Defining the landscape of ATP-competitive inhibitor resistance residues in protein kinases.

Kinases are involved in disease development and modulation of their activity can be therapeutically beneficial. Drug-resistant mutant kinases are valuable tools in drug discovery efforts, but the prediction of mutants across the kinome is challenging. Here, we generate deep mutational scanning data to identify mutant mammalian kinases that drive resistance to clinically relevant inhibitors. We aggregate these data with subsaturation mutagenesis data and use it to develop, test and validate a framework to prospectively identify residues that mediate kinase activity and drug resistance across the kinome. We validate predicted resistance mutations in CDK4, CDK6, ERK2, EGFR and HER2. Capitalizing on a highly predictable residue, we generate resistance mutations in TBK1, CSNK2A1 and BRAF. Unexpectedly, we uncover a potentially generalizable activation site that mediates drug resistance and confirm its impact in BRAF, EGFR, HER2 and MEK1. We anticipate that the identification of these residues will enable the broad interrogation of the kinome and its inhibitors.

Genome-wide functional screening identifies CDC37 as a crucial HSP90-cofactor for KIT oncogenic expression in gastrointestinal stromal tumors.

Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5-7 weeks, at which point assessment of relative hairpin abundance identified the HSP90 cofactor, CDC37, as one of the top six GIST-specific essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (>20 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib-resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.

A trinuclear intermediate in the copper-mediated reduction of O2: four electrons from three coppers.

The reaction of metal complexes with dioxygen (O2) generally proceeds in 1:1, 21, or 41 (metal:O2) stoichiometry. A discrete, structurally characterized 31 product is presented. This mixed-valence trinuclear copper cluster, which contains copper in the highly oxidized trivalent oxidation state, exhibits O2 bond scission and intriguing structural, spectroscopic, and redox properties. The relevance of this synthetic complex to the reduction of O2 at the trinuclear active sites of multicopper oxidases is discussed.

Electronic absorption spectroscopy of copper proteins.

We have seen from the previous discussion that absorption spectral studies in the ligand field region probe the energy splittings of the d orbitals and that this relates to the geometry of the metal center. The energies and intensities of ligand-to-metal charge transfer transitions sensitively probe bonding interactions of the ligand with the metal center. Charge transfer transitions can be used both qualitatively to observe ligand binding to a metal center, owing to the requirement of orbital overlap for significant charge transfer intensity, and quantitatively to define the electron donor ability of that ligand and experimentally evaluate the results of electronic structure calculations. Studies of the intensities of peaks at the ligand K edge can define the covalent interaction of the ligand with the metal valence orbitals, whereas copper K-edge spectroscopy is a powerful probe of metal ion oxidation state and the ligand field geometry of d10 cuprous sites that are inaccessible through other spectroscopic methods. Absorption spectral studies in all regions are strongly complemented by CD, variable temperature MCD, and single-crystal polarized absorption spectroscopies, which should also be pursued whenever possible to obtain detailed electronic structural insight of relevance to catalysis.