A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity

2008 ◽  
Vol 389 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Jian-Qiang Fan
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Protein Misfolding ◽  
Enzyme Inhibitors ◽  
Lysosomal Storage ◽  
New Paradigm ◽  
Inherited Disorders ◽  
Pharmacological Chaperone ◽  
Mutant Proteins ◽  
Enzyme Active Site

Abstract Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.

Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants

2006 ◽  
Vol 290 (4) ◽  
pp. C1076-C1082 ◽  
Author(s):  
Gary Hin-Fai Yam ◽  
Nils Bosshard ◽  
Christian Zuber ◽  
Beat Steinmann ◽  
Jürgen Roth
Keyword(s):  
Enzyme Activity ◽  
Fabry Disease ◽  
Competitive Inhibitor ◽  
Apparent Difference ◽  
Residual Enzyme Activity ◽  
Lysosomal Storage ◽  
Electron Microscopic ◽  
Pharmacological Chaperone ◽  
Mutant Proteins

Fabry disease is a lysosomal storage disorder caused by deficiency of α-galactosidase A (α-Gal A) resulting in lysosomal accumulation of glycosphingolipid globotriosylceramide Gb3. Misfolded α-Gal A variants can have residual enzyme activity but are unstable. Their lysosomal trafficking is impaired because they are retained in the endoplasmic reticulum (ER) by quality control. Subinhibitory doses of the competitive inhibitor of α-Gal A, 1-deoxygalactonojirimycin (DGJ), stabilize mutant α-Gal A in vitro and correct the trafficking defect. We showed by immunolabeling that the chaperone-like action of DGJ significantly reduces the lysosomal Gb3 storage in human Fabry fibroblasts harboring the novel mutations T194I and V390fsX8. The specificity of the DGJ effect was proven by RNA interference. Electron microscopic morphometry demonstrated a reduction of large-size, disease-associated lysosomes and loss of characteristic multilamellar lysosomal inclusions on DGJ treatment. In addition, the pre-Golgi intermediates were decreased. However, the rough ER was not different between DGJ-treated and untreated cells. Pulse-chase experiments revealed that DGJ treatment resulted in maturation and stabilization of mutant α-Gal A. Genes involved in cell stress signaling, heat shock response, unfolded protein response, and ER-associated degradation show no apparent difference in expression between untreated and DGJ-treated fibroblasts. The DGJ treatment has no apparent cytotoxic effects. Thus our data show the usefulness of a pharmacological chaperone for correction of the lysosomal storage in Fabry fibroblasts harboring different mutations with residual enzyme activity. Pharmacological chaperones acting on misfolded, unstable mutant proteins that exhibit residual biological activity offer a convenient and cost-efficient therapeutic strategy.

scholarly journals Kinetics of protein modification reactions. Plot of fractional enzyme activity versus extent of protein modification in cases where all modifiable groups are essential for enzyme activity

1984 ◽  
Vol 223 (1) ◽  
pp. 259-262 ◽  
Author(s):  
E T Rakitzis
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Protein Modification ◽  
Enzyme Molecule ◽  
Single Group ◽  
Initial Portion ◽  
Enzyme Active Site ◽  
Kinetics Of

The plot of fractional enzyme activity versus extent of protein modification, for cases where all enzyme modifiable groups of a certain kind are essential for activity, is found to be nearly independent of the number, per enzyme active site, of modifiable groups involved. Such plots usually, by a fallacious extension of the initial portion of the plot on the extent-of-modification axis, are interpreted to mean the modification of one single group per enzyme active site (or per enzyme molecule). The possible relevance of these findings to cases in the literature is discussed.

scholarly journals Structural basis of pharmacological chaperoning for human β-galactosidase

