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LY3295668 chemical characterization

shRNA suppressor screening data for H446 and MDA-MB-468 cells (tabs 1, 2) and RNA expression level associations with LY3295668 Abs IC50 values from 493 cancer cell lines (tab 3).

Supplementary figures

Abs IC50 values (µM) for 36 small molecule inhibitors across a panel of cancer cell lines.

Abs IC50 values (µM) for LY3295668 across a panel of 580 cell lines.

Supplementary Tables 1-3, Figures 1-4 from SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

Supplementary Figure 1 from SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition. [Mol Cancer Ther 2009;8(12):3181–90]

Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1mut cancer cells and leads to durable regression of RB1mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. Significance: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors. See related commentary by Dick and Li, p. 169. This article is highlighted in the In This Issue feature, p. 151

Loss-of-function mutations in the retinoblastoma gene

As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu

The application of x-ray absorption spectroscopy methods to both materials and life sciences is well appreciated. However, the power of extended x-ray absorption fine structure (EXAFS) spectroscopy as a quantitative structural technique has largely been limited by its application to the microscopically homogeneous systems, in which the local environment around each absorbing atom in the sample is the same. The growing interest in time-resolved EXAFS studies of systems in physics, chemistry, biology, and materials science has reintroduced the requirement for an analytical tool to probe heterogeneous mixtures in situ. While long being recognized as a premiere technique for this role, EXAFS studies of mixtures have been particularly difficult due to the strong model dependence and correlations between parameters in the fit. To circumvent these drawbacks, we introduce two new techniques in EXAFS analysis: the principal component analysis and the residual phase analysis. Using a test case of a heterogeneous mi...

A hydrophobic nanostructured composite has been created through the addition of an organic monolayer to the external surface of a smectite clay. This film inhibits the free exchange of water in to and out of the interlayer located between the aluminosilicate sheets of the clay. The effect of the presence of such a monolayer, which is formed from octadecyltrimethoxysilane, on iron(III) cations exchanged into the clay interlayer has been examined using X-ray absorption spectroscopy. Mild hydrothermal conditions have been used to increase the rate of potential reactions involving the ferric ions. The addition of the organic coating results in partial reduction of the iron to the ferrous state, a process that is furthered by hydrothermal treatment of the hydrophobic clay. In contrast, the iron in the simple ion-exchanged clay aggregates into small iron−oxygen clusters but is not reduced. These results are discussed in the context of the reduction potential of iron(III) and the susceptibility of that iron to h...

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and cytoplasm. The yeast NPC is an eight-fold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins (Nups). Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal “FG” repeats containing a Gle2p-binding sequence motif (GLEBS motif) and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by Small Angle X-ray Scattering (SAXS). Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiaeNup145N, and human Nup98 are discussed.

The X-ray structure of a putative BenF-like (gene name: PFL1329) protein from Pseudomonas fluorescens Pf-5 (PflBenF) has been determined at 2.6Å resolution. X-ray crystallography revealed a canonical 18-stranded β-barrel fold that forms a central pore with a diameter of ∼4.6Å, which is consistent with the size and physicochemical properties of the presumed aromatic acid substrate, benzoate. Detailed comparisons with the previously-determined structure of Pseudomonas aeruginosa OpdK, a vanillate influx channel, revealed an arginine-rich aromatic acid selectivity filter of nearly identical structure composed of seven highly conserved residues Arg∼Asp∼Arg∼Arg∼Ser∼Asp∼Arg (R∼D∼R∼R∼S∼D∼R sequence motif, where ∼ denotes intervening residues) that define the narrowest part of the pore.

Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of ~30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9Å resolution.

X-ray specular reflectivity has been used to characterize the structure of silicon--silicon-oxide surfaces coated with chemisorbed hydrocarbon monolayer films (alkylsiloxanes). Using synchrotron radiation the reflectivity was followed over 9 orders of magnitude, from grazing incidence to an incident angle of \ensuremath{\varphi}\ensuremath{\approxeq}6.5\ifmmode^\circ\else\textdegree\fi{}, or q=(4\ensuremath{\pi}/\ensuremath{\lambda})sin(\ensuremath{\varphi})=0.8 A${\r{}}^{\mathrm{\ensuremath{-}}1}$, allowing a spatial resolution of features approximately \ensuremath{\pi}/0.8\ensuremath{\approxeq}4.0 A\r{} along the surface normal. Analysis was performed by fitting the data to reflectivities calculated from models of the surface electron density and by calculating Patterson functions directly from the data. Although the measured reflectivities could be equally well described by different sets of model parameters, the electron densities calculated from these different parameters were remarkably alike. Inspection of the electron densities allowed identification of a layer of ${\mathrm{SiO}}_{2}$ (\ensuremath{\approxeq}17-A\r{} thick), a layer of head-group region where the alkylsiloxane adsorbs to the ${\mathrm{SiO}}_{2}$, and the hydrocarbon layer. Fitting the data also required that the various interfaces have different widths. The fact that the same local hydrocarbon density of 0.85 g/${\mathrm{cm}}^{3}$ was observed for both fully formed and partially formed monolayers with alkane chains of varying length excluded a model in which the partially formed monolayer was made up of separated islands of well-formed monolayers. Measurements before and after chemical reaction of a monolayer in which the alkyl chain was terminated by an olefinic group demonstrated the ability to use x-ray reflectivity to characterize chemical changes. The effects of radiation damage on these types of measurements are described.

