Butaselen

Hepatocellular carcinoma (HCC) has the third highest cancer-related mortality rate globally. The immunosuppressive microenvironment of HCC limits effective treatment options. HCC cells and associated microenvironmental factors suppress NK and T cell infiltration and cytotoxic activities. The abnormal number or function of NK and T cells leads to a lack of immune surveillance. Recently, immunotherapy targeting PD-1 and PD-L1 has been shown to activate functionally exhausted cytotoxic immune cells in some solid tumors. However, the response rate and therapeutic efficacy against solid tumors with little lymphocyte infiltration are limited, especially for HCC. Therefore, new targets and therapeutics that induce tumor cell apoptosis and overcome the problem of depletion of immune cells, thereby inhibiting the immune escape of HCC cells, are urgently required. Butaselen (2-bis[2-(1,2-benzisothiazol-2(2H)-ketone)]butane), an organic molecule containing selenium, is a new type of thioredoxin reductase inhibitor. In this study, we found that butaselen promoted NK and T cell activity and infiltration in the tumor microenvironment in HCC-bearing mice by enhancing the expression of CXCR3, NKG2D, and their respective ligands. When used alone, it can significantly inhibit tumor growth and exert a synergistic effect in combination with PD-1 blockade. We suggested the role of the thioredoxin reductase system in the regulation of the tumor immunosuppressive microenvironment and developed a new effective therapeutic molecule for HCC, revealing the mechanism of butaselen in inhibiting tumor cell immune escape.

Butaselen is an ebselen analog that is under clinical trials for treating hepatic and pulmonary fibrosis. Our previous studies showed that butaselen is mainly present in human plasma in the form of M2, a free Se-methylated metabolite.

This study aimed to investigate the metabolic mechanisms of butaselen.

Butaselen was incubated with human plasma. Butaselen immediately disappeared, and the butaselen-HSA (human serum albumin) adduct was detected by HPLC-HRMS, showing that butaselen covalently binds to HSA. The butaselen-HSA adduct was precipitated using acetonitrile and then incubated with PBS, Cys, and GSH for 1 hour. The product was M1, a reduced form of butaselen. The results indicated that HSA, Cys, and GSH can reduce the butaselen-HSA covalent bond. The binding site for butaselen could be the cysteine-34 residue of HSA through pronase and trypsin hydrolysis. Incubating butaselen with cysteine, butaselen-Cys, butaselen-2Cys, and M1 were generated, indicating the covalent binding and reduction of butaselen by cysteine. We incubated liver microsomes and cytosol with butaselen, 6.22 and 246 nM M2 were generated, respectively. The results demonstrated that cytosolic enzymes are mainly involved in M2 production. The amount of M2 in the liver cytosol decreased from 246 nM to 2.21 nM when 10 mM m-anisic acid (a specific TPMT enzyme inhibitor) was added, showing that TPMT is responsible for M2 formation.

Butaselen was covalently bound to HSA, and the binding site was the cysteine-34 residue of HSA. The butaselen-HSA adduct was reduced by free thiol compounds to generate M1. M1 was further metabolized to M2 by cytosolic TPMT. This study provides a basis for studying the pharmacokinetics of selenium-containing drugs.