scholarly journals Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

2021 ◽  
Author(s):  
Sanjay Bhattarai ◽  
Sujan Devkota ◽  
Michael S Wolfe
Keyword(s):  
Transition State ◽  
Active Site ◽  
Transmembrane Domain ◽  
Competitive Inhibition ◽  
Solid Phase ◽  
Substrate Recognition ◽  
Solid Phase Peptide Synthesis ◽  
Rational Drug Design ◽  
Binding Assays ◽  
Secretase Activity

γ-Secretase, a membrane-embedded aspartyl protease complex with presenilin as the catalytic component, cleaves within the transmembrane domain (TMD) of its many substrates, which include the amyloid precursor protein (APP) of Alzheimer's disease (AD). APP TMD is processively cut by γ-secretase through endoproteolysis followed by tricarboxypeptidase "trimming", the latter function being deficient with mutations causing hereditary AD. Toward better understanding of substrate recognition and hydrolytic reactions catalyzed by this enzyme within its hydrophobic milieu, we recently developed a prototype substrate TMD mimetic as a chemical tool for structural analysis (Bhattarai S et al. J. Am. Chem. Soc., 2020, 142(7): 3351-3355). This TMD mimetic--composed of a helical peptide inhibitor (HPI) connected through a linker to a transition-state analog inhibitor (TSA)--simultaneously engages the substrate docking exosite and the active site and is pre-organized to trap the protease transition state of carboxypeptidase trimming. In this study, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate. New HPI-TSA conjugates were synthesized using an earlier established divergent strategy, which involved the construction of a tripeptidomimetic building block followed by solid-phase peptide synthesis (SPPS). For each class of structural probe, SAR analysis led to discovery of highly potent prototypes with stoichiometric or low-nanomolar inhibition. These TMD mimetics exhibited non-competitive inhibition of γ-secretase activity and occupy both docking and active site as demonstrated by enzyme cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with γ-secretase, ultimately aiding rational drug design.

scholarly journals Effect of N-methylation on the modulation by synthetic peptides of the activity of the complement-factor-B-derived serine proteinase CVFBb

1990 ◽  
Vol 270 (2) ◽  
pp. 531-537 ◽  
Author(s):  
A Berkovich ◽  
M C O'Keefe ◽  
P Hensley ◽  
L Caporale
Keyword(s):  
Active Site ◽  
Solid Phase ◽  
Alternative Pathway ◽  
Substrate Recognition ◽  
Serine Proteinase ◽  
Fold Increase ◽  
Catalytic Triad ◽  
Complement Factor ◽  
Peptide Substrate ◽  
Scissile Bond

Although they share the active-site catalytic triad of less-specific enzymes such as trypsin and chymotrypsin, the serine proteinases of the complement and coagulation cascades each cleave a highly restricted set of substrates. Peptides with sequences similar to that at which C3 is cleaved by the alternative-pathway complement proteinase CVFBb were synthesized by solid-phase methodology and examined for their effects on the activity of this enzyme as measured by three different types of assays. It was found that a peptide methylated at the scissile bond was a far more effective inhibitor of the cleavage of the protein substrate C5 and of the lysis of guinea-pig erythrocytes by the alternative pathway than was the equivalent unmethylated peptide. Whereas the unmethylated peptide inhibited cleavage of the peptide substrate, the methylated peptide actually stimulated cleavage in this assay. This stimulation was found to be due to a 2.8-fold increase in kcat; the dissociation constant for the substrate was not altered significantly. One model consistent with this behaviour is that the binding of the activator peptide in the extended substrate-recognition region stabilizes a catalytically more active conformation of the active site. A small peptide substrate may have access to such an activated active site, whereas the larger substrate, C5, may be excluded from the site. These results demonstrate that the observed effect of a given compound on activity of an enzyme with an extended substrate-recognition region may depend upon the substrate.

scholarly journals Peptides Fixing Industrial Textile Dyes: A New Biochemical Method in Wastewater Treatment

