Analisi cristallografiche statiche e dinamiche finalizzate allo sviluppo di inibitori specifici e selettivi per la terapia epigenetica del cancro

Responsabili di progetto

Rocco Caliandro, Eric Di Luccio

Accordo

COREA DEL SUD - NRF - National Research Foundation of Korea

Bando

CNR-NRF 2016-2017

Dipartimento

Scienze chimiche e tecnologie dei materiali

Area tematica

Scienze chimiche e tecnologie dei materiali

Stato del progetto

Nuovo

Proposta di ricerca

The development of epigenetic therapies is the latest frontier in the fight against cancer. Enzymes responsible for histone post-translational modifications (PTM) are novel candidate therapeutic targets in cancer: histones are the stage of diverse post-translational modifications that ultimately regulates the gene transcription, and covalent histone modifications are key in chromatin regulatory mechanisms [1]. One such histone modification, lysine methylation, can have both activating and repressive functions on transcriptional events. Histone lysine methyltransferases (HMTases) are transcriptional co-regulators that target specific lysines on histone H3 and H4, and can transfer up to three methyl groups (Kme1, Kme2, and Kme3) [2]. However, the molecular mechanisms of histone mark recognition remain unclear. Dysfunctions in the regulation of histone methylation are linked to pathological conditions including oncogenesis and tumor progression. An increasing number of epigenetic modifiers such as HMTases are bona fide oncoproteins found aberrantly expressed in several cancers, suggesting potential for novel therapeutic strategies. In particular, MMSET/NSD2 is emerging as a target for therapeutic interventions against multiple myeloma, especially t(4;14) myeloma that is associated with a significantly worse prognosis than other biological subgroups. Multiple myeloma is the second most common hematological malignancy in the United States, after non-Hodgkin lymphoma and remains an incurable malignancy in mature. Thus, novel therapeutic strategies are sorely needed. DNA-methylation inhibitors, histone-deacetylase and histone-methyltransferase inhibitors are being developed as the utilization of epigenetic targets is emerging as an effective and valuable approach to chemotherapy as well as chemoprevention of cancer.
Very few lead compounds have been described to selectively inhibit HMTase enzymes. This is mostly due to the lack of structural information on HMTase in order to support the design of selective inhibitors. HMTase inhibitors are scarce but one of the most efficient strategies seem to target the L-lysine-[histone] or L-arginine-[histone] binding pocket [3]. HMTases need to accommodate different histone substrates of differential methylation state. Understanding the structural basis of substrate specificity of HMTase is indispensable for designing inhibitors.
In this context, detailed knowledge of the three-dimensional structure of HMTases and their binding modes with candidate inhibitors is fundamental both in understanding chromatin dynamics at atomic details and the dramatic changes seen in diseases, especially cancers, and in developing more potent and selective inhibitors [4]. Crystallography is the technique of choice for these achievements, due to its unique ability to provide details at atomic resolution and to identify weak binding sites.
Although routinely applied to proteins, solving a protein crystal structure is far for being straightforward in case of poorly diffracting crystals, low resolution data, low homology target sequences, while low occupancy ligands could be missed from the electron density map.
To overcome these limitations, more efficient phasing methods are continuously developed, to push forward the data resolution limit for the application of direct or Patterson methods [5], or to reach molecular replacement solutions even by using distant models [6]. Moreover, computational techniques are able to increase the experimental data resolution, with the aim of improving the crystal structure solution and better defining the details of the experimental electron density map [7].
Besides such advancements, the major drawback of the crystallographic analysis, i.e. its static nature, is being overcome by a novel technique, called Modulated Enhanced Diffraction (MED). It is based on the use of different datasets, taken on the same crystal system at different times, while some external stimulus is changed. The combined offline analysis of the diffraction patterns is able to extract the contribution from the varying part of the crystal system. Such analysis can be used to find the substructure of "active" atoms, and to study the kinetic of the response of the crystal system to the external stimulus [8].
The aim of this project is to combine the crystallographic and structural biology expertise of the Caliandro's group with the biochemistry and pharmacology expertise of the di Luccio's group to foster the structure-based drug design of both specific and selective HMTase inhibitors.
The structural investigations will be carried out at synchrotron sources on crystallized enzymes and adducts enzymes-inhibitor, obtained either by soaking or co-crystallization. Single datasets will be processed by programs developed by the Institute of Crystallography to solve the protein crystal structures [9]. A fragment-based approach [10] will be used to develop more potent and selective inhibitors (sub-micro molar level), starting from predefined scaffolds. The MED approach will instead be used to monitor the ligand binding dynamics to the target proteins, by using the soaking time or the ligand concentration as varying external stimuli. Crystals of various constructs of human and yeast HMTase have been already obtained in the di Luccio lab, and will be used as targets in this project.

[1] Morishita M, di Luccio E Biochim Biophys Acta 1816,158, 2011
[2] Morishita M, Mevius D, di Luccio E BMC Struct Biol 12,14, 2014
[3] di Luccio E J Cancer Prev 20,113, 2015
[4] Morishita M, di Luccio E Biochem Biophys Res Commun 412,214, 2011
[5] Caliandro R, Carrozzini B et al Acta Cryst A 69,98, 2013
[6] Caliandro R, Carrozzini B et al Acta Cryst A65 512 2009
[7] Caliandro R, Carrozzini B et al J Appl Cryst 40,931, 2007
[8] Caliandro R, Chernyshov D et al J Appl Cryst 45,458, 2012
[9] Burla MC, Caliandro R et al J Appl Cryst 48, 306, 2015
[10] Caliandro R, Belviso DB et al Fut Med Chem 5,1121, 2113

Obiettivi della ricerca

The project aims at elucidating the crystal structures of HMTases complexed with candidate inhibitors to develop lead compounds by structure-based drug design.
Static structural investigations will be performed to map the binding modes of potential inhibitors and to identify new potent and selective lead compounds by a fragment-based approach. Crystals of the target protein will be soaked with small molecule putative inhibitors isolated by virtual ligand screening and examined at high-throughput synchrotron beamlines. Bound inhibitors in the crystal structures at different binding pockets (histone or AdoMet co-factor sites) will be studied and further derived and optimized to form lead compounds.
The ligand binding dynamics will be analyzed by using the MED technique. As a first step, cryo-trapped protein-ligand intermediates will be produced by varying the soaking time or the ligand concentration, and analyzed one after the other by X-ray diffraction. As a second step the ligand concentration will be used as stimulus for in-situ modifications, and several measurements will be carried out on the same crystal, hit at different points.
Overall, the project will allow discovering new inhibitors for histone modifications which can affect transcriptional events. These results will open new routes for collaborating with pharmaceutical companies interested into targeting epigenetic regulators for cancer therapy, and will allow preparing competitive proposals for forthcoming calls.

Ultimo aggiornamento: 08/06/2025