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  • 1
    In: Biotechnology Journal, July 2018, Vol.13(7), pp.n/a-n/a
    Description: Trastuzumab is a recombinant, humanized monoclonal antibody glycoprotein that selectively targets the extracellular domain of human epidermal growth factor receptor 2 protein (Her2) and is an approved anticancer drug. In this study, the authors demonstrate that amino acid‐based advanced formulation development enables ideal balancing between high physical and chemical stability of highly concentrated therapeutic antibody formulations with low viscosities using the model antibody trastuzumab. This work is of significant importance for the manufacturing of stable highly concentrated therapeutic antibodies.
    Keywords: Antibodies ; Biotherapeutics ; Chromatography ; Medical Biotechnology ; Protein Aggregation
    ISSN: 1860-6768
    E-ISSN: 1860-7314
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  • 2
    Language: English
    In: The Journal of biological chemistry, 01 October 2010, Vol.285(40), pp.30686-97
    Description: Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). Sapropterin dihydrochloride, the synthetic form of BH(4), was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH(4), a previously unknown finding. Analyses of structurally preactivated variants substantiated BH(4)-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.
    Keywords: Biopterin -- Analogs & Derivatives ; Coenzymes -- Chemistry ; Phenylalanine -- Chemistry ; Phenylalanine Hydroxylase -- Chemistry
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 3
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(4), p.e93852
    Description: The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central β-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: The Journal of organic chemistry, 05 April 2002, Vol.67(7), pp.2087-92
    Description: The two epimeric covalent hydrates A and B of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine are metabolically stable analogues of hypothetical intermediates proposed in the reactions catalyzed by riboflavin synthase and lumazine synthase. To confirm the stereochemical assignments previously based solely on results for epimer B, a (15)N[(19)F] REDOR NMR study was performed on the complex formed from epimer A and a recombinant, uniformly (15)N-labeled F22W mutant of Bacillus subtilis lumazine synthase. The results indicate that the fluorines of the ligands are closer to the side chain nitrogens of Arg127 and farther away from the side chain nitrogens of Lys135 in epimer B than in epimer A. These results are consistent with the assignment of the earlier 7R configuration of epimer A and the 7S configuration of epimer B.
    Keywords: Bacillus Subtilis -- Enzymology ; Multienzyme Complexes -- Metabolism ; Pteridines -- Chemistry
    ISSN: 0022-3263
    E-ISSN: 15206904
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  • 5
    Language: English
    In: Journal of Biological Chemistry, 04/13/2001, Vol.276(15), pp.11524-11530
    Description: Conserved amino acid residues of riboflavin synthase from Escherichia coli were modified by site-directed mutagenesis. Replacement or deletion of phenylalanine 2 afforded catalytically inactive proteins. S41A and H102Q mutants had substantially reduced reaction velocities. Replacements of various other conserved polar residues had little impact on catalytic activity. super(19)F NMR protein perturbation experiments using a fluorinated intermediate analog suggest that the N-terminal sequence motif MFTG is part of one of the substrate-binding sites of the protein.
    Keywords: Escherichia Coli ; Protein Structure ; N.M.R. ; Phenylalanine ; Site-Directed Mutagenesis ; Riboflavin Synthase ; Active Sites ; Enzymes ; Amino Acid Sequence ; Amino Acid Sequence;
    ISSN: 0021-9258
    E-ISSN: 1083-351X
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  • 6
    In: Biochemistry, July 27, 1999, Vol.38(30), p.9735(1)
    Description: The role of the intraflavin and the 4'-ribityl-hydroxyl-N(1) hydrogen bonds in the stabilization of reduced forms of human electron-transfer flavoprotein (ETF) was investigated. This was done by synthesizing 4'-deoxy-FAD and reconstituting ETF with the FAD analogue. The 4'-ribityl-hydroxyl-N(1) hydrogen bond was found to be essential for stabilizing the anionic semiquinone and anion hydroquinone oxidation states of ETF and may provide a route for electron transfer between the ETF flavin and the ETF-ubiquinone oxidoreductase flavin.
    Keywords: Proteins -- Research ; Flavonoids -- Research ; Hydrogen Bonds -- Research ; Oxidation-reduction Reactions -- Research
    ISSN: 0006-2960
    E-ISSN: 1943295X
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  • 7
    In: Human Molecular Genetics, 2011, Vol. 20(13), pp.2628-2641
    Description: 4 in vitro in vivo 4 4 4 4 4 The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH 4 cofactor) in the context of enzyme function in vitro and in vivo , we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH 4 concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH 4 loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH 4 ratio. Finally, evaluation of data from BH 4 loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH 4 -responsive PAH deficiency.
    Keywords: Data Processing ; Cofactors ; Kinetics ; Landscape ; Enzymes ; Phenylalanine 4-Monooxygenase ; Genotypes ; Mutation ; Drugs ; Phenylalanine ; Phenylketonuria ; Human Genetics;
    ISSN: 0964-6906
    E-ISSN: 1460-2083
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  • 8
    Language: English
    In: Vaccine, 17 April 2014, Vol.32(19), pp.2231-2240
    Description: Currently, the need for cooled storage and the impossibility of terminal sterilisation are major drawbacks in vaccine manufacturing and distribution. To overcome current restrictions a preclinical safety and efficacy study was conducted to evaluate new influenza A vaccine formulations regarding thermal resistance, resistance against irradiation-mediated damage and storage stability. We evaluated the efficacy of novel antigen stabilizing and protecting solutions (SPS) to protect influenza A(H1N1)pdm09 split virus antigen under experimental conditions and . Original or SPS re-buffered vaccine (Pandemrix) was spray-dried and terminally sterilised by irradiation with 25 kGy (e-beam). Antigen integrity was monitored by SDS-PAGE, dynamic light scattering, size exclusion chromatography and functional haemagglutination assays. screening experiments revealed a number of highly stable compositions containing glycyrrhizinic acid (GA) and/or chitosan. The most stable composition was selected for storage tests and assessment of seroconversion in non-human primates (Macaca fascicularis) using a prime-boost strategy. Redispersed formulations with original adjuvant were administered intramuscularly. Storage data revealed high stability of protected vaccines at 4 °C and 25 °C, 60% relative humidity, for at least three months. Animals receiving original Pandemrix exhibited expected levels of seroconversion after 21 days (prime) and 48 days (boost) as assessed by haemagglutination inhibition and microneutralisation assays. Animals vaccinated with spray-dried and irradiated Pandemrix failed to exhibit seroconversion after 21 days whereas spray-dried and irradiated, SPS-protected vaccines elicited similar seroconversion levels to those vaccinated with original Pandemrix. Boost immunisation with SPS-protected vaccine resulted in a strong increase in seroconversion but had only minor effects in animals treated with non SPS-protected vaccine. In conclusion, utilising the SPS formulation technology, spray-drying and terminal sterilisation of influenza A(H1N1)pdm09 split virus vaccine is feasible. Findings indicate the potential utility of such formulated vaccines e.g. for needle-free vaccination routes and delivery to countries with uncertain cold chain facilities.
    Keywords: Thermal Stability ; Viruses ; Irradiation ; Excipients ; Stabilising and Protecting Solution (Sps) ; Dry Powder Vaccine ; Medicine ; Biology ; Veterinary Medicine ; Pharmacy, Therapeutics, & Pharmacology
    ISSN: 0264-410X
    E-ISSN: 1873-2518
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  • 9
    Language: English
    In: The American Journal of Human Genetics, 2008, Vol.83(1), pp.5-17
    Description: A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiopterin (BH ), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten gene mutations identified in patients with BH -responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, I65S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H). Residual enzyme activity was generally high, but allostery was disturbed in almost all cases and pointed to altered protein conformation. This was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding—with particular impact on the regulatory domain—observed in most variants. Three-dimensional modeling revealed the involvement of functionally relevant amino acid networks that may communicate misfolding throughout the protein. Our results substantiate the view that PAH deficiency is a protein-misfolding disease in which global conformational changes hinder molecular motions essential for physiological enzyme function. Thus, PKU has evolved from a model of a genetic disease that leads to severe neurological impairment to a model of a treatable protein-folding disease with loss of function.
    Keywords: Biology
    ISSN: 0002-9297
    E-ISSN: 1537-6605
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  • 10
    In: Human Molecular Genetics, 2012, Vol. 21(8), pp.1877-1887
    Description: Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH 4 ), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH 4 shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institute's chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice ( Pah enu1 ). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.
    Keywords: Medicine ; Biology;
    ISSN: 0964-6906
    E-ISSN: 1460-2083
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