2014 ◽  
Vol 70 (a1) ◽  
pp. C843-C843
Author(s):  
Hironori Suzuki ◽  
Umeharu Ohto ◽  
Katsumi Higaki ◽  
Teresa Mena-Barragán ◽  
Matilde Aguilar-Moncayo ◽  
...  
Keyword(s):  
Active Site ◽  
Neurodegenerative Disorder ◽  
Lysosomal Storage Diseases ◽  
Japanese Patients ◽  
Structural Basis ◽  
Lysosomal Storage ◽  
Active Site Residues ◽  
Recognition Mechanism ◽  
Gm1 Gangliosidosis ◽  
Pharmacological Chaperone

GM1-gangliosidosis and Morquio B are rare lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme human β-Galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation (ERAD) due to mutations in the β-Gal gene. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding, prevent ERAD and promote trafficking to the lysosome. Here, we present the enzymological properties of wild-type human β-Gal and two representative mutations in GM1 gangliosidosis Japanese patients (R201C and I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we determined the crystal structures of β-Gal in complex with these compouds and two structurally related analogues to elucidate the detailed atomic view of the recognition mechanism. All compounds bind to the active site of β-Gal with the sugar moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity and the PC potential are strongly affected by the mono or bicyclic structure of the core as well as the orientation, the nature and the length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.

scholarly journals α-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin

1980 ◽  
Vol 187 (2) ◽  
pp. 345-352 ◽  
Author(s):  
N I Tarasova ◽  
G I Lavrenova ◽  
V M Stepanov
Keyword(s):  
Enzyme Activity ◽  
Carboxy Group ◽  
Active Site ◽  
Protein Modification ◽  
Phenacyl Bromide ◽  
Enzyme Molecule ◽  
Noticeable Effect ◽  
Porcine Pepsin ◽  
Enzyme Active Site ◽  
Phenacyl Bromides

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.

scholarly journals Conserved Active-Site Residues Associated with OAS Enzyme Activity and Ubiquitin-Like Domains Are Not Required for the Antiviral Activity of goOASL Protein against Avian Tembusu Virus

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 371 ◽  
Author(s):  
Shun Chen ◽  
Chao Yang ◽  
Jinyue Zhang ◽  
Zhen Wu ◽  
Mingshu Wang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Antiviral Activity ◽  
Active Site ◽  
Antiviral Effect ◽  
Dependent Manner ◽  
Active Site Residues ◽  
Oligoadenylate Synthetase ◽  
Mutant Proteins ◽  
Tembusu Virus

Interferon (IFN)-induced 2′-5′-oligoadenylate synthetase (OAS) proteins exhibit an extensive and efficient antiviral effect against flavivirus infection in mammals and birds. Only the 2′-5′-oligoadenylate synthetase-like (OASL) gene has been identified thus far in birds, except for ostrich, which has both OAS1 and OASL genes. In this study, we first investigated the antiviral activity of goose OASL (goOASL) protein against a duck-origin Tembusu virus (DTMUV) in duck embryo fibroblast cells (DEFs). To investigate the relationship of conserved amino acids that are related to OAS enzyme activity and ubiquitin-like (UBL) domains with the antiviral activity of goOASL, a series of mutant goOASL plasmids was constructed, including goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T. Interestingly, all these mutant proteins significantly inhibited the replication of DTMUV in DEFs in a dose-dependent manner. Immunofluorescence analysis showed that the goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T proteins were located not only in the cytoplasm where DTMUV replicates but also in the nucleus of DEFs. However, the goOASL and goOASL mutant proteins were mainly colocalized with DTMUV in the cytoplasm of infected cells. Our data indicated that goOASL could significantly inhibit DTMUV replication in vitro, while the active-site residues S64, D76, D78 and D144, which were associated with OAS enzyme activity, the UBL domains were not required for the antiviral activity of goOASL protein.

scholarly journals The molecular basis of the effect of temperature on enzyme activity

2009 ◽  
Vol 425 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Roy M. Daniel ◽  
Michelle E. Peterson ◽  
Michael J. Danson ◽  
Nicholas C. Price ◽  
Sharon M. Kelly ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Classical Model ◽  
Active Form ◽  
Enzyme Reaction ◽  
Effect Of Temperature ◽  
Enzyme Active Site ◽  
Temperature Ranges ◽  
The Difference ◽  
Two State Model

