| Full text | |
| Author(s): |
Vilela, Adriana Ferreira Lopes
[1]
;
Narciso dos Reis, Vitor Eduardo
[1]
;
Cardoso, Carmen Lucia
[1]
Total Authors: 3
|
| Affiliation: | [1] Univ Sao Paulo, Grp Cromatog Bioafinidade & Prod Nat, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Ribeirao Preto - Brazil
Total Affiliations: 1
|
| Document type: | Journal article |
| Source: | FRONTIERS IN CHEMISTRY; v. 9, JUL 8 2021. |
| Web of Science Citations: | 0 |
| Abstract | |
We have developed a dual enzymatic system assay involving liquid chromatography-mass spectrometry (LC-MS) to screen AChE and BACE1 ligands. A fused silica capillary (30 cm x 0.1 mm i.d. x 0.362 mm e.d.) was used as solid support. The co-immobilization procedure encompassed two steps and random immobilization. The resulting huAChE+BACE1-ICER/MS was characterized by using acetylcholine (ACh) and JMV2236 as substrates. The best conditions for the dual enzymatic system assay were evaluated and compared to the conditions of the individual enzymatic system assays. Analysis was performed in series for each enzyme. The kinetic parameters (K-Mapp) and inhibition assays were evaluated. To validate the system, galantamine and a beta-secretase inhibitor were employed as standard inhibitors, which confirmed that the developed screening assay was able to identify reference ligands and to provide quantitative parameters. The combination of these two enzymes in a single on-line system allowed possible multi-target inhibitors to be screened and identified. The innovative huAChE+BACE1-ICER/MS dual enzymatic system reported herein proved to be a reliable tool to identify and to characterize hit ligands for AChE and BACE1 in an enzymatic competitive environment. This innovative system assay involved lower costs; measured the product from enzymatic hydrolysis directly by MS; enabled immediate recovery of the enzymatic activity; showed specificity, selectivity, and sensitivity; and mimicked the cellular process. (AU) | |
| FAPESP's process: | 14/50249-8 - Green chemistry: sustainable synthetic methods employing benign solvents, safer reagents, and bio-renewable feedstock |
| Grantee: | Arlene Gonçalves Corrêa |
| Support Opportunities: | Research Grants - Research Centers in Engineering Program |
| FAPESP's process: | 16/02873-0 - Acetylcholinesterase and beta-secretase 1: a study of co-immobilization conditions for screening of ligands |
| Grantee: | Adriana Ferreira Lopes Vilela |
| Support Opportunities: | Scholarships in Brazil - Doctorate |
| FAPESP's process: | 19/05363-0 - Enzyme ligand screening: an on-flow approach via LC-MS/MS |
| Grantee: | Carmen Lúcia Cardoso |
| Support Opportunities: | Regular Research Grants |