DhHP-6 ameliorates hepatic oxidative stress and insulin resistance in type 2 diabetes mellitus through the PI3K/AKT and AMPK pathway.

Insulin resistance is one major features of type 2 diabetes mellitus (T2DM). Deuterohemin-ßAla-His-Thr-Val-Glu-Lys (DhHP-6), a novel microperoxidase mimetic designed and synthesized based on microperoxidase 11 (MP-11), can scavenge reactive oxygen species (ROS) in vivo. In our previous studies, we showed that oral DhHP-6 could reduce blood glucose and improve insulin resistance. To investigate the mechanisms of how DhHP-6 ameliorates oxidative stress and insulin resistance, we established T2DM mouse models and glucosamine-induced HepG2 cell insulin resistance models. The results suggested that DhHP-6 decreased blood glucose, increased antioxidant enzyme activity, and inhibited glycogen synthesis in T2DM mice. In addition, DhHP-6 improved insulin resistance by activating phosphatidylinositol 3-kinase (PI3K)/AKT, and AMP-activated protein kinase (AMPK) pathway in T2DM mice. Furthermore, DhHP-6 also activated PI3K/AKT and AMPK pathway in glucosamine-induced HepG2 cells. However, LY294002 did not completely inhibit AKT phosphorylation, and partially inhibited AMPK phosphorylation, whilst compound C only partially reduced AMPK phosphorylation, and also partially inhibited AKT phosphorylation, suggesting that AKT and AMPK interact to improve insulin resistance. Thus, these data suggest that DhHP-6 attenuates insulin resistance via the PI3K/AKT and AMPK pathway.

The design and characterization of a hypersensitive glucose sensor: two enzymes co-fixed on a copper phosphate skeleton.

In this study, we first reported a new glucose sensor for the preparation of a glucose oxidase (GOx)-conjugated novel artificial peroxidase mimic deuterohemin peptide (Dh-Ala-His-Thr-Val-Glu-Lys, DhHP-6) (GOx&DhHP-6-Cu3(PO4)2) in order to determine the glucose concentration. The same catalytic environment (pH 7.0, 35 °C) between glucose oxidase (GOx) and DhHP-6 ensured their utilization in catalytic cascade reactions. DhHP-6 and GOx were co-fixed on a copper phosphate skeleton, which ensured resistance toward harsh environments and improved storage stability. Meanwhile, the sensor reduced the diffusion of hydrogen peroxide (H2O2) in solutions by immobilization. GOx&DhHP-6-Cu3(PO4)2 possessed a wide linear range of 5-2000 µM, and a limit of detection of 0.5 µM. Based on the above-mentioned results, a new method to rapidly detect glucose concentrations was successfully developed using GOx&DhHP-6-Cu3(PO4)2 nanoflowers with high sensitivity and an easily applied methodology.

Oral DhHP-6 for the Treatment of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is associated with pancreatic ß-cell dysfunction which can be induced by oxidative stress. Deuterohemin-ßAla-His-Thr-Val-Glu-Lys (DhHP-6) is a microperoxidase mimetic that can scavenge reactive oxygen species (ROS) in vivo. In our previous studies, we demonstrated an increased stability of linear peptides upon their covalent attachment to porphyrins. In this study, we assessed the utility of DhHP-6 as an oral anti-diabetic drug in vitro and in vivo. DhHP-6 showed high resistance to proteolytic degradation in vitro and in vivo. The degraded DhHP-6 product in gastrointestinal (GI) fluid retained the enzymatic activity of DhHP-6, but displayed a higher permeability coefficient. DhHP-6 protected against the cell damage induced by H2O2 and promoted insulin secretion in INS-1 cells. In the T2DM model, DhHP-6 reduced blood glucose levels and facilitated the recovery of blood lipid disorders. DhHP-6 also mitigated both insulin resistance and glucose tolerance. Most importantly, DhHP-6 promoted the recovery of damaged pancreas islets. These findings suggest that DhHP-6 in physiological environments has high stability against enzymatic degradation and maintains enzymatic activity. As DhHP-6 lowered the fasting blood glucose levels of T2DM mice, it thus represents a promising candidate for oral administration and clinical therapy.

