Identification of mutant Asp251Gly/Gln307His of cytochrome P450 BM3 for the generation of metabolites of diclofenac, ibuprofen and tolbutamide.

The soluble, catalytically self-sufficient cytochrome P450 BM3 from Bacillus megaterium is a good candidate as biocatalyst for the synthesis of drug metabolites. To this end, error-prone polymerase chain reaction (PCR) was used to generate a library of P450 BM3 mutants with novel activities toward drugs. The double mutant Asp251Gly/Gln307His (A2) with activities towards diclofenac, ibuprofen and tolbutamide was identified by screening with the alkali method. This is based on the detection of NADPH oxidation during enzymatic turnover on whole Escherichia coli cells heterologously expressing the P450 BM3 mutants in the presence of the target substrates. The three drugs screened are marker substrates of human liver cytochromes P450 belonging to the 2C subfamily. Interestingly the mutations Asp251Gly/Gln307His are located on the protein surface and they are not directly involved in substrate binding and turnover. Dissociation constants and K(M) values of mutant A2 for diclofenac, ibuprofen and tolbutamide are in the micromolar range. Catalysis leads to hydroxylations in specific positions, producing 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide, respectively.

An overview of the progress on double umbilical cord blood transplantation.

Umbilical cord blood transplantation has been increasingly used over the past years for both malignant and non-malignant hematologic and other diseases as an alternative to mismatched-related or matched-unrelated bone marrow or peripheral blood hematopoietic stem cell transplantation. A disadvantage of cord blood is its low cell content which limits cord blood transplantation to generally low weight recipients, such as children. Various alternatives have been used to overcome this limitation, including co-infusion of two partially HLA-matched cord blood units. According to Eurocord Registry data, this strategy has been applied in approximately 993 adult patients with hematologic diseases since the first double umbilical cord blood transplantation in 1999. In fact, since 2005, the number of adult patients receiving double umbilical cord blood transplantation has surpassed the number of adults transplanted with single cord blood units. The engraftment rate is comparable for both single and double umbilical cord blood transplantation, although the latter is accompanied by a higher incidence of grade II acute graft-versus-host disease and lower leukemia relapse for patients in first complete remission. In the majority of patients undergoing double umbilical cord blood transplantation, transient chimerism, due to the presence of cells from both donor units early post transplant, is replaced by sustained dominance of one unit from which long-term hematopoiesis is derived. Although the biology and the factors that determine unit dominance have not been clarified, the implication of immune-mediated mechanisms has been reported. Preliminary data have demonstrated the safety of double umbilical cord blood transplantation. Ongoing clinical trials and prolonged follow up of the patients will clarify the immunology and determine the efficacy of this approach. We present here a brief overview of the clinical experience on double umbilical cord blood transplantation and its underlying biology.

Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity.

Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand-gated ion-channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (alpha1-10, beta1-4, gamma, delta, and epsilon). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non-neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high-resolution X-ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high-resolution X-ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype-specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.

Expression of human AChR extracellular domain mutants with improved characteristics.

The muscle nicotinic acetylcholine receptor (AChR) has a central role in neuromuscular transmission, and is the major target in the autoimmune disease myasthenia gravis (MG). We created mutants of the extracellular domains (ECDs) of the human α1, ß1, δ and ε AChR subunits, whereby their Cys-loop was exchanged for that of the acetylcholine binding protein. The mutants were expressed in Pichia pastoris and had improved solubility resulting in 2- to 43-fold higher expression yields compared to the wild type. An additional mutant was created for the α1 ECD restoring its glycosylation site within the Cys-loop and its α-bungarotoxin binding ability. Furthermore, we constructed dimeric and pentameric concatamers of the mutant ECDs. All concatamers were successfully expressed as soluble secreted proteins, although the pentamers had about 10-fold lower expression than the dimers and were more susceptible to fragmentation. Initial crystallizations with the mutant ECDs were promising, and we reproducibly obtained crystals of the ß1 ECD, diffracting at ~12 Å. Further optimization is underway to obtain crystals suitable for high resolution crystallography. The proteins described herein are useful tools in structural studies of the human muscle AChR and can be used in applications requiring high yields such as therapeutic adsorbents for MG autoantibodies.

Hydroxylation of non-substituted polycyclic aromatic hydrocarbons by cytochrome P450 BM3 engineered by directed evolution.

Chrysene and pyrene are known toxic compounds recalcitrant to biodegradation. Here directed evolution allowed us to identify two new mutants of cytochrome P450 BM3 that are able to hydroxylate both compounds. Random mutagenesis has been used to generate libraries of mutants of P450 BM3 active toward polycyclic aromatic hydrocarbons (PAHs) PAHs. After two rounds of error-prone PCR and backcross with parental DNA, three mutants were identified for improved activity toward pyrene and for the first time a new activity toward chrysene in comparison to the wild type enzyme. The mutants show higher affinity and coupling efficiency for chrysene with faster rates of product formation compared to the wild type. Furthermore, the mutants are able to hydroxylate chrysene in different positions, producing four metabolites, 1-, 3-, 4-, and 6-hydroxychrysene, and to hydroxylate pyrene to 1-hydroxypyrene. The majority of the mutation sites are found to be far from the active site, demonstrating the power of directed evolution in identifying mutations difficult to predict with a rational design approach. The different product profiles obtained for the different P450 BM3 mutants indicate that substrate orientation in the catalytic pocket of the protein can be modified by protein engineering. The mutants can be used for metabolic engineering for safe and cost-effective sustainable production of hydroxylated PAHs for industrial purposes as well as for the assessment of their carcinogenic activity in mammals.

Double cord blood transplantation: co-operation or competition?

Over the last two decades umbilical cord blood (UCB) transplantation (UCBT) is increasingly used for a variety of malignant and benign hematological and other diseases. The main factor that limits the use of UCB to low weight recipients, mainly children and adolescents, is its low progenitor cell content. Various alternatives have been exploited to overcome this difficulty, including the transplantation of two UCB units (double umbilical cord blood transplantation, dUCBT). Following dUCBT, donor(s) hematopoietic stem cells (HSC) can be detected in the peripheral blood of the recipient as soon as 14 days post-transplantation. Sustained engraftment of HSC from one or both donors can be observed- dominance or mixed chimerism respectively, although single donor unit dominance has been observed in over 85% of patients. The underlying biology, which accounts for the interactions both between the two infused UCB units- cooperative or competitive, and with the recipient's immune system, has not been elucidated.

Wild-type CYP102A1 as a biocatalyst: turnover of drugs usually metabolised by human liver enzymes.

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119-131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography-mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The KM values calculated for these compounds were in the millimolar range: 1.21+/-0.07 mM for chlorzoxazone, 2.52 +/- 0.08 mM for aniline, 0.81+/-0.04 mM for propranolol. The values of vmax for chlorzoxazone and propranolol were 46.0+/-9.0 and 7.6+/-3.4 nmol min-1 nmol-1, respectively. These values are higher then those measured for the human enzymes. The vmax value for aniline was 9.4+/-1.3 nmol min-1 nmol-1, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.