1,2,4-thiadiazole: a novel Cathepsin B inhibitor

A novel class of Cathepsin B inhibitors has been developed with a 1,2,4-thiadiazole heterocycle as the thiol trapping pharmacophore. Several compounds with different dipeptide recognition sequence (i.e., P1′–P2′=Leu-Pro-OH or P2–P1=Cbz-Phe-Ala) at the C5 position and with different substituents (i.e., OMe, Ph, or COOH) at the C3 position of the 1,2,4-thiadiazole ring have been synthesized and tested for their inhibitory activities. The substituted thiadiazoles 3a–h inhibit Cat B in a time dependent, irreversible manner. A mechanism based on active-site directed inactivation of the enzyme by disulfide bond formation between the active site cysteine thiol and the sulfur atom of the heterocycle is proposed. Compound 3a (K_i=2.6 μM, k_i/K_i=5630 M⁻¹ s⁻¹) with a C3 methoxy moiety and a Leu-Pro-OH dipeptide recognition sequence, is found to be the most potent inhibitor in this series. The enhanced inhibitory potency of 3a is a consequence of its increased enzyme binding affinity (lower K_i) rather than its increased intrinsic reactivity (higher k_i). In addition, 3a is inactive against Cathepsin S, is a poor inhibitor of Cathepsin H and is >100-fold more selective for Cat B over papain.     

Sialylated endogenous glycoconjugates in plant cells

Bioengineered plants are emerging as promising systems for the production of therapeutically valuable proteins. It has been commonly accepted that plants do not perform mammalian-like post-translational modifications, particularly sialylation of glycoconjugates, and no evidence has previously been reported to suggest that they have such capabilities. Here we report the presence of sialylated glycoconjugates in suspension-cultured cells of Arabidopsis thaliana and suggest that a genetic and enzymatic basis for sialylation exists in plants.     

Myocardial ischemia/reperfusion injury in the inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) plays an important role in the inflammatory process of certain major cardiac disorders including myocardial infarction and allograft rejection. However, the role of iNOS in acute myocardial ischemia has not been well defined. We determined the effects of genetically disruption of the intact iNOS system on cardiac tolerance to ischemia/reperfusion injury. Adult male wild-type (WT) and iNOS knockout (KO) B6,129 mice were subjected to 20 min global ischemia and 30 min reperfusion in a Langendorff isolated perfused heart model (37 degrees C, n = 10/each group). Ventricular contractile function, heart rate, coronary flow, and leakage of intracellular enzymes (CK and LDH) were not significantly different between the groups during pre-ischemia as well as reperfusion period (P > 0.05). Myocardial infarct size was also not significantly different between WT (20.2+/-2.0% of risk area) and KO mice (23.5+/-3.8%; Mean+/-SEM, P > 0.05). However, the post-ischemic heart rate was significantly preserved in KO as compared to WT (P < 0.05). We conclude that disruption of iNOS gene does not exacerbate ischemia/ reperfusion injury in the heart.     

Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.     

Occult breast carcinoma in reduction mammaplasty specimens: 14-year experience

Reduction mammaplasty is commonly performed for bilateral macromastia, congenital asymmetry, or as a contralateral symmetry procedure in breast reconstruction following mastectomy for cancer. Occult carcinoma has been detected in 0.06 percent to 0.4 percent of breast reduction specimens. The purpose of this study was to examine the incidence of breast cancer in breast reductions performed in one institution over a 14-year period. The authors reviewed their experience with 800 reduction mammaplasties performed between 1988 and 2001. Six cancers were detected (0.8 percent). Of these cancers, three were invasive (0.4 percent) and three were ductal carcinoma in situ (0.4 percent). Stratified by indication for surgery, there was a trend toward higher detection rates in the reconstruction group (1.2 percent) compared with the macromastia (0.7 percent) or congenital asymmetry (0 percent) groups. Mammography was performed preoperatively in these patients and all results were negative for masses or suspicious microcalcification. Pathological diagnosis was guided by gross specimen evaluation in two patients and specimen radiography in one patient. Reduction mammaplasty has a small but definite risk of finding cancer in the resection specimen.     

Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T=3 and T=1 capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by RNA-protein, protein-protein and calcium-mediated protein-protein interactions. The removal of calcium has been proposed to be a prerequisite for the disassembly of the virus. The crystal structure of native T=3 SeMV capsid revealed that residues D146 and D149 from one subunit and Y205, N267 and N268 of the neighboring subunit form the calcium-binding site (CBS). The CBS environment is found to be identical even in the recombinant CP-NDelta65 T=1 capsids. Here, we have addressed the role of calcium and the residues involved in calcium co-ordination in the assembly and stability of T=3 and T=1 capsids by mutational analysis. Deletion of N267 and N268 did not affect T=3 or T=1 assembly, although the capsids were devoid of calcium, suggesting that assembly does not require calcium ions. However, the stability of the capsids was reduced drastically. Site-directed mutagenesis revealed that either a single mutation (D149N) or a double mutation (D146N-D149N) of SeMV coat protein affected drastically both the assembly and stability of T=3 capsids. On the other hand, the D146N-D149N mutation in CP-NDelta65 did not affect the assembly of T=1 capsid, although their stability was reduced considerably. Since the major difference between the T=3 and T=1 capsids is the absence of the N-terminal arginine-rich motif (N-ARM) and the beta-annulus from the subunits forming the T=1 capsids, it is possible that D149 initiates the N-ARM-RNA interactions that lead to the formation of the beta-annulus, which is essential for T=3 capsid assembly.     

