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.

These results demonstrate that satraplatin and JM-118 have preclinical antitumor activity in human prostate cancer and other tumor types as well, including several cell lines displaying drug resistance to cisplatin, docetaxel and mitoxantrone. In addition, the results suggest that PSA should be further evaluated as a relevant marker of clinical response in patients with prostate cancer treated with satraplatin.

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.

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Antibody-dependent cell-mediated cytotoxicity plays an important role in the macrophage-mediated destruction of target cells. While the selectivity is based on antibody specificity, the lytic attack is triggered by Fc receptor-mediated respiratory burst. To mimic IgG opsonization, a chimeric antibody-like molecule, containing human IgG1 Fc, was expressed on the surface of mammalian cells. The transmembrane domain of the human transferrin receptor was fused in-frame to the N-terminus of the second and third domains of human IgG1 heavy-chain constant region. This fusion molecule was designed to take advantage of the type II membrane anchor property of the transferrin receptor to express the Fc portion of the molecule in a reverse orientation, such that the Fc portion projected away from the cell surface. This is in contrast to the conventional cell surface IgG, which is anchored by a C-terminal type I transmembrane domain. The cell surface expressed reverse Fc no longer activated complement, but retained Fc receptor-binding capability and activated superoxide production by macrophages. This activity was completely blocked by an FcgammaR I-specific monoclonal antibody.