In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.

Organic small molecules generally act by perturbing the function of one or more cellular target proteins, the identification of which is essential to an understanding of the molecular basis of drug action. Here we describe the application of methotrexate-linked small molecule ligands to a mammalian three-hybrid interaction trap for proteome-wide identification of small molecule targets, quantification of the targeting potency of unmodified small molecules for such targets in intact cells, and screening for inhibitors of small molecule-protein interactions. During the course of this study we also identified the pyrido[2,3-d]pyrimidine PD173955, a known SRC kinase inhibitor, as a potent inhibitor of several ephrin receptor tyrosine kinases. This finding could perhaps be exploited in the design of inhibitors for this kinase subfamily, members of which have been implicated in the pathogenesis of various diseases, including cancer.

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Levuglandin E2 (LGE2), a rearrangement product derived from the prostaglandin endoperoxide, PGH2, causes repair-resistant DNA-protein cross-links and cell death (LD50 = 230 nM) in V79 Chinese hamster lung fibroblasts. The half-life for sequestration of LGE2 by covalent binding to cellular nucleophiles is at least an hour for 10 microM LG. This suggests that the in vivo production and distribution of free LGs should be measurable on this time scale. Following removal of the LGE2 and the return of the cultures to normal growth medium, additional DNA-protein cross-links continued to form over the ensuing 6-24 h. The results suggest that LG adducts to DNA or protein are not repaired, but react further at sites on protein or DNA in close proximity to the initial adducts, forming cross-links in a slow phase of the process.

The identification of molecular determinants of tumor cell survival is an important objective in cancer research. Here, we describe a small-molecule kinase inhibitor (RGB-286147), which, besides inhibiting tumor cell cycle progression, exhibits potent cytotoxic activity toward noncycling tumor cells, but not nontransformed quiescent fibroblasts. Extensive yeast three-hybrid (Y3H)-based proteome/kinome scanning with chemical dimerizers revealed CDK1/2/3/5/7/9 and the less well-characterized CDK-related kinases (CRKs) p42/CCRK, PCTK1/3, and PFTK1 as its predominant targets. Thus, RGB-286147 is a proteome-wide CDK/CRK-specific kinase inhibitor whose further study could yield new insight into molecular determinants of tumor cell survival. Our results also suggest that the [1, 3, 6]-tri-substituted-pyrazolo[3,4-d]-pyrimidine-4-one kinase inhibitor scaffold is a promising template for the rational design of kinase inhibitors with potential applications to disease indications other than cancer, such as neurodegeneration, cardiac hypertrophic growth, and AIDS.

Iso[4]-levuglandin E 2 (iso[4]-LGE 2 , 5-acetyl-6-formyl-9-hydroxy-7(E),11(Z)-heptadecadienoic acid) is a structural isomer of levuglandin E 2 (8-acetyl-9-formyl-12-hydroxy-5(Z),10(E)-heptadecadienoic acid) that we postulate to be generated during autoxidation of arachidonic acid or arachidonate esters. To facilitate detection and identification of iso[4]-LGE 2 in biological samples, a succinct total synthesis was developed that depends on conjugate addition of a higher order vinyl cyanocuprate to a γ-alkoxy enone to provide a key carbon-carbon bond-forming step. Bismethoxime pentafluorobenzyl ester trimethylsilyl ether derivatives of LGE 2 and iso[4]-LGE 2 were prepared and shown to be suitable for mass spectral identification of these highly oxidized lipids.

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