Global Proteomics Investigation of SAMT-247 Targets: An Antiviral Thioester that Acetylates Zinc Finger Proteins

Abstract

Covalent modification of target proteins is a well-established mechanism of action for small molecule inhibitors. Cysteine residues in particular have been exploited for their reactivity toward electrophilic molecules. SAMT-247 is a mercaptobenzamide thioester that covalently acetylates cysteines in the zinc-coordinating domains of the HIV nucleocapsid protein. This SAMT-247-promoted reaction leads to loss of zinc binding by the protein, with concomitant loss of protein structure and function. Although it has low cytotoxicity in animal models, recent studies have indicated that it affects other protein targets in uninfected cells, for example leading to increased immune cell functions. In this study, global proteomics approaches have been used to better understand other protein targets of SAMT-247. Minimal effects are observed when unstimulated THP-1 monocyte cells were treated with SAMT-247. In contrast, thermal proteome profiling identified 170 proteins with altered thermal stability when THP-1 cells were stimulated with phorbol 12-myristate 13-acetate/Ionomycin (PMA/Iono) before SAMT-247 treatment. Among the affected proteins, 81 contain a zinc-coordinating domain and/or have been shown to have a reactive cysteine residue. Among these, several play a role in cellular metabolism, and Seahorse assays demonstrated that SAMT-247 significantly increased the anti-metabolic and pro-glycolytic effect of PMA/Iono in THP-1 cells. Two of the most-affected proteins were ZC3H7A, a microRNA-binding protein with four zinc finger domains, and MGMT, a DNA damage repair protein with a reactive cysteine. Both proteins were modified by SAMT-247 when tested alone or in the presence of THP-1 cell lysate, indicating that they are bona fide targets of the inhibitor. The low activity of SAMT-247 in unstimulated THP-1 cells is consistent with its low cytotoxicity. The increased effects of SAMT-247 in stimulated immune cells suggests that this molecule could be developed to target diseases other than HIV.