Continuous Spectrometric Assays for Glutaminyl Cyclase Activity

doi:10.1006/abio.2001.5560

Analytical Biochemistry

Volume 303, Issue 1, 1 April 2002, Pages 49-56

https://doi.org/10.1006/abio.2001.5560 Get rights and content

Abstract

The enzymatic conversion of one chromogenic substrate, $l$ -glutamine-p-nitroanilide, and two fluorogenic substrates, $l$ -glutaminyl-2-naphthylamide and $l$ -glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.

References (24)

W.H.J. Busby et al.
An enzyme(s) that converts glutaminyl-peptides into pyroglutamyl-peptides: Presence in pituitary, brain, adrenal medulla, and lymphocytes
J. Biol. Chem.
(1987)
P.A. Sykes et al.
Evidence for tissue-specific forms of glutaminyl cyclase
FEBS Lett.
(1999)
S.A. Hinke et al.
Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon
J. Biol. Chem.
(2000)
A.P. Consalvo et al.
A rapid fluorometric assay for N-terminal glutaminyl cyclase activity using high-performance liquid chromatography
Anal. Biochem.
(1988)
J.B. Koger et al.
Assay of glutaminylpeptide cyclase
Methods Enzymol.
(1989)
R.C.J. Bateman
A spectrophotometric assay for glutaminyl-peptide cyclizing enzymes
J. Neurosci. Methods
(1989)
M.M. Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)
M.Y. Gololobov et al.
Steady-state kinetics of glutamine cyclotransferase
Arch. Biochem. Biophys.
(1994)
K. Fujiwara et al.
The substrate specificity of pyrrolidone carboxylyl peptidase from Bacillus amyloliquefaciens
Biochim. Biophys. Acta
(1979)
R. Wynn et al.
Chemical modification of protein thiols: Formation of mixed disulfides
Methods Enzymol.
(1995)

M. Messer

Enzymatic cyclization of $l$ -glutamine and $l$ -glutaminyl peptides

Nature

(1963)

Cited by (49)