A deuterohemin peptide protects a transgenic Caenorhabditis elegans model of Alzheimer's disease by inhibiting Aß1-42 aggregation.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disease and is the most common cause of dementia in the elderly. The main hallmark of AD is the deposition of insoluble amyloid (Aß) outside the neuron, leading to amyloid plaques and neurofibrillary tangles in the brain. Deuterohemin-Ala-His-Thr-Val-Glu-Lys (DhHP-6), a novel porphyrin-peptide, has both microperoxidase activity and cell permeability. In the present study, DhHP-6 efficiently inhibited the aggregation of Aß and reduced the ß-sheet percentage of Aß from 89.1% to 78.3%. DhHP-6 has a stronger affinity (KD = 100 ±â€¯12 µM) for binding with Aß at Phe4, Arg5, Val18, Glu11 and Glu22. In addition, DhHP-6 (100 µM) significantly prolonged lifespan, alleviated paralysis and reduced Aß plaque formation in the Aß1-42 transgenic Caenorhabditis elegans CL4176 model of AD. Our results demonstrate that DhHP-6 is a potential drug candidate that efficiently protects a transgenic C. elegans model of Alzheimer's disease by inhibiting Aß aggregation.

A Novel Peroxidase Mimics and Ameliorates Alzheimer's Disease-Related Pathology and Cognitive Decline in Mice.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, which is characterized by the accumulation of amyloid ß (Aß) plaques, oxidative stress, and neuronal loss. Therefore, clearing Aß aggregates and reducing oxidative stress could be an effective therapeutic strategy for AD. Deuterohemin-AlaHisThrValGluLys (DhHP-6), a novel deuterohemin-containing peptide mimetic of the natural microperoxidase-11 (MP-11), shows higher antioxidant activity and stability compared to the natural microperoxidases. DhHP-6 possesses the ability of extending lifespan and alleviating paralysis in the Aß1-42 transgenic Caenorhabditis elegans CL4176 model of AD, as shown in our previous study. Therefore, this study was aimed at exploring the neuroprotective effect of DhHP-6 in the APPswe/PSEN1dE9 transgenic mouse model of AD. DhHP-6 reduced the diameter and fiber structure of Aß1-42 aggregation in vitro, as shown by dynamic light scattering and transmission electron microscope. DhHP-6 exerted its neuroprotective effect by inhibiting Aß aggregation and plaque formation, and by reducing Aß1-42 oligomers-induced neurotoxicity on HT22 (mouse hippocampal neuronal) and SH-SY5Y (human neuroblastoma) cells. In the AD mouse model, DhHP-6 significantly ameliorated cognitive decline and improved spatial learning ability in behavioral tests including the Morris water maze, Y-maze, novel object recognition, open field, and nest-building test. Moreover, DhHP-6 reduced the deposition of Aß plaques in the cerebral cortex and hippocampus. More importantly, DhHP-6 restored the morphology of astrocytes and microglia, and significantly reduced the levels of pro-inflammatory cytokines. Our findings provide a basis for considering the non-toxic, peroxidase mimetic DhHP-6 as a new candidate drug against AD.

Heme-binding of bovine lactoferrin: the potential presence of a heme-binding capacity in an ancestral transferrin gene.

Lactoferrin (Lf) and transferrin (Tf) are iron-binding proteins that can bind various metal ions. This study demonstrates the heme-binding activity of bovine Lf and Tf using biotinylated hemin. When both proteins were coated on separate plate wells, each directly bound biotinylated hemin. On the other hand, when biotinylated hemin was immobilized on an avidin-coated plate, soluble native Lf bound to the immobilized biotinylated hemin whereas native Tf did not, suggesting that a conformational change triggered by coating on the plate allows the binding of denatured Tf with hemin. Incubation of Lf with hemin-agarose resulted in negligible binding of Lf with biotinylated hemin. Lf in bovine milk also bound to immobilized biotinylated hemin. These results demonstrate that bovine Lf has specific heme-binding activity, which is different from Tf, suggesting that either Tf lost heme-binding activity during its evolution or that Lf evolved heme-binding activity from its Tf ancestral gene. Additionally, Lf in bovine milk may bind heme directly, but may also bind heme indirectly by interaction with other milk iron- and/or heme-binding proteins.

Eficacia y seguridad de hemina humana para el tratamiento de la crisis aguda de porfiria aguda intermitente / Efficacy and safety of human hemin for the treatment of acute intermittent acute porphyria crisis

INTRODUCCIÓN: El presente informe expone la evaluación del uso de hemina humana para el tratamiento de la crisis aguda de porfiria aguda intermitente. La porfiria aguda intermitente (PAI) es una enfermedad metabólica hereditaria causada por la deficiencia de la tercera enzima de la síntesis del grupo Hem (porfobilinógeno deaminasa) y que conlleva a la acumulación de los precursores: ácido δ-aminolevulínico (ALA) y porfobilinógeno (PBG). Los pacientes afectados sufren de crisis agudas (ataques) caracterizados por una combinación de dolor abdominal, alteraciones mentales leves y disfunción autonómica, que algunas veces puede complicarse con neuropatía periférica y encefalopatía severa. OBJETIVO: El objetivo del presente dictamen es la evaluación de la mejor evidencia disponible acerca de la hemina humana en el tratamiento de las crisis agudas de PAI, en términos de disminución de secuelas neurológicas, calidad de vida y reducción de las crisis. TECNOLOGÍA SANITARIA DE INTERÉS: La hemina humana se aísla y purifica de un concentrado de glóbulos rojos humanos. El mecanismo de acción postulado es la disminución de la síntesis de precursores de porfirina vía retroalimentación negativa. METODOLOGÍA: Se realizó una búsqueda de la literatura con respecto a la eficacia y seguridad de hemina humana para el tratamiento de la crisis aguda de de porfiria aguda intermitente. Esta búsqueda se realizó utilizando los meta-buscadores: Translating Research into Practice (TRIPDATABASE), National Library of Medicine (PubMed-MEDLINE) y Health Systems Evidence. Adicionalmente, se amplió la búsqueda revisando la evidencia generada por grupos internacionales que realizan revisiones sistemáticas (RS), evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC), tales como la Cochrane Group, The National Institute for Health and Care Excellence (NICE), the Agency for Health care Research and Quality (AHRQ), The Canadian Agency for Drugs and Technologies in Health (CADTH) y The Scottish Medicines Consortium (SMC) y la Haute Autorite de Sante (HAS). Esta búsqueda se completó ingresando a la página web www.clinicaltrials.gov, para así poder identificar ensayos clínicos en elaboración o que no hayan sido publicados aún, y así disminuir el riesgo de sesgo de publicación. RESULTADOS: El uso de hemina humana en ataques de PAI fue evaluado en un solo ensayo aleatorizado pequeño. Se compararon los datos pareados de nueve pacientes que usaron hemina humana o placebo en diferentes episodios de ataques de PAI. Aunque se observó una mayor diferencia en la disminución de ALA y PBG en orina cuando el paciente usó hemina humana, esto no se reflejó en mayor mejora del alivio del dolor, en menor requerimiento de analgésicos o en menor estancia hospitalaria, respecto al placebo. Este estudio fue criticado porque la administración de hemina humana no fue inmediatamente al ingreso hospitalario del paciente (se administró al tercer día de hospitalización). Por ello, se ha incluido en la evaluación de este dictamen estudios en los que se administró hemina humana al ingreso del paciente, aunque no fueron controlados. La primera serie incluyó a 51 casos de ataques agudos de PAI de los pacientes que recibieron hemina humana después de un promedio de 1.6 días de ser hospitalizados. Este reporte, concluye que el dolor y los niveles urinarios de ALA y PBG disminuyeron. Sin embargo, no se describe el método ni la escala con la que se midió el dolor, ni tampoco se ha descrito la naturaleza de los analgésicos, sus dosis y/o equivalencias, que permitan estimar el cambio producido con la intervención. Además, los niveles de precursores de porfirinas no fueron medidos de manera uniforme con la misma escala y los autores no reportaron estos valores para un mismo punto