Molecular insights into protein synthesis with proline residues

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono‐ and diprolyl‐tRNAs. These structures provide a high‐resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.     

Effects of Ascorbic Acid and Analogs on the Activity of Testicular Hyaluronidase and Hyaluronan Lyase on Hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

A comparative study of ATP analogs for phosphorylation-dependent kinase–substrate crosslinking

Kinase-catalyzed protein phosphorylation is an important post-translational modification that regulates a variety of cellular functions. Identification of the many substrates of a specific kinase is critical to fully characterize cell biology. Unfortunately, kinase–substrate interactions are often transient, which makes their identification challenging. Here, the transient kinase–substrate complex was stabilized by covalent crosslinking using γ-phosphate modified ATP analogs. Building upon prior use of an ATP-aryl azide photocrosslinking analog, we report here the creation of an ATP-benzophenone photocrosslinking analog. ATP-benzophenone displayed a higher conversion percentage but more diffuse crosslinking compared to the ATP-aryl azide analog. A docking study was also performed to rationalize the conversion and crosslinking data. In total, the photocrosslinking ATP analogs produced stable kinase–substrate complexes that are suitable for future applications characterizing cell signaling pathways.     

Monoval-LdC: Efficient Prodrug of 2′-Deoxy-β-L-cytidine (L-dC), A Potent and Selective Anti-HBV Agent

Abstract

In order to improve the oral bioavailability of LdC, valinyl esters were prepared as prodrugs. We report here the syntheses of the 3′-mono-, 5′-mono, and 3′,5′-di-O-valinyl esters of LdC. The comparison of their ease of synthesis, their physicochemical properties, as well as their pharmacokinetic parameters in cynomologus monkeys has revealed 3′-mono-O-valinyl derivative as the most promising of the studied prodrugs. This compound is being developed as a new anti-HBV agent.     

Structural models of viral insulin-like peptides and their analogs

The insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF1R), and the insulin/IGF1 hybrid receptors (hybR) are homologous transmembrane receptors. The peptide ligands, insulin and IGF1, exhibit significant structural homology and can bind to each receptor via site-1 and site-2 residues with distinct affinities. The variants of the Iridoviridae virus family show capability in expressing single-chain insulin/IGF1 like proteins, termed viral insulin-like peptides (VILPs), which can stimulate receptors from the insulin family. The sequences of VILPs lacking the central C-domain (dcVILPs) are known, but their structures in unbound and receptor-bound states have not been resolved to date. We report all-atom structural models of three dcVILPs (dcGIV, dcSGIV, and dcLCDV1) and their complexes with the receptors (μIR, μIGF1R, and μhybR), and probed the peptide/receptor interactions in each system using all-atom molecular dynamics (MD) simulations. Based on the nonbonded interaction energies computed between each residue of peptides (insulin and dcVILPs) and the receptors, we provide details on residues establishing significant interactions. The observed site-1 insulin/μIR interactions are consistent with previous experimental studies, and a residue-level comparison of interactions of peptides (insulin and dcVILPs) with the receptors revealed that, due to sequence differences, dcVILPs also establish some interactions distinct from those between insulin and IR. We also designed insulin analogs and report enhanced interactions between some analogs and the receptors.     

Stereoselectivity in central analgesic action of tocainide and its analogs

The antiarrhythmic drug tocainide (5a) and some related chiral α-amino and α-imino anilides (5b–e) were synthesized in optically active form. The antinociceptive effects of the different stereoisomers of these compounds were examined and it was found that the analgesic effect of tocainide is due only to its (?)-(R)-enantiomer. Benzyl replacement for methyl group at the stereogenic centre of tocainide causes loss of activity while both enantiomers of the αiminoxilidide 5e and of the strictly related tocainide analog 5d produce an analgesic effect without any stereoselectivity. Pharmacological tests and [³H] quinuclidinyl benzilate ([³H]QNB) binding assay, taken together, seem to show that the antinociceptive effect of (?)-(R)-tocainide, like the analgesia induced by lidocaine, procaine, and mexiletine, is due to a central presynaptic cholinergic mechanism of action. © 1993 Wiley-Liss, Inc.     

Polypeptide-assisted oligomerization of analogs in dilute aqueous solution

Poly (Leu-Lys) was shown to assist the oligomerization of activated nucleotide diphosphates. Short oligomers of pdGp are formed in dilute solution. Activated oligomers can complex to the polypeptide and polymerize to form longer oligomers. Oligomers up to the 18-mer can be obtained under these conditions from 1 × 10^–3 M ImpdGpIm in the absence of a preformed polynucleotide template. The total yield of polymerization remains limited although 50% more pyrophosphate bonds are formed in the presence of polypeptide. However, the elongation effect is more significant since the yield of oligomers longer than the decamer is increased by a factor of 60. The possible prebiotic implications of these experiments are discussed.Abbreviations pdGp the 3,5-bisphosphate of dG - ImpdGpIm the 3,5-bisphosphoimidazolide of dG - KEDTA ethylenediamine tetraacetic acid, potassium salt - Bis-Tris bis(2-hydroxymethyl)iminotris(hydroxymethyl)methane Correspondence to: B. Barbier     

Differential Effects of Fluoronorepinephrines on Phosphatidylinositol Turnover in Rat Pinealocytes

(+/-)-Norepinephrines (NAs), substituted with fluorine at positions 2, 5, and 6 of the ring, were compared with the unsubstituted compound with respect to their capacity for eliciting increased incorporation of [32P]orthophosphate into phosphatidylinositol of pinealocytes. 5F-NA and 6F-NA were approximately equipotent with NA, whereas 2F-NA required a higher concentration and gave lower maximum stimulation. Inhibition of these effects by prazosin confirmed the participation of alpha 1-adrenergic receptors. The results are comparable with those reported for alpha 1-adrenergic receptor-mediated events in other systems and different from the beta-adrenergic receptor-mediated elevation of cyclic AMP in pinealocytes. These and earlier results emphasize the importance of the hydroxyl group at position 3 of the ring and at the beta-position of the side chain in catecholamine activation of the pineal alpha-adrenergic receptors, which are involved in alterations of phospholipid metabolism.     

Inhibition of a Membrane-Bound Enkephalin-Degrading Aminopeptidase by Bestatin Analogs

Abstract: A variety of bestatin analogs were examined as potent inhibitors of a membrane-bound enkephalin-degrading aminopeptidase that was purified from monkey brain. Bestatinyl amino acid derivatives showed strong inhibition of this enzyme. The most effective was bestatin- l -Arg AcOH, with a K _i value of 0.21 × 10⁻⁸ M with Leu-enkephalin as substrate. It exhibited competitive kinetics and was about 100-fold more potent than bestatin. This compound seems to be useful for pharmacological and other studies.