Recombinant Enzyme Preparations Research Articles

Search for Inhibitors of Mycobacterium tuberculosis Transketolase in a Series of Sulfo-Substituted Compounds.

As a result of the computer screening of a library of sulfo-substituted compounds, molecules capable of binding to the active site of transketolase from Mycobacterium tuberculosis were identified. An experimental verification of the inhibitory activity of the most promising compound, STK045765, against a highly purified recombinant enzyme preparation was carried out. It was shown that the STK045765 molecule competes for the binding site of the pyrophosphate group of the thiamine diphosphate cofactor and, at a micromolar concentrations, is able to suppress the activity of mycobacterial transketolase. The discovered furansulfonate scaffold may serve as the basis for the creation of anti-tuberculosis drugs.

Synergistic hydrolysis of filter paper by recombinant cellulase cocktails leveraging a key cellobiase, Cba2, of Cellulomonas biazotea.

Cellulomonas biazotea, a Gram-positive cellulolytic bacterium isolated from soil, is capable of producing a complete cellulase complex exhibiting endoglucanase, exoglucanase, and cellobiase activities. Despite the presence of a full complement of all three types of cellulases, samples prepared from both cell lysates and culture media of C. biazotea showed only weak synergistic activities formed among the cellulase components, as reflected by their inefficient performance in filter paper hydrolysis. However, when the five previously characterized recombinant cellobiases of C. biazotea were mixed individually or in different combinations with recombinant enzyme preparations (CenA/Cex) containing an endoglucanase, CenA, and an exoglucanase, Cex, of another Cellulomonas species, C. fimi, the cellulase cocktails exhibited not only much higher but also synergistic activities in filter paper hydrolysis. Among the 5 C. biazotea cellobiases studied, Cba2 was shown to perform 2.8 to 3.8 times better than other homologous isozymes when acting individually with CenA/Cex. More noteworthy is that when Cba2 and Cba4 were added together to the reaction mixture, an even better synergistic effect was achieved. The filter paper activities resulting from Cba2 and Cba4 interacting with CenA/Cex are comparable to those obtained from some commercial fungal cellulase mixtures. To our knowledge, our results represent the first demonstration of synergistic effects on filter paper hydrolysis achieved using recombinant bacterial cellulases.

Bone involvement in Gaucher disease type 1: a differential diagnosis of multiple fractures in a young patient. Case report

Gaucher disease type 1 (GD1) is a rare inherited disease caused by mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase, leading to decreased enzyme activity and the consequential accumulation of its substrate, glucosylceramide, within macrophages. Bone involvement in GD1 may include avascular necrosis, bone infarcts, fragility fractures, lytic or sclerotic bone lesions and osteomyelitis. Bone pain can manifest as an acute and very painful bone crisis or a chronic pain that might vary in intensity, usually caused by fractures or avascular necrosis with progressive collapse of bone and joints. Hence, this report aims to describe a case of Gaucher disease type 1 and discuss its diagnosis, bone involvement and management, since late diagnosis or inadequate therapy can lead to clinical worsening, irreversible mobility impairment and increased morbidity. AGR, a 32-year-old male patient, was admitted to the Rheumatology Service with a previous history of diffuse bone pain, episodic hyperemia in the lower limbs since the age of 9 and two low-impact fractures. He had an initial non-confirmed diagnostic hypothesis of chronic recurrent multifocal osteomyelitis and was taking dipyrone and tramadol to reduce pain. His physical examination evinced overweight (body mass index: 29.7 kg/m2) and hepatosplenomegaly. Laboratory tests shown thrombocytopenia, with platelet count 103,000/mm3 (reference value [RV]: 140,000-450,000/mm3), serum phosphorus 4.7 mg/dL (RV: 2.7-4.5 mg/dL), C-reactive protein 88.8 mg/dL (RV: < 5 mg/dL) and ferritin 1336 ng/mL (RV: 30-400 ng/mL). Biochemical and bone turnover markers were normal, including serum total calcium and magnesium, parathyroid hormone, 25-OH vitamin D, alkaline phosphatase, procollagen type-I N-terminal propeptide and C-terminal telopeptide of type I collagen. Conventional radiographies and magnetic resonance imaging were performed and confirmed previous fractures in the right humerus and the right acetabulum. They also showed sclerotic lesions in the left femur and Erlenmeyer flask deformities in the distal femurs and the proximal tibias. Bone scintigraphy indicated a bone infarct area in the left tibia. Areal bone mineral density (BMD) evaluated by bone densitometry showed a left total femur Z-score of +4.3 and left femoral neck Z-score of +4.5. Differently, the bone microarchitecture and volumetric BMD, evaluated by a high-resolution peripheral quantitative computed tomography (HR-pQCT) at distal radius and distal tibia, demonstrated an impairment of cortical compartment with lower cortical thickness and lower cortical volumetric BMD, possibly contributing to the low-impact fractures. Considering the compatible radiological findings, associated with hepatosplenomegaly and thrombocytopenia, Gaucher disease type 1 became the major hypothesis. It was confirmed after enzymatic dosage, which showed low residual glucocerebrosidase activity and high serum chitotriosidase. Enzyme replacement therapy was started with taliglucerase alfa, a recombinant enzyme preparation that aims to supply the lacking glucocerebrosidase in lysosomes. After 6 months of treatment, patient reported a significant improvement in bone pain and general quality of life. Therefore, clinicians should be alert to young patients with multiple low-impact fractures and further evaluate other possible bone involvement, as GD1 may be a differential diagnosis. Since it needs specific treatment, early diagnosis can be crucial to avoid permanent bone damage and morbidity.

Critical Differences between Enzyme Sources in Sensitivity to Detect Time-Dependent Inactivation of CYP2C8.

Clopidogrel acyl-β-d-glucuronide is a mechanism-based inhibitor of cytochrome P450 2C8 in human liver microsomes (HLMs). However, time-dependent inactivation (TDI) of CYP2C8 could not be detected in an earlier study in human recombinant CYP2C8 (Supersomes). Here, we investigate whether different enzyme sources exhibit differences in detection of CYP2C8 TDI under identical experimental conditions. Inactivation of CYP2C8 by amiodarone (100 μM), clopidogrel acyl-β-d-glucuronide (100 μM), gemfibrozil 1-O-β-glucuronide (100 μM), and phenelzine (100 μM) was investigated in HLMs and three recombinant human CYP2C8 preparations (Supersomes, Bactosomes, and EasyCYP Bactosomes) using amodiaquine N-deethylation as the marker reaction. Furthermore, the inactivation kinetics of CYP2C8 by clopidogrel glucuronide (5-250 μM) was determined in Supersomes and Bactosomes. Amiodarone caused weak TDI in all enzyme preparations tested, while the extent of inactivation by clopidogrel glucuronide, gemfibrozil glucuronide, and phenelzine varied markedly between preparations, and even different Supersome lots. Both glucuronides caused strong inactivation of CYP2C8 in HLMs, Bactosomes and in one Supersome lot (>50% inhibition), but significant inactivation could not be reliably detected in other Supersome lots or EasyCYP Bactosomes. In Bactosomes, the concentration producing half of kinact (KI) and maximal inactivation rate (kinact) of clopidogrel glucuronide (14 μM and 0.054 minute-1) were similar to those determined previously in HLMs. Phenelzine caused strong inactivation of CYP2C8 in one Supersome lot (91% inhibition) but not in HLMs or other recombinant CYP2C8 preparations. In conclusion, different enzyme sources and different lots of the same recombinant enzyme preparation are not equally sensitive to detect inactivation of CYP2C8, suggesting that recombinant CYPs should be avoided when identifying mechanism-based inhibitors.

Assays of D-Amino Acid Oxidase Activity.

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

Using an Inducible Promoter of the Glucoamylase Gene to Construct New Multienzyme Complexes from Penicillium verruculosum

New recombinant strains of Penicillium verruculosum are created using a new plasmid construct based on an inducible promoter of glucoamylase gene (gla1) that secretes heterologous xylanase E (XylE) from P. canescens. New biocatalysts are produced that contain cellulolytic enzyme preparations (EPs) enriched with XylE. The amount of XylE in the recombinant EPs varies in the optimum range of 11–24% of the total protein while generally preserving the P. verruculosum cellulose complex. The hydrolytic activity of the new EPs with respect to polymeric plant-derived substrates exceeds that of EPs produced using other expression systems and commercial preparations. The new EP glaX-17 surpasses in particular a control EP based on the recipient strain by 13% in the efficiency of hydrolyzing aspen wood and is 20% more efficient than the commercial EP Accelerase Duet. The new EP glaX-17 displays 25% greater efficiency (35–43%) during the hydrolysis of wheat bran than the commercial EP Accelease Duet. The effectiveness is demonstrated of using the new gla1 promoter for the production of EPs (biocatalysts) while preserving the balanced cellulose complex of the strain and optimum yield of heterologous XylE required for the deep hydrolysis of xylan-containing plant biomass.

The Application of the Inducible Promoter of Glucoamylase Gene for Preparation of New Multienzyme Complexes Penicillium verruculosum

A new plasmid construction based on the inducible promoter of glucoamylase gene ( gla1 ) was used for the obtaining of new recombinant strains Penicillium verruculosum that secrete heterological xylanase E (XylE) P. canescens . New biocatalysts were developed which are XylEenriched cellulolytic enzyme preparations (EP). The content of XylE in recombinant EP varied within the optimal range from 11 to 24 % of the general protein pool at the preservation (in total) of the cellulase complex P. verruculosum . In comparison to EP prepared using the other expression systems and commercial preparations, the new EP demonstrated a higher hydrolytic activity with respect to polymers of plant substrates. For example, the efficiency of new EP glaX-17 to hydrolysis of aspenwood was higher by 13 % than that of the control EP agent and by 20 % than that of the commercial EP Accelerase Duet. In hydrolysis of wheat bran, glaX-17 was 35–43 % and 25 % more efficient than the control EP and the commercial EP Accelerase Duet, respectively. The efficiency of the new gla1 promoter for the production of EP (biocatalysts) at the preserved balanced cellulase complex of the strain and optimal yield of heterological XylE (that are necessary for complete hydrolysis of xylan-containing plant biomass) was demonstrated.

Preparing catalyst based on recombinant cellulolytic enzyme specimen Penicillium verruculosum and its use in the paper industry

The effective application of a biocatalyst based on recombinant enzyme preparations obtained using Penicillium verruculosum is studied for the modification of marketable bleached sulfate hardwood cellulose. The effect of treatment with cellulase complex on the structure, morphology, and surface state of cellulose fibers is evaluated. After biocatalytic exposure, cellulose is shown to have an improved capacity for beating and the formation of bonds in paper sheets, along with greater strength than the initial cellulose. Application of the enzymatic modification of cellulose in the paper industry during biotechnological processes allows a range of problems in the efficient use of energy and resources to be solved.

Functional expression of recombinant goat chymosin in Pichia pastoris bioreactor cultures: A commercially viable alternate

Abstract Recombinant goat chymosin (RGC), a promising alternate of bovine/calf chymosin, has better catalytic, proteolytic and thermostable characteristics. Limited information exists about its expression in different hosts. Pichia pastoris (P. pastoris) has ability to grow to very high cell densities and improved protein production has been demonstrated in scale up studies. A modified methanol feeding strategy based on flask level optimizations was successfully used for expressing the goat prochymosin in recombinant methylotrophic yeast P. pastoris Mut+ bioreactor cultures. Additionally, a comparative analysis of RGC expression in flask versus bioreactor cultures was performed. Bioreactor cultures yielded an approximate 8 times increase in cell density compared to flask cultures prior to induction and an increase of 24% in expression level of chymosin was also recorded. Functional characterization demonstrated that the enzyme produced even at early stages was found to be correctly folded, intact and functional. The recombinant enzyme preparation has 42.4% residual activity at 60 °C and retained 69% activity at pH 6.5. An expression strategy for scale up production of RGC, which can act as a lead for production of industrially viable enzyme preparation, is outlined in the present study.

