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Brahma Ghosh,
Alexey O Benyumov, Phalguni Ghosh,
Yan Jia,
Svetlana Avdulov,
Peter S Dahlberg,
Mark Peterson,
Karen Smith,
Vitaly A Polunovsky,
Peter B Bitterman,
Carston R Wagner
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ABSTRACT: Normal growth and development depends upon high fidelity regulation of cap-dependent translation initiation, a process that is usurped and redirected in cancer to mediate acquisition of malignant properties. The epithelial-to-mesenchymal transition (EMT) is a key translationally regulated step in the development of epithelial cancers and pathological tissue fibrosis. To date, no compounds targeting EMT have been developed. Here we report the synthesis of a novel class of histidine triad nucleotide binding protein (HINT)-dependent pronucleotides that interdict EMT by negatively regulating the association of eIF4E with the mRNA cap. Compound eIF4E inhibitor-1 potently inhibited cap-dependent translation in a dose-dependent manner in zebrafish embryos without causing developmental abnormalities and prevented eIF4E from triggering EMT in zebrafish ectoderm explants without toxicity. Metabolism studies with whole cell lysates demonstrated that the prodrug was rapidly converted into 7-BnGMP. Thus we have successfully developed the first nontoxic small molecule able to inhibit EMT, a key process in the development of epithelial cancer and tissue fibrosis, by targeting the interaction of eIF4E with the mRNA cap and demonstrated the tractability of zebrafish as a model organism for studying agents that modulate EMT. Our work provides strong motivation for the continued development of compounds designed to normalize cap-dependent translation as novel chemo-preventive agents and therapeutics for cancer and fibrosis.
ACS Chemical Biology 05/2009; 4(5):367-77. · 6.45 Impact Factor
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ABSTRACT: One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired by-products. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR-eIF4E protein by using affinity and anion exchange chromatography. Fluorescence quenching experiments indicated the cap-analog, 7MeGTP, bound to DHFR-eIF4E and eIF4E with a dissociation constant (K(d)) of 6+/-5 and 10+/-3 nM, respectively. Recombinant eIF4E and DHFR-eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 microM. Nevertheless increased concentrations of eIF4E and DHFR-eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 microM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR-eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs.
Protein Expression and Purification 09/2008; 60(2):132-9. · 1.59 Impact Factor
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ABSTRACT: Cap-dependent translation is initiated by the binding of eIF4E to capped mRNA (m(7)GpppN). We have prepared a small library of 7-methyl guanosine nucleoside and nucleotide analogs and evaluated their ability to inhibit eIF4E binding to 7-methyl GTP with a competitive eIF4E binding immunoassay. 5'-H-Phosphonate derivatives in which the 2'- and 3'-riboside hydroxyls were tethered together by an isopropylidene group were shown to be a new class of inhibitors of eIF4E binding to capped mRNA.
Bioorganic & Medicinal Chemistry Letters 05/2005; 15(8):2177-80. · 2.55 Impact Factor
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ABSTRACT: The binding of pyridine by V(II) in aqueous solution shows evidence for the late onset of cooperativity. The K(1) governing formation of [V(py)](2+) (lambda(max) = 404 nm, epsilon(max) = 1.43 +/- 0.3 M(-1) cm(-1)) was determined spectrophotometrically to be 11.0 +/- 0.3 M(-)(1), while K(1) for isonicotinamide was found to be 5.0 +/- 0.1 M(-1). These values are in the low range for 3d M(2+) ions and indicate that V(II).py back-bonding is not significant in the formation of the 1:1 complex. Titration of 10.5 mM V(II) with pyridine in aqueous solution showed an absorption plateau at about 1 M added pyridine, indicating a reaction terminus. Vanadium K-edge EXAFS analysis of 63 mM V(II) in 2 M pyridine solution revealed six first-shell N/O ligands at 2.14 A and 4 +/- 1 pyridine ligands per V(II). UV/vis absorption spectroscopy indicated that the same terminal V(II) species was present in both experiments. Model calculations showed that in the absence of back-bonding only 2.0 +/- 0.2 and 2.4 +/- 0.2 pyridine ligands would be present, respectively. Cooperativity in multistage binding of pyridine by [V(aq)](2+) is thus indicated. XAS K-edge spectroscopy of crystalline [V(O(3)SCF(3))(2)(py)(4)] and of V(II) in 2 M pyridine solution each exhibited the analogous 1s --> (5)E(g) and 1s --> (5)T(2g) transitions, at 5465.5 and 5467.5 eV, and 5465.2 and 5467.4 eV, respectively, consistent with the EXAFS analysis. In contrast, [V(py)(6)](PF(6))(2) and [V(H(2)O)(6)]SO(4) show four 1s --> 3d XAS transitions suggestive of a Jahn-Teller distorted excited state. Comparison of the M(II)[bond]N(py) bond lengths in V(II) and Fe(II) tetrapyridines shows that the V(II)[bond]N(py) distances are about 0.06 A shorter than predicted from ionic radii. For [VX(2)(R-py)(4)] (X = Cl(-), CF(3)SO(3)(-); R = 4-Et, H, 3-EtOOC), the E(1/2) values of the V(II)/V(III) couples correlate linearly with the Hammett sigma values of the R group. These findings indicate that pi back-bonding is important in [V(py)(4)](2+) even though absent in [V(py)](2+). The paramagnetism of [V(O(3)SCF(3))(2)(py)(4)] in CHCl(3), 3.8 +/- 0.2 mu(B), revealed that the onset of back-bonding is not accompanied by a spin change. Analysis of the geometries of V(II) and Fe(II) tetrapyridines indicates that the ubiquitous propeller motif accompanying tetrapyridine ligation may be due to eight dipole interactions arising from the juxtaposed C-H edges and pi clouds of adjoining ligands, worth about -6 kJ each. However, this is not the source of the cooperativity in the binding of multiple pyridines by V(II) because the same interactions are present in the Fe(II)-tetrapyridines, which do not show cooperative ligand binding. Cooperativity in the binding of pyridine by V(II) is then assigned by default to V(II)-pyridine back-bonding, which emerges only after the first pyridine is bound.
