3-Chloro-5-Substituted-1,2,4-Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers

Abstract

The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8.

Keywords: biotin switch assay; covalent inactivators; nucleophilic aromatic substitution; proteomic studies; thiol modification.

Scheme 1

Synthesis of 2‐chloro‐5‐substituted thiadiazole derivatives: i) RBr (a–h), H₂O, acetone, 0 °C, r. t., 24 h, yield 55–88 %; ii) SO₂Cl₂, CHCl₃, 0 °C, r. t., 24 h, yield 43–78 %; iii) 1.2 eq 30 % H₂O₂ (aq), 0 °C, r. t., 24 h, 25–90 %; iv) 3 eq 30 % H₂O₂ (aq), 0 °C, r. t., 24 h, yield 31–87 %.

Figure 1

TNB^2‐ reactivity assay and covalent HDAC8 labelling. A) is showing the general reaction scheme of the used TNB^2‐ reactivity assay and the structures of the used compounds as cysteine modifiers. B) Reactivity of the four studied cysteine modifier against TNB2‐ followed by decrease in absorbance at 412 nm. Time dependent increase in labeling degree of HDAC8 bound cysteines for N‐benzyl maleimide (C) and 5h (D), as determined from quantitative HPLC/ESI‐QTOF measurements

Figure 2

Reaction scheme for the reaction of 5‐SO₂‐TDZs, 5‐SO‐TDZs and 5‐S‐TDZs with free cysteine thiols. 5‐SO₂‐TDZs are reacting faster than 5‐SO‐TDZs, whilst 5‐S‐TDZs are showing no reactivity against free thiols.

Figure 3

Dot Blot for cysteine reactivity of different TDZs with HDAC8. HDAC8 cysteines were blocked with indicated TDZs and N‐ethylmaleimide (NEM) as a common thiol blocking agent. A DMSO control without thiol blocking agent was also prepared. Unblocked thiols were subsequently blocked with NEM‐biotin. Brightness of dots indicate HRP activity of Streptavidin‐HRP conjugate bound to biotin labeled thiols.