The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group.

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Synthesis of 6- and 7-membered cyclic enaminones: scope and mechanism.

Six- and seven-membered cyclic enaminones can be prepared using common, environmentally benign reagents. Amino acids are used as synthetic precursors allowing diversification and the incorporation of chirality. The key reaction in this multistep process involves deprotection of Boc-amino ynones and subsequent treatment with methanolic K(2)CO(3) to induce cyclization. A ß-amino elimination side reaction was identified in a few labile substrates that led to either loss of stereochemical purity or degradation. This process can be mitigated in specific cases using mild deprotection conditions. NMR and deuterium-labeling experiments provided valuable insight into the workings and limitations of this reaction. Although disguised as a 6-endo-dig cyclization, the reagents employed in the transformation play a direct role in bond-making and bond-breaking, thus changing the mode of addition to a 6-endo-trig cyclization. This method can be used to construct an array of monocyclic and bicyclic scaffolds, many of which are found in well-known natural products (e.g., indolizidine, quinolizidine, and Stemona alkaloids).

The CCR2/MCP-1 Chemokine Pathway and Lung Adenocarcinoma.

Host anti-tumor immunity can be hindered by various mechanisms present within the tumor microenvironment, including the actions of myeloid-derived suppressor cells (MDSCs). We investigated the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine lung cancer models. Phenotypic profiling revealed maximal expression of CCR2 by tumor-resident MDSCs, and MCP-1 by transplanted TC1 tumor cells, respectively. Use of CCR2-knockout (CCR2-KO) mice showed dependence of tumor growth on CCR2 signaling. Tumors in CCR2-KO mice had fewer CCR2low MDSCs, CD4 T cells and Tregs than WT mice, and increased infiltration by CD8 T cells producing IFN-γ and granzyme-B. Effects were MDSC specific, since WT and CCR2-KO conventional T (Tcon) cells had comparable proliferation and production of inflammatory cytokines, and suppressive functions of WT and CCR2-KO Foxp3+ Treg cells were also similar. We used a thioglycolate-induced peritonitis model to demonstrate a role for CCR2/MCP-1 in trafficking of CCR2+ cells to an inflammatory site, and showed the ability of a CCR2 antagonist to inhibit such trafficking. Use of this CCR2 antagonist promoted anti-tumor immunity and limited tumor growth. In summary, tumor cells are the prime source of MCP-1 that promotes MDSC recruitment, and our genetic and pharmacologic data demonstrate that CCR2 targeting may be an important component of cancer immunotherapy.

Synthesis of a quinolone library from ynones.

A library of 72 quinolones was synthesized from substituted anthranilic acids, using ynone intermediates. These masked ß-dicarbonyl synthons allowed cyclization under milder conditions than previously reported quinolone syntheses.

Paclitaxel succinate analogs: Anionic and amide introduction as a strategy to impart blood-brain barrier permeability.

A focused library of TX-67 (C10 hemi-succinate) analogs has been prepared, including C7 regioisomers, esters, amides, and one-carbon homologs. These were prepared to investigate whether the lack of TX-67 interaction with P-glycoprotein (Pgp) is due to the presence of the carboxylic acid moiety and whether this phenomenon was restricted to C10 analogs. Tubulin stabilization ability, cytotoxicity, and Pgp interactions were evaluated. All carboxylic acid analogs and several of the amides had no apparent interactions with Pgp at the concentrations used, whereas the ester variants displayed characteristics of Pgp substrates. Furthermore, it was demonstrated that hydrogen-bonding properties were significant with respect to Pgp interactions. Calculations of logD and cross-sectional areas revealed that these analogs are predicted to partition into the membrane and can compete for Pgp binding sites. The anionic and amide introduction strategy may allow for delivery of paclitaxel into the CNS and may be a potential approach for the delivery of other, structurally complex and lipophilic non-CNS permeable drugs.

Enantiospecific synthesis and cytotoxicity of 7-(4-methoxyphenyl)-6-phenyl-2,3,8,8a-tetrahydroindolizin-5(1H)-one enantiomers.

An enantiospecific synthesis was developed to generate both enantiomers of 7-(4-methoxyphenyl)-6-phenyl-2,3,8,8a-tetrahydroindolizin-5(1H)-one. A biological assay utilizing the HCT-116 colon cancer cell line to determine the cytotoxicity of these analogs revealed that only the (R)-enantiomer exhibited appreciable cytotoxicity with an IC(50) value of 0.2 microM.

Microwave-assisted Suzuki-Miyaura couplings on α-iodoenaminones.

A systematic study of α-iodination and subsequent Suzuki-Miyaura couplings between non-attenuated enaminones and a wide range of aromatic boronic acids is reported. The microwave-assisted variant of this transformation furnished the α-arylenaminones in significantly shorter reaction times and slightly improved yields as compared to conventional heating.

Amino acid-derived enaminones: a study in ring formation providing valuable asymmetric synthons.

A new reaction for the preparation of enaminones has been discovered. This method employs beta-amino acids as starting materials to allow diversification as well as incorporation of chirality. The beta-amino acids, once converted to ynones, are readily cyclized to the desired six-membered enaminone via a two-step, one-pot protocol. Although disguised as a 6-endo-dig cyclization, the reagents employed in the transformation play a direct role in bond making and bond breaking, thus changing the mode of addition.

Single-site chemical modification at C10 of the baccatin III core of paclitaxel and Taxol C reduces P-glycoprotein interactions in bovine brain microvessel endothelial cells.

A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core that reduces interaction with P-glycoprotein in bovine brain microvessel endothelial cells is described. Modification and derivatization of the C10 position were carried out using a substrate controlled hydride addition to a key C9 and C10 diketone intermediate. The analogs were tested for tubulin assembly and cytotoxicity, and were shown to retain potency similar to paclitaxel. P-glycoprotein interaction was examined using a rhodamine assay and it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel and Taxol C can reduce interaction with the P-glycoprotein transporter that may allow for increased permeation of taxanes into the brain.

[Bis(2-methoxyethyl)amino]sulfur trifluoride, the Deoxo-Fluor reagent: application toward one-flask transformations of carboxylic acids to amides.

The use of the Deoxo-Fluor reagent is a versatile method for acyl fluoride generation and subsequent one-flask amide coupling. It provides mild conditions and facile purification of the desired products in good to excellent yields. We have explored the utility of this reagent for the one-flask conversion of acids to amides and Weinreb amides and as a peptide-coupling reagent.

Stabilization of the cyclin-dependent kinase 5 activator, p35, by paclitaxel decreases beta-amyloid toxicity in cortical neurons.

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-beta (Abeta) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Abeta toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Abeta peptides, inhibits Abeta-induced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Abeta-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Abeta-induced activation of the cytosolic cdk5-p25 complex, but not the membrane-associated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood-brain barrier, we assessed the potential of taxanes to attenuate Abeta toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood-brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Abeta-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Abeta toxicity and neurofibrillary pathology in AD and other tauopathies.