temporal, sino el valor más bajo durante el tratamiento. Estas limitaciones metodológicas impiden la interpretación de los datos. Así, el estudio más reciente respecto a hemina humana fue una descripción de los registros, pero este estudio no fue diseñado para medir la eficacia de hemina humana. CONCLUSIONES: La porfiria aguda intermitente (PAI) es una enfermedad metabólica hereditaria causada por la deficiencia de la tercera enzima de la síntesis del grupo Hem (porfobilinógeno deaminasa). La hemina humana fue aprobada por las agencias reguladoras de medicamentos bajo la denominación de medicamentos para enfermedades huérfanas, y aunque su aprobación se remonta a más de tres décadas atrás, no se ha podido identificar una evaluación sobre su eficacia y seguridad en los sitios web correspondientes. El ECA de Harrick et al., 1989 comparó datos pareados de nueve pacientes que usaron hemina humana o placebo en diferentes episodios de ataques de PAI. En este estudio se utilizó una escala categórica para puntuar el dolor (de 0 a 5) y se midió el uso de analgésicos. Aunque, se observó una mayor diferencia en la disminución de ALA y PBG en orina cuando el paciente uso hemina, esto no se reflejó en una mayor mejora del alivio del dolor, en menor requerimiento de analgésicos o en menor estancia hospitalaria. Respecto a los datos de seguridad, los escasos estudios han descrito muy escuetamente los eventos adversos observados y por un breve periodo. A pesar de ello, se han reportado EA serios de daño vascular con el uso de hemina humana, que incluyen flebitis y trombosis venosa. En un estudio, se reportaron de manera incompleta eventos adversos serios que incluyeron flebitis severa, bacteriemia severa y el otro caso no fue descrito. Además, en ninguno de estos estudios se ha reportado el daño renal reversible al que hizo referencia la carta de advertencia de la FDA. Por tanto, la información disponible del uso de hemina humana es incompleta e insuficiente respecto a eventos adversos serios, que no permite saber cuál es el perfil de seguridad de hemina humana. Hasta el momento, el uso de hemina humana se basa en reportes de series de casos. La mayoría de ellos recogieron información de manera retrospectiva y sin uso estandarizado de escalas de medición del dolor, que mostraran cambios respecto al basal en el dolor y los niveles. Además, no existe evidencia acerca de si hemina humana reduce las secuelas neurológicas asociadas con la acumulación de precursores de porfirinas, o si mejora la calidad de vida de los pacientes o reduce la mortalidad. Actualmente se desconoce cuál es el beneficio de agregar hemina humana al tratamiento habitual de las crisis agudas de PAI. Ya que ningún estudio ha establecido la eficacia clínica de hemina humana durante la crisis aguda de PAI. El único cambio observado fue en la disminución del dolor, para el cual se usó analgésicos opioides según requerimiento individualizado. Además, no se observó ningún efecto en las secuelas neurológicas u otros desenlaces clínicos relevantes como la calidad de vida o mortalidad. Por otro lado, con la poca información disponible y en corto tiempo de observación, el uso de hemina se asoció con riesgo de producir eventos adversos serios vasculares. Por tanto, es poco claro el balance riesgo beneficio que pueda ofrecer este medicamento. Ante un desfavorable balance riesgo-beneficio, el alto costo de este medicamento no justifica su incierto beneficio potencial, por lo que su implementación no se presenta con un perfil de costo-oportunidad favorable. Así, con los datos de un estudio aleatorizado, doble ciego y controlado con placebo, que aún se encuentra en fase de reclutamiento, se podrá saber si el uso de hemina mejora los desenlaces clínicos y si existe una correlación con los cambios bioquímicos. El Instituto de Evaluación de Tecnologías en Salud e Investigación IETSI no aprueba el uso de hemina humana para el tratamiento de los ataques de porfiria aguda intermitente.