T=1 capsid structures of Sesbania mosaic virus coat protein mutants: determinants of T=3 and T=1 capsid assembly

Sesbania mosaic virus particles consist of 180 coat protein subunits of 29kDa organized on a T=3 icosahedral lattice. N-terminal deletion mutants of coat protein that lack 36 (CP-NDelta36) and 65 (CP-NDelta65) residues from the N terminus, when expressed in Escherichia coli, produced similar T=1 capsids of approximate diameter 20nm. In contrast to the wild-type particles, these contain only 60 copies of the truncated protein subunits (T=1). CP-NDelta65 lacks the "beta-annulus" believed to be responsible for the error-free assembly of T=3 particles. Though the CP-NDelta36 mutant has the beta-annulus segment, it does not form a T=3 capsid, presumably because it lacks an arginine-rich motif found close to the amino terminus. Both CP-NDelta36 and CP-NDelta65 T=1 capsids retain many key features of the T=3 quaternary structure. Calcium binding geometries at the coat protein interfaces in these two particles are also nearly identical. When the conserved aspartate residues that coordinate the calcium, D146 and D149 in the CP-NDelta65, were mutated to asparagine (CP-NDelta65-D146N-D149N), the subunits assembled into T=1 particles but failed to bind calcium ions. The structure of this mutant revealed particles that were slightly expanded. The analysis of the structures of these mutant capsids suggests that although calcium binding contributes substantially to the stability of T=1 particles, it is not mandatory for their assembly. In contrast, the presence of a large fraction of the amino-terminal arm including sequences that precede the beta-annulus and the conserved D149 appear to be indispensable for the error-free assembly of T=3 particles.     

Rieske business: structure-function of Rieske non-heme oxygenases

Rieske non-heme iron oxygenases (RO) catalyze stereo- and regiospecific reactions. Recently, an explosion of structural information on this class of enzymes has occurred in the literature. ROs are two/three component systems: a reductase component that obtains electrons from NAD(P)H, often a Rieske ferredoxin component that shuttles the electrons and an oxygenase component that performs catalysis. The oxygenase component structures have all shown to be of the alpha3 or alpha3beta3 types. The transfer of electrons happens from the Rieske center to the mononuclear iron of the neighboring subunit via a conserved aspartate, which is shown to be involved in gating electron transport. Molecular oxygen has been shown to bind side-on in naphthalene dioxygenase and a concerted mechanism of oxygen activation and hydroxylation of the ring has been proposed. The orientation of binding of the substrate to the enzyme is hypothesized to control the substrate selectivity and regio-specificity of product formation.     

Specific targeting of hepatitis C virus NS3 RNA helicase. Discovery of the potent and selective competitive nucleotide-mimicking inhibitor QU663

Hepatitis C virus (HCV) infection is an emerging global epidemic, and no effective cure is yet available. Interferon-alpha (INFalpha) and pegylated INFs, in combination or otherwise with ribavirin, have proven to be effective in no more than 50% of chronically infected patients. New and better therapeutic strategies are therefore needed. HCV nonstructural protein 3 (NS3) RNA helicase (h) is a promising target for developing new therapeutics. QU663 was discovered as a potent new selective inhibitor of the helicase reaction of HCV NS3 (K(i) = 0.75 microM), competing with the nucleic acid substrate without affecting ATPase function, even at high concentrations. QU663 is one of a new generation of small-molecule nucleotide-mimicking inhibitors which are potential anti-HCV agents. A thorough molecular modeling study was carried out to explain the molecular basis of NS3h inhibition by QU663. The resulting three-dimensional interaction model is discussed.     

Bioprospecting in plants for engineered proteins

For more than two decades, bioengineered plants have produced protein therapeutics for human and animal use. Almost all proteins produced by other existing systems, including antibodies, vaccines and plasma proteins, have now been manufactured in plants. Considering the limitations of microbial and mammalian reactor-based protein-production technologies and the impending bottleneck in manufacturing capacity, plants are now emerging as an attractive alternative system with which to supply the growing need for protein-based therapeutics. However, full realization of the promise of plant-derived engineered proteins requires that we confront the dual challenges of bioequivalence and product consistency, challenges that are largely related to post-translational protein modifications (PTMs) that are crucial to the structure and function of most eukaryotic proteins. Among the protein PTMs, the foremost challenge for bioactivity and acceptance by the pharmaceutical and biotechnology industries and regulatory agencies is glycosylation. Advances made in recent years that 'humanize' plant glycosylation pathways combined with the discovery of terminal sialic acids (SAs) in plants now make feasible the bioengineering in plants of glycoproteins that have mammalian-like glycosylation.