The use of principal component (factor) analysis for the interpretation of EXAFS spectra is described. The components derived from EXAFS spectra share mathematical properties with the original spectra. As a result, the abstract components can be analyzed using standard EXAFS methodology to yield bond distances and other coordination parameters. The number of components that must be analyzed is usually less than the number of original spectra. The method is demonstrated using a series of spectra from aqueous solutions of uranyl ions.

X-ray absorption spectroscopy has often been used to examine the local environment around an absorbing cation in both the solid and the liquid state. In order to examine the properties of a transition-metal ion under conditions in which the degree of solvation can be regulated, the authors have placed cupric ions within a smectite clay. Smectite clays consist of negatively charged sheets of aluminosilicates which are separated by an interlayer whose thickness changes upon absorption of solvent. In the native clay used for these experiments, bentonite, the calcium was replaced with copper by stirring the clay in a 0.1 M aqueous solution of CuCl[sub 2]. For the experiments described here, Cu K-edge spectra were acquired at the National Synchrotron Light Source and Stanford Synchrotron Radiation Laboratory in both transmission and fluorescence modes. Four samples of Cu-bentonite powder, dried from water, methanol, ethanol, and ethylene glycol, were examined, as well as slurries of these samples in their respective solvents. The normalized X-ray absorption near edge (XANES) spectra are obtained for copper ion in the Cu-bentonite dried from methanol and for the Cu-clay as a slurry in the same solvent. The spectrum of the dry material clearly shows the electronic 1s-4p transitionmore » as a resolved shoulder on the absorption edge at 8986 eV. This transition, which is characteristic of a copper ion with square planar coordination, has broadened and almost disappeared in the slurry. The observed change suggests that as the solvent penetrates into the clay, some of the solvent molecules enter the coordination sphere of the copper(II) ion. The addition of two axial oxygen ligands shifts the coordination geometry of the Cu(II) from a square planar configuration to a distorted octahedron. There is an obvious change in the EXAFS data between the dry clay and the slurry which cannot be readily discerned by examination of the radial distributions alone. 23 refs., 2 figs., 1 tab.« less

Long-chain alkyltrichlorosilanes, C13Si (CH2)nX, adsorb from solution onto silicon-silicon dioxide (Si/SiO2) substrates and form ordered alkylsiloxane monolayer films. These films were characterized by wettability, ellipsometry, and XPS. Except for very short chains (n = 0,1,2), the wetting of these monolayers was approximately independent of chain length. The presence of small amounts of water was necessary for the formation of these films. The alkylsiloxane monolayers were stable in common organic solvents, water, and acid, but were destroyed by prolonged exposure to base. Simple reactions on vinyl-terminated monolayers generated alcohol-, carboxylic acid-, and bromine- terminated films whose contact angles were lower than the starting monolayers, but whose lengths were largely unchanged. Measurements of the contact angle of acid-terminated interfaces as a function of pH indicated that ionization of soluble carboxylic acids. Monolayers containing mixtures of methyl- and carboxyl-functionalities exhibited wetting properties that mirrored the composition of the interface.

The use of sulfur-containing organic monolayer films improves adhesion between gold and silicon dioxide. The structures of these monolayers were analyzed using contact angle, ellipsometry, and XPS. The zone of adhesive failure was at or near the gold-monolayer interface.

The thicknesses of C10-C18 alkylsiloxane monolayers on silicon-silicon dioxide substrates have been measured with ellipsometry and low-angle X-ray reflection. Although, for any given sample, thicknesses measured by the two methods agree to within experimental error, ellipsometric measurements are systematically larger by approximately 2 {angstrom}. This difference may result from variations in the sensitivity of the two techniques to the structure of the interface between silicon dioxide and the alkylsiloxane monolayer. The x-ray reflectivity measurements provide evidence that these organic monolayers do not build up as island structures and demonstrate that the approximate area projected by each alkyl group in the plane of the monolayer is {approximately} 21 {plus minus}3 {angstrom}{sup 2}. Preliminary studies indicate that this technique can be used to follow the changes in the structure of a monolayer which result from chemical transformations. The influence of damage that is induced by x-ray radiation on these measurements is discussed.