DhHP­6 attenuates cerebral ischemia­reperfusion injury in rats through the inhibition of apoptosis.

As a novel reactive oxygen species (ROS) scavenger, deuterohemin His peptide­6 (DhHP­6) has been demonstrated to prolong the lifespan of Caenorhabditis elegans and has also exhibited protective effects in myocardial ischemia­reperfusion injury. Whether similar effects occur during cerebral ischemia­reperfusion (CIR) injury remains to be elucidated. The present study evaluated the function of DhHP­6 and its underlying mechanisms in a middle cerebral artery occlusion (MCAO) model in rats. The focal transient MCAO model was implemented using the Longa method of ischemia for 2 h followed by reperfusion for 22 h in male Wistar rats. DhHP­6 was administered at the onset of reperfusion via intraperitoneal injection. The infarct volume, brain edema, brain apoptosis and neurological function were evaluated 24 h following stroke. To further determine the role of DhHP­6 in CIR injury, the levels of ROS and malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH­Px), and the protein expression levels of B­cell lymphoma 2 (Bcl­2)­associated X protein (Bax), cleaved caspase­3, cytochrome c, Bcl­2 and phosphorylated­Akt/Akt were measured in ischemic cortex tissues. The results demonstrated that DhHP­6 significantly improved infarct volume, brain edema and neurological deficits, and reduced the percentage of TUNEL­positive cells. The levels of ROS and MDA were decreased, whereas no significant changes in the activities of SOD, CAT and GSH­Px were observed. The levels of Bax, cleaved caspase­3, and cytochrome c were downregulated, whereas the levels of Bcl­2 and p­Akt/Akt were upregulated. The results of the present study indicated that DhHP­6 may offer therapeutic potential for cerebral ischemia. The neuroprotective effects of DhHP­6 maybe mediated by its anti­oxidative properties, anti­apoptotic activities, or activation of the phosphoinositide 3­kinase/Akt survival pathway.

Deuterohemin-Peptide Enzyme Mimic-Embedded Metal-Organic Frameworks through Biomimetic Mineralization with Efficient ATRP Catalytic Activity.

An enzyme mimic harboring iron porphyrin (DhHP-6) embedded in zeolite imidazolate framework-8 (ZIF-8) was constructed through a biomimetic mineralization approach to obtain composite DhHP-6@ZIF-8. The composite was then used as a catalyst in the atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMA500) in which poly(PEGMA500) could be synthesized with monomer conversion of 76.1% and Mn of 45 900 g/mol, stronger than that obtained when using free DhHP-6 as a catalyst. More importantly, it could efficiently overcome the drawbacks of free DhHP-6 and achieve the easy separation of DhHP-6 from the catalytic system and the elimination of iron residues in the synthesized polymer. In addition, it exhibited an enhanced recyclability with monomer conversion of 75.7% after five cycles and favorable stability during the ATRP reaction with <3.0% of DhHP-6 release within 100 h. Thus, the enzyme mimic-ZIF-8 composite developed through biomimetic mineralization can be potentially used as an effective catalyst for preparing well-defined polymers with biomedical applications.

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b.

A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O2 and H2O2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O2 to the ferric state. The subsequent second-order reaction between the ferric complex and H2O2 is slow, pH-dependent, and further decelerated by D2O2 (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H2O2. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H2O2 cleavage is therefore unclear. From a cellular perspective, the use of H2O2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.

Hydrogen peroxide-mediated conversion of coproheme to heme b by HemQ-lessons from the first crystal structure and kinetic studies.

Heme biosynthesis in Gram-positive bacteria follows a recently described coproporphyrin-dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme-HemQ from Listeria monocytogenes) at 1.69 Å resolution and the conversion of coproheme to heme b followed by UV-vis and resonance Raman spectroscopy as well as mass spectrometry. The ferric five-coordinated coproheme iron of HemQ is weakly bound by a neutral proximal histidine H174. In the crystal structure of the resting state, the distal Q187 (conserved in Firmicutes HemQ) is H-bonded with propionate p2 and the hydrophobic distal cavity lacks solvent water molecules. Two H2 O2 molecules are shown to be necessary for decarboxylation of the propionates p2 and p4, thereby forming the corresponding vinyl groups of heme b. The overall reaction is relatively slow (kcat /KM = 1.8 × 102 m-1 ·s-1 at pH 7.0) and occurs in a stepwise manner with a three-propionate intermediate. We present the noncovalent interactions between coproheme and the protein and propose a two-step reaction mechanism. Furthermore, the structure of coproheme-HemQ is compared to that of the phylogenetically related heme b-containing chlorite dismutases. DATABASE: Structural data are available in the PDB under the accession number 5LOQ.

Heme Oxygenase 1 Up-Regulates Glomerular Decay Accelerating Factor Expression and Minimizes Complement Deposition and Injury.

