Probing the Interactions of Sulfur-Containing Histidine Compounds with Human Gamma-Glutamyl Transpeptidase.

Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(Nπ)-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo-l-norleucine (DON), and are not toxic for human embryonic cells. We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors.

The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation.

Protein-templated fragment ligations have been established as a powerful method for the assembly and detection of optimized protein ligands. Initially developed for reversible ligations, the method has been expanded to irreversible reactions enabling the formation of super-additive fragment combinations. Here, protein-induced Mannich ligations are discovered as a biocatalytic reaction furnishing inhibitors of the transcription factor STAT5. STAT5 protein catalyzes multicomponent reactions of a phosphate mimetic, formaldehyde, and 1H-tetrazoles yielding protein ligands with greatly increased binding affinity and ligand efficiency. Reactions are induced under physiological conditions selectively by native STAT5 but not by other proteins. Formation of ligation products and (auto-)inhibition of the reaction are quantified and the mechanism is investigated. Inhibitors assembled by STAT5 block specifically the phosphorylation of this protein in a cellular model of acute myeloid leukemia (AML), DNA-binding of STAT5 dimers, expression of downstream targets of the transcription factor, and the proliferation of cancer cells in mice.

Small Molecules Targeting Human N-Acetylmannosamine Kinase.

N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. In search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. By applying different orthogonal assays for their validation we identified four potential MNK-specific inhibitors with IC50 values in the low-micromolar range. Molecular modelling of the inhibitors into the active site of MNK supports their binding to the sugar or the ATP-binding pocket of the enzyme or both. These compounds are promising for downregulation of the sialic acid content of glycoconjugates and for studying the functional contribution of sialic acids to disease development.

Fluorescent mimetics of CMP-Neu5Ac are highly potent, cell-permeable polarization probes of eukaryotic and bacterial sialyltransferases and inhibit cellular sialylation.

Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion of cells, bacteria, and virus particles. The synthesis of neuraminic acid containing glycosides is accomplished by intracellular sialyl transferases. Therefore, the chemical manipulation of cellular sialylation could be very important to interfere with cancer development, inflammations, and infections. The development and applications of the first nanomolar fluorescent inhibitors of sialyl transferases are described herein. The obtained carbohydrate-nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed that the anionic mimetics intruded rapidly and efficiently into cells in vesicles and translocated to cellular organelles surrounding the nucleus of CHO cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation.

Theoretical and experimental evaluation of a CYP106A2 low homology model and production of mutants with changed activity and selectivity of hydroxylation.

Steroids are important pharmaceutically active compounds. In contrast to the liver drug-metabolising cytochrome P450s, which metabolise a variety of substrates, steroid hydroxylases generally display a rather narrow substrate specificity. It is therefore a challenging goal to change their regio- and stereoselectivity. CYP106A2 is one of only a few bacterial steroid hydroxylases and hydroxylates 3-oxo-Delta4-steroids mainly in 15beta-position. In order to gain insights into the structure and function of this enzyme, whose crystal structure is unknown, a homology model has been created. The substrate progesterone was then docked into the active site to predict which residues might affect substrate binding. The model was substantiated by using a combination of theoretical and experimental investigations. First, numerous computational structure evaluation tools assessed the plausibility of its protein geometry and its quality. Second, the model explains many key properties of common cytochrome P450s. Third, two sets of mutants have been heterologously expressed, and the influence of the mutations on the catalytic activity towards deoxycorticosterone and progesterone has been studied experimentally: the first set comprises six mutations located in the structurally variable regions of this enzyme that are very difficult to predict by cytochrome P450 modelling (K27R, I86T, E90V, I71T, D185G and I215T). For these positions, no participation in the active-site formation was predicted, or could be experimentally demonstrated. The second set comprises five mutants in substrate recognition site 6 (S394I, A395L, T396R, G397P and Q398S). For these residues, participation in active-site formation and an influence on substrate binding was predicted by docking. These mutants are based on an alignment with human CYP11B1, and in fact most of these mutants altered the active-site structure and the hydroxylation activity of CYP106A2 dramatically.

The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation.

Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono- and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3Dstructure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-alpha-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly conserved amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly- but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors.

Use of high-performance liquid chromatography/electrospray ionization collision-induced dissociation mass spectrometry for structural identification of monohydroxylated progesterones.

For the structural identification of monohydroxylated progesterones synthesized by microorganisms, a method was developed using a combination of high-performance liquid chromatography and electrospray ionization collision-induced dissociation mass spectrometry (HPLC/ESI-CIDMS). The retention times and MS/MS spectra of 11 different standards at 30 eV were collected and compared. The identification of D-ring-hydroxylated progesterones (15beta-, 16alpha-, 17alpha- and 21-OH-P) using ESI-CIDMS was not possible. However, they were separated chromatographically using a 65:35 mixture of water and acetonitrile containing 0.5% acetic acid. The other hydroxylated progesterones (2alpha-, 6beta-, 7beta-, 9alpha-, 11alpha-, 11beta-, and 19-OH-P) could be identified by comparison of eight fragments. The complete separation of 11 standards was achieved chromatographically. The developed assay was evaluated by the identification of monohydroxylated progesterones produced by CYP106A2 from Bacillus megaterium ATCC 13368.

Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry.

Two previously uncharacterised products, produced by recombinant CYP106A2 of Bacillus megaterium ATCC 13368 using progesterone as substrate, were identified. For this purpose a combination of comparative HPLC and electrospray ionisation collision induced dissociation mass spectrometry (ESI CID MS) was established and applied for rapid identification of the steroids, which were identified as 11alpha-hydroxyprogesterone and 9alpha-hydroxyprogesterone. The pharmaceutical relevance of these steroids is discussed. Furthermore, the hydroxylation activity was quantified for all monohydroxylation products (15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone). The V(max) values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone were determined as 337.3+/-43.7, 22.3+/-0.9, 17.5+/-0.9, and 6.5+/-0.3nmol product/min/nmol CYP106A2, respectively.

Deletions in the loop surrounding the iron-sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450(scc) (CYP11A1).

The redox active iron-sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe-2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe-2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein.