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Design of a dual-function agent by fusing a designed anti-VEGF-A binder and CPG-2 enzyme
Ali Etemadi1, Mohammad Hossein Karimi-Jafari2, Babak Negahdari1
1Medical Biotechnology Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Abstract:
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.
Insights
Researchers developed a dual-action cancer therapy agent by linking an anti-VEGF antibody with the CPG-2 enzyme. This novel agent targets VEGF-A and activates chemotherapy prodrugs within tumor cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapy
Background:
- Vascular Endothelial Growth Factor A (VEGF-A) is a key target in anti-cancer therapies.
- Carboxypeptidase G2 (CPG-2) is an enzyme that activates prodrugs into cytotoxic agents within tumor cells.
Purpose of the Study:
- To design a novel dual-activity agent combining anti-VEGF functionality with CPG-2 enzyme activity.
- To enable targeted delivery of activated chemotherapy to tumors expressing VEGF-A.
Main Methods:
- Computational docking and Rosetta design were used to engineer anti-VEGF binders.
- Yeast display and flow cytometry were employed for binder selection and screening.
- Enzyme activity assays and protein purification techniques (IMAC, SEC) were performed.
Main Results:
- Successfully designed and selected binders that exhibit specific binding to VEGF-A.
- Confirmed the enzymatic activity of CPG-2 in the presence of the substrate methotrexate (MTX).
- Demonstrated that the dual-activity agent can target VEGF-A and activate prodrugs.
Conclusions:
- The developed dual-activity agent shows promise for targeted cancer therapy.
- This approach facilitates combination therapy by targeting VEGF-A and activating prodrugs via CPG-2.
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