An essential histidine in bacterial cytochrome c peroxidases

The cytochrome c peroxidase from the bacterium Pciracoccus denitrificans is a relative of the extensively characterised enzyme from Pseudomonas aeruginosa. This study investigates the role of an essential histidine residue in the enzyme mechanism of bacterial peroxidases.Cytochrome c peroxidase from Paracoccus denitrificans was modified with the histidine-specific reagent diethylpyrocarbonate. The reaction can be followed spectroscopically and, at low excess of reagent, one mol of histidine was modified in the oxidised enzyme. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. Modification of this easily modifiable histidine was associated with loss of the enzyme's ability to form the active state. With time, the modification reversed and the ability to form the active mixed valence state was recovered. However the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity and a slow transfer of the ethoxyformyl group to another amino acid is proposed. The presence of CN" bound to the active peroxidatic site of the enzyme completely protected the essential histidine from modification.In its active form cytochrome c peroxidase is a dimer, with Ca2+ situated at the interface between the two monomers. Under conditions where the dimer is the dominant species modification of only 0.5 mol histidine abolishes enzyme activityLimited subtilisin treatment of the native enzyme resulted in cleavage at a single peptide bond. Although the two fragments remain tightly associated, the cleaved enzyme is inactive. Modification with radiolabeled diethylpyrocarbonate and subsequent subtilisin treatment, followed by tryptic digestion of a 9k fragment, showed that radioactivity was located in a peptide containing a single histidine 275.With the benefit of four homologous sequences and the use of secondary structure prediction analysis we can determine that histidine 275 is indeed conserved in the four sequences and is preceded by a remarkably unvaried a-helical region suggestive of functional importance. It is proposed that this conserved residue acts as both a catalytic active site residue and a conduit for intermolecular electron transfer in the active mixed-valence high spin-state

Interspecies evaluation of a physiologically based pharmacokinetic model to predict the biodistribution dynamics of dendritic nanoparticles.

The exposure of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) was determined in mouse, rat and dog, with the aim of investigating interspecies differences facilitating clinical translation. Plasma area under the curves (AUCs) were found to be dose proportional across species, while dose normalized concentration time course profiles in plasma, liver and spleen were superimposable in mouse, rat and dog. A physiologically based pharmacokinetic (PBPK) model, previously developed for mouse, was evaluated as a suitable framework to prospectively capture concentration dynamics in rat and dog. The PBPK model, parameterized either by considering species-specific physiology or using alternate scaling methods such as allometry, was shown to capture exposure profiles across species. A sensitivity analysis highlighted API systemic clearance as a key parameter influencing released API levels. The PBPK model was utilized to simulate human exposure profiles, which overlaid dose-normalized data from mouse, rat and dog. The consistency in measured interspecies exposures as well as the capability of the PBPK model to simulate observed dynamics support its use as a powerful translational tool

Prediction of human renal clearance from preclinical species for a diverse set of drugs that exhibit both active secretion and net re- absorption

Number of words in abstract: 184 Number of words in introduction: 552 Number of words in discussion: 822 Abbreviations: ADME, absorption, distribution, metabolism and excretion; afe, average fold error; CLr, renal clearance; CLp, plasmatic clearance; fu, unbound plasma fraction; GFR, glomerular filtration rate; KBF, kidney blood flow; NSAID, non-steroidal anti-inflammatory drug; OAT, organic anion transporter; OATP, organic anion transport protein; PPB, plasma protein binding; rmse, root mean square error. DMD # 37267 Abstract Identifying any extra-hepatic excretion phenomenon in preclinical species is crucial for an accurate prediction of the pharmacokinetics in man. This is particularly the case for drugs with a small volume of distribution, as they require an especially low total clearance in order to be suitable for a once-a-day dosing regimen in man. In this study, three animal scaling techniques were applied for the prediction of the human renal clearance of 36 diverse drugs that show active secretion or net re-absorption

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The exposure of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) was determined in mouse, rat and dog, with the aim of investigating interspecies differences facilitating clinical translation. Plasma area under the curves (AUCs) were found to be dose proportional across species, while dose normalized concentration time course profiles in plasma, liver and spleen were superimposable in mouse, rat and dog. A physiologically based pharmacokinetic (PBPK) model, previously developed for mouse, was evaluated as a suitable framework to prospectively capture concentration dynamics in rat and dog. The PBPK model, parameterized either by considering species-specific physiology or using alternate scaling methods such as allometry, was shown to capture exposure profiles across species. A sensitivity analysis highlighted API systemic clearance as a key parameter influencing released API levels. The PBPK model was utilized to simulate human exposure profiles, which overlaid dose-normalized data from mouse, rat and dog. The consistency in measured interspecies exposures as well as the capability of the PBPK model to simulate observed dynamics support its use as a powerful translational tool.</div

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The exposure of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) was determined in mouse, rat and dog, with the aim of investigating interspecies differences facilitating clinical translation. Plasma area under the curves (AUCs) were found to be dose proportional across species, while dose normalized concentration time course profiles in plasma, liver and spleen were superimposable in mouse, rat and dog. A physiologically based pharmacokinetic (PBPK) model, previously developed for mouse, was evaluated as a suitable framework to prospectively capture concentration dynamics in rat and dog. The PBPK model, parameterized either by considering species-specific physiology or using alternate scaling methods such as allometry, was shown to capture exposure profiles across species. A sensitivity analysis highlighted API systemic clearance as a key parameter influencing released API levels. The PBPK model was utilized to simulate human exposure profiles, which overlaid dose-normalized data from mouse, rat and dog. The consistency in measured interspecies exposures as well as the capability of the PBPK model to simulate observed dynamics support its use as a powerful translational tool.</div

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The exposure of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) was determined in mouse, rat and dog, with the aim of investigating interspecies differences facilitating clinical translation. Plasma area under the curves (AUCs) were found to be dose proportional across species, while dose normalized concentration time course profiles in plasma, liver and spleen were superimposable in mouse, rat and dog. A physiologically based pharmacokinetic (PBPK) model, previously developed for mouse, was evaluated as a suitable framework to prospectively capture concentration dynamics in rat and dog. The PBPK model, parameterized either by considering species-specific physiology or using alternate scaling methods such as allometry, was shown to capture exposure profiles across species. A sensitivity analysis highlighted API systemic clearance as a key parameter influencing released API levels. The PBPK model was utilized to simulate human exposure profiles, which overlaid dose-normalized data from mouse, rat and dog. The consistency in measured interspecies exposures as well as the capability of the PBPK model to simulate observed dynamics support its use as a powerful translational tool.</div

A single histidine is required for activity of cytochrome c peroxidase from Paracoccus denitrificans

Wellcome TrustThe diheme cytochrome e peroxidase from Paracoccus denitrificans was modified with the histidine-specific reagent diethyl pyrocarbonate. At low excess of reagent, 1 mol of histidine was modified in the oxidized enzyme, and modification was associated with loss of the ability to form the active state. With time, the modification reversed, and the ability to form the active state was recovered. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. However, the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity, and we propose a slow transfer of the ethoxyformyl group to an unidentified amino acid. The presence of CN- bound to the active peroxidatic site of the enzyme led to complete protection of the essential histidine from modification. Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (residues 251-338) showed that radioactivity was located in a peptide containing a single histidine at position 275. We propose that this conserved residue, in a highly conserved region, is central to the function of the active mixed-valence state.publishersversionpublishe