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (ΔHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of ΔG*cat made on the basis of the two-state (‘Classical’) model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

scholarly journals Pharmacological Chaperones: A Therapeutic Approach for Diseases Caused by Destabilizing Missense Mutations

2020 ◽  
Vol 21 (2) ◽  
pp. 489 ◽  
Author(s):  
Ludovica Liguori ◽  
Maria Monticelli ◽  
Mariateresa Allocca ◽  
Bruno Hay Mele ◽  
Jan Lukas ◽  
...  
Keyword(s):  
Small Molecules ◽  
Specific Binding ◽  
Total Activity ◽  
Missense Mutations ◽  
Lysosomal Storage ◽  
Pharmacological Chaperone ◽  
Mutant Proteins ◽  
Pharmacological Chaperones ◽  
Protein Mutants ◽  
Competitive Inhibitors

The term “pharmacological chaperone” was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein.

scholarly journals The Organ Distribution, Characterization and Modification of Acetylcholinesterase Activity in Adult African Grasshopper: Zonocerus sp Linn.

2019 ◽  
pp. 1-9
Author(s):  
E. A. Fajemisin ◽  
O. S. Bamidele ◽  
S. O. Ogunsola ◽  
E. A. Aiyenuro
Keyword(s):  
Enzyme Activity ◽  
Protein Content ◽  
Active Site ◽  
Ache Activity ◽  
Specific Activity ◽  
Acetylcholinesterase Activity ◽  
Organ Distribution ◽  
Enzyme Active Site ◽  
University Of Technology ◽  
Tryptophan Residues

Aim: To determine the organ distribution and characterization of acetylcholinesterase in the adult African variegated grasshoppers – Zonocerus variegatus and Zonocerus elegans. (Zonocerus Sp. Linn) Place and Duration of the Study: The insect model: African variegated grasshoppers are gotten from the Open green fields at the Federal University of Technology, Akure, Nigeria, and research was carried out between March and June, 2016 in the Enzymology laboratory, Biochemistry department, Federal University of Technology, Akure, Nigeria. Methodology: Twenty (20) adults variegated grasshoppers were taken from the Open field in the University community, and taken to the Biology department for Identification. After identification, the specimen was weighed, freeze, dissected into fractions (Head, Thorax and Abdomen) and then homogenized to get the crude protein extract. The crude enzyme extract is further purified using the Ion-exchange chromatography with column bed packed with DEAE – Sephadex A50. The protein content of the purified AChE was determined using the Lowry method while the Acetylcholinesterase activity was determined by the Ellman’s assay procedures. The characterization of AChE was tested by modifying agent such as N-Bromo Succinamide (NBS) which confirms the presence of key aromatic proteins involve in catalysis at the active site of the enzyme. Results: The protein concentration according to their fractions: Head (35.7%), Thorax (29.2%), and Abdomen (35.1%). The AChE activity according to their fractions: Head (38.6%), Thorax (23.7%), and Abdomen (37.7%). The specific activity which relates the AChE activity to protein content is given: Head (28.8%), Thorax (40.4%), and Abdomen (30.8%). From the Organ distribution and AChE activity, it was observed that the Head Fractions has the Highest protein content, and Enzyme activity. Comparatively, there are slight differences in the Enzyme activity of the Head and Abdominal fractions which represents the two peaks in the AChE chart. As well, the thorax has the highest specific activity. The modification by the chemical agent NBS shows a drastic decrease (about 50%) in Enzyme activity and characterize enzyme active site with aromatic proteins especially tryptophan residues. Conclusion: Research findings shows the dominance of AChE protein in the Head region, hence high enzyme activity (useful for nervous coordination) as well as presence of tryptophan residues at the enzyme active site. The importance of research is useful in enzymology, neuroscience and public health.

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