Endogenous and synthetic MMP inhibitors in CNS physiopathology.

Matrix metalloproteinases (MMPs, including the membrane-type MMPs (MT-MMPs)), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin motifs belong to the metzincins, a subclass of metalloproteinases that contain a Met residue and a Zn(2+) ion at the catalytic site necessary for enzymatic reaction. MMP proteolytic activity is mainly controlled by their natural tissue inhibitors of metalloproteinase (TIMP). A number of synthetic inhibitors have been developed to control deleterious MMP activity. The roles of MMPs and some of their ECM substrates in CNS physiology and pathology are covered by other chapters of the present volume and will thus not be addressed in depth. This chapter will focus (i) on the endogenous MMP inhibitors in the CNS, (ii) on MMP and TIMP regulations in three large classes of neuropathologic processes (inflammatory, neurodegenerative, and infectious), and (iii) on synthetic inhibitors of MMPs and the perspective of their use in different brain diseases.

Iron-Promoted Nucleophilic Additions to Diimine-Type Ligands: A Synthetic and Structural Study

We report here three examples of the reactivity of protic nucleophiles with diimine-type ligands in the presence of FeII salts. In the first case, the iron-promoted alcoholysis reaction of one nitrile group of the ligand 2,3-dicyano-5,6-bis(2-pyridyl)-pyrazine (L1) permitted the isolation of an stable E-imido−ester, [Fe(L1‘)2](CF3SO3)2 (1), which has been characterized by spectroscopic studies (IR, ES-MS, Mössbauer), elemental analysis, and crystallographically. Compound 1 consists of mononuclear octahedrally coordinated FeII complexes where the FeII ion is in its low-spin state. The iron-mediated nucleophilic attack of water to the asymmetric ligand 2,3-bis(2-pyridyl)pyrido[3,4-b]pyrazine (L2) has also been studied. In this context, the crystal structures of two hydration−oxidation FeIII products, [Fe(L2‘)2](ClO4)3·3CH3CN (2) and trans-[FeL2‘‘Cl2] (3), are described. Compounds 2 and 3 are both mononuclear FeIII complexes where the metals occupy octahedral positions. In principle, L2 is expected to coordinate to metal ions through its bipyridine-type units to form a five-membered ring; however, this is not the case in compounds 2 and 3. In 2, the ligand coordinates through its pyridines and through the hydroxyl group attached to the pyrazine imino carbon after hydration, that is, in an N,O,N tridentate manner. In compound 3, the ligand has suffered further transformations leading to a very stable diamido complex. In this case, the metal ion achieves its octahedral geometry by means of two pyridines, two amido N atoms, and two axial chlorine atoms. Magnetic susceptibility measurements confirmed the spin state of these two FeIII species:  compounds 2 and 3 are low-spin and high-spin, respectively.

Synthetic studies towards a novel, chemical stable, abasic site analogue of DNA

We synthesized the phosphinate 7 via photoaddition of methanol to the alpha, beta unsaturated deoxyribono lactone as the key step, followed by an Arbusov reaction for the introduction of phosphorous. Precursor 7 serves for the synthesis and incorporation into DNA of a novel chemically stable abasic site analogue that might act as an inhibitor for DNA glycosylases

106: Synthetic preimplantation factor (sPIF*) promotes neuroprotection by modulating PKA/PKC kinases

OBJECTIVE: Survivors of premature birth suffer from long term disabilities. Synthetic PreImplantation Factor (sPIF*) modulates inflammatory responses and reverses neuroinflammation. Proteinkinase A (PKA) and protein kinase C (PKC) are crucial signaling molecules. PKA up-regulates IL-10 and brain-derived neurotrophic factor (BDNF) expression, which exert neuroprotective effects. Anti-apoptotic phosphorylation of Bad is mediated by PKA. PKC phosphorylates GAP-43, a marker for neuronal plasticity and structural recovery. We explored sPIF protective role in neuronal (N2a) cells and in a rat model of encephalopathy of prematurity. *proprietary. STUDY DESIGN: Cells were subjected to LPS and treated with sPIF or scrambled sPIF. Neonatal rats (postnatal day 3: P3) were subjected to LPS, ligation of carotid artery, and hypoxia (8% O2, 65min; n¼ 30). sPIF (0.75mg/kg twice daily) was injected (P6-13) and brains harvested at P13. sPIF’s potential and mechanisms were evaluated using immunohistochemistry, ELISA, Western Blot, and qRT-PCR. Data were analyzed using two-tailed Student’s t-test. P<0.05 wasconsidered statistically significant. RESULTS: In vitro sPIF increased PKA/PKC activity in time dependent manner (Fig. 1A). sPIF induced higher IL-10, BDNF, and GAP-43 and lower CASP3, BAD, and TNF-a mRNA levels (Fig. 1B,C). sPIF increased pGap-43/Gap-43 and decreased pBad/Bad ratio while decreasing Bad (Fig. 1 D,E). In brain tissue sPIF treatment resulted in rescued neuronal number (NeuN positive cells) and reduced apoptosis (Casp-3 positive cells) with decreased glial (Iba-1 positive cells) activation (Fig. 2A,B). The Iba-1 morphology changed from predominantly amoeboid to ramified state. Additionally sPIF increased IL-10 mRNA levels (Fig. 2C) and pGap-43/Gap-43 ratio (Fig. 2D). CONCLUSION: sPIF modulates PKA/PKC pathways reducing apoptosis and inflammatory responses while increasing neuronal plasticity and survival. The identified PKA/PKC regulatory axis strengthens the potential of sPIF in reducing the burden of prematurity.

A synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes.

The 3' processing of histone pre-mRNAs is a nuclear event in which the U7 small nuclear ribonucleoprotein (snRNP) participates as an essential trans-acting factor. We have constructed a chimeric histone-U7 RNA that when injected into the cytoplasm of Xenopus laevis oocytes assembles into a snRNP-like particle and becomes cleaved at the correct site(s). RNP assembly is a prerequisite for cleavage, but, since neither the RNA nor the RNP appreciably enter the nucleus, cleavage occurs mostly, if not exclusively, in the cytoplasm. Consistent with this, cleavage also occurs in enucleated oocytes or in oocytes which have been depleted of U7 snRNPs. Thus all necessary components for cleavage must be present in the oocyte cytoplasm. The novel cleavage occurs in cis, involving only a single molecule of chimeric RNA with its associated proteins. This reaction is equally dependent upon base pairing interactions between histone spacer sequences and the 5'-end of the U7 moiety as the natural in trans reaction. These results imply that U7 is the only snRNP required for histone RNA processing. Moreover, the chimeric RNA is expected to be useful for further studies of the cleavage and assembly mechanisms of U7 snRNP.

Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.

In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.

A Synthetic Factor XIIa Inhibitor Blocks Selectively Intrinsic Coagulation Initiation.

Coagulation factor XII (FXII) inhibitors are of interest for the study of the protease in the intrinsic coagulation pathway, for the suppression of contact activation in blood coagulation assays, and they have potential application in antithrombotic therapy. However, synthetic FXII inhibitors developed to date have weak binding affinity and/or poor selectivity. Herein, we developed a peptide macrocycle that inhibits activated FXII (FXIIa) with an inhibitory constant Ki of 22 nM and a selectivity of >2000-fold over other proteases. Sequence and structure analysis revealed that one of the two macrocyclic rings of the in vitro evolved peptide mimics the combining loop of corn trypsin inhibitor, a natural protein-based inhibitor of FXIIa. The synthetic inhibitor blocked intrinsic coagulation initiation without affecting extrinsic coagulation. Furthermore, the peptide macrocycle efficiently suppressed plasma coagulation triggered by contact of blood with sample tubes and allowed specific investigation of tissue factor initiated coagulation.

[Synthetic philosophy] ...

[v.2] & [v.5] have New York imprint only

Learning Models of Gene Expression from Synthetic DNA Sequences

Thesis (Ph.D.)--University of Washington, 2016-06

Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and other malignancies. WT1 is one of the earliest genes expressed during kidney development. WT1 proteins can activate and repress putative target genes in vitro, although the in vivo relevance of such target genes often remains unverified. To better understand the role of WT1 in tumorigenesis and kidney development, we need to identify downstream target genes. In this study, we have expression pro. led human embryonic kidney 293 cells stably transfected to allow inducible WT1 expression and mouse mesonephric M15 cells transfected with a WT1 antisense construct to abolish endogenous expression of all WT1 isoforms to identify WT1-responsive genes. The complementary overlap between the two cell lines revealed a pronounced repression of genes involved in cholesterol biosynthesis by WT1. This pathway is transcriptionally regulated by the sterol responsive element-binding proteins (SREBPs). Here, we provide evidence that the C-terminal end of the WT1 protein can directly interact with SREBP, suggesting that WT1 may modify the transcriptional function of SREBPs via a direct protein-protein interaction. Therefore, the tumour suppressor activities of WT1 may be achieved by repressing the mevalonate pathway, thereby controlling cellular proliferation and promoting terminal differentiation.

The chemistry and biology of human relaxin-3

A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin-3. Like human relaxin-1 and relaxin-2, relaxin-3 is predicted to consist of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin-1 and relaxin-2, however, relaxin-3 could not be successfully prepared by simple combination of the individual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S-protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin-3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin-3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin-3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin-1 or relaxin-2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three-dimensional structure of relaxin-3 and to identify key structural differences between the human relaxins.

Quantitative fluorescence spectra and quantum yield map of synthetic pheomelanin

Spectroscopic studies of pheomelanin and its constituents have been sparse. These data present what is by far the most complete description of the fluorescence characteristics of synthetic pheomelanin. Emission spectra between 260 and 600 nm were acquired,for excitation wavelengths between 250 and 500 nm at 1-nm intervals. A quantum yield map is also presented, correcting the fluorescence intensities for differences in species concentration and molar absorptivity. These fluorescence features exhibit interesting similarities and differences to eumelanin, and these data are interpreted with respect to possible chemical structures. Overall, these data suggest that pheomelanin oligomers may be more tightly coupled than those of eumelanin. Finally, the quantum yield is shown to be on the order of 10(-4) and exhibit a complex dependence on excitation energy, varying by a factor of 4 across the energies employed here. (c) 2006 Wiley Periodicals, Inc.