Selective A1-adenosine receptor antagonists identified using yeast Saccharomyces cerevisiae functional assays.

Evaluation of a biased "library" of pyrrolo[2,3-d]pyrimidines using yeast-based functional assays expressing human A1- and A2a-adenosine receptors, led to the A1 selective antagonist 4b. A direct correlation between yeast functional activity and binding data was established. Practical compounds with polar residues at C-4 of the pyrrolopyrimidine system required H-bond donor functionality for high potency.

Design and synthesis of SH3 domain binding ligands: modifications of the consensus sequence XPpXP.

Spirolactam-based Pro-Pro mimetics incorporated in the consensus sequence XPpXP, lead to effective nonpeptide ligands of SH3 domains.

Design and synthesis of a pyridone-based phosphotyrosine mimetic.

A novel pyridone-based tyrosine analog, 6, has been designed to mimic the binding interaction of SH2 domains with phosphotyrosine (pTyr) containing peptides. Synthesis of 6 features a key Pd catalyzed coupling of beta-iodoalanine with phosphonomethyl 4-pyridone triflate.

Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae.

Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of filarial parasites and other nematode species. We have investigated the possible involvement of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro. The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine, and N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-al anine benzylamide. None of the inhibitors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100 to 200 microM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylcadaverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide epsilon-(gamma-glutamyl)lysine. These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed reactions.

Matrilysin-inhibitor complexes: common themes among metalloproteases.

Matrix metalloproteases are a family of enzymes that play critical roles in the physiological and pathological degradation of the extracellular matrix. These enzymes may be important therapeutic targets for the treatment of various diseases where tissue degradation is part of the pathology, such as cancer and arthritis. Matrilysin is the smallest member of this family of enzymes, all of which require zinc for catalytic activity. The first X-ray crystal structures of human matrilysin are presented. Inhibitors of metalloproteases are often characterized by the chemical group that interacts with the active site zinc of the protein. The structures of matrilysin complexed with hydroxamate (maximum resolution 1.9 A), carboxylate (maximum resolution 2.4 A), and sulfodiimine (maximum resolution 2.3 A) inhibitors are presented here and provide detailed information about how each functional group interacts with the catalytic zinc. Only the zinc-coordination group is variable in this series of inhibitors. Examination of these inhibitor-matrilysin complexes emphasizes the dominant role the zinc-coordinating group plays in determining the relative potencies of the inhibitors. The structures of these matrilysin-inhibitor complexes also provide a basis for comparing the catalytic mechanism of MMPs and other metalloproteins.

Study of noncovalent enzyme-inhibitor complexes and metal binding stoichiometry of matrilysin by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was used to study the noncovalent metallo-enzyme-inhibitor complexes of matrilysin (a matrix metalloproteinase of mass 18,720 u) under gentle experimental conditions and to determine the metal ion association stoichiometries in both the free enzyme and the complexes. The metal association stoichiometries of the free matrilysin were found to be highly sensitive to solution pH changes. At pH 2.2 the enzyme existed as metal-free apo-matrilysin and was not capable of binding an inhibitor. At pH 4.5-7.0 the enzyme associated specifically with zinc and calcium cations and became active in inhibitor binding. Although the stoichiometries of the metal cofactors varied (zero to two zinc and/or calcium ions) in the free enzyme dependent on solution pH, the predominant form of the enzyme-inhibitor complexes in the pH range of 4.5-7.0, in contrast, always had the metal association stoichiometry of 2Zn + 2Ca, which was the same stoichiometry the most active free metallo-enzyme had at the optimal pH of 7. At the activity onset pH of 4.5 matrilysin existed mostly as apo-enzyme (but in a conformation different from the denatured one at pH 2.2) and bound to an inhibitor slowly (time constant ∼ 2.5 min) to form the noncovalent metallo-enzyme-inhibitor complex. Of the two inhibitors studied, the one with the higher solution binding constant also produced larger ion signals for the noncovalent complex in the solvent-free gas phase, which pointed to the feasibility of the use of ESI-MS for inhibitor screening studies.

Inactivation of a cytosolic phospholipase A2 by thiol-modifying reagents: cysteine residues as potential targets of phospholipase A2.

The cytosolic phospholipase A2 (cPLA2) from the human monocytic cell line U937 contains nine cysteine residues and is subject to oxidation. Iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid) were used to explore the susceptibility of cysteine residues to thiol modification agents as outlined in Schemes 2 and 3. In the absence of thiol reducing agents such as DTT, cPLA2 takes up only 2.8 equiv of [1-14C]iodoacetamide at pH 8.03/37 degrees C. With DTT present, cPLA2 is in its fully reduced form, and 4-5 equiv of acetamide are taken up without altering enzyme activity to give IA-cPLA2. A single equivalent of DTNB suffices to inactivate IA-cPLA2, giving a TNB-labeled enzyme, with the loss of activity correlating with release of an equivalent of 5-thio-2-nitrobenzoate. The TNB-labeled enzyme is quite stable up to 33 degrees C; enzyme activity is recoverable with DTT, even after this disulfide-enzyme adduct is incubated with iodoacetamide at pH 9.5, conditions that inactivate the free enzyme. At pH 9.5/37 degrees C, a single equivalent of 14C-labeled iodoacetamide is incorporated by IA-cPLA2 concomitant with complete loss of enzyme activity. Amino acid analysis of the 14C-labeled enzyme indicates that only cysteine residues are labeled. Lys-C digestion of labeled enzyme with 2 M guanidine at pH 8.0 yields a 40-mer peptide. Amino acid sequencing establishes that the label resides primarily in Cys324, although Cys331 is also labeled. These results identify a region of the enzyme that is susceptible to labeling by group modification reagents and may represent a suitable target for small molecule inhibitors.

Solid-state 13C NMR study of a transglutaminase-inhibitor adduct.

We have used solid-state 13C NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-halo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assumption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazole ring. The tetrahedral intermediate formed could then break down with the loss of the halide group and the subsequent formation of a stable imino thioether adduct. We have compared the 13C CPMAS spectra of the chloro-, bromo-, and (ethylthio)dihydroisozazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensitive to the nature of the heteroatom. Subtraction of the natural-abundance 13C solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad peak at 156 ppm. The chemical shift of this peak is very close to that observed for a model 3-ethylthio compound and suggests the formation of a stable imino thioether enzyme adduct. Similar results were obtained for lyophilized enzyme adducts and for frozen solutions of the enzyme adduct in the absence and presence of Ca2+. We have also compared these results with those obtained by solution NMR on an aqueous solution of the enzyme-inhibitor complex. The 13C-labeled C-3 resonance was not observed in this case.

A new class of mechanism-based inhibitors of transglutaminase enzymes inhibits the formation of cross-linked envelopes by human malignant keratinocytes.

A series of tyrosinamidomethyl dihydrohaloisoxazole compounds, designed as mechanism-based inhibitors of bovine epidermal transglutaminase enzyme, was examined for effects on the formation of cross-linked envelopes by human SCC-9 malignant keratinocytes. Compounds inhibited ionophore-induced envelope formation in a manner that reflected their capacity to inhibit transglutaminase activity. Preincubation and inhibitor wash-out studies indicated that the inhibitor must be present at the time of cell activation by ionophore in order to inhibit envelope formation. The stereospecific nature of the inhibitory activity of these compounds on both transglutaminase activity and cross-linked envelope formation makes this class of compounds an important tool in the study of transglutaminase-mediated events at the cellular level.