Nucleosome acidic patch-targeting binuclear ruthenium compounds induce aberrant chromatin condensation

ARTICLE DOI: 10.1038/s41467-017-01680-4 OPEN Nucleosome acidic patch-targeting binuclear ruthenium compounds induce aberrant chromatin condensation 1, 1234567890 Gabriela E. Davey1, Zenita Adhireksan1, Zhujun Ma1, Tina Riedel2, Deepti Sharma Sivaraman Padavattan1, Daniela Rhodes1,3,4, Alexander Ludwig 1, Sara Sandin1,3, Benjamin S. Murray 5, Paul J. Dyson2 & Curt A. Davey1,3 The ‘acidic patch’ is a highly electronegative cleft on the histone H2A–H2B dimer in the nucleosome. It is a fundamental motif for protein binding and chromatin dynamics, but the cellular impact of targeting this potentially therapeutic site with exogenous molecules remains unclear. Here, we characterize a family of binuclear ruthenium compounds that selectively target the nucleosome acidic patch, generating intra-nucleosomal H2A-H2B cross-links as well as inter-nucleosomal cross-links. In contrast to cisplatin or the progenitor RAPTA-C anticancer drugs, the binuclear agents neither arrest specific cell cycle phases nor elicit DNA damage response, but rather induce an irreversible, anomalous state of condensed chromatin that ultimately results in apoptosis. In vitro, the compounds induce misfolding of chromatin fibre and block the binding of the regulator of chromatin condensation 1 (RCC1) acidic patch-binding protein. This family of chromatin-modifying molecules has potential for applications in drug development and as tools for chromatin research. 1 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. 2 Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. 3 NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921, Singapore. 4 Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921, Singapore. 5 School of Mathematics and Physical Sciences, University of Hull, Hull HU6 7RX, UK. Gabriela E. Davey, Zenita Adhireksan, Zhujun Ma and Tina Riedel contributed equally to this work. Correspondence and requests for materials should be addressed to B.S.M. (email: ) or to P.J.D. (email: paul.dyson@epfl.ch) or to C.A.D. (email: ) NATURE COMMUNICATIONS | 8: 1575 | DOI: 10.1038/s41467-017-01680-4 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01680-4 H istone proteins package eukaryotic DNA into chromatin, yielding a structural hierarchy, in which nucleosomes comprise the basic repeating units1. Each nucleosome consists of a core region, composed of ~146 bp wrapped around a histone octamer, which comprises two copies each of four different core histone proteins. The H3–H4 histone tetramer organizes largely the central DNA of the nucleosome core, while the two H2A–H2B histone dimers organize the outer DNA regions. Outside of the core region, each nucleosome harbours a variable length of linker DNA, typically 10–90 bp, which can be associated with a fifth type of histone protein, linker histone. There are at least six distinct epigenetic features of chromatin that are modulated in a site-specific and cell state-dependent manner to achieve precise control of genomic activities, notably transcription. These entail mostly attributes that relate to individual nucleosomes, including histone octamer occupancy/positioning on the double helix and an enormity of potential posttranslational modifications to the histone proteins, in addition to substitutions with different histone variants and methylation of the DNA2, 3. Moreover, histone composition and DNA sequence can also impact nucleosome stability and dynamics properties4, 5. An additional epigenetic feature of chromatin regulatory structure entails consolidation of nucleosomes into higher states of compaction through linker histone association. The pronounced changes in the gene expression profiles that yield different cell types and states means that there are a multitude of epigenetic distinctions in chromatin, which underlie disease and differentiation. In cancer cells for instance, aberrant nucleosome occupancy, histone post-translational modifications and DNA methylation work in unison to silence tumour suppressor genes while activating other genes that enable tumour development and progression2, 3. This suggests the possibility that compounds capable of recognizing distinguishing structural or chemical features of chromatin could allow the targeting of vulnerable points in cancer cells6. But on top of this, studying such compounds can also provide basic insights into molecular recognition and chromatin activity7–9. In studying metal-based anticancer agents that were initially expected to act therapeutically by forming DNA adducts, we had found that certain ruthenium and osmium compounds have in fact a preference to form adducts at defined histone protein sites in the nucleosome10, 11. This had prompted us to explore further the activities of histone-associating metalloagents as they can have certain advantages over purely organic compounds. Heavy metal centres allow distinct characteristics of coordination geometry, oxidation state and ligand exchange for fine-tuning reactivity and affinity properties of the compound12, and whether one is focused on the structural biology, biochemistry or cellular localization, the presence of heavy atoms can make visualization and quantification more accessible. The RAPTA [(η6-arene)Ru(PTA)Cl2] (PTA = 1,3,5-triaza-7phosphaadamantane) antimetastasis, antitumour and antiangiogenic compounds13–15 have shown promise for use in chromatin research as they have a proclivity to generate protein adducts in cellular chromatin7. Steric access limitations to the ruthenium centre from the presence of both arene and PTA ligands disfavours DNA adduct formation7, with RAPTA adducts forming in the nucleosome core mainly at two adjacent sites, RU1 and RU2, within a highly electronegative cleft region on the surface of the H2A–H2B dimer9, 10. This region, known as the acidic patch because it comprises a preponderance of glutamate/ aspartate residues, coincides with a key binding platform for nuclear factor association and chromatin compaction1, 16, 17. Given the distinct cellular impact and molecular targeting properties of the RAPTA compounds7, 9, 10, 13–15, here we investigate the activity of binuclear compounds that are 2 N P Cl Ru Cl H N Ru Cl Cl O H N O O O O P N N N PEG 318 ± 59 Ru Cl N N P N N C10 Ru Cl 29.5 ± 2.6 O Cl Cl N P Ru N H O N N C2 183.5 ± 15.1 P N N N Cl Ru Cl O Cl O H (R) N Ru N P (R) N N N RR H 38.7± 10.3 P N N N Cl H3N Cl N P N H H N Cl Cl Ru O N Cl Cl O H N Cl N N cisplatin RAPTA-C Ru N P Cl N N Pt 35.7 ± 0.6 1893 ± 51 H3N Cl Fig. 1 Structures and cytotoxicity parameters of compounds used throughout the study. Cell growth inhibition, IC50, values (μM; HeLa cells, 40 h) are sho (...truncated)