Chemical Neurobiology of the Histone Lysine Demethylase KDM1A

Abstract

Epigenetic mechanisms regulate gene expression and mediate interactions between genetic factors and environmental exposures. The enzymes responsible for epigenetic regulation may thus be important therapeutic targets for multifactorial neurological syndromes. KDM1A, the first histone lysine demethylase to be discovered, regulates the maturation of neurons and is inactivated by non-selective monoamine oxidase inhibitors such as the antidepressant tranylcypromine. This thesis entails the development of small-molecule tools to study KDM1A in a neurobiological context, with application towards the development of new therapeutic agents.

We leveraged the chemical scaffold of tranylcypromine to generate novel KDM1A inhibitors. In chapter 2, we profile these analogs using biochemical, cellular, and in vivo assays. We show that RN1 potently inhibits KDM1A, exhibits high brain uptake, and affects the behavior of mice in a novel object recognition assay. Thermal shift assays reveal engagement of KDM1A by tranylcypromine in the brains of systemically-treated rats, suggesting that inhibition of KDM1A by non-selective antidepressants in a clinical setting warrants further examination.

We sought to discover new mechanisms of KDM1A inhibition in order to gain further selectivity versus the monoamine oxidases. In chapter 3, we present outcomes of a high-throughput screen and secondary assays which reveal a predominant mode of KDM1A inhibition based on thiol-reactivity, and widespread contamination of test compounds by elemental sulfur. We show that KDM1A is inhibited by the FDA-approved drug disulfiram, and disclose two novel scaffolds for medicinal chemistry development.

In chapter 4, we further profile the thiol-reactivity of KDM1A and show that catalytically-generated hydrogen peroxide negatively regulates demethylase activity. MALDI-TOF mass spectrometry indicates that hydrogen peroxide blocks labeling of cysteine 600, which we propose forms an intramolecular disulfide bond with cysteine 618. This activity-dependent regulation is unique among histone-modifying enzymes but consistent with redox sensitivity of epigenetic regulators. KDM1A may use this thiol/disulfide switch as a mechanism to sense other cellular oxidants, such as the monoamine neurotransmitter dopamine.