Cytochrome P460 Cofactor Maturation Involves a Peroxide- Dependent Post-Translational Modification

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide as part of the biogeochemical nitrogen cycle. They bear unique “heme P460” cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, This proenzyme undergoes complete maturation to active enzyme with spectroscopic properties that match wild-type cyt P460. Together, these data indicate that the cofactor is primed to undergo this covalent modification by virtue of the protein fold. A putative mechanism analogous to that used by heme oxygenases to degrade hemes is proposed.

2-Sulfonylpyrimidines as Privileged Warheads for the Development of S. aureus Sortase A Inhibitors

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with emerging multiresistant isolates causing a significant burden to public health systems. We identified 2-sulfonylpyrimidines as a new class of potent inhibitors against S. aureus sortase A acting by covalent modification of the active site cysteine 184. Series of derivatives were synthesized to derive structure-activity relationship (SAR) with the most potent compounds displaying low micromolar KI values. Studies on the inhibition selectivity of homologous cysteine proteases showed that 2-sulfonylpyrimidines reacted efficiently with protonated cysteine residues as found in sortase A, though surprisingly, no reaction occurred with the more nucleophilic cysteine residue from imidazolinium-thiolate dyads of cathepsin-like proteases. By means of enzymatic and chemical kinetics as well as quantum chemical calculations, it could be rationalized that the SNAr reaction between protonated cysteine residues and 2-sulfonylpyrimidines proceeds in a concerted fashion, and the mechanism involves a ternary transition state with a conjugated base. Molecular docking and enzyme inhibition at variable pH values allowed us to hypothesize that in sortase A this base is represented by the catalytic histidine 120, which could be substantiated by QM model calculation with 4-methylimidazole as histidine analog.

A covalent modification strategy for di-alkyne tagged metal-organic frameworks to access efficient heterogeneous catalysts toward C-C bond formation

Organic and inorganic building blocks construct a class of metal-organic frameworks (MOFs) that exhibit tremendous chemical tunability. In this study, a novel Zirconium-based MOF UiO-66-(alkyne)2 with a di-alkyne tag was...

Proteomics

Proteomics is a branch of molecular biology that deals with the identification and quantification of proteins in living objects, as well as the analysis of protein functions and their interactions. Proteomics is studied by proteins that are expressed in a given cell, tissue or organism over a period of time (under certain conditions). It is known that information about the primary structure of a protein (the sequence of amino acid residues in a protein) is contained in a structural gene in the form of a codon sequence (genetic code). On the other hand, less than 10% of genes are functionally active (expressed) in the somatic cells of our body. Moreover, a distinct tissue-specific expression of genes is observed. This, in turn, leads to the peculiarities of the qualitative composition of the synthesized proteins in various tissues. No less important is the fact that the total amount of proteins synthesized by our tissues is much greater than the total number of structural genes containing information about their original structure. This phenomenon is explained by the activity of such mechanisms as alternative splicing and a wide variety of post-translational peptide processing pathways (covalent modification of a polypeptide synthesized on the ribosome) in health and disease. Thus, even a brief review of the semantic content of the term "proteomics" indicates an extremely complex system of protein molecules in our body, which plays a fundamental role in maintaining homeostasis and is involved in the formation of adaptive responses in response to adverse changes in the internal and external environment.

Selective Metal Coordination in Antifouling Coatings

<p>Marine biofouling is the accumulation of biological material (e.g. microorganisms, soft- and hard-fouling organisms) on the surface of an object submerged in seawater, and it remains a worldwide problem for shipping industries. The fouling of ship hulls results in a reduction of speed and manoeuvrability due to frictional drag, as well as increased fuel consumption and accelerated corrosion, and the exorbitant expenses and losses of efficiency attributed to biofouling have prompted the development of antifouling coatings. Current antifouling paints use copper as a biocidal agent, but copper-based paints are increasingly being banned due to environmental concerns about the non-target effects of leached copper. This project aims to circumvent these concerns and tightening regulations via a revolutionary concept: the development of marine antifouling paints that incorporate Cu(II)-selective ligands to draw the biocidal ingredient (i.e. Cu(II)) from seawater. A multistage strategy emerged for the development of this technology. First, criteria were established for the project’s ideal ligand, and ligands were synthesised or selected based on these criteria. Second, the ligands were incorporated in coatings through covalent modification of the paint binder or additives. Third, methodology was developed and implemented to test each coating’s ability to coordinate and retain Cu(II), as well as its subsequent ability to prevent microfouling by marine bacteria. The suitability of two ligand classes was assessed: acylhydrazones and tetraaza macrocycles, specifically cyclen. Unlike the acylhydrazones, cyclen met the established criteria and was initially evaluated as a curing agent and/or surface-modifier in a two-pack epoxy system with resin Epikote™ 235. However, the Cu(II)-loading by these coatings was relatively low, being at most ~0.05% w/w, and the modification of silica, a common paint additive, with cyclen was explored as an alternative formulation route. The method for the functionalisation of silica with cyclen was optimised, and the maximum Cu(II)-loading achieved by the product was 2.60% w/w. The cyclen-functionalised silica was incorporated on the surface of an epoxy coating, and a bacterial adherence assay was developed to assess the cellular attachment of marine bacterium Vibrio harveyi to this coating, which was found to be undeterred. Yet, the development of the strategy and testing methodology by which the project’s goals may be achieved provides a solid foundation for future work.</p>

Selective Metal Coordination in Antifouling Coatings

<p>Marine biofouling is the accumulation of biological material (e.g. microorganisms, soft- and hard-fouling organisms) on the surface of an object submerged in seawater, and it remains a worldwide problem for shipping industries. The fouling of ship hulls results in a reduction of speed and manoeuvrability due to frictional drag, as well as increased fuel consumption and accelerated corrosion, and the exorbitant expenses and losses of efficiency attributed to biofouling have prompted the development of antifouling coatings. Current antifouling paints use copper as a biocidal agent, but copper-based paints are increasingly being banned due to environmental concerns about the non-target effects of leached copper. This project aims to circumvent these concerns and tightening regulations via a revolutionary concept: the development of marine antifouling paints that incorporate Cu(II)-selective ligands to draw the biocidal ingredient (i.e. Cu(II)) from seawater. A multistage strategy emerged for the development of this technology. First, criteria were established for the project’s ideal ligand, and ligands were synthesised or selected based on these criteria. Second, the ligands were incorporated in coatings through covalent modification of the paint binder or additives. Third, methodology was developed and implemented to test each coating’s ability to coordinate and retain Cu(II), as well as its subsequent ability to prevent microfouling by marine bacteria. The suitability of two ligand classes was assessed: acylhydrazones and tetraaza macrocycles, specifically cyclen. Unlike the acylhydrazones, cyclen met the established criteria and was initially evaluated as a curing agent and/or surface-modifier in a two-pack epoxy system with resin Epikote™ 235. However, the Cu(II)-loading by these coatings was relatively low, being at most ~0.05% w/w, and the modification of silica, a common paint additive, with cyclen was explored as an alternative formulation route. The method for the functionalisation of silica with cyclen was optimised, and the maximum Cu(II)-loading achieved by the product was 2.60% w/w. The cyclen-functionalised silica was incorporated on the surface of an epoxy coating, and a bacterial adherence assay was developed to assess the cellular attachment of marine bacterium Vibrio harveyi to this coating, which was found to be undeterred. Yet, the development of the strategy and testing methodology by which the project’s goals may be achieved provides a solid foundation for future work.</p>

Characterisation of Zampanolide as a Microtubule-Stabilizing Agent

<p>Microtubule-stabilizing agents (MSAs) are extremely important chemotherapeutic drugs since microtubules (MTs) are one of the most successful cancer drug targets. Currently there are four MSAs that are clinically used for the treatment of cancer. Cancer cells, however, can develop resistance towards these drugs, the most common being over-expression of the P-glycoprotein drug efflux pump. Zampanolide (ZMP), a novel secondary metabolite isolated from a marine sponge consists of a 20-membered macrolide ring with an unusual N-acyl-hemiaminal side chain. It is a potent MSA with similar cellular effects to the clinically relevant MSAs, Taxol®, Taxotere® and Ixempra®. ZMP has a small number of stereogenic centers and therefore is relatively easier to synthesize than other macrolide natural products. Using established cancer cell lines and isolated bovine brain tubulin ZMP in the present study was further characterized as a potential anti-cancer compound and was shown to have significant advantages over currently used MSAs. These studies provided insight into how this important drug class induces MT assembly, suggesting strategies for the development of new generation MSAs for use in the clinic. ZMP and its less active analog dactylolide competed with paclitaxel for binding to MTs and represented a novel MSA chemotype. Unlike traditional taxoid site ligands, ZMP remained significantly more cytotoxic in cell lines with mutations in the taxoid binding site, and behaved in an unusual manner in vitro. This was later found to be due to its mechanism of binding which involved covalent modification of two amino acids in the taxoid binding site, histidine 229 as the major product and asparagine 228 as the minor product. Alkylation of both these luminal site residues was also detected in unassembled tubulin, providing the first direct evidence that the taxoid binding site exists in unassembled tubulin and suggesting that the induction of MT nucleation by MSAs may proceed through an allosteric mechanism. X-ray crystallography data confirmed the presence of this binding site in unassembled tubulin and indicated that covalent modification occurs at C9 of ZMP with the NE2 of the histidine side chain. The potent stabilization of MTs observed with ZMP occurred due to its side chain interaction with the stabilizing M-loop of β-tubulin. In unassembled tubulin the M-loop is unordered. Upon ZMP binding, it is restructured into a short, well-defined helix. It is this restructuring that leads to the potent stabilization by ZMP and most likely other MSAs, including those currently used in the clinic. This information provides a basis for structure-guided drug engineering to design and develop new generation MSAs with potent stabilizing activity. In addition, covalent binding of ZMP means that it is able to avoid drug efflux pumps and thus evade the main mechanism of resistance presented to MSAs in the clinic. It was shown by studying structure-activity relationships that there are a number of key chemical motifs in ZMP responsible for its potent activity. Simpler analog structures that retain significant stabilizing activity could be used as lead compounds for further drug development. Moreover, MSAs have clinically relevant anti-angiogenic and vascular-disrupting properties, and ZMP was also shown to potently inhibit cell migration and thus have possible benefits as a vasculature-targeting compound. It was concluded that ZMP is a potent covalently-binding MSA in both cells and in vitro. Given these promising results, further preclinical development of the compound is warranted.</p>

Characterisation of Zampanolide as a Microtubule-Stabilizing Agent

<p>Microtubule-stabilizing agents (MSAs) are extremely important chemotherapeutic drugs since microtubules (MTs) are one of the most successful cancer drug targets. Currently there are four MSAs that are clinically used for the treatment of cancer. Cancer cells, however, can develop resistance towards these drugs, the most common being over-expression of the P-glycoprotein drug efflux pump. Zampanolide (ZMP), a novel secondary metabolite isolated from a marine sponge consists of a 20-membered macrolide ring with an unusual N-acyl-hemiaminal side chain. It is a potent MSA with similar cellular effects to the clinically relevant MSAs, Taxol®, Taxotere® and Ixempra®. ZMP has a small number of stereogenic centers and therefore is relatively easier to synthesize than other macrolide natural products. Using established cancer cell lines and isolated bovine brain tubulin ZMP in the present study was further characterized as a potential anti-cancer compound and was shown to have significant advantages over currently used MSAs. These studies provided insight into how this important drug class induces MT assembly, suggesting strategies for the development of new generation MSAs for use in the clinic. ZMP and its less active analog dactylolide competed with paclitaxel for binding to MTs and represented a novel MSA chemotype. Unlike traditional taxoid site ligands, ZMP remained significantly more cytotoxic in cell lines with mutations in the taxoid binding site, and behaved in an unusual manner in vitro. This was later found to be due to its mechanism of binding which involved covalent modification of two amino acids in the taxoid binding site, histidine 229 as the major product and asparagine 228 as the minor product. Alkylation of both these luminal site residues was also detected in unassembled tubulin, providing the first direct evidence that the taxoid binding site exists in unassembled tubulin and suggesting that the induction of MT nucleation by MSAs may proceed through an allosteric mechanism. X-ray crystallography data confirmed the presence of this binding site in unassembled tubulin and indicated that covalent modification occurs at C9 of ZMP with the NE2 of the histidine side chain. The potent stabilization of MTs observed with ZMP occurred due to its side chain interaction with the stabilizing M-loop of β-tubulin. In unassembled tubulin the M-loop is unordered. Upon ZMP binding, it is restructured into a short, well-defined helix. It is this restructuring that leads to the potent stabilization by ZMP and most likely other MSAs, including those currently used in the clinic. This information provides a basis for structure-guided drug engineering to design and develop new generation MSAs with potent stabilizing activity. In addition, covalent binding of ZMP means that it is able to avoid drug efflux pumps and thus evade the main mechanism of resistance presented to MSAs in the clinic. It was shown by studying structure-activity relationships that there are a number of key chemical motifs in ZMP responsible for its potent activity. Simpler analog structures that retain significant stabilizing activity could be used as lead compounds for further drug development. Moreover, MSAs have clinically relevant anti-angiogenic and vascular-disrupting properties, and ZMP was also shown to potently inhibit cell migration and thus have possible benefits as a vasculature-targeting compound. It was concluded that ZMP is a potent covalently-binding MSA in both cells and in vitro. Given these promising results, further preclinical development of the compound is warranted.</p>