Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose.

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.

Inhibition of human DNA topoisomerase IB by a cyclometalated gold III compound: analysis on the different steps of the enzyme catalytic cycle.

A gold(III) compound [Au(C^N^C)(IMe)]CF(3)SO(3) (Gold III) has been reported to have anticancer properties as it is able to reduce topoisomerase IB activity in vitro and suppress tumor growth in nude mice model. Here we have investigated the mechanism of inhibition of human topoisomerase IB activity by this compound, analyzing the various steps of the catalytic cycle. DNA supercoiled relaxation and the cleavage reaction are inhibited, but Gold III does not perturb the religation reaction, in contrast to what has been observed for camptothecin. Pre-incubation of enzyme with the inhibitor before adding DNA substrate increases the inhibitory effect. In addition, when Gold III is preincubated with the enzyme it prevents the stabilization of the cleavable complex by camptothecin. The analysis of the DNA-topoisomerase binding reaction indicates that the compound acts as a topoisomerase I inhibitor by preventing the enzyme-DNA interaction.

Evidences of a natively unfolded state for the human topoisomerase IB N-terminal domain.

The N-terminal domain of human topoisomerase IB has been expressed, purified and characterized by spectroscopic techniques. CD spectra as a function of concentration and pH indicate that the domain does not possess any defined secondary structure. The protein is probably in a natively unfolded state since its denaturation curve is indicative of a non-cooperative transition. Evidence of a partially folded structure comes from the fluorescence spectrum of ANS, whose intensity increases in presence of the domain. Indication of a partial structural arrangement of the domain comes also from the endogenous fluorescence of tryptophans that is centred at 350 nm in the native and shifts to 354 nm in the fully denaturated protein. Interestingly despite the poor structural degree, as also confirmed by a predictive approach, the domain efficiently binds DNA, suggesting that the absence of a defined 3D structure has a functional meaning that permits the domain to be available for the interaction with different molecular partners.

Conjugated eicosapentaenoic acid inhibits human topoisomerase IB with a mechanism different from camptothecin.

Conjugated eicosapentaenoic acid (cEPA) has been found to have antitumor effects which has been ascribed to their ability to inhibit DNA topoisomerases and DNA polymerases. We here show that cEPA inhibits the catalytic activity of human topoisomerase I, but unlike camptothecin it does not stabilize the cleavable complex, indicating a different mechanism of action. cEPA inhibits topoisomerase by impeding the catalytic cleavage of the DNA substrate as demonstrated using specific oligonucleotide substrates, and prevents the stabilization of the cleavable complex by camptothecin. Preincubation of the inhibitor with the enzyme is required to obtain complete inhibition. Molecular docking simulations indicate that the preferred cEPA binding site is proximal to the active site with the carboxylic group strongly interacting with the positively charged K443 and K587. Taken together the results indicate that cEPA inhibitor does not prevent DNA binding but inhibits DNA cleavage, binding in a region close to the topoisomerase active site.

Role of flexibility and long range communication on the function of human topoisomerase I.

Eukaryotic topoisomerase I is an essential enzyme that regulates the changes in DNA topology, relaxing the superhelical tension associated with DNA replication, transcription and recombination. Human topoisomerase I is of significant medical interest being the only target of the antitumor drug camptothecin. The enzyme undergoes large conformational changes during its catalytic cycle and the knowedge of the degree of flexibility of the different regions provides an useful guide to the understanding of such movements. Molecular dynamics simulation is a well consolidated method for the investigation of structural and dynamic properties of proteins and nucleic acids and has been successfully applied to study the dynamical properties of the DNA-human topoisomerase complex. This review highlights some structural and dynamic properties of topoisomerase, obtained by MD simulations, that permits to explain the importance of flexibility in the modulation of the functional properties of the enzyme and in the transmission of communication between domains located far away one from each other.

A natural anticancer agent thaspine targets human topoisomerase IB.

The different steps of the topoisomerase I catalytic cycle have been analyzed in the presence of the plant alkaloid thaspine (1- (2-(Dimethylamino)ethyl)-3,8-dimethoxychromeno[5,4,3-cde]chromene-5,10-dione), known to induce apoptosis in colon carcinoma cells. The experiments indicate that thaspine inhibits both the cleavage and the religation steps of the enzyme reaction. The inhibition is reversible and the effect is enhanced upon pre-incubation. Molecular docking simulations of thaspine over topoisomerase I, in the presence or absence of the DNA substrate, show that thaspine, when interacting with the enzyme alone in the closed or in the open state, can bind in proximity of the active residues preventing the cleavage reaction, whilst when docked with the enzyme-DNA cleavable complex intercalates between the DNA bases in a way similar to that found for camptothecin, explaining its religation inhibition. These results unequivocally demonstrate that thaspine targets human topoisomerase I .

Role of Flexibility in Protein-DNA-Drug Recognition: The Case of Asp677Gly-Val703Ile Topoisomerase Mutant Hypersensitive to Camptothecin.

Topoisomerases I are ubiquitous enzymes that control DNA topology within the cell. They are the unique target of the antitumor drug camptothecin that selectively recognizes the DNA-topoisomerase covalent complex and reversibly stabilizes it. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant with enhanced CPT sensitivity have been investigated. The mutant displays a lower religation rate of the DNA substrate when compared to the wild-type protein. Analyses of the structural dynamical properties by molecular dynamics simulation show that the mutant has reduced flexibility and an active site partially destructured at the level of the Lys532 residue. These results demonstrate long-range communication mechanism where reduction of the linker flexibility alters the active site geometry with the consequent lowering of the religation rate and increase in drug sensitivity.