Synthetic pyridyl-alanyl decapeptides having antiovulatory activity

Pyridyl-alanyl decapeptides have been effectively synthesized and found to have antiovulatory activity. The exemplary [N-Ac-D-2-Nal.sup.1,pCl-D-Phe.sup.2, D-3-Pal.sup.3,D-Arg.sup.6,D-Ala.sup.10 ]-LHRH has very high potency to inhibit ovulation, both parenterally and orally. Also, this exemplary pyridyl-alanyl-decapeptide showed an unexpected prolonged duration of antiovulatory activity. Such pyridyl-alanyl-decapeptides are useful to control reproduction.Board of Regents, University of Texas Syste

Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m7G to m2,7G, and (2) conversion of m2,7G to m2,2,7G. hTgs1 is unable to methylate G or m2G, signifying that both steps require an m7G cap. hTgs1 utilizes a broad range of m7G nucleotides, including mono-, di-, tri-, and tetraphosphate derivatives as well as cap dinucleotides with triphosphate or tetraphosphate bridges. In contrast, Giardia lamblia Tgs (GlaTgs2) exemplifies a different clade of guanine-N2 methyltransferase that synthesizes only a dimethylguanosine (DMG) cap structure and cannot per se convert DMG to TMG under any conditions tested. Methylation of benzyl7G and ethyl7G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key π-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members

Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency

Synthetic analogs of the N7-methylated guanosine triphosphate cap at the 5′ end of eukaryotic mRNAs and snRNAs have played an important role in understanding their splicing, intracellular transport, translation, and turnover. We report here a new series of N7-benzylated dinucleoside tetraphosphate analogs, b(7)Gp(4)G, b(7)m(3′-)(O)Gp(4)G, and b(7)m(2)Gp(4)G, that extend our knowledge of the role of the cap in translation. We used these novel analogs, along with 10 previously synthesized analogs, to explore five parameters: binding affinity to eIF4E, inhibition of cap-dependent translation in a rabbit reticulocyte lysate system, efficiency of incorporation into RNAs during in vitro transcription (% capping), orientation of the analog in the synthetic mRNA (% correct orientation), and in vitro translational efficiency of mRNAs capped with the analog. The 13 cap analogs differed in modifications of the first (distal) and second (proximal) guanine moieties, the first and second ribose moieties, and the number of phosphate residues. Among these were analogs of the naturally occurring cap m(3)(2,2,7)Gp(3)G. These compounds varied by 61-fold in affinity for eIF4E, 146-fold in inhibition of cap-dependent translation, 1.4-fold in % capping, and 5.6-fold in % correct orientation. The most stimulatory analog enhanced translation 44-fold compared with uncapped RNA. mRNAs capped with b(7)m(2)Gp(4)G, m(7)Gp(3)m(7)G, b(7)m(3′-)(O)Gp(4)G, and m(7)Gp(4)m(7)G were translated 2.5-, 2.6-, 2.8-, and 3.1-fold more efficiently than mRNAs capped with m(7)Gp(3)G, respectively. Relative translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to translational efficiency

Charge Distribution in 7-Methylguanine Regarding Cation-π Interaction with Protein Factor eIF4E

Electric charge distribution in mRNA 5′ cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-π stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5′-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring

Weak binding affinity of human 4EHP for mRNA cap analogs

Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5′ cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs