Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2\u27, 3\u27 -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2\u27-5\u27 and 3\u27-5\u27 phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2\u27, 3\u27 -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2\u27-5\u27 and 3\u27-5\u27 phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors

Asymptotics and Dimensional Dependence of the Number of Critical Points of Random Holomorphic Sections

We prove two conjectures from [M. R. Douglas, B. Shiffman and S. Zelditch, Critical points and supersymmetric vacua, II: Asymptotics and extremal metrics. J. Differential Geom. 72 (2006), no. 3, 381-427] concerning the expected number of critical points of random holomorphic sections of a positive line bundle. We show that, on average, the critical points of minimal Morse index are the most plentiful for holomorphic sections of {\mathcal O}(N) \to \CP^m and, in an asymptotic sense, for those of line bundles over general K\"ahler manifolds. We calculate the expected number of these critical points for the respective cases and use these to obtain growth rates and asymptotic bounds for the total expected number of critical points in these cases. This line of research was motivated by landscape problems in string theory and spin glasses.Comment: 14 pages, corrected typo

1,3-Allylic Strain as a Strategic Diversification Element For Constructing Libraries of Substituted 2-Arylpiperidines

Flipping diversity—Minimization of 1,3-allylic strain is a recurring element in the design of a stereochemically- and spatially-diverse collection of 2-arylpiperidines. Here, stereochemicallydiverse scaffolding is first constructed using A1,3 strain to guide the regioselective addition of nucleophiles, which serve as handles for further substitution. N-substitution with alkyl and acyl substituents again leverages A1,3 strain to direct each stereoisomer to two different conformer populations, doubling the number of library member

Catalytic Enantioselective Decarboxylative Protonation

We report a highly enantioselective, general catalytic system for the facile synthesis of tertiary stereocenters by protonation adjacent to cyclic ketones. The method relies on catalytic decarboxylative protonation of readily accessible racemic quaternary β-ketoesters. A range of substituted cycloalkanone compounds can be accessed through this process with high levels of enantioselectivity

Mechanistic origin of the stereodivergence in decarboxylative allylation

A stereochemical test has been used to probe the mechanism of decarboxylative allylation. This probe suggests that the mechanism of DcA reactions can change based on the substitution pattern at the α-carbon of the nucleophile, however reaction via stabilized malonate nucleophiles is the lower energy pathway. Lastly, this mechanistic proposal has predictive power and can be used to explain chemoselectivities in decarboxylative reactions that were previously confounding

Human oncoprotein Musashi-2 N-terminal RNA recognition motif backbone assignment and identification of RNA-binding pocket

RNA-binding protein Musashi-2 (MSI2) is a key regulator in stem cells, it is over-expressed in a variety of cancers and its higher expression is associated with poor prognosis. Like Musashi-1, it contains two N-terminal RRMs (RNA-recognition Motifs, also called RBDs (RNA-binding Domains)), RRM1 and RRM2, which mediate the binding to their target mRNAs. Previous studies have obtained the three-dimensional structures of the RBDs of Musashi-1 and the RBD1:RNA complex. Here we show the binding of MSI2-RRM1 to a 15nt Numb RNA in Fluorescence Polarization assay and time resolved Fluorescence Resonance Energy Transfer assay. Using nuclear magnetic resonance (NMR) spectroscopy we assigned the backbone resonances of MSI2-RRM1, and characterized the direct interaction of RRM1 to Numb RNA r(GUAGU). Our NMR titration and structure modeling studies showed that MSI2-RRM1 and MSI1-RBD1 have similar RNA binding events and binding pockets. This work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 specific and MSI1/MSI2 dual inhibitors

Design of Substrate Transmembrane Mimetics as Structural Probes for γ-Secretase

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemical Society (JACS), copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/jacs.9b13405.γ-Secretase is a membrane-embedded aspartyl protease complex central in biology and medicine. How this enzyme recognizes transmembrane substrates and catalyzes hydrolysis in the lipid bilayer is unclear. Inhibitors that mimic the entire substrate transmembrane domain and engage the active site should provide important tools for structural biology, yielding insight into substrate gating and trapping the protease in the active state. Here we report transmembrane peptidomimetic inhibitors of the γ-secretase complex that contain an N-terminal helical peptide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety targeted to the active site. Both regions are required for stoichiometric inhibition of γ-secretase. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrate that both the docking exosite and the active site are engaged by the bipartite inhibitors. The solution conformations of these potent transmembranemimetic inhibitors are similar to those of bound natural substrates, suggesting these probes are preorganized for high-affinity binding and should allow visualization of the active γ-secretase complex, poised for intramembrane proteolysis, by cryo-electron microscopy.NIH R01 grant GM 122894NIH grant P30GM110761NIH grant P41GM11113