Protease Inhibition Mechanism of Camelid-like Synthetic Human Antibodies.
Biochemistry
; 59(40): 3802-3812, 2020 10 13.
Article
em En
| MEDLINE
| ID: mdl-32997500
ABSTRACT
Macromolecular protease inhibitors and camelid single-domain antibodies achieve their enzymic inhibition functions often through protruded structures that directly interact with catalytic centers of targeted proteases. Inspired by this phenomenon, we constructed synthetic human antibody libraries encoding long CDR-H3s, from which highly selective monoclonal antibodies (mAbs) that inhibit multiple proteases were discovered. To elucidate their molecular mechanisms, we performed in-depth biochemical characterizations on a panel of matrix metalloproteinase (MMP)-14 inhibitory mAbs. Assays included affinity and potency measurements, enzymatic kinetics, a competitive enzyme-linked immunosorbent assay, proteolytic stability, and epitope mapping followed by quantitative analysis of binding energy changes. The results collectively indicated that these mAbs of convex paratopes were competitive inhibitors recognizing the vicinity of the active cleft, with their significant epitopes scattered across the north and south rims of the cleft. Remarkably, identified epitopes were the surface loops that were highly diverse among MMPs and predominately located at the prime side of the proteolytic site, shedding light on the mechanisms of target selectivity and proteolytic resistance. Substrate sequence profiling and paratope mutagenesis further suggested that mAb 3A2 bound to the active-site cleft in a canonical (substrate-like) manner, by direct interactions between 100hNLVATP100m of its CDR-H3 and subsites S1-S5' of MMP-14. Overall, synthetic mAbs carrying convex paratopes can achieve efficient inhibition and thus hold great therapeutic promise for effectively and safely targeting biomedically important proteases.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Metaloproteinase 14 da Matriz
/
Inibidores de Metaloproteinases de Matriz
/
Anticorpos Monoclonais
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos