20S proteasome as novel biological target for organochalcogenanes

Highlights

• 20S proteasome inhibition by hypervalent chalcogen compounds was studied.

• Organoselenuranes and organotelluranes in cellular assays were evaluated.

• Organotelluranes were identified as more potent inhibitors than organoselenanes.

• High inhibitory potency and specificity toward the β2 catalytic site was observed.

Abstract

A series of hypervalent selenium- and tellurium-containing compounds (organoselenuranes and organotelluranes) was evaluated aiming novel inhibitors of a threonine protease, namely the 20S proteasome (20S PT). In vitro assays demonstrated high inhibitory potency and specificity of these compounds toward the β2 catalytic site of the 20S PT. Organotelluranes were identified as more potent inhibitors than organoselenuranes since their IC₅₀ ranged from 3.5 to 16 μM while for organoselenuranes the IC₅₀ ranged from 16 to 35 μM indicating great potential to be explored in 20S proteasome inhibition. Cellular assays with those compounds were employed to verify the cytotoxicity and ability to inhibit 20S proteasome in cell. These assays demonstrated that organoselenuranes are capable of maintaining their selectivity in cell while the organotelluranes became inactive under cellular conditions. Stability studies of the organochalcogenanes were performed by ⁷⁷Se and ¹²⁵Te NMR analysis. It was observed that organotelluranes are stable under enzymatic assay conditions and, organoselenuranes, the structures responsible for inhibitory activity are cyclized organoselenuranes.

Introduction

Biological investigations on selenium- and tellurium-containing compounds have increased in last decade. These compounds were thought to be deleterious but, recent studies have been proven otherwise and beneficial pleiotropic activities have been described. Antioxidative (as GPx mimetics) [1], anti-inflammatory [2], [3], neuroprotective [4], cancer prevention [5], pro-apoptotic [6] and immunomodulatory activities [7] are some of the biological properties described for selenium and tellurium-containing compounds. And that includes structure-based design of small molecules as enzyme inhibitors such as calpains, papain, cathepsins, protein tyrosine phosphatases [8]. The biological response for selenium- and tellurium-containing compounds commonly relies on an interaction with some sulfhydryl enzymes [9]. Pioneering demonstrated that inorganic telluranes are able to inhibit thiol-dependent enzymes but are inactive against oxygen-nucleophilic (O-nucleophilic) proteases (e.g. serine proteases) [10]. After that, the potential of chalcogenanes (organic and inorganic) has focused on thiol-dependent enzymes. Even when it was demonstrated that organotelluranes are more reactive species (more powerful inhibitors of thiol-dependent proteases) [11] than inorganic ones the possible activity of organotelluranes (and organoselenuranes) against other proteases was not explored. In this way, we decided to explore these possibilities, by demonstrating that organoselenuranes are capable of inhibiting cysteine cathepsins, a thiol-dependent proteases family [12] and we also established the structure–activity relationships involving both organoselenuranes and organotelluranes and cysteine cathepsins V and S [13]. Based on ours previous reports, we have considered the possibility that chalcogenated organic compounds can inhibit another class of protease, since, as aforementioned, only inorganic telluranes were evaluated against serine proteases [10]. To the best of our knowledge, interactions of selenium- and tellurium-containing organic compounds with non-thiol-dependent proteases are yet unknown. For that, we chosen as target the proteasome, a threonine protease (O-nucleophilic protease) which is responsible for all non-lysosomal cellular proteolysis in cell [14]. This enzyme is a multi-catalytic protease with a proteolytic core (20S proteasome, 20S PT), which contains three catalytic sites (β1, β2 and β5), each one presenting a nucleophilic N-terminus threonine residue [15]. The catalytic sites have different specificities: β1 presenting a caspase-like activity, β2 trypsin-like activity and β5 chymotrypsin-like activity [16]. This protease is involved in the most of intracellular processes of eukaryotic cells such as signal transduction, immune response, metabolism, cell death, besides protein turnover for cell quality control and regulatory purposes [17]. The most important proteasome inhibitors described in the literature can be divided into 5 classes: peptide aldehydes, peptide vinyl sulfones, peptide boronates, peptide epoxyketones and β-lactones [18]. Moreover, these inhibitors present unsuitable properties for future clinical investigation [19] and new classes of compounds capable of inhibiting proteasome are still required. In view of the importance of 20S PT and a lack of a study of organic selenium and tellurium compounds with thiol-independent proteases, we decided to explore the reactivity of organoselenuranes and organotelluranes (Fig. 1) against an O-nucleophilic protease (20S PT).