Discovery of potent indazole-based human glutaminyl cyclase (QC) inhibitors as Anti-Alzheimer's disease agents
2022, European Journal of Medicinal Chemistry
The toxic pyroglutamate form of amyloid-β (pE-Aβ) is important for the pathogenesis of early Alzheimer's disease (AD); therefore, reducing pE-Aβ by inhibiting glutaminyl cyclase (QC) provides a promising strategy for developing disease-modifying AD drugs.
In this study, potent and selective QC inhibitors with desirable drug-like properties were discovered by replacing the 3,4-dimethoxyphenyl group in a QC inhibitor with a bioisosteric indazole surrogate. Among them, 3-methylindazole-6-yl and 3-methylindazole-5-yl derivatives with an N-cyclohexylurea were identified as highly potent inhibitors with IC₅₀ values of 3.2 nM and 2.3 nM, respectively, both of which were approximately 10-fold more potent than varoglutamstat. In addition, the three inhibitors significantly reduced pE-Aβ_3-40 levels in an acute animal model after intracerebroventricular (icv) injection and were selective for hQC. Further in vitro pharmacokinetic and toxicity studies, including those investigating cytotoxicity, hERG inhibition, blood–brain barrier (BBB) permeability and metabolic stability, indicated that N-(3-methylindazole-6-yl)-N’-(cyclohexyl)urea derivative exhibited the most promising efficacy, selectivity and drug-like profile; thus, it was evaluated for its in vivo efficacy in an AD model.
Discovery of highly potent human glutaminyl cyclase (QC) inhibitors as anti-Alzheimer's agents by the combination of pharmacophore-based and structure-based design
2021, European Journal of Medicinal Chemistry
The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the treatment of early Alzheimer's disease (AD) by reducing the amount of the toxic pyroform of β-amyloid (Aβ_Ν3pE) in the brains of AD patients. In this work, we identified potent QC inhibitors with subnanomolar IC₅₀ values that were up to 290-fold higher than that of PQ912, which is currently being tested in Phase II clinical trials.
Among the tested compounds, the cyclopentylmethyl derivative (214) exhibited the most potent in vitro activity (IC₅₀ = 0.1 nM), while benzimidazole (227) showed the most promising in vivo efficacy, selectivity and druggable profile. 227 significantly reduced the concentration of pyroform Aβ and total Aβ in the brain of an AD animal model and improved the alternation behavior of mice during Y-maze tests. The crystal structure of human QC (hQC) in complex with 214 indicated tight binding at the active site, supporting that the specific inhibition of QC results in potent in vitro and in vivo activity. Considering the recent clinical success of donanemab, which targets Aβ_Ν3pE, small molecule-based QC inhibitors may also provide potential therapeutic options for early-stage AD treatment.
A Unique Carboxylic-Acid Hydrogen-Bond Network (CAHBN) Confers Glutaminyl Cyclase Activity on M28 Family Enzymes
2021, Journal of Molecular Biology
Proteins with sequence or structure similar to those of di-Zn exopeptidases are usually classified as the M28-family enzymes, including the mammalian-type glutaminyl cyclases (QCs). QC catalyzes protein N-terminal pyroglutamate formation, a posttranslational modification important under many physiological and pathological conditions, and is a drug target for treating neurodegenerative diseases, cancers and inflammatory disorders. Without functional characterization, mammalian QCs and their orthologs remain indistinguishable at the sequence and structure levels from other M28-family proteins, leading to few reported QCs. Here, we show that a low-barrier carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity and discriminates QCs from M28-family peptidases. We demonstrate that the CAHBN-containing M28 peptidases deposited in the PDB are indeed QCs. Our analyses identify several thousands of QCs from the three domains of life, and we enzymatically and structurally characterize several. For the first time, the interplay between a CAHBN and the binuclear metal-binding center of mammalian QCs is made clear. We found that the presence or absence of CAHBN is a key discriminator for the formation of either the mono-Zn QCs or the di-Zn exopeptidases. Our study helps explain the possible roles of QCs in life.
Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis
2021, Journal of Biological Chemistry
Citation Excerpt :
Kinetic measurements using the artificial fluorometric substrate H-Gln-AMC (43) clearly show that all three recombinant bacteroidal enzymes exhibit QC activity (Table 1; Fig. S1A).
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer’s disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
Glutaminyl cyclase inhibitor exhibits anti-inflammatory effects in both AD and LPS-induced inflammatory model mice
2019, International Immunopharmacology
Citation Excerpt :
For AD mice, one day after the behavioral testing, mice were sacrificed by CO2 inhalation; the brains were removed, divided into 3 groups (cortex, hippocampus, hemisphere) and homogenized in chilled PBS (m/v 1:9) complemented with 1% (v/v) protease inhibitors (Roche, Basel, Switzerland). After centrifugation, the supernatants were used for QC activity assay according to the report [47]. Briefly, reactions were started by the addition of supernatants into the assay buffer (30 U/mL glutamate dehydrogenase, 3.84 mM Gln-Gln, 14 mM α-Ketoglutaric acid, 0.3 mM NADH, 150 mM NaCl, 50 mM Tris, pH 8.0) at 25 °C.
Up-regulated glutaminyl cyclase (QC) plays crucial roles in the initiation of Alzheimer's disease (AD) and kinds of chronic diseases mediated by inflammation. QC is supposed as a novel target for the therapeutics of these diseases. Here, we explored the anti-inflammation effects of diphenyl conjugated imidazole (DPCI) derivatives which were previously designed, synthesized and evaluated as novel QC inhibitors for AD treatment in our lab. Behavioral tests, QC activity assay, histology and ELISA analysis were conducted on both AD and lipopolysaccharides (LPS)-induced inflammatory model mice. It was shown that behavioral and cognitive performance in AD mice treated with the selected compound DPCI-23 were enhanced notably. QC activity, the formation of pE-Aβ and Aβ plaques and the activation of astrocytes and microglia cells in AD mice brains were inhibited, and the levels of inflammatory factors such as IL-6, IL-1β and TNF-α in serum were reduced remarkably. Furthermore, elevated QC activity in inflammatory mice brains was also inhibited, and levels of IL-1β, IL-1ra, TNF-α and CCL2 in serum, kidneys and brains together with the activated astrocytes and microglia cells in brains were all repressed significantly after the treatment of DPCI-23. These findings observed in this research demonstrated the anti-inflammation potency of DPCI-23 in modal of AD and inflammation by inhibiting QC activity, and may contribute to the employment of QC inhibitors for the prevention and treatment of AD and other inflammatory diseases.
Structure-activity relationship investigation of Phe-Arg mimetic region of human glutaminyl cyclase inhibitors
2018, Bioorganic and Medicinal Chemistry
Citation Excerpt :
The pyrimidine 67 was synthesized from amine 53 by reacting with 2-chloropyrimidine. As previously reported, to evaluate the human QC inhibition of the synthesized compounds we performed the QC activity assays employing a fluorogenic substrate, Gln-AMC (l-glutamine 7-amido-4-methylcoumarin), and pyroglutamyl peptidase (pGAP) as an auxiliary enzyme.25 First, we investigated a series of 3-aminoalkyloxy-4-methoxyphenyl analogues represented as template B (Table 1).
Glutamyl cyclase (QC) is a promising therapeutic target because of its involvement in the pathogenesis of Alzheimer’s disease. In this study, we developed novel QC inhibitors that contain 3-aminoalkyloxy-4-methoxyphenyl and 4-aminoalkyloxyphenyl groups to replace the previously developed pharmacophore. Several potent inhibitors were identified, showing IC₅₀ values in a low nanomolar range, and were further studied for in vitro toxicity and in vivo activity. Among these, inhibitors 51 and 53 displayed the most potent Aβ_N3pE−40-lowering effects in in vivo acute model with reasonable BBB penetration, without showing cytotoxicity and hERG inhibition. The molecular modeling analysis of 53 indicated that the salt bridge interaction and the hydrogen bonding in the active site provided a high potency. Given the potent activity and favorable BBB penetration with low cytotoxicity, we believe that compound 53 may serve as a potential candidate for anti-Alzheimer’s agents.

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¹: To whom correspondence should be addressed at probiodrug AG, Weinbergweg 22, 06120 Halle, Germany. Fax: 49 345 5559901. E-mail: [email protected].

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Regular ArticleContinuous Spectrometric Assays for Glutaminyl Cyclase Activity

Abstract

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Anal. Biochem.

Methods Enzymol.

J. Neurosci. Methods

Anal. Biochem.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

Methods Enzymol.

Enzymatic cyclization of l-glutamine and l-glutaminyl peptides

Nature

Regular Article
Continuous Spectrometric Assays for Glutaminyl Cyclase Activity

Enzymatic cyclization of $l$ -glutamine and $l$ -glutaminyl peptides