Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine

Abstract

BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.

Chemical Epigenetics: The Impact of Chemical and Chemical Biology Techniques on Bromodomain Target Validation

“…As this residue is not present in other bromodomain KAc binding pockets the compounds show good selectivity.I nM V4;11 cells these covalent inhibitors exerted more durable anti-proliferative effects and decreased the mRNAexpression of several BETdependent genes compared to reversible BET-bromodomain inhibition. [114] This is the first example of amethionine residue being targeted for covalent inhibition, and the methionineepoxide combination offers the possibility that further selective covalent ligands for other proteins can be developed by exploiting this pairing.…”

“… Covalent crosslinking probes that undergo conjugate addition with cysteine residues ( 58 – 59 ) or alkylate a methionine residue with their epoxide warhead ( 60 ) …”

Chemische Epigenetik: der Einfluss chemischer und chemo‐biologischer Techniken auf die Zielstruktur‐Validierung von Bromodomänen

“… Kovalent vernetzende Sonden, die mit Cysteinresten eine Michael‐artige Addition eingehen ( 58 und 59 ) oder über einen Epoxid‐Warhead Methioninreste alkylieren ( 60 ) …”

“…Da dieser Rest nicht in anderen Bromodomänen‐KAc‐Bindetaschen vorhanden ist, zeigen diese Stoffe gute Selektivität. Verglichen mit reversiblen Bromodomänen‐Inhibitoren üben diese kovalenten Inhibitoren in MV4;11‐Zellen länger antiproliferative Effekte aus und verringern die andauernde mRNA‐Expression von verschiedenen BET‐abhängigen Genen . Dies ist das erste Beispiel eines Met‐Restes als Zielstruktur für kovalente Inhibition.…”

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…Methionine does not possess the inherent nucleophilicity of cysteine, explaining its limited attention to date. Two recent publications have utilised hypervalent iodine reagents and redox‐active oxiridine systems to covalently modify methionine residues in native proteomes, thus interest in this residue is likely to increase …”