Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides.

The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in an Escherichia coli strain which also overexpressed the rare argU tRNA. The protease was isolated from inclusion bodies, refolded, and exhibited a kcat/Km = 3280 M-1 s-1 using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease with kinact/Kinact values as high as 23,400 M-1 s-1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to the kinact/Kinact rate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.

Glutathione adducts, not carbamylated lysines, are the major modification of lens alpha-crystallins from renal failure patients.

alpha-Crystallins from the water-soluble and the water-insoluble, guanidine-soluble portions of lenses from four renal failure patients and two normal donors of similar age were isolated and enzymatically digested into peptides. Molecular weights of the peptides, determined by fast atom bombardment mass spectrometry, indicated modifications specifically associated with renal failure. The only modifications observed in the alpha-crystallins from renal failure patients, but not in the normal old lenses, were glutathione adducts to Cys 131 and Cys 142. These adducts were present in the lenses of all four renal failure patients, but not in the two normal old lenses. The four lenses from the renal failure patients were searched for evidence of carbamylation at lysyl or cysteinyl residues: carbamylation was not detected. Because the same mass spectrometric methods had previously demonstrated sufficient sensitivity and specificity to detect as little as 5% modification in the examination of in vitro carbamylated bovine lenses, these results indicated that carbamylation is not a major modification of the lens alpha-crystallins of renal failure patients.

Asimin, asiminacin, and asiminecin: novel highly cytotoxic asimicin isomers from Asimina triloba.

Activity-directed fractionation of the stem bark extracts of the North American paw paw tree, Asimina triloba (Annonaceae), has yielded three further acetogenins: asimin (2), asiminacin (3), and asiminecin (4). 2-4 are structural isomers of asimicin (1), which is a potent inhibitor of mitochondrial NADH:ubiquinone oxidoreductase, and thus exhibits potent antitumor and pesticidal effects. 2-4 have the same carbon skeleton and configurations as those of 1, but they have the third hydroxyl group located at C-10, C-28, and C-29, respectively, rather than at C-4. The determinations of the hydroxyl group locations were largely based on mass spectral analyses of TMSi and TMSi-d9 derivatives. 2-4 all showed highly potent cytotoxicities (ED50 values as low as < 10(-12) micrograms/mL) with notable selectivities for the HT-29 human colon cancer cell line. The presence of a third hydroxyl at C-4, C-10, C-28, or C-29, as in 1-4, greatly enhances the bioactivity of 4-deoxyasimicin (5).

Post-translational modifications of water-soluble human lens crystallins from young adults.

Post-translational modifications of the water-soluble human lens crystallins from young adult donors were identified and located using electrospray ionization mass spectrometric analysis of the intact proteins and fast atom bombardment mass spectrometry of enzymatic digests. Peptides corresponding to all of the sequences of alpha A-, alpha B-, and beta B2-crystallins were found, permitting the entire sequences to be searched for modifications. The major portions of these three crystallins were not modified. Modifications of alpha A-crystallin that were detected included 2 phosphorylated Ser residues (1 of which appears to be unique to human lenses), deamidation at some Gln and Asn residues, a disulfide bond between Cys-131 and Cys-142, and loss of the COOH-terminal Ser residue. Three phosphorylated Ser residues, but no deamidation, were found in alpha B-crystallin. The molecular weights of neither the intact protein nor the peptides in the enzymatic digests indicated any post-translational modification of the principal beta-crystallin, beta B2. The molecular weights of the other beta- and gamma-crystallins for which sequences have been published suggested the presence of post-translational modifications or errors in the published sequences. Although enough peptides were found to establish the presence of specific proteins, peptides corresponding to all portions of these proteins were not found, and elucidation of these structures is not yet complete. This mass spectrometric characterization of the total water-soluble proteins from normal young adult lenses provides a reference data base for future investigations of the modifications present in aged and cataractous lenses.

Biologically active acetogenins from stem bark of Asimina triloba.

In continuing our research with cytotoxic and pesticidal components from the stem bark of the North American paw paw, Asimina triloba, the novel cytotoxic monotetrahydrofuran Annonaceous acetogenins, cis- and trans-annonacin-A-one, cis- and trans-gigantetrocinone and cis-isoannonacin, in addition to the known compounds, trans-isoannonacin and squamolone, have been identified. Brine shrimp lethality testing was used to direct the fractionation. The structures were elucidated by spectral analysis and/or chemical synthesis. These acetogenins have potent cytotoxicities against the human tumour cell lines of A-549 (lung carcinoma), MCF-7 (breast carcinoma) and HT-29 (colon adenocarcinoma).

30-, 31-, and 32-hydroxybullatacinones: bioactive terminally hydroxylated annonaceous acetogenins from Annona bullata.

From Annona bullata, three more pairs of new ketolactone Annonaceous acetogenins were isolated by bioactivity-directed isolation. They are hydroxylated adjacent bistetrahydrofuran (THF) acetogenins and are named (2,4-cis and trans)-32-hydroxybullatacinone (1 and 2), (2,4-cis and trans)-31-hydroxybullatacinone (3 and 4), and (2,4-cis and trans)-30-hydroxybullatacinone (5 and 6). The structures were elucidated by analysis of the 1H- and 13C-nmr spectra of 1-6 and their acetates and the ms of their tri-trimethylsilyl (TMSi) derivatives as compared with bullatacinone [7]. This is the first time that Annonaceous acetogenins with OH groups at successive positions near the end of the aliphatic chain have been reported. All of the new compounds showed potent activities in the brine shrimp lethality test and against human solid tumor cells in culture, with selectivities exhibited especially toward the colon cancer cell line (HT-29).