Computational characterisation of the selectivity and binding mode of mono- and binuclear transition metal based anticancer compounds

The rising resistance and the severe side effects associated with classical anticancer drugs such as cisplatin or mercaptopurines has triggered the development of novel antitumourous agents based on transition metal (e.g. ruthenium, osmium, and gold) organometallic complexes. In parallel, the tools of computational biochemistry have become more and more powerful and can nowadays provide important insights into the mechanistic origins of many systems of medicinal relevance creating a synergetic working environment between experimental and theoretical approaches.

In this dissertation, a range of computational methods to study molecules from a quantum mechanical and/or classical mechanical point of view have been applied to characterise key properties of biological drug-target complexes. All of the computational research presented herein has been performed in tight collaboration with experimental groups, which determined X-ray structures of drug-target adducts and characterised the anticancer properties of the synthesised novel anticancer drugs studied here.

In particular, the interactions of a small ruthenium-based complex acting as an DNA semi-intercalator,[(eta^6-5,8,9,10-tetrahydroanthracene)Ru(II)(ethylenediamine)X] with X representing a leaving group such as Cl-, were characterised exposing different binding modes of the compounds with free vs. nucleosomal DNA.

Additionally, classical molecular dynamics simulations were used to complete a characterisation of the localisation of the linker of a novel binuclear antitumourous compound, that was designed in such a way as to take advantage of newly discovered allosteric pathways present in the nucleosome core particle with the aim of creating a synergistic effect in the uptake the two moieties. In addition to the localisation of the linker, the effects of the bound compound on the allostery of the nucleosome core particle has been explored to compare the mode of action of the newly synthesised binuclear compound with that of non-linked analogue with synergistic properties. Moreover, a similar study has been performed on a herpes virus peptide, (Met-Ala-Pro-Pro-Gly-Met-Arg-Leu-Arg-Ser-Gly-Arg-Ser-Thr-Gly-Ala-Pro-Leu-Thr-Arg- Gly-Ser), binding to the nucleosome core particle to observe whether or not it would be able to trigger allosteric pathways.

Finally, some fundamental issues related to the selectivity of transition metal based anticancer compounds for specific binding sites on the nucleosome core particle were tackled by simulating the approach of three different ruthenium and osmium-based complexes, [(eta^6-cymene)Ru(II)(1,3,5-triaza-7-phosphoadamantane)X_2], [(eta^6-cymene)Ru(II)(N-phenylpyridine-2-carbothioamide)X] and [(eta^6-cymene)Os(II)( N-phenylpyridine-2-carbothioamide)X], from the bulk solvent to their bound state with the histone proteins with the help of classical and mixed quantum mechanical/molecular mechanical molecular dynamics simulations. A determination and analysis of the corresponding free energy profiles were used to provide some rationalisation of the observed preference of different compounds for specific binding sites. All the computational studies performed here and the insights gained can provide some guidance for the rational design of anticancer compounds with specific binding properties.

Computational Investigation of Intracellular Signalling Cascades

Siri Camee van Keulen

In order to adjust to changes in the environment, cells can communicate via signalling cascades. A common pathway for intercellular communication is the G-protein-coupled- receptor (GPCR) signal transduction cascade which is triggered by a (chemical or physical) extracellular signal, such as light or small molecules. This external interaction induces conformational changes that can lead either to GPCR activation or deactivation. In the active form, GPCRs can induce the activation of trimeric G proteins, which results in the dissociation of the Galpha from the beta and gamma subunits. Subsequently, active Galpha can interact with other cytosolic proteins, such as adenylyl cyclase (AC), which converts adenosine triphosphate to cyclic adenosine monophosphate (cAMP) upon stimulation. AC activation can be induced via stimulatory Galpha subunit (Galpha_s) interactions, while inhibition is related to inhibitory Galpha (Galpha_i) association. X-ray crystallography is crucial for the computational study of the GPCR signal transduction pathway. However, sometimes important molecules or moieties can become lost or cannot be incorporated during crystallisation procedures, which can have a significant effect on a protein’s conformation and function. In this thesis, three steps in the GPCR signal transduction cascade have been studied, rhodopsin activation (i), the conformation of active Galpha_i (ii) and the interaction between Galpha_i and AC (iii). In all three cases some groups or molecules that are absent in the X-ray structures play a vital role in the function of the proteins. In the first project, rhodopsin’s early intermediates after photon exposure are investigated together with a deprotonation reaction that takes place in the later stages of the activation pathway. The classical molecular dynamics (MD) and mixed quantum mechanics/molecular mechanics MD results suggest that photon exposure induces active site rearrangements which increase the stability of rhodopsin’s deprotonated Schiff base state. A bridging water molecule in the active site that is absent in the X-ray structure appears to play an important role during the deprotonation mechanism, which demonstrates the dependence of the protein’s function on its environment. The second study investigates the conformation of the active soluble form of Galpha_i, which has not yet been fully resolved via experimental studies due to crystallisation difficulties of the lipidated N-terminus. To investigate the effect of lipidation via classical MD simulations, a model of soluble lipid-bound Galpha_i was constructed. The simulations show that Galpha_i can form a hydrophobic pocket for the lipid on the protein surface. Other regions of the protein, important for protein-protein interaction, are adjusted as well. Hence, the post-translational modification appears to have a significant impact on Galpha_i’s active conformation and function, which was not captured via the unlipidated X-ray structures. The last project is devoted to a complex of active soluble Galpha_i and AC (Galpha_i :AC). While the interaction site for Galpha_s on AC is known, the interaction site for Galpha_i has not yet been identified. Classical MD simulations were performed on a docked Galpha_i:AC complex to understand the inhibition mechanisms as well as the differentiation between AC stimulation/inhibition. The results show that Galpha_i binding not only leads to AC inhibition via impeding ATP binding but also by preventing re-activation via Galpha_s through closure of the subunit’s interaction site on AC. Keywords: GPCR signal transduction pathway, G-protein-coupled recepter, rhodopsin, G protein, complex formation, cis-trans isomerisation, adenylyl cyclase.

