Covalent inhibition of histone deacetylase 8 by 3,4-dihydro-2H-pyrimido[1,2-c][1,3]benzothiazin-6-imine

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 3; pp. 577 - 585
• Main Authors: Muth, Marius, Jänsch, Niklas, Kopranovic, Aleksandra, Krämer, Andreas, Wössner, Nathalie, Jung, Manfred, Kirschhöfer, Frank, Brenner-Weiß, Gerald, Meyer-Almes, Franz-Josef
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2019
• Subjects: active sites; Binding mechanism; childhood; computational methodology; Covalent inhibitor; crystal structure; Enzyme inhibition; HDAC8; histone deacetylase; Non-hydroxamate inhibitor; reaction mechanisms; reducing agents; Selective inhibition; sulfides; T-cell lymphoma; tandem mass spectrometry; therapeutics; thiocyanates; thiols; zinc; Enzyme inhibition; Non-hydroxamate inhibitor; Selective inhibition; Covalent inhibitor; HDAC8; Binding mechanism
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2019.01.001

HDAC8 is an established target for T-cell lymphoma and childhood neuroblastoma. Benzothiazine-imines are promising HDAC8 inhibitors with unknown binding mechanism lacking a usual zinc binding group. In this study high-resolution and quantitative HPLC-coupled ESI-MS/MS techniques are combined with crystal structure determination and a variety of biochemical and computational methods to elucidate the reaction mechanism between benzothiazine-imine 1 and HDAC8. 1) 1 is a covalent inhibitor of HDAC8; 2) inhibition is reversible in the presence of reducing agents; 3) C153 in the active site and C102 are involved in the inhibition mechanism; 4) 1 modifies various cysteines in HDAC8 forming either thiocyanates or mixed disulfides with 3; 5) 1 and 5 dock in close proximity to C153 within the active site. This is supposed to accelerate covalent inactivation particularly in HDAC8 and suggested as major determinant for the observed nanomolar potency and selectivity of 1. 1 and its analogs are interesting model compounds but unsuitable for therapeutic treatment due to their high unselective reactivity towards thiol groups. However, the postulated preceding non-covalent binding mode of 1 opens a door to optimized next generation compounds that combine potent and selective non-covalent recognition with low reactivity towards C153 at the active site of HDAC8. 1 represents a completely new class of inhibitors for HDAC8. Initial non-covalent interaction at the bottom of the active site is suggested to be the key for its selectivity. Further optimization of non-covalent interaction and thiol-reactivity provides opportunities to develop therapeutic useful covalent HDAC8 inhibitors. •PD 404,182 is a isoenzyme selective HDAC8 inhibitor.•PD 404,182 reacts chemically with thiols yielding a thiophenol.•The mechanism-based inhibition of HDAC8 involves transient formation of a mixed disulfide.•PD 404,182 eventually modifies Cys153 yielding the corresponding isothiocyanate.