Inorganic Chemistry 07/2002; 41(12):3269-79. · 4.60 Impact Factor
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04/2002;
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ABSTRACT: The reaction of in situ generated Cp(2)V(OTf)(2) (Cp = cyclopentadienyl; OTf = O(3)SCF(3)) with excess 1,10-phenanthroline and 2,2'-bipyridine yields the d(1) vanadocene coordination compounds [Cp(2)V(phen)][OTf](2) (1) and [Cp(2)V(bpy)][OTf](2) (2), respectively. The compounds have been characterized by UV-vis and EPR spectroscopy and by cyclic voltammetry. The complexes have relatively low vanadium(IV)-vanadium(III) reduction potentials (-0.62 V vs Cp(2)Fe(+/0) in acetonitrile). Structures of 1 and 2 have been determined by X-ray crystallography. Compound 1 crystallized in a monoclinic system, space group P2(1)/n, with a = 10.2763(5) Å, b = 18.1646(9) Å, c = 13.5741(7) Å, beta = 99.4150(10) degrees, and Z = 4. Refinement of its structure by full-matrix least-squares techniques gave final residuals R = 0.040 and R(w) = 0.096. Compound 2 crystallized in a monoclinic system, space group P2(1)/c, with a = 10.6451(6) Å, b = 18.3863(10) Å, c = 12.6993(7) Å, beta = 98.6220(10) degrees, and Z = 4. Refinement of its structure by full-matrix least-squares techniques gave final residuals R = 0.046 and R(w) = 0.101. The two nitrogen atoms and centroids of the two cyclopentadienyl rings for both compounds occupy a distorted tetrahedral geometry around the vanadium(IV) center. The chelated ring plane is inclined closer to one of the neighboring Cp rings with the tilt more evident in 1 ( approximately 8 degrees ) than 2 ( approximately 4 degrees ). The membrane interactions of these compounds and the titanium analogues, [Cp(2)Ti(phen)][OTf](2) (3) and [Cp(2)Ti(bpy)][OTf](2) (4), have been studied with zwitterionic unilamellar liposomes as artificial membranes. We show that the ability of metallocenes to enhance the permeability of a liposomal membrane depends on the hydrophobicity, as well as the size and planarity of the ancillary chelated ligands, but not the nature of the central metal ion. Also provided is evidence that metallocene-induced permeability changes in artificial membranes are not caused by lipid peroxidation.
Inorganic Chemistry 09/1999; 38(16):3730-3737. · 4.60 Impact Factor
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ABSTRACT: In a systematic search for vanadocene complexes with sperm immobilizing activity as a new class of contraceptive agents, we identified V(η5-C5H5)2((C2H5)2NCS2)(BF4) (=[VCp2(DeDtc)] (BF4)) as the most potent and stable spermicidal compound. Here we report the detailed biologic and physicochemical characterization of this lead spermicidal compound by computer-assisted sperm analysis, electron paramagnetic resonance spectroscopy, electrochemistry, and X-ray crystallography. [VCp2(DeDtc)](BF4) crystallized in the monoclinic space group P21/c, with unit cell dimensions a=7.0877(4) Å, b=22.2881(14) Å, c=11.8021(7) Å, β=94.107(1)°, V=1859.6(2) Å3. The final structure of [VCp2(DeDtc)] (BF4) had an R factor of 0.0581 for 3191 independent reflections. The two sulfur atoms of the dithiocarbamate and centroids of the cyclopentadienyl rings in this vanadocene complex with unique contraceptive potential occupy four tetrahedral – like coordination sites about the central metal atom.
Journal of Inorganic Biochemistry.
