TY - JOUR
T1 - Design of a dual-function agent by fusing a designed anti-VEGF-A binder and CPG-2 enzyme
AU - Etemadi, Ali
AU - Karimi-Jafari, Mohammad Hossein
AU - Negahdari, Babak
AU - Asgari, Yazdan
AU - Reza Khorramizadeh, Mohammad
AU - Mohammadian, Farideh
AU - Mazloomi, Mohammadali
N1 - Publisher Copyright:
© 2023 Informa UK Limited, trading as Taylor & Francis Group.
PY - 2023/1/1
Y1 - 2023/1/1
N2 - 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.
AB - 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.
KW - cancer
KW - CPG-2
KW - cytotoxic moieties
KW - De novo
KW - methotrexate
KW - VEGF
UR - http://www.scopus.com/inward/record.url?scp=85146253638&partnerID=8YFLogxK
U2 - 10.1080/07391102.2022.2162584
DO - 10.1080/07391102.2022.2162584
M3 - Article
C2 - 36629035
AN - SCOPUS:85146253638
SN - 0739-1102
VL - 41
SP - 11463
EP - 11470
JO - Journal of Biomolecular Structure and Dynamics
JF - Journal of Biomolecular Structure and Dynamics
IS - 21
ER -