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3‐Chloro‐5‐Substituted‐1,2,4‐Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers
(2022)
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.
HDAC8 is an important target in several indication areas including childhood neuroblastoma. Several isozyme selective inhibitors of HDAC8 with L-shaped structures have been developed. A theoretical study has suggested that methionine 274 (M274) would act as a “switch” that controls a transient binding pocket, which is induced upon binding of L-shaped inhibitors. This hypothesis was experimentally examined in this study. The thermostability and functionality of HDAC8 wildtype and mutant variants with exchanged M274 were analyzed using biophysical methods. Furthermore, the binding kinetics of L-shaped and linear reference inhibitors of these HDAC8 variants were determined in order to elucidate the mode of interaction. Exchange of M274 has considerable impact on enzyme activity, but is not the decisive factor for selective recognition of HDAC8 by L-shaped inhibitors.
Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors.
The kinetics and mechanism of drug binding to its target are critical to pharmacological efficacy. A high throughput (HTS) screen often results in hundreds of hits, of which usually only simple IC50 values are determined during reconfirmation. However, kinetic parameters such as residence time for reversible inhibitors and the kinact/KI ratio, which is the critical measure for evaluating covalent inactivators, are early predictive measures to assess the chances of success of the hits in the clinic. Using the promising cancer target human histone deacetylase 8 as an example, we present a robust method that calculates concentration-dependent apparent rate constants for the inhibition or inactivation of HDAC8 from dose–response curves recorded after different pre-incubation times. With these data, hit compounds can be classified according to their mechanism of action, and the relevant kinetic parameters can be calculated in a highly parallel fashion. HDAC8 inhibitors with known modes of action were correctly assigned to their mechanism, and the binding mechanisms of some hits from an internal HDAC8 screening campaign were newly determined. The oxonitriles SVE04 and SVE27 were classified as fast reversible HDAC8 inhibitors with moderate time-constant IC50 values of 4.2 and 2.6 µM, respectively. The hit compound TJ-19-24 and SAH03 behave like slow two-step inactivators or reversible inhibitors, with a very low reverse isomerization rate.