A growth-based assay using fluorescent protein emission to screen for S-adenosylmethionine synthetase inhibitors.
Drug Dev Res
; 85(1): e22122, 2024 Feb.
Article
em En
| MEDLINE
| ID: mdl-37819020
ABSTRACT
The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Criptosporidiose
/
Cryptosporidium
Limite:
Humans
Idioma:
En
Revista:
Drug Dev Res
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos