Supplementary Table 1 from Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

Supplementary Figure 2 from Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

Supplementary Methods, Figure Legends 1-3 from Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

Supplementary Figure 3 from Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

Supplementary Figure 4 from Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

Supplementary Figure 3 from Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

Supplementary Table 2 from Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

PIK-75 is a phosphoinositide-3-kinase (PI3K) α-isoform-selective inhibitor with high potency. Although published structure-activity relationship data show the importance of the NO2 and the Br substituents in PIK-75, none of the published studies could correctly determine the underlying reason for their importance. In this publication, we report the first X-ray crystal structure of PIK-75 in complex with the kinase GSK-3β. The structure shows an unusual U-shaped conformation of PIK-75 within the active site of GSK-3β that is likely stabilized by an atypical intramolecular Br⋅⋅⋅NO2 halogen bond. NMR and MD simulations show that this conformation presumably also exists in solution and leads to a binding-competent preorganization of the PIK-75 molecule, thus explaining its high potency. We therefore suggest that the site-specific incorporation of halogen bonds could be generally used to design conformationally restricted bioactive substances with increased potencies.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

AbstractPIK‐75 ist ein α‐Isoform‐selektiver Phosphoinositid‐3‐Kinase(PI3K)‐Inhibitor mit hoher Potenz. Obwohl veröffentlichte SAR‐Daten die Wichtigkeit der NO2‐ und Br‐Substituenten in PIK‐75 zeigen, konnte bisher keine Studie die zugrundeliegende Ursache der Bedeutung korrekt bestimmen. In dieser Zuschrift beschreiben wir die erste Kristallstruktur von PIK‐75 im Komplex mit der Kinase GSK‐3β. Die Struktur zeigt eine ungewöhnliche U‐förmige Konformation von PIK‐75 innerhalb des aktiven Zentrums von GSK‐3β, die wahrscheinlich durch eine atypische intramolekulare Br⋅⋅⋅NO2‐Halogenbindung stabilisiert wird. NMR‐ und MD‐Simulationen zeigen, dass diese Konformation wahrscheinlich ebenfalls in Lösung vorliegt und zu einer bindungskompetenten Konformation des PIK‐75‐Moleküls führt, was wiederum seine hohe Potenz erklärt. Wir schlagen daher vor, dass der ortsspezifische Einbau von intramolekularen Halogenbindungen im Allgemeinen verwendet werden könnte, um konformationell eingeschränkte bioaktive Substanzen mit erhöhter Potenz zu generieren.

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

Nat. Chem. Biol. 11, 571–578 (2015); published online 13 July 2015; corrected online 20 August 2015 In the author list, affiliation and correspondence information for Cheryl Arrowsmith was missing. Her name should be accompanied by affiliation number 3 (signifying the Structural Genomics Consortium,University of Toronto, Toronto, Ontario, Canada) and by an asterisk indicating her status as a corresponding author.

TP53 (which encodes p53 protein) is the most frequently mutated gene among all human cancers. Prevalent p53 missense mutations abrogate its tumour suppressive function and lead to a 'gain-of-function' (GOF) that promotes cancer. Here we show that p53 GOF mutants bind to and upregulate chromatin regulatory genes, including the methyltransferases MLL1 (also known as KMT2A), MLL2 (also known as KMT2D), and acetyltransferase MOZ (also known as KAT6A or MYST3), resulting in genome-wide increases of histone methylation and acetylation. Analysis of The Cancer Genome Atlas shows specific upregulation of MLL1, MLL2, and MOZ in p53 GOF patient-derived tumours, but not in wild-type p53 or p53 null tumours. Cancer cell proliferation is markedly lowered by genetic knockdown of MLL1 or by pharmacological inhibition of the MLL1 methyltransferase complex. Our study reveals a novel chromatin mechanism underlying the progression of tumours with GOF p53, and suggests new possibilities for designing combinatorial chromatin-based therapies for treating individual cancers driven by prevalent GOF p53 mutations.

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.

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.

Abelson (Abl) tyrosine kinase is an important cellular enzyme that is rendered constitutively active in the breakpoint cluster region (BCR)-Abl fusion protein, contributing to several forms of leukemia. Although inhibiting BCR-Abl activity with imatinib shows great clinical success, many patients acquire secondary mutations that result in resistance to imatinib. Second-generation inhibitors such as dasatinib and nilotinib can overcome the majority of these mutations but fail to treat patients with an especially prevalent T315I mutation at the gatekeeper position of the kinase domain. However, a combination of nilotinib with an allosteric type IV inhibitor was recently shown to overcome this clinically relevant point mutation. In this study, we present the development of a direct binding assay that enables the straightforward detection of allosteric inhibitors which bind within the myristate pocket of Abl. The assay is amenable to high-throughput screening and exclusively detects the binding of ligands to this unique allosteric site.