Design of a dual-function agent by fusing a designed anti-VEGF-A binder and CPG-2 enzyme

Ali Etemadi, Mohammad Hossein Karimi-Jafari, Babak Negahdari, Yazdan Asgari, Mohammad Reza Khorramizadeh, Farideh Mohammadian, Mohammadali Mazloomi

Résultats de recherche: Contribution à un journalArticleRevue par des pairs

Résumé

Anti-VEGF therapies are common for the treatment of cancer. Carboxypeptidase G (CPG-2) enzyme is a zinc-dependent metalloenzyme that metabolizes non-toxic synthetic ‘benzoic mustard prodrugs’ to cytotoxic moieties in tumor cells. In this study, we designed a dual-activity agent by combining a designed anti-VEGF- and CPG-2 enzyme to convert methotrexate (MTX). VEGF-A was docked against a set of scaffolds, and suitable inverse rotamers were made. Rosetta design was used for the interface design. The top 1200 binders were chosen by flow cytometry and displayed in yeast. The activity of CPG-2 enzyme was analyzed at different temperature conditions and in the presence of the substrate, MTX. Optimal binders were selected and protein was eluted using immobilized metal affinity chromatography and size-exclusion chromatography. Both, native PAGE and on-yeast flow cytometry confirmed the binding of the binder to VEGF-A. The activity of truncated enzymes was slightly lower than that of full-length enzymes linked to VEGF-A. The method should be generally useful as a dual-activity agent for targeting VEGF-A and combination therapy with the enzyme CPG-2 for metabolizing non-toxic prodrugs to cytotoxic moieties. Communicated by Ramaswamy H. Sarma.

langue originaleAnglais
Pages (de - à)11463-11470
Nombre de pages8
journalJournal of Biomolecular Structure and Dynamics
Volume41
Numéro de publication21
Les DOIs
étatPublié - 1 janv. 2023
Modification externeOui

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