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Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an experimental strategy to regulate the intracellular concentration of any protein of interest by using an engineered destabilizing protein domain and a cell-permeable small molecule. Destabilizing domains have general utility to confer instability to a wide range of proteins including integral transmembrane proteins. This study reports a destabilizing domain system based on the ligand binding domain of the estrogen receptor that can be regulated by one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen., (© 2012 American Chemical Society)

Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough analysis of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP.rapamycin.FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Analysis of the data shows that rapamycin binds to FRB with moderate affinity (K(d) = 26 +/- 0.8 microM). The FKBP12.rapamycin complex, however, binds to FRB 2000-fold more tightly (K(d) = 12 +/- 0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiological conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.

The cell-permeable dihydrofolate reductase inhibitor methotrexate was covalently linked to a ligand for the protein FKBP to create a bifunctional molecule called MTXSLF. The covalent tether between the two ligands was designed to be prohibitively short, so that unfavorable protein-protein interactions between DHFR and FKBP preclude formation of a trimeric complex. In vitro and in vivo experiments demonstrate that MTXSLF is an effective inhibitor of human DHFR, but that efficacy is decreased in the presence of human FKBP due to the high concentration of FKBP and its tight affinity for MTXSLF. MTXSLF also inhibits Plasmodium falciparum DHFR in vitro, but a low concentration of the weaker binding Plasmodium FKBP has no effect on the inhibitory potency of MTXSLF in vivo. These studies illustrate a potentially general strategy for modulating the biological activity of synthetic molecules that depends on the ligand-binding properties of a nontarget protein.

Ustiloxin D and phomopsin A are potent antimitotic agents that bind to tubulin and interfere with cellular microtubule function. A synthetic strategy has been developed to allow access to both of the natural products as well as a variety of variants of the ustiloxin and phomopsin family members in order to provide sufficient quantities for biological studies. Herein we report the enantioselective total synthesis of ustiloxin D using a longest linear sequence of 20 steps. Four of the five stereocenters were set using catalytic asymmetric methodologies. In particular, Evans's new Al-catalyzed asymmetric aldol reaction facilitated access to both syn and anti products corresponding to the different benzylic stereochemistries found in ustiloxins and phomopsins. In addition, due to its high functional group tolerance, Trost's Pd-mediated etherification was used to construct the chiral tertiary alkyl-aryl ether. Taken together, these synthetic strategies allow us to use densely functionalized intermediates to realize an efficient synthesis of ustiloxin D.