Complement-activation controllers, including decay accelerating factor (DAF), are gaining emphasis as they minimize injury in various dysregulated complement-activation disorders, including glomerulopathies. Heme oxygenase (HO)-1 overexpression or induction has been shown to attenuate injury in complement-dependent models of glomerulonephritis. This study investigated whether up-regulation of DAF by heme oxygenase 1 (HO-1) is an underlying mechanism by using Hmox-1-deficient rats (Hmox1+/-; Hmox1-/-) or rats with HO-1 overexpression targeted to glomerular epithelial cells (GECHO-1), which are particularly vulnerable to complement-mediated injury owing to their terminally differentiated nature. Constitutively expressed DAF was decreased in glomeruli of Hmox1-/- rats and augmented in glomeruli of GECHO-1 rats. In GECHO-1 rats with anti-glomerular basement membrane antibody mediated, complement-dependent injury, complement component C3 fragment b (C3b) deposition was reduced, whereas proteinuria was diminished. In glomeruli of wild-type rats, the natural Hmox substrate, hemin, induced glomerular DAF. This effect was attenuated in glomeruli of Hmox1-/- rats and augmented in glomeruli of GECHO-1 rats. Hemin analogues differing in either metal or porphyrin ring functionalities, acting as competitive Hmox-substrate inhibitors, also increased glomerular DAF and reduced C3b deposition after spontaneous complement activation. In the presence of a DAF-blocking antibody, the reduction in C3b deposition was reversed. These observations establish HO-1 as a physiologic regulator of glomerular DAF and identify hemin analogues as inducers of functional glomerular DAF able to minimize C3b deposition.

Electron self-exchange in hemoglobins revealed by deutero-hemin substitution.

Hemoglobins (phytoglobins) from rice plants (nsHb1) and from the cyanobacterium Synechocystis (PCC 6803) (SynHb) can reduce hydroxylamine with two electrons to form ammonium. The reaction requires intermolecular electron transfer between protein molecules, and rapid electron self-exchange might play a role in distinguishing these hemoglobins from others with slower reaction rates, such as myoglobin. A relatively rapid electron self-exchange rate constant has been measured for SynHb by NMR, but the rate constant for myoglobin is equivocal and a value for nsHb1 has not yet been measured. Here we report electron self-exchange rate constants for nsHb1 and Mb as a test of their role in hydroxylamine reduction. These proteins are not suitable for analysis by NMR ZZ exchange, so a method was developed that uses cross-reactions between each hemoglobin and its deutero-hemin substituted counterpart. The resulting electron transfer is between identical proteins with low driving forces and thus closely approximates true electron self-exchange. The reactions can be monitored spectrally due to the distinct spectra of the prosthetic groups, and from this electron self-exchange rate constants of 880 (SynHb), 2900 (nsHb1), and 0.05M(-1) s(-1) (Mb) have been measured for each hemoglobin. Calculations of cross-reactions using these values accurately predict hydroxylamine reduction rates for each protein, suggesting that electron self-exchange plays an important role in the reaction.

Identification of amino acid residues involved in hemin binding in Porphyromonas gingivalis hemagglutinin 2.

Porphyromonas gingivalis (P. gingivalis) is a major etiological agent in the development and progression of chronic periodontitis. It produces cysteine proteases (gingipains), including a lysine-specific gingipain and two arginine-specific gingipains. Heme binding and uptake are fundamental to the growth and virulence of P. gingivalis. The recombinant hemagglutinin 2 domain (rHA2) of gingipain binds hemin with high affinity. The aim of the present work was to identify the key residues involved in its hemin-binding activity. A functional rHA2 was expressed and bound to hemin-agarose, and then digested with endopeptidases. The peptides bound to hemin-agarose were identified by mass spectrometry and the amino acids were assessed by mutation and peptide binding inhibition analysis. The DHYAVMISK sequence was identified in peptides derived from both Asp-N and Lys-C endopeptidase digestions of rHA2. A monoclonal antibody, mAb QB, was produced and its epitope was associated with the DGFPGDHYAVMISK peptide within the HA2 domain. Hemin was shown to competitively inhibit the immunoreactivity of rHA2 or the peptide to mAb QB. The peptide DHYAVMISK inhibited hemin-binding activity; although, this inhibition was not seen when the peptide contained the H1001E mutation (DEYAVMISK). Based on these results, we propose that residue His1001 is involved in the hemin-binding mechanism of the P. gingivalis rHA2 and the peptide containing this residue, DHYAVMISK, may be an inhibitor of hemin binding.

