Enantioselective synthesis of tranylcypromine analogues as lysine demethylase (LSD1) inhibitors

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Abstract

Asymmetric cyclopropanation of styrenes by tert-butyl diazoacetate followed by ester hydrolysis and Curtius rearrangement gave a series of tranylcypromine analogues as single enantiomers. The o,- m- and p-bromo analogues were all more active than tranylcypromine in a LSD1 enzyme assay. The m- and p-bromo analogues were micromolar growth inhibitors of the LNCaP prostate cancer cell line as were the corresponding biphenyl analogues prepared from the bromide by Suzuki crosscoupling.

Introduction

Eukaryotic DNA is packaged together with histone proteins to form chromatin in which genes are usually silenced. The DNA needs to unwind from the histone complex to be transcriptionally active and this interaction between DNA and histones is orchestrated by three sets of players. Firstly, there are the ‘writers’, enzymes that introduce post-translational modifications into DNA and histone proteins and thereby alter their affinity for one another. Next, there are corresponding ‘erasers’ that remove these modifications and return chromatin to its native state. Finally, ‘readers’ recognize and respond to the pattern of dynamic chromatin post-transcriptional modification, referred to as the ‘epigenetic code’.1

Among the post-translational modifications, methylation is unique in that it occurs both in DNA (primarily at the C-5 position of cytosine) and in histone proteins (at lysine and arginine residues). It is also unique in that it was long considered to be an irreversible modification although there is no chemical reason why demethylation would be impossible. In 2004, the first lysine demethylase LSD1 (Lysine Specific Demethylase) was described.2 Soon thereafter in 2006, a second family of lysine and arginine demethylases containing the JmjC domain was identified that are iron-dependent α-ketoglutarate dioxygenases.3 In 2009, enzymes that convert 5-methylcytosine in DNA to 5-hydroxymethylcytosine were reported and there is evidence that DNA repair enzymes will replace the modified base by cytosine thus effecting an indirect demethylation.4

Currently, nearly 30 human lysine demethylases within the LSD and JmjC families are known.5 LSDs comprising LSD1 and LSD2 are homologous to monoamine oxidases (MAOs) and demethylate mono- and dimethyllysine residues. The reaction involves electron transfer between the FAD cofactor and the nitrogen lone pair. This is why trimethyllysine is not a substrate, while mono- or dimethyllysine are oxidized to an iminium ion that is hydrolytically decomposed to formaldehyde (Fig. 1). The best characterized LSD substrate is histone H3 on K4 and K9 residues but there are additional non-histone client proteins of importance. LSD1 mediated demethylation of the tumor suppressor p53 inhibits its function by preventing interaction with the co-activator 53BP1,6 while demethylation of the DNA methyltransferase Dnmt1 is necessary for maintaining DNA methylation activity.7

Overexpression of LSDs is observed in neuroblastoma, prostate, breast and bladder cancers where it is believed to repress transcription of repair pathways that would normally lead to apoptosis instead of proliferation.8 Thus, LSD inhibitors are of interest as anticancer agents as well as potentially applicable to other human diseases that exhibit misregulated gene expression. The homology between LSDs and MAOs, a clinically validated target, suggests that LSDs are druggable. Indeed, screening known MAO inhibitors has uncovered micromolar LSD inhibitors among which the best known is the antidepressant drug tranylcypromine (trans-2-phenylcyclopropylamine, Parnate).9 The compound acts as an irreversible inhibitor forming a covalent adduct with the FAD cofactor. X-ray structures of this complex bound within the LSD1 active site are not completely unambiguous, and it is likely that more than one adduct is formed (Fig. 2).10

Recently, several groups have reported the evaluation of tranylcypromine analogues as LSD inhibitors. Tranylcypromine itself is clinically used as a racemate and the same is true of the analogues described (Fig. 3).10(c), 11 Here, we report our independent results in developing a high-throughput LSD1 assay and the synthesis of tranylcypromine analogues by a route that provides compounds as single enantiomers.

Section snippets

LSD1 assays

We expressed His-tagged full length recombinant human LSD1 using a plasmid kindly provided by Shi.2 The purified enzyme was assayed by Shi’s immunoblot method. Briefly, the enzyme was incubated with purified histones or a methylated histone peptide substrate and activity detected with a commercial H3K4(me2) antibody. In our hands, this did not give reproducible results. SELDI-TOF mass spectrometric analysis of the reaction mixture was more reliable (Fig. 4) and we observed both single and

Conclusions

We report the first enantioselective synthesis of tranylcypromine analogues designed as LSD inhibitors. Our results indicate very little difference between the two enantiomers of tranylcypromine in LSD1 inhibition. The bromo substituted tranylcypromine analogues 4ce were superior to tranylcypromine in the enzyme assay regardless of the position of bromination, with the para-bromo derivative showing the highest activity. We have prepared the biphenyl derivatives 4fh and show that the meta or

Expression and purification of full length human LSD1

The plasmid pet15b-His-tagged full length human LSD1 was kindly provided by Fei Lan in Dr. Yang Shi’s laboratory.2 The plasmid was transformed into BL21 RIPL Codon Plus (DE3) bacteria (Stratagene) and protein expression was induced with 0.1 mM IPTG for 3 h at room temperature. The bacterial pellet was lysed (40 mM Tris–HCl pH 8.0, 300 mM NaCl, 0.2 % Triton X100, 5% glycerol, 10 μg/ml DNase I and 10 mM MgCl2 in the presence of protease inhibitors), sonicated on ice and centrifuged. The supernatant was

Acknowledgements

We are grateful to Cancer Research UK for a studentship to H.B. We thank Cancer Research UK, the Southampton Cancer Research UK Centre and Experimental Cancer Medicine Centre and COST Action TD0905 ‘Epigenetics: Bench to Bedside’ for financial support. We thank Dr. Bashar Zeidan and Professor Paul Townsend at the Faculty of Medicine, University of Southampton for assistance with mass spectrometric studies, and Dr. Wynne Aherne and Dr. Kathy Boxall at the Institute of Cancer Research for a gift

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