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Inhibitor Design (inhibitor + design)
Selected AbstractsA Macrophage Cell Model for Selective Metalloproteinase Inhibitor DesignCHEMBIOCHEM, Issue 13 2008Faith E. Jacobsen Abstract The desire to inhibit zinc-dependent matrix metalloproteinases (MMPs) has, over the course of the last 30 years, led to the development of a plethora of MMP inhibitors that bind directly to the active-site metal. With one exception, all of these drugs have failed in clinical trials, due to many factors, including an apparent lack of specificity for MMPs. To address the question of whether these inhibitors are selective for MMPs in a biological setting, a cell-based screening method is presented to compare the relative activities of zinc, heme iron, and non-heme iron enzymes in the presence of these compounds using the RAW264.7 macrophage cell line. We screened nine different zinc-binding groups (ZBGs), four established MMP inhibitors (MMPis), and two novel MMP inhibitors developed in our laboratory to determine their selectivities against five different metalloenzymes. Using this model, we identified two nitrogen donor compounds,2,2,-dipyridylamine (DPA) and triazacyclononane (TACN),as the most selective ZBGs for zinc metalloenzyme inhibitor development. We also demonstrated that the model could predict known nonspecific interactions of some of the most commonly used MMPis, and could also give cross-reactivity information for newly developed MMPis. This work demonstrates the utility of cell-based assays in both the design and the screening of novel metalloenzyme inhibitors. [source] Inhibitor design for ribonuclease A: the binding of two 5,-phosphate uridine analoguesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Vicky G. Tsirkone In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5,-phosphate (U5P) and uridine 5,-diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with Ki values of 4.0 and 0.65,mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purine-binding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily. [source] X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibitionIUBMB LIFE, Issue 6 2010Koustav Maity Abstract Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2, substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2, position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2, and 4, unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2, and 4,. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2, and 4, positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. © 2010 IUBMB IUBMB Life, 467,476, 2010 [source] Backbone-modified amylin derivatives: implications for amyloid inhibitor design and as template for self-assembling bionanomaterials,JOURNAL OF PEPTIDE SCIENCE, Issue 11 2007Ronald C. Elgersma Abstract This report reviews our approach to the design, synthesis and structural/morphological analysis of backbone-modified amylin(20,29) derivatives. Depending on the position in the peptide backbone and the type of amide bond isostere/modification, the amylin(20,29) peptides behave either as inhibitors of amyloid fibril formation, which are able to retard amyloid formation of native amylin(20,29), or as templates for the formation of self-assembled supramolecular structures. Molecular fine-tuning of the hydrogen-bond accepting/donating properties allows the control over the morphology of the supramolecular aggregation motifs such as helical ribbons and tapes, ribbons progressing to closed peptide nanotubes, (twisted) lamellar sheets or amyloid fibrils. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitorsPROTEIN SCIENCE, Issue 3 2010Susmita Khamrui Abstract For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed "mini-proteins," containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECIL -WCIS, ETIL -WCIS, and STIL -WCIS, where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECIL -WCIS and STIL -WCIS behave like good inhibitors, ETIL -WCIS behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETIL -WCIS shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETIL -WCIS leading to a loss of canonical conformation, explaining its substrate-like behavior. Incorporation of this barrier back in ETIL -WCIS through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop-scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design. [source] Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysisPROTEIN SCIENCE, Issue 6 2008Dalei Wu Abstract Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr. [source] Structural analysis of mycobacterial branched-chain aminotransferase: implications for inhibitor designACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Alina Castell The branched-chain aminotransferase (BCAT) of Mycobacterium tuberculosis has been characterized as being essential to the survival of the bacterium. The enzyme is pyridoxal 5,-phosphate-dependent and belongs to the aminotransferase IIIa subfamily, to which the human BCATs also belong. The overall sequence similarity is high within the subfamily and the sequence identity among the active-site residues is high. In order to identify structurally unique features of M. tuberculosis BCAT, X-ray structural and functional analyses of the closely related BCAT from M. smegmatis were carried out. The crystal structures include the apo form at 2.2,Å resolution and a 1.9,Å structure of the holo form cocrystallized with the inhibitor O -benzylhydroxylamine (Obe). The analyses highlighted the active-site residues Tyr209 and Gly243 as being structurally unique characteristics of the mycobacterial BCATs relative to the human BCATs. The inhibitory activities of Obe and ammonium sulfate were verified in an inhibition assay. Modelling of the inhibitor Obe in the substrate pocket indicated potential for the design of a mycobacterial-specific inhibitor. [source] Structure of 3-deoxy- manno -octulosonate cytidylyltransferase from Haemophilus influenzae complexed with the substrate 3-deoxy- manno -octulosonate in the ,-configurationACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2008Hye-Jin Yoon The enzyme 3-deoxy- manno -octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy- d - manno -octulosonate (or 2-keto-3-deoxy- manno -octonic acid; KDO) by forming CMP-KDO. CKS is unique to Gram-negative bacteria and is an attractive target for the development of antibacterial agents. The crystal structure of CKS from Haemophilus influenzae in complex with the substrate KDO has been determined at 2.30,Å resolution by combining single-wavelength anomalous diffraction and molecular-replacement methods. The two monomers in the asymmetric unit differ in the conformation of their C-terminal ,-helix (Ala230,Asn254). The KDO bound to the active site exists as the ,-pyranose form in the 5C2 chair conformation. The structure of CKS from H. influenzae in complex with KDO will be useful in structure-based inhibitor design. [source] Crystallization and initial crystallographic analysis of phosphoglucosamine mutase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Ritcha Mehra-Chaudhary The enzyme phosphoglucosamine mutase catalyzes the conversion of glucosamine 6-phosphate to glucosamine 1-phosphate, an early step in the formation of the nucleotide sugar UDP- N -acetylglucosamine, which is involved in peptidoglycan biosynthesis. These enzymes are part of the large ,- d -phosphohexomutase enzyme superfamily, but no proteins from the phosphoglucosamine mutase subgroup have been structurally characterized to date. Here, the crystallization of phosphoglucosamine mutase from Bacillus anthracis in space group P3221 by hanging-drop vapor diffusion is reported. The crystals diffracted to 2.7,Å resolution under cryocooling conditions. Structure determination by molecular replacement was successful and refinement is under way. The crystal structure of B. anthracis phosphoglucosamine mutase should shed light on the substrate-specificity of these enzymes and will also serve as a template for inhibitor design. [source] Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase complexed with 6-phosphogluconateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Scott Cameron Two crystal structures of recombinant Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase (Gs6PDH) in complex with the substrate 6-phosphogluconate have been determined at medium resolution. Gs6PDH shares significant sequence identity and structural similarity with the enzymes from Lactococcus lactis, sheep liver and the protozoan parasite Trypanosoma brucei, for which a range of structures have previously been reported. Comparisons indicate that amino-acid sequence conservation is more pronounced in the two domains that contribute to the architecture of the active site, namely the N-terminal and C-terminal domains, compared with the central domain, which is primarily involved in the subunit,subunit associations required to form a stable dimer. The active-site residues are highly conserved, as are the interactions with the 6-phosphogluconate. There is interest in 6PDH as a potential drug target for the protozoan parasite T. brucei, the pathogen responsible for African sleeping sickness. The recombinant T. brucei enzyme has proven to be recalcitrant to enzyme,ligand studies and a surrogate protein might offer new opportunities to investigate and characterize 6PDH inhibitors. The high degree of structural similarity, efficient level of expression and straightforward crystallization conditions mean that Gs6PDH may prove to be an appropriate model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species. [source] Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase IACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2005Antonio Ariza Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0,Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way. [source] Interaction of the Catalytic Domain of Inositol 1,4,5-Trisphosphate 3-Kinase A with Inositol Phosphate AnaloguesCHEMBIOCHEM, Issue 8 2005Alexandra Poinas Dr. Abstract The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D -2-deoxy-Ins(1,4,5)P3 was devised. D -2-Deoxy-Ins(1,4,5)P3 and D -3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D -2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding. [source] | ||||||||||