Investigation of bn-44 peptide fragments using high resolution mass spectrometry and isotope labeling.

An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic bn-44 ions instead of bn-28 (an) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH3CHO (44.0262) from a Thr side chain; (2) loss of CO2 (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel bn-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway.

Determination of a deuterohemin-peptide conjugate in rat plasma by liquid chromatography-tandem mass spectrometry and application to a preclinical pharmacokinetic study.

The deuterohemin-peptide conjugate (DhHP-6) is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species. This paper reports the development of a sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of DhHP-6 in rat plasma using triptorelin as an internal standard (IS). 50µL plasma was used in sample preparation, and a simple protein precipitation procedure with acetonitrile was involved. Satisfactory peak shapes of analyte and IS were obtained on an Agilent HC-C18 column by using a gradient elution with 10mM ammonium acetate-0.5% formic acid (v:v) and acetonitrile, there was no significant interference impacting the determination. A calibration curve obtained from this method was linear within the concentration range 10-3000ng/mL with intra- and inter-day precisions of 4.2-6.8% and 3.2-8.9%, respectively and accuracy of -1.3% to 2.1%. The recovery was above 80% with low matrix effects. The method was successfully applied to support a preclinical pharmacokinetic study in rat.

Chitosan-g-hematin: enzyme-mimicking polymeric catalyst for adhesive hydrogels.

Phenol derivative-containing adhesive hydrogels has been widely recognized as having potential for biomedical applications, but their conventional production methods, utilizing a moderate/strong base, alkaline buffers, the addition of oxidizing agents or the use of enzymes, require alternative approaches to improve their biocompatibility. In this study, we report a polymeric, enzyme-mimetic biocatalyst, hematin-grafted chitosan (chitosan-g-hem), which results in effective gelation without the use of alkaline buffers or enzymes. Furthermore, gelation occurs under mild physiological conditions. Chitosan-g-hem biocatalyst (0.01%, w/v) has excellent catalytic properties, forming chitosan-catechol hydrogels rapidly (within 5 min). In vivo adhesive force measurement demonstrated that the hydrogel formed by the chitosan-g-hem activity showed an increase in adhesion force (33.6 ± 5.9 kPa) compared with the same hydrogel formed by pH-induced catechol oxidation (20.6 ± 5.5 kPa) in mouse subcutaneous tissue. Using the chitosan-g-hem biocatalyst, other catechol-functionalized polymers (hyaluronic acid-catechol and poly(vinyl alcohol)-catechol) also formed hydrogels, indicating that chitosan-g-hem can be used as a general polymeric catalyst for preparing catechol-containing hydrogels.

DhHP-6 extends lifespan of Caenorhabditis elegans by enhancing nuclear translocation and transcriptional activity of DAF-16.

Earlier studies have demonstrated that Deuterohaemin-AlaHisThrValGluLys (DhHP-6), a novel porphyrin-peptide, increases lifespan and enhances stress resistance of Caenorhabditis elegans. To explore the possible mechanisms, in this study we investigated the roles of SIR-2.1 and DAF-16 in DhHP-6's function using wild-type and various other mutant strains of C. elegans. DhHP-6's effect was dependent upon DAF-16, and it did not extend the lifespan of the loss-of-function daf-16 mutant strain (daf-16(mu86) I). DhHP-6 enhanced DAF-16 translocation from cytoplasm to nuclei; and it increased DAF-16's transcriptional activity, likely by activating the SIR-2.1/DAF-16 complex. DhHP-6's effect was also dependent upon SIR-2.1, and it did not increase the lifespan of the worms with SIR-2.1 deacetylase activity inhibited by niacin amide (SIR-2.1 inhibitor) and SIR-2.1 RNA interference (RNAi). Niacin amide and RNAi increased DAF-16's nuclear localization; but they decreased DAF-16's transcriptional activity, likely by preventing the formation of the SIR-2.1/DAF-16 complex. These results suggest that DhHP-6 extends the lifespan of C. elegans via SIR 2.1 and DAF-16, and they provide new insights into the molecular mechanisms of aging.

Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis.

We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of (1)H NMR and GC-MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent δ-ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with B. vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC-MS and (1)H NMR.

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.