Targeting Her2-insYVMA with Covalent Inhibitors-A Focused Compound Screening and Structure-Based Design Approach.

Mutated or amplified Her2 serves as a driver of non-small cell lung cancer or mediates resistance toward the inhibition of its family member epidermal growth factor receptor with small-molecule inhibitors. To date, small-molecule inhibitors targeting Her2 which can be used in clinical routine are lacking, and therefore, the development of novel inhibitors was undertaken. In this study, the well-established pyrrolopyrimidine scaffold was modified with structural motifs identified from a screening campaign with more than 1600 compounds, which were applied against wild-type Her2 and its mutant variant Her2-A775_G776insYVMA. The resulting inhibitors were designed to covalently target a reactive cysteine in the binding site of Her2 and were further optimized by means of structure-based drug design utilizing a set of obtained complex crystal structures. In addition, the analysis of binding kinetics and absorption, distribution, metabolism, and excretion parameters as well as mass spectrometry experiments and western blot analysis substantiated our approach.

RASPELD to Perform High-End Screening in an Academic Environment toward the Development of Cancer Therapeutics.

The identification of compounds for dissecting biological functions and the development of novel drug molecules are central tasks that often require screening campaigns. However, the required architecture is cost- and time-intensive. Herein we describe the devices and technologies that comprise a Robotics-Assisted Screening Platform for Efficient Ligand Discovery (RASPELD), which we set up in an academic laboratory. RASPELD provides semi-automated high-end screening, and it can be maintained by graduate students. We demonstrate its successful application in biochemical and cellular screens for the identification and validation of bioactive chemical entities as candidate cancer-relevant inhibitors. Specifically, we examined the interaction between a transcription factor, Nrf2, and its key regulator, Keap1. We also examined drug-resistant mutants of the epidermal growth factor receptor (EGFR). Screening campaigns with more than 30 000 compounds were performed in a reasonable period of time. We identified the molecule RSL6586 as a starting point for hit optimization, which is currently ongoing.

FLiK: a direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations.

Despite the hundreds of kinase inhibitors currently in discovery and preclinical phases, the number of FDA-approved kinase inhibitors remains very low by comparison, a discrepancy which reflects the challenges which accompanies kinase inhibitor development. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibit kinase activity, but the highly conserved nature of the ATP-binding site often contributes to the poor inhibitor selectivity. To address this problem, we developed a high-throughput screening technology that can discriminate for inhibitors, which stabilize inactive kinase conformations by binding within allosteric pockets in the kinase domain. Here, we describe how to use the Fluorescence Labels in Kinases approach to measure the K(d) of ligands as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries in high throughput.

Highly enantioselective catalytic synthesis of neurite growth-promoting secoyohimbanes.

Natural products endowed with neuromodulatory activity and their underlying structural scaffolds may inspire the synthesis of novel neurotrophic compound classes. The spirocyclic secoyohimbane alkaloid rhynchophylline is the major component of the extracts of Uncaria species used in Chinese traditional medicine for treatment of disorders of the central nervous system. Based on the structure of rhynchophylline, a highly enantioselective and efficient organocatalyzed synthesis method was developed that gives access to the tetracyclic secoyohimbane scaffold, embodying a quaternary and three tertiary stereogenic centers in a one-pot multistep reaction sequence. Investigation of a collection of the secoyohimbanes in primary rat hippocampal neurons and embryonal stem cell-derived motor neurons led to discovery of compounds that promote neurite outgrowth and influence the complexity of neuronal network formation.

Fluorescence labels in kinases: a high-throughput kinase binding assay for the identification of DFG-out binding ligands.

Despite the hundreds of kinase inhibitors currently in discovery and pre-clinical phases, the number of kinase inhibitors which have been approved and are on the market remains low by comparison. This discrepancy reflects the challenges which accompany the development of kinase inhibitors which are relatively specific and less toxic. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibiting kinase activity, but the highly conserved nature of the ATP-binding site contributes to poor inhibitor selectivity, issues which have particularly hampered the development of novel kinase inhibitors. We developed a high-throughput screening technology that can discriminate for inhibitors which stabilize the inactive "DFG-out" kinase conformation by binding within an allosteric pocket adjacent to the ATP-binding site. Here, we describe how to use this approach to measure the K (d) of ligands, as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries at high throughput.

Synthesis and biological evaluation of 4-anilinoquinolines as potent inhibitors of epidermal growth factor receptor.

The mutant receptor tyrosine kinase EGFR is a validated and therapeutically amenable target for genotypically selected lung cancer patients. Here we present the synthesis and biological evaluation of a series of 6- and 7-substituted 4-anilinoquinolines as potent type I inhibitors of clinically relevant mutant variants of EGFR. Quinolines 3a and 3e were found to be highly active kinase inhibitors in biochemical assays and were further investigated for their biological effect on EGFR-dependent Ba/F3 cells and non-small cell lung cancer (NSCLC) cell lines.

High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38alpha.

Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases between inactive and enzymatically competent conformations and vice versa. The development of novel kinase inhibitors is mainly fostered by high-throughput screening initiatives where the small molecule perturbation of the phosphorylation reaction is measured to identify inhibitors. Such setups require enzymatically active kinase preparations and present a risk of solely identifying classical ATP-competitive Type I inhibitors. Here we report the high-throughput screening of a library of approximately 35000 small organic molecules with an assay system that utilizes enzymatically inactive human p38alpha MAP kinase to detect stabilizers of the pharmacologically more desirable DFG-out conformation. We used protein X-ray crystallography to characterize the binding mode of hit compounds and reveal structural features which explain how these ligands stabilize and/or induce the DFG-out conformation. Lastly, we show that although some of the hit compounds were confirmed by protein X-ray crystallography, they were not detected in classic phosphorylation assays, thus validating the unique sensitivity of the assay system used in this study and highlighting the potential of screening with inactive kinase preparations.

Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors.

Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less conserved allosteric sites would be expected to offer new opportunities for scaffold development. Because no high-throughput method was previously available, we developed a fluorescence-based kinase binding assay for identifying and characterizing ligands which stabilize the inactive kinase conformation. Here, we present a description of the development and validation of this assay using the serine/threonine kinase p38alpha. By covalently attaching fluorophores to the activation loop of the kinase, we were able to detect conformational changes and measure the K(d), k(on), and k(off) associated with the binding and dissociation of ligands to the allosteric pocket. We report the SAR of a synthesized focused library of pyrazolourea derivatives, a scaffold known to bind with high affinity to the allosteric pocket of p38alpha. Additionally, we used protein X-ray crystallography together with our assay to examine the binding and dissociation kinetics to characterize potent quinazoline- and quinoline-based type II inhibitors, which also utilize this binding pocket in p38alpha. Last, we identified the b-Raf inhibitor sorafenib as a potent low nanomolar inhibitor of p38alpha and used protein X-ray crystallography to confirm a unique binding mode to the inactive kinase conformation.

Predicting drug susceptibility of non-small cell lung cancers based on genetic lesions.

Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non-small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition.