5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity

Daniel Paprocki,Jarosław Poznański,Elżbieta Speina,Maria Winiewska-Szajewska,Róża Kucharczyk

doi:10.1038/s41598-021-03136-8

Abstract

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2. Herein, we study its new analogs: 5,6-diiodo- and 5,6-diiodo-4,7-dibromo-1H-benzotriazole. We used biophysical (MST, ITC) and biochemical (enzymatic assay) methods to describe the interactions of halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 (hCK2α). To trace the biological activity, we measured their cytotoxicity against four reference cancer cell lines and the effect on the mitochondrial inner membrane potential. The results obtained lead to the conclusion that iodinated compounds are an attractive alternative to brominated ones. One of them retains the cytotoxicity against selected cancer cell lines of the reference TBBt with a smaller side effect on mitochondrial activity. Both iodinated compounds are candidate leaders in the further development of CK2 inhibitors.

Highlights

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2
Protein kinase CK2 is highly pleiotropic and constitutively active serine/threonine kinase, which catalyzes the phosphorylation of numerous protein substrates, which are often related to gene expression or protein synthesis
Microscale thermophoresis (MST), which enables direct estimation of binding affinity, Kd, in a pseudo-titration mode has already been applied in detailed thermodynamic studies of the interactions between kinase hCK2α and its ligands[12,17]

Summary

Introduction

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2. One of them retains the cytotoxicity against selected cancer cell lines of the reference TBBt with a smaller side effect on mitochondrial activity Both iodinated compounds are candidate leaders in the further development of CK2 inhibitors. We study the properties of two iodinated compounds: 5,6-diiodo-1H-benzotriazole (HIIH) and 4,7-dibromo-5,6-diiodo-1H-benzotriazole (BIIB) Both can be obtained from the commercially available 4,5-diiodobenzene-1,2-diamine[19] under a single-step protocol, avoiding direct site-specific bromination of benzotriazole. Their binding affinities to hCK2α are comparable to that of TBBt, but HIIH displayed better physicochemical properties, being less hydrophobic, less acidic and much more soluble in aqueous m edium[20]. Since its cytotoxicity can be explained by the suppression of activation of apoptotic proteins, and by the decrease of mitochondrial membrane potential[25], we monitored how our compounds affect the mitochondrial inner membrane potential, which may be further related to their toxicity, especially in context of cardiotoxicity[26]

Methods

Results

Conclusion