EPFL2017

In silico DNA-binding and rational design of ruthenium-arene anticancer drugs

Christian Gossens

Organometallic ruthenium(II)-arene (RA) antitumour compounds of the general type [Ru(II)(η6-arene)(X)2(pta)], RA-pta, and [Ru(II)(η6-arene)X(en)], RA-en, (X=leaving group; pta=1,3,5-triaza-7-phosphaadamantane, en=ethylenediamine) have been investigated computationally. The main focus has been i.e. the hydrolysis properties, ligand exchange reactions, binding to DNA and the analysis of the resulting structural perturbations. The goal of this thesis was to elucidate key steps in the reactivity of the above drugs that are not readily accessible by experimental techniques. The methodologies employed cover tailor made force fields in classical molecular dynamics (MD), density functional theory (DFT) MD simulations in implicit and explicit solvent as well as combined classical/quantum (QM/MM) MD simulations. For RA-pta compounds, binding energies between the metal centres and the surrounding ligands were calculated. The calculated energies rationalize the experimentally observed tendencies for arene loss, and show that the pta ligands are relatively strongly bound. Exchange of metal centre, methylation or protonation of the pta-ligand, or change of the arene result in significant differences in the metal-arene binding energies while leaving the metalphosphine bond strength essentially unchanged. Significantly lower binding energies and reduced hapticity are predicted for the exchange of arene by nucleobases. The latter show higher binding energies for nitrogen π-bonding than for p-bonding. No influence on the ruthenium-arene interaction was observed for RA-pta complexes bearing arenes with functionalized side chains. A combined DFT/continuum electrostatics approach has been used to estimate the protonation states and absolute pKa values of a series of RA-pta compounds and their hydrolysis products. Our results suggest that the selective in vivo activity towards cancer cells and the observed pH dependent DNA damage is due to a pH dependent activation/deactivation mechanism. In the context of rational drug design, it is hypothesised that analogues with fluorinated arene rings should result in improved selectivity towards hypoxic cancer cells. For RA-en complexes, we rationalized the chemoselectivity towards guanine. The calculated DFT binding energies for the three investigated nucleobases (G, A, C) decreases in the order G(N7) >> C(O2) ∼ C(N3) > A(N7) > G(O6) > OH2. The G(N7) complex is the most stable product due to a C6=O-HNen hydrogen bond while the corresponding C6-NH2-HNen interaction in adenine is repulsive. A study of the reaction of [(η6-benzene)Ru(en)(OH2)]2+ (1) towards the nucleobases G, A and C reveals a strong preference for a formation of an H-bonded cis vs. trans reactant adduct. Only guanine can form a thermally stable trans reactant adduct. The potential reaction sites of adenine and cytosine might loose their nucleophilicity by protonation while simultaneously the aqua leaving group of 1 is converted into an unreactive hydroxo ligand. For the reaction of 1 with guanine three different reaction pathways were identified, however, a "direct trans" reaction pathway is probably the most biologically relevant. Using classical and QM/MM MD we showed that both investigated ruthenium compounds, RA-pta and RA-en, can bind to the major groove of duplex DNA and are stable in this position. The DNA is highly flexible, adapts very fast and widens the major groove to accommodate the ruthenium complex. The local and global structural changes of the DNA (e.g. bending towards the major groove) observed for the RA-pta series are similar to those reported for cisplatin. A severe perturbation of the Watson-Crick base pairing adjacent to the binding site of RA-en was observed that can be linked to recent experimental results. Finally, a tailor made force field for RA-en was derived from our QM/MM trajectories using a force matching approach.

EPFL2007

Small Molecule Based Approaches to Inhibit Macrophage Migration Inhibitory Factor (MIF) Activities and Elucidate its Role in Health and Disease

Hajer Ouertatani-Sakouhi

Macrophage migration inhibitory factor (MIF) is a major mediator in innate immunity and inflammation and a potential therapeutic target in multiple inflammatory, infectious and autoimmune diseases including cancer. Current therapeutic strategies for targeting MIF focus on modulating its biological activities using anti-MIF neutralizing antibodies or developing inhibitors of its tautomerase activity. Although the identity of its natural substrate remains unknown, several small molecule inhibitors have been reported to be effective inhibitors of MIF tautomerase activity in vitro. All these inhibitors were identified by rational design and structure-activity studies based on the structure of the catalytic site and/or non physiologic substrates of MIF. The lack of suitable high-throughput screening (HTS) assays has hindered the screening of chemical libraries and discovery of more diverse inhibitors. Motivated by the goal of discovering diverse classes of MIF inhibitors that modulate MIF activity specific targeting of its biochemical, conformation and quaternary structure properties, both activity based endpoint and kinetic HTS assays were developed, optimized and performed on diverse set of libraries. Using the endpoint assay, out of 15440 compounds screened, twelve novel inhibitors of MIF's catalytic activity (∼ 0.1% hit rate) with IC50s in the range of 1.5 to 15.5 µM were identified and validated. The interaction site and mechanism of action of all these inhibitors were defined using structure activity studies and a battery of biochemical and biophysical methods, including mass spectrometry, light scattering, and NMR. The effect on MIF biological activities was also examined on MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Using the kinetic-based activity assay, we screened 80,000 small molecules and identified and validated thirteen novel inhibitors of MIF catalytic activity