2019 ◽  
Vol 2019 ◽  
pp. 1-7
Author(s):  
Amor Mosbah ◽  
Habib Chouchane ◽  
Soukaina Abdelwahed ◽  
Alaeddine Redissi ◽  
Manel Hamdi ◽  
...  
Keyword(s):  
Textile Industry ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Pollution Prevention ◽  
Textile Wastewater ◽  
Textile Dye ◽  
Binding Assays ◽  
Biochemical Method ◽  
Phase Synthesis

The aim of the present work was the development of a new biological method for the treatment of textile industry effluents, which is cheaper, more profitable, and eco-friendly. This method is essentially based on the synthesis of dye-fixing peptides. The use of peptides synthesized via a solid-phase synthesis to fix a reference textile dye like “Cibacron blue” (CB) and the performance analysis of binding assays were the main objectives of this study. For this reason, two peptides P1 (NH2-C-G-G-W-R-S-Q-N-Q-G-NH2) and P2 (NH2-C-G-G-R-R-Y-Q-P-D-S-NH2) binding with the CB dye were synthesized by the solid-phase peptide synthesis (SPPS) technique. The obtained results showed significant fixation yields of CB-peptides of 91.5% and 45.9%, respectively, and consequently, their interesting potential as a tool for a new biochemical method in the pollution prevention of textile wastewater.

scholarly journals Antibody-catalyzed formation of a 14-membered ring lactone

2002 ◽  
Vol 80 (12) ◽  
pp. 1643-1645 ◽  
Author(s):  
Michael D Pungente ◽  
Larry Weiler ◽  
Hermann J Ziltener
Keyword(s):  
Monoclonal Antibody ◽  
Transition State ◽  
Active Site ◽  
Large Scale ◽  
Competitive Inhibition ◽  
Membered Ring ◽  
Catalytic Antibodies ◽  
Transition State Analogue ◽  
Catalytic Experiment ◽  
Ether Extraction

Monoclonal antibody (MAb) F123, raised against a macrocyclic phosphonate transition-state analogue, catalyzed an intramolecular transesterification of the corresponding hydroxy ester to give a 14-membered ring lactone. The MAb reaction displayed enzyme-like Michaelis–Menten kinetics with a Km of 255 µM and a kcat of 0.01 min–1 based on p-nitrophenol release and calculated on an active-site basis. Substrate specificity and competitive inhibition by a transition state analogue (Ki = 3 µM) demonstrated that the catalytic activity was associated with binding in the antibody-combining site. The lactone product was isolated from a large-scale catalytic experiment through ether extraction and identified by gas chromatography – mass spectroscopy.Key words: macrocyclization, lactones, catalytic antibodies.

A Strategy for Thioamide Incorporation During Fmoc Solid-Phase Peptide Synthesis with Robust Stereochemical Integrity

2019 ◽  
Author(s):  
luis camacho III ◽  
Bryan J. Lampkin ◽  
Brett VanVeller
Keyword(s):  
Amino Acid ◽  
Functional Groups ◽  
Peptide Synthesis ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Side Chains ◽  
Amino Acid Side Chains ◽  
Peptide Elongation

We describe a method to protect the sensitive stereochemistry of the thioamide—in analogy to the protection of the functional groups of amino acid side chains—in order to preserve the thioamide moiety during peptide elongation.<br>

Analytical Methods for Solid Phase Peptide Synthesis

2004 ◽  
Vol 8 (4) ◽  
pp. 291-301 ◽  
Author(s):  
Giuseppina Sabatino ◽  
Mario Chelli ◽  
Alberto Brandi ◽  
Anna Papini
Keyword(s):  
Peptide Synthesis ◽  
Analytical Methods ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis

Structure-Based Design, Synthesis, Biological Evaluation and Molecular Docking Study of 4-Hydroxy-N'-methylenebenzohydrazide Derivatives Acting as Tyrosinase Inhibitors with Potentiate Anti-Melanogenesis Activities

2020 ◽  
Vol 16 (7) ◽  
pp. 892-902 ◽  
Author(s):  
Aida Iraji ◽  
Mahsima Khoshneviszadeh ◽  
Pegah Bakhshizadeh ◽  
Najmeh Edraki ◽  
Mehdi Khoshneviszadeh
Keyword(s):  
Molecular Docking ◽  
Active Site ◽  
Competitive Inhibition ◽  
Docking Study ◽  
Biological Evaluation ◽  
Primary Response ◽  
Ratio Method ◽  
Molecular Docking Study ◽  
Metal Chelating

Background: Melanogenesis is a process of melanin synthesis, which is a primary response for the pigmentation of human skin. Tyrosinase is a key enzyme, which catalyzes a ratelimiting step of the melanin formation. Natural products have shown potent inhibitors, but some of these possess toxicity. Numerous synthetic inhibitors have been developed in recent years may lead to the potent anti– tyrosinase agents. Objective: A number of 4-hydroxy-N'-methylenebenzohydrazide analogues with related structure to chalcone and tyrosine were constructed with various substituents at the benzyl ring of the molecule and evaluate as a tyrosinase inhibitor. In addition, computational analysis and metal chelating potential have been evaluated. Methods: Design and synthesized compounds were evaluated for activity against mushroom tyrosinase. The metal chelating capacity of the potent compound was examined using the mole ratio method. Molecular docking of the synthesized compounds was carried out into the tyrosine active site. Results: Novel 4-hydroxy-N'-methylenebenzohydrazide derivatives were synthesized. The two compounds 4c and 4g showed an IC50 near the positive control, led to a drastic inhibition of tyrosinase. Confirming in vitro results were performed via the molecular docking analysis demonstrating hydrogen bound interactions of potent compounds with histatidine-Cu+2 residues with in the active site. Kinetic study of compound 4g showed competitive inhibition towards tyrosinase. Metal chelating assay indicates the mole fraction of 1:2 stoichiometry of the 4g-Cu2+ complex. Conclusion: The findings in the present study demonstrate that 4-Hydroxy-N'- methylenebenzohydrazide scaffold could be regarded as a bioactive core inhibitor of tyrosinase and can be used as an inspiration for further studies in this area.

Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

1999 ◽  
Vol 73 (5) ◽  
pp. 3737-3743 ◽  
Author(s):  
Alfred Klausegger ◽  
Birgit Strobl ◽  
Gerhard Regl ◽  
Alexandra Kaser ◽  
Willem Luytjes ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Solid Phase ◽  
Hepatitis Virus ◽  
Bovine Brain ◽  
Brain Extract ◽  
Binding Assays ◽  
Bovine Coronavirus ◽  
Close Relationship ◽  
Influenza C Virus ◽  
Cloned Gene

ABSTRACT We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substratesp-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α1macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

Fmoc Solid Phase Peptide Synthesis

1999 ◽  
Keyword(s):  
Peptide Synthesis ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Peptide Chain ◽  
Standard Approach ◽  
Side Chain ◽  
Complex Structures ◽  
Protein Conjugation ◽  
Chemoselective Ligation ◽  
Routine Production

In the years since the publication of Atherton and Sheppard's volume, the technique of Fmoc solid-phase peptide synthesis has matured considerably and is now the standard approach for the routine production of peptides. The basic problems outstanding at the time of publication of this earlier work have now been, for the most part, solved. As a result, innovators in the field have focussed their efforts to develop methodologies and chemistry for the synthesis of more complex structures. The focus of this new volume is much broader, and covers not only the essential procedures for the production of linear peptides but also more advanced techniques for preparing cyclic, side-chain modified, phospho- and glycopeptides. Many other methods also deserving attention have been included: convergent peptide synthesis; peptide-protein conjugation; chemoselective ligation; and chemoselective purification. The difficult preparation of cysteine and methionine-containing peptides is also covered, as well as methods for overcoming aggregation during peptide chain assembly and a survey of available automated instrumentation.

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