Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligandbinding site (Phe-36 3 Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (FM) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a ''reverse dimerization'' regulatory system involving F M fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated FM domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.

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By incorporating an N-hydroxyurea functionality onto diaryltetrahydrofurans, a novel series of compounds was investigated as dual 5-lipoxygenese (5-LO) inhibitor and platelet-activating factor (PAF) receptor antagonist. These dual functional compounds were evaluated in vitro for 5-LO inhibition in RBL cell extracts and human whole blood, and PAF receptor antagonism in a receptor binding assay. PAF-induced hemoconcentration and arachidonic acid- and TPA-induced ear edema in mice were used to determine in vivo activities. The structure-activity relationship analysis to define a preclinical lead is presented. (+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl- N-h ydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (40, CMI-392) was selected for further study. In the arachidonic acid-induced mouse ear edema model, 40 was more potent than either zileuton (a 5-LO inhibitor) or BN 50739 (a PAF receptor antagonist), and it demonstrated the same inhibitory effect as a physical combination of the latter two agents. These results suggest that a single compound which both inhibits leukotriene synthesis and blocks PAF receptor binding may provide therapeutic advantages over single-acting agents. The clinical development of compound 40 is in progress.

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