhibitor with excellent in vivo efficacy and selectivity. Therefore, skepinone-L is a valuable probe for chemical biology research, and it may foster the development of a unique class of kinase inhibitors.

ALK mutations conferring differential resistance to structurally diverse ALK inhibitors.

PURPOSE: EML4-ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4-ALK-positive tumors. EXPERIMENTAL DESIGN: By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684. RESULTS: Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4-ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066. CONCLUSIONS: Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4-ALK fusions and ALK mutations.

Identification of Ustilago maydis Aurora kinase as a novel antifungal target.

Infestation of crops by pathogenic fungi has continued to have a major impact by reducing yield and quality, emphasizing the need to identify new targets and develop new agents to improve methods of crop protection. Here we present Aurora kinase from the phytopathogenic fungus Ustilago maydis as a novel target for N-substituted diaminopyrimidines, a class of small-molecule kinase inhibitors. We show that Aurora kinase is essential in U. maydis and that diaminopyrimidines inhibit its activity in vitro. Furthermore, we observed an overall good correlation between in vitro inhibition of Aurora kinase and growth inhibition of diverse fungi in vivo. In vitro inhibition assays with Ustilago and human Aurora kinases indicate that some compounds of the N-substituted diaminopyrimidine class show specificity for the Ustilago enzyme, thus revealing their potential as selective fungicides.

Synthesis and biological evaluation of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase and epidermal growth factor receptor.

Here we present the synthesis and biological activity of a series of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase (MLCK) and the epidermal growth factor receptor kinase (EGFR). The inhibitory effect of these molecules was found to be dependent on the nature of the substituents at the 7-position of the isoquinoline scaffold.

Characterization of irreversible kinase inhibitors by directly detecting covalent bond formation: a tool for dissecting kinase drug resistance.

Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants.

Fluorophore labeling of the glycine-rich loop as a method of identifying inhibitors that bind to active and inactive kinase conformations.

Targeting protein kinases with small organic molecules is a promising strategy to regulate unwanted kinase activity in both chemical biology and medicinal chemistry research. Traditionally, kinase inhibitors are identified in activity-based screening assays using enzymatically active kinase preparations to measure the perturbation of substrate phosphorylation, often resulting in the enrichment of classical ATP competitive (Type I) inhibitors. However, addressing enzymatically incompetent kinase conformations offers new opportunities for targeted therapies and is moving to the forefront of kinase inhibitor research. Here we report the development of a new FLiK (Fluorescent Labels in Kinases) binding assay to detect small molecules that induce changes in the conformation of the glycine-rich loop. Due to cross-talk between the glycine-rich loop and the activation loop in kinases, this alternative labeling approach can also detect ligands that stabilize inactive kinase conformations, including slow-binding Type II and Type III kinase inhibitors. Protein X-ray crystallography validated the assay results and identified a novel DFG-out binding mode for a quinazoline-based inhibitor in p38alpha kinase. We also detected the high-affinity binding of a clinically relevant and specific VEGFR2 inhibitor, and we provide structural details of its binding mode in p38alpha, in which it stabilizes the DFG-out conformation. Last, we demonstrate the power of this new FLiK labeling strategy to detect the binding of Type I ligands that induce conformational changes in the glycine-rich loop as a means of gaining affinity for the target kinase. This approach may be a useful alternative to develop direct binding assays for kinases that do not adopt the DFG-out conformation while also avoiding the use of expensive kits, detection reagents, or radioactivity frequently employed with activity-based assays.

Displacement assay for the detection of stabilizers of inactive kinase conformations.

Targeting protein kinases with small molecules outside the highly conserved ATP pocket to stabilize inactive kinase conformations is becoming a more desirable approach in kinase inhibitor research, since these molecules have advanced pharmacological properties compared to compounds exclusively targeting the ATP pocket. Traditional screening approaches for kinase inhibitors are often based on enzyme activity, but they may miss inhibitors that stabilize inactive kinase conformations by enriching the active state of the kinase. Here we present the development of a kinase binding assay employing a pyrazolourea type III inhibitor and enzyme fragment complementation (EFC) technology that is suitable to screen stabilizers of enzymatically inactive kinases. To validate this assay system, we report the binding characteristics of a series of kinase inhibitors to inactive p38alpha and JNK2. Additionally, we present protein X-ray crystallography studies to examine the binding modes of potent quinoline-based DFG-out binders in p38alpha.