Metal-responsive control of the expression of genes involved in metal metabolism and metal homeostasis allows an organism to tightly regulate the free or bioavailable concentration of beneficial metal ions, such as zinc, copper, and iron, within an acceptable range, while efficiently removing nonbeneficial or toxic metals. Emerging evidence also suggests that metal homeostasis is intimately coupled to the oxidative stress response in many cell types. The expression of genes that encode metallothioneins in all vertebrate cells is strongly induced by potentially toxic concentrations of zinc and cadmium, as well as in response to strong oxidizing agents, including hydrogen peroxide. This induction requires a cis-acting DNA element, termed a metal response element (MRE), and MRE-binding transcription factor-1 (MTF-1), a Cys2-His2 zinc finger protein. This review summarizes recent progress that has been made toward understanding the structure, function, and metalloregulation of mammalian MTF-1.

Staphylococcus aureus pI258 CadC is a metal sensor protein that regulates the expression of the cad operon which encodes metal ion resistance proteins involved in the efficient efflux of Cd(II), Pb(II), Zn(II) and, according to one report, Bi(III) ions. In this paper, direct evidence is presented that Bi(III) binds to CadC and negatively regulates cad operator/promoter (O/P) binding. Optical absorption spectroscopy reveals that dimeric CadC binds approximately 0.8 mol equivalents of Bi(III) per CadC monomer to form a coordination complex characterized by three S(-)-->Bi(III) ligand-to-metal charge transfer transitions, with the longest wavelength absorption band centered at 415 nm (epsilon(415)=4000 M(Bi)(-1) cm(-1)). UV-Vis absorption spectra of wild-type and mutant Cys-->Gly (Ser) substitution CadC mutants compared to [Bi(DTT)(2)], [Bi(GSH)(3)] and [Bi(NAC)](3) model complexes reveal that Cys7, Cys11, Cys60 and Cys58 directly coordinate Bi(III) in a tetrathiolate coordination complex. The apparent affinity derived from a Bi(III)-displacement optical titration with Cd(II) is estimated to be K(Bi)< or =10(12) M(-1). Apo-CadC binds with high affinity [ K(a)=1.1(+/-0.3)x10(9) M(-1); 0.40 M NaCl, pH 7.0, 25 degrees C] to a 5'-fluorescein-labeled cad O/P oligonucleotide,while the binding of one molar equivalent of Bi(III) per CadC monomer (Bi(1)-CadC) reduces the affinity by approximately 170-fold. Strikingly, Bi(III)-responsive negative regulation of cad O/P binding is abrogated for Bi(1)-C60G CadC and severely disrupted in Bi(1)-C7G CadC, whose relative affinity is reduced only 10-fold. The mechanism of Bi(III)-responsive metalloregulation is discussed, based on the findings presented here. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0336-9.

Metal-response element (MRE)-binding transcription factor-1 (MTF-1) is a zinc-regulated transcriptional activator of metallothionein (MT) genes in mammalian cells. The MRE-binding domain of MTF-1 (MTF-zf) has six canonical Cys(2)-His(2) zinc finger domains that are distinguished on the basis of their apparent affinities for zinc and their specific roles in MRE-binding. In this paper, pulsed alkylation of the zinc-liganding cysteine thiolate pairs with the sulfhydryl-specific alkylating reagent d(5)-N-ethylmaleimide (d(5)-NEM) is used as a residue-specific probe of the relative stabilities of the individual zinc finger coordination complexes in Zn(6) MTF-zf. A chase with excess H(5)-N-ethylmaleimide (H(5)-NEM) to fully derivatize MTF-zf concomitant with complete proteolysis, followed by MALDI-TOF mass spectrometry allows quantitation of the mole fraction of d(5),d(5)-, d(5),H(5)-, and H(5),H(5)-NEM derivatized peptides corresponding to each individual zinc finger domain as a function of d(5)-NEM pulse time. This experiment establishes the hierarchy of cysteine thiolate reactivity in MTF-zf as F5 > F6 >> F1 > F2 approximately F3 approximately F4. The apparent second-order rate of reaction of F1 thiolates is comparable to that determined for the DNA binding domain of Sp1, Zn(3) Sp1-zf, under identical solution conditions. The reactivities of all Cys residues in MTF-zf are significantly reduced when bound to an MREd-containing oligonucleotide. An identical experiment carried out with Zn(5) MTF-zf26, an MTF-zf domain lacking the N-terminal F1 zinc finger, reveals that MTF-zf26 binds to the MREd very weakly, and is characterized by strongly increased reactivity of nonadjacent F4 thiolates. These findings are discussed in the context of existing models for metalloregulation by MTF-1.

AbstractmTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.