Abstract B171: Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines

Abstract Background: Cancer cells preferentially channel glucose towards lactate production even when oxygen is plentiful, a process termed “aerobic glycolysis” or the Warburg effect. The entire metabolic machinery is reprogrammed in cancer cells wherein glutamine utilization is increased via elevation of glutaminase activity thereby generating the necessary substrates required for eventual ATP synthesis and energy production. Targeting glutaminase therefore represents a potential therapeutic strategy to curb malignant transformation of cells and combat cancer progression. Herein, we describe the biological and pharmacokinetic properties of RP10107, a novel small molecule glutaminase inhibitor, with scope to be further developed as a clinical candidate for solid tumors. Methods: Inhibition of glutaminase (GLS1) activity was determined in rodent brain lysates and recombinant human enzyme preparations using a colorimetric or fluorometric procedure. Selectivity over liver glutaminase (GLS2) was confirmed using mouse liver mitochondrial lysates. Viability was assessed using the colorimetric MTT reagent after incubation of RP10107 with cell lines representative of breast, lung, and colorectal cancer. Glutamate concentrations in cell lines following treatment with RP10107 was estimated by LC-MS/MS. Expression of GLS-1, cMyc, pS6, and p-mTOR were estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Pharmacokinetic profile of RP10107 in plasma after single dose oral administration (10 mg/kg) or IV injection (1 mg/kg) was determined in mice. Results: RP10107 inhibited glutaminase (GLS1) activity in mouse brain lysate, rat brain lysate, and recombinant human enzyme with IC50 of 21.2, 18.2, and 26.4 nM, respectively. In contrast, activity in liver mitochondrial preparations (GLS2) was minimal (<10% inhibition) even at 10 μM concentration. RP10107 caused a dose-dependent inhibition in proliferation of breast, lung, and colorectal cell lines. Response was the highest in MDA-MB-231 (GI50 = 27 nM) with half-maximal growth inhibition for ZR.75.1, MCF-7, A549, H441, HCT-8, and HCT-116 observed at <2 μM RP10107. Reduction in cell viability was accompanied by a dose-dependent increase in the ratio of glutamine to glutamate with >50% response noticed at 100 nM in MDA-MB-231 cells. While p-mTOR expression levels were not affected, addition of RP10107 caused a reduction in cMyc and pS6 (>50% at 100-1000 nM) in lung and breast cancer cell lines. Pharmacokinetic studies of RP10107 in mice indicated high oral bioavailability (62%) with maximum plasma concentrations (Cmax) exceeding 10 μM. Conclusions: RP10107 is a potent and selective GLS1 inhibitor that inhibited growth of solid tumor cell lines with a concomitant increase in glutamine to glutamate ratio. Reduction in glutaminase activity resulted in downregulation of cMyc and pS6 in breast and lung cancer cells. Based on the in vitro efficacy and excellent pharmacokinetic profile, RP10107 is current being tested in various mouse xenograft models. Citation Format: Srikant Viswanadha, Prashant Bhavar, Gayatriswaroop Merikapudi, Babu Govindrajulu, Satyanarayana Eleswarapu, Sridhar Veeraraghavan, Swaroop Vakkalanka. Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B171.

In Vivo and In Vitro Characterization of a Novel MAO-B Inhibitor Radioligand, 18F-Labeled Deuterated Fluorodeprenyl.

The aim of this study was to radiolabel a novel bis-deuterium substituted l-deprenyl analog (fluorodeprenyl-D2) with (18)F and to evaluate its potential to visualize and quantify monoamine oxidase (MAO) B activity in vivo. The precursor compound ( 5A: + 5B: ) and reference standard ( 6: ) were synthesized in multistep syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used to determine inhibitory concentrations of 50%. Radiolabeling was accomplished by a nucleophilic substitution reaction. Whole-hemisphere autoradiography was performed with (18)F-fluorodeprenyl-D2. A PET study was performed on a cynomolgus monkey. Radiometabolites were measured in monkey plasma using high-performance liquid chromatography. The 50% inhibitory concentration of compound 6 for MAO-B was 227 ± 36.8 nM. Radiolabeling was accomplished with high radiochemical yield, purity, and specific radioactivity. The autoradiography binding density of (18)F-fluorodeprenyl-D2 was consistent with known MAO-B expression in the human brain. In vivo, (18)F-fluorodeprenyl-D2 showed favorable kinetic properties, with relatively fast washout from the brain. Regional time-activity curves were better described by the 2-tissue-compartment model. Administration of a 1 mg/kg dose of l-deprenyl yielded 70% inhibition of MAO-B in all regions. Radiometabolite studies demonstrated 20% unchanged radioligand at 120 min after injection. (18)F-fluorodeprenyl-D2 showed less irreversibility than did previously reported MAO-B radioligands. The results suggest that (18)F-fluorodeprenyl-D2 is a suitable PET radioligand for visualization of MAO-B activity in the human brain.

Properties of enzyme preparations and homogeneous enzymes - endoglucanases EG2 Penicillium verruculosum and LAM Myceliophthora thermophila.

The genes of endoglucanases EG2 (36.2kDa) Penicillium verruculosum and LAM (30.8kDa) Myceliophthora thermophila were cloned in P. verruculosum recombinant strain. New enzyme preparations with highly stable activity against β-glucan and laminarin were obtained and investigated, homogeneous enzymes EG2 (EC 3.2.1.4) and LAM (EC 3.2.1.6) being purified and characterized. For β-glucan, the EG2 Km value was found to be 10 times higher than that for LAM; however, EG2 demonstrated greater processivity due to its higher kcat. The pH and temperature optima of EG2 and LAM activity against barley β-glucan overlapped and were 4.3-4.9 and 61-67°C, respectively, and EG2 appeared to be more stable than LAM. Oligosaccharides with degree of polymerization 2-10 were formed by hydrolysis of β-glucan and laminarin by the studied enzymes. The recombinant enzyme preparations were faster and more effective in decreasing the reduced viscosity of wholegrain barley extract than some commercial enzyme preparations. Thus, the new enzyme preparations seem to be rather perspective as feed additives for degradation of non-starch polysaccharides in grain animal feed.

Reduction of Plasma Globotriaosylsphingosine Levels After Switching from Agalsidase Alfa to Agalsidase Beta as Enzyme Replacement Therapy for Fabry Disease.

Agalsidase alfa and agalsidase beta, recombinant enzyme preparations for treatment of Fabry disease (FD), have different approved dosing schedules: 0.2mg/kg and 1.0mg/kg every other week (EOW), respectively. This open-label, multicenter, exploratory phase 4 study evaluated plasma globotriaosylsphingosine (lyso-GL-3) and plasma and urine globotriaosylceramide (GL-3) levels at baseline and 2, 4, and 6 months after the switch from agalsidase alfa (0.2mg/kg EOW for ≥12 months) to agalsidase beta (1.0mg/kg EOW) in 15 male patients with FD. Immunoglobulin (Ig)G antidrug antibody titers were assessed, and safety was monitored throughout the study. Plasma lyso-GL-3 concentrations decreased significantly within 2 months after switch and reductions continued through month 6 (mean absolute changes, -12.8, -16.1, and -16.7ng/mL at 2, 4, and 6 months, respectively; all P < 0.001). The mean percentage reduction from baseline was 39.5% (P < 0.001) at month 6. For plasma GL-3, the mean absolute change from baseline (-0.9μg/mL) and percentage reduction (17.9%) at month 6 were both significant (P < 0.05). Urine GL-3 measurements showed intra-patient variability and changes from baseline were not significant. No clinical outcomes were assessed in this 6-month study, and, therefore, no conclusions can be drawn regarding the correlation of observed reductions in glycosphingolipid concentrations with clinically relevant outcomes. There were no differences in IgG antidrug antibody titers between the two enzymes. The switch from agalsidase alfa to agalsidase beta was well tolerated. Plasma lyso-GL-3 and GL-3 levels reduced after switching from agalsidase alfa to agalsidase beta, indicating that agalsidase beta has a greater pharmacodynamic effect on these markers at the recommended dose. These data further support the use of agalsidase beta 1.0mg/kg EOW as enzyme replacement therapy in FD.

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.

Application of complex biocatalysts based on recombinant Penicillium verruculosum enzyme preparations in the hydrolysis of semichemical hardwood pulp

A comparative study of the efficiency of the biocatalysts based on recombinant enzyme preparations obtained from the Penicillium verruculosum fungus in the hydrolysis of semichemical hardwood pulp is performed. The activity of the biocatalysts with respect to different types of semichemical pulp and the dependence of the depth of exhaustive hydrolysis on its beating and drying is determined. It is shown that semichemical pulp after cooking with green liquor displays high reactivity in enzymatic hydrolysis with cellulase complex and is of clear interest as a substrate for scaling the bioengineering processes of renewable plant feedstock conversion.

Synthesis and evaluation of [18F]fluororasagiline, a novel positron emission tomography (PET) radioligand for monoamine oxidase B (MAO-B)

The aim of this study was to synthesize and evaluate a novel fluorine-18 labeled analogue of rasagiline (6) as a PET radioligand for monoamine oxidase B (MAO-B). The corresponding non-radioactive fluorine-19 ligand, (1S,2S)-2-fluoro-N-(prop-2-yn-1-yl)indan-1-amine (4), was characterized in in vitro assays.The precursor compound (3aS,8aR)-3-(prop-2-yn-1-yl)-3,3a,8,8a-tetrahydroindeno[1,2-d][1,2,3]oxathiazole 2,2-dioxide (3) and reference standard 4 were synthesized in multi-step syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used in order to determine IC50 values for compound 4 by use of an enzymatic assay employing kynuramine as substrate. Radiolabeling was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulphamidate group. Human whole hemisphere autoradiography (ARG) was performed with [18F]fluororasagiline. Blocking experiments with pirlindole (MAO-A), l-deprenyl and rasagiline (MAO-B) were conducted to demonstrate the specificity of the binding. A positron emission tomography (PET) study was carried out in a cynomolgus monkey where time activity curves for whole brain and regions with high and low MAO-B activity were recorded. Radiometabolites were measured in monkey plasma using gradient HPLC.Compound 4 inhibited MAO-B with an IC50 of 27nM and MAO-A with an IC50 of 2.3μM. Radiolabeling of precursor 3 and subsequent hydrolysis of the protecting group towards (1S,2S)-2-[18F]fluoro-N-(prop-2-yn-1-yl)indan-1-amine (6) was successfully accomplished with an radiochemical yield of 40–70%, a radiochemical purity higher than 99% and a specific radioactivity higher than 200GBq/μmol. ARG demonstrated selective binding for [18F]fluororasagiline (6) to MAO-B containing brain regions, for example, striatum. The initial uptake in the monkey brain was 250% SUV at 4min post injection. The highest amounts of radioactivity were observed in the striatum and thalamus as expected whereas in the cortex and cerebellum lower levels were observed. Metabolite studies demonstrated 30% unchanged radioligand at 90min post injection.Our investigations demonstrated that the new ligand [18F]fluororasagiline (6) binds specifically to MAO-B in vitro and has a MAO-B specific binding pattern in vivo. Thus, it could serve as a novel potential candidate for human PET studies.

Enhanced storage stability of recombinant enzyme preparation of α-CGTase from Paenibacillus macerans by chemical additives

Enhanced storage stability of recombinant enzyme preparation of α-CGTase from Paenibacillus macerans by chemical additives

CYP3A5-mediated metabolism of midazolam in recombinant systems is highly sensitive to NADPH-cytochrome P450 reductase activity

Data from in vitro drug metabolism studies with recombinant enzyme systems are frequently used to predict human drug metabolism in vivo. However, for the CYP3A probe substrate midazolam (MDZ), considerable variability in enzyme kinetic parameters has been observed in different in vitro studies. The aim of this study was to explore the effect of varying activities of the electron donor NADPH-cytochrome P450 reductase (CPR) on CYP3A5-mediated metabolism of MDZ.Microsomes with similar levels of CYP3A5 but 12-fold difference in CPR activity showed a 30-fold difference in intrinsic clearance for the formation of 1′-OH-MDZ. Significantly higher Km and lower Vmax for the formation of 1′-OH-MDZ were found in microsomes with low CPR activity compared with microsomes with higher CPR activity (P = 0.024 and 0.001). In the microsomes with lowest CPR activity, the formation of 1′-OH-MDZ displayed Michaelis–Menten kinetics, whereas substrate inhibition was observed in the two preparations with higher CPR activity.The present study shows that the CPR activity in different recombinant enzyme preparations is crucial for in vitro CYP3A5-mediated clearance of MDZ. This suggests that the CPR activity of enzyme preparations could be an important factor for the ability of in vitro data to predict human drug metabolism in vivo.

Phenol-oxidizing laccases from the termite gut

cDNAs encoding two gut laccase isoforms ( RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma – mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

Alglucosidase alfa: Long term use in the treatment of patients with Pompe disease

Pompe disease is a lysosomal storage disorder characterized by muscle weakness and cardiomyopathy. It shows a broad variability regarding the clinical severity as well as the age of onset. In the past, two different recombinant enzyme preparations have been developed for the treatment of Pompe patients: alpha-glucosidase, produced in rabbit milk, and alpha-glucosidase, produced in Chinese hamster ovary (CHO) cell lines. The CHO enzyme received marketing approval in 2006 after it was proven to be effective in ameliorating muscle strength and improving heart function. The other has not been approved. The clinical efficacy of this enzyme preparation could be confirmed by several clinical trials in patients with different age of onset and disease severity. Enzyme replacement therapy, however, has its limitations due to unsatisfactory access of recombinant alpha-glucosidase to the muscle cells and due to the formation of antibodies. To overcome these therapeutic restraints, the development of a more effective enzyme preparation may become necessary.