Mechanistic Exploration of Methionine 274 Acting as a “Switch” of the Selective Pocket Involved in HDAC8 Inhibition: An in Silico Study

Abstract

The overexpression of histone deacetylase 8 (HDAC8) causes several diseases, and the selective inhibition of HDAC8 has been touted as a promising therapeutic strategy due to its fewer side effects. However, the mechanism of HDAC8 selective inhibition remains unclear. In this study, flexible docking and in silico mutation were used to explore the structural change of methionine (M274) during HDAC8 binding to inhibitors, along with the reason for this change. Meanwhile, steered and conventional molecular dynamics simulations were employed to explore the stability of the structural change. The findings suggest that M274 acts as a “switch” to control the exposure of the HDAC8‐selective pocket. The structure of M274 changes from flipped‐out to flipped‐in only when L‐shaped inhibitors bind to HDAC8. This structural change forms a groove that allows these inhibitors to enter the selective pocket. In other HDACs, a leucine residue replaces M274 in situ, and the same structural change is not observed. The findings reveal the mechanism of selective HDAC8 inhibition and provide guidance for the development of novel selective inhibitors.

Methionine 274 Is Not the Determining Factor for Selective Inhibition of Histone Deacetylase 8 (HDAC8) by L-Shaped Inhibitors

“…This observation contradicted the postulation of a computational study which suggested that leucine instead of methionine is unable to act as a switch that opens a side pocket for the binding of HDAC8-selective L-shaped inhibitors [ 21 ]. We anticipated that the replacement of methionine by the smaller amino acid alanine in HDAC8 M274A would create a constitutively open HDAC8-selective pocket between the L1- and L6-loop, with facilitated access for L-shaped inhibitors.…”

“…By analyzing crystal structures of L-shaped inhibitors bound to smHDAC8 from Schistosoma mansoni , a special selective binding pocket was observed between the catalytic tyrosine and the L1–L6 loop [ 20 ]. A computational study suggested that this selective binding pocket is a transient binding pocket, which opens upon binding of L-shaped inhibitors, and is controlled by movement of M274 [ 21 ]. Moreover, this structural change was not observed when M274 was replaced by leucine in the same computational study, leading to the hypothesis that M274 is the key factor responsible for selective HDAC8 inhibition [ 21 ].…”

“…A computational study suggested that this selective binding pocket is a transient binding pocket, which opens upon binding of L-shaped inhibitors, and is controlled by movement of M274 [ 21 ]. Moreover, this structural change was not observed when M274 was replaced by leucine in the same computational study, leading to the hypothesis that M274 is the key factor responsible for selective HDAC8 inhibition [ 21 ].…”

“…In other HDAC isozymes, a L1–L6 lock sterically prevents L-shaped inhibitor binding, which might explain why L-shaped inhibitors preferentially bind to HDAC8. In a computational study using flexible docking and molecular dynamics simulations, Yao et al suggested that the flexibility of methionine 274 (M274) and the L-shaped structure of inhibitors might be the most important determinants for selective inhibition of HDAC8 [ 21 ]. Binding of L-shaped inhibitors was proposed to induce a conformational change of M274 from flipped-out to flipped-in conformation, which should open the HDAC8-selective pocket.…”

“…In other HDAC isozymes, methionine is replaced by leucine, which lacks the flexibility of M274 in molecular dynamics simulations. Consequently, L-shaped inhibitors were assumed to be unable to induce opening of the selectivity pocket between the L1 and L6 loops [ 21 ]. Since selective L-shaped inhibitors bind in very similar poses to wildtype smHDAC8 and smHDAC8 H292M in crystal structures, where the critical H292 is exchanged with methionine [ 20 ], it appears disputable whether the postulated function of this amino acid as a pivotal switch to control the exposure of the selective sub-pocket would be the determining mechanism for selective HDAC8 inhibition.…”