Identification and characterization of a small molecule that activates thiosulfate sulfurtransferase and stimulates mitochondrial respiration

Abstract

The enzyme Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), is a positive genetic predictor of diabetes type 2 and obesity. As increased TST activity protects against the development of diabetic symptoms in mice, an activating compound for TST may provide therapeutic benefits in diabetes and obesity. We identified a small molecule activator of human TST through screening of an inhouse small molecule library. Kinetic studies in vitro suggest that two distinct isomers of the compound are required for full activation as well as an allosteric mode of activation. Additionally, we studied the effect of TST protein and the activator on TST activity through mitochondrial respiration. Molecular docking and molecular dynamics (MD) approaches supports an allosteric site for the binding of the activator, which is supported by the lack of activation in the Escherichia coli. mercaptopyruvate sulfurtransferase. Finally, we show that increasing TST activity in isolated mitochondria increases mitochondrial oxygen consumption.

Keywords: diabetes; mitochondria; thiosulfate sulfurtransferase.

FIGURE 1

(a) Activity of TST in the presence of 1 mM test compound as a percentage of base activity. (b) Chemical structure of Hit 1. (c) Chemical structure of Hit 2.

FIGURE 2

(a) DSF screening result of TST without compound. This curve was used as a reference to judge the TST stabilizing ability of the potential activators that were screened. (b) DSF results when testing the stability of TST in presence of Hit 1. The melting point was increased to 72°C. (c) DSF results when testing the stability of TST in presence of Hit 2. The melting point was increased to 76°C. (d) DSF results of HIT 1 without protein. (e) DSF results of HIT 2 without protein.

FIGURE 3

(a) Hit 2 contains two stereo‐centers (indicated by red asterisks) caused by the non‐stereoselective reaction of homocysteine 1, cyclopropanecarbaldehyde 2 and benzyl isocyanide 3 in 2,2,2‐Trifluoroethanol (TFE) yielding the diastereomeric compound 4. (b) To establish the role of these stereo‐centers the synthesis was repeated using either S‐homo‐cysteine or R‐homo‐cysteine to yield compounds Dia1 and Dia2, respectively. (c) While the identity of the first stereo center (*1: Figure 1a) can be thereby be determined, assignment of the second stereocenter (*2) cannot be made from the data from the chiral column alone.

FIGURE 4

(a) Activity assay of hTST with Hit 1 and Hit 2 at concentrations from 0.244 to 500 μM. (b, c) Activity assay of hTST with Dia 1 and Dia 2, at concentrations from 0.244 to 500 μM. (d–g) Activity assay of hTST with Iso1, Iso 2, Iso 3, and Iso 4 at concentrations from 0.244 to 500 μM. (h) Activity assay of Hit 2 with hTST, bTST, and ecMST at concentrations from 0.244 to 500 μM. (i) MST binding assay of hTST to Hit 2. The concentration‐response curve showed the interaction between the RED‐tris‐NTA dye labeled hTST and Hit 2, each measurement was performed in triplicate. F _norm, normalized fluorescence.

FIGURE 5

Steady‐state kinetic characterization of the hTST versus the Sodium Thiosulfate concentration of activator Hit 2. (a) The Michaelis–Menten plots and (b) the Lineweaver–Burk plots show the relation of hTST activity versus the Sodium Thiosulfate concentration at three selected concentrations of Hit 2 (∙) 0 μM, (▪) 17.1 μM, and (▴) 34 μM in the presence of 60 mM KCN substrate. Steady‐state kinetic characterization of the hTST versus the KCN concentration of Hit 2 activator. (c) The Michaelis–Menten plots, and (d) the Lineweaver–Burk plots show the relation of hTST activity versus the KCN concentration at three selected concentrations of Hit 2 (∙) 0 μM, (▪) 17.1 μM, and (▴) 34 μM in the presence of 60 mM Sodium Thiosulfate substrate. (e) Scheme 1. Kinetic model for non‐essential activation. (f) Equation 1 for the enzyme kinetics according to the model in Scheme 1. v is the reaction velocity, V _max is the maximal reaction velocity, [S] is the substrate concentration and K _m is the Michaelis–Menten constant, [A] is the activator concentration. α and β respectively, are the parameters to describe the change in the affinity of substrate binding and the change in the catalytic constant.

FIGURE 6

Re‐plot of 1/∆ slopes and 1/∆ y‐intercept versus concentration of Hit 2.

FIGURE 7

Hit 2 increases the uncoupled respiration in isolated mitochondria. (a) Representative oxygen slope of isolated mitochondria. Definition of mitochondrial states (1, 2, 3, 3u) by addition of indicated substances in a representative measurement. (b) Quantification of state 2. (c) state 3 (ADP stimulated respiration). (d) state 3u (ETS, uncoupler FCCP titrations) of mitochondria pre‐treated with vehicle (Ethanol, depicted as Ctr) or Hit 2 (50 μM). Vehicle values are set at 100. Data are shown as mean ± SEM, n = 3–4 technical replicates; n = 7 biological replicates. p‐values indicating statistically significant differences between the mean values are defined as follows: **p < 0.01, ***p < 0.001 drug versus control.

FIGURE 8

Identification of the binding site of Hit 2 binding site in hTST. (a) The hTST structure (gray) and the CRKGVT motive (blue and yellow sticks) with the catalytic Cys248 (yellow sticks). (b) Docking result of Hit 2 in hTST, with the catalytic site highlighted in red surface. Site 1 is labeled and represented by a dashed circle. (c) MD and MDpocket analysis identified highly conserved pockets; site 1 is shown as a dashed circle. The frequency of appearance of the binding site is indicated by the blue‐red color scale. (d) Structural alignment of modeled hTST(black), bovine TST (PDB 1RHD, cyan), and E. coli MST (PDB 1URH, red), the location of site 1 is represented as contrasted colored surface.

FIGURE 9

Binding energy and molecular interactions of compoun1 with hTST. Pairwise RMSD of Hit 2 binding poses over the course of 200 ns of simulation. The black box denotes Hit 2's stable binding to site 1 (100 ns). (b) Hit 2's energetic decomposition based on MM/PBSA calculations. (c) Table of the contribution of energy components computed from MM/PBSA and calculated binding energy of Hit 2 to hTST. (d) Energy contribution per residue. Val2, Leu6, Val5, Arg8, Ala9, Pro226, and Tyr262 have the most negative values. (e) The types and frequency of interactions that support stable Hit 2 with hTST interactions. (f) The per‐residue energy decomposition is mapped in 3D by the hTST structure. (g) The molecular interactions and potential binding mode of one isomer of Hit 2: ((2R)‐N‐benzyl‐2‐cyclopropyl‐2‐{[(3S)‐2‐oxothiolan‐3‐yl]amino}acetamide), are depicted in a representative frame from Lig‐hTST MD (green sticks). hTST carbon residues are colored according to the per‐residue decomposition scale, with water molecules represented by blue spheres, hydrogen bonds represented by blue dashes, π‐stacking represented by magenta dashes, and hydrophobic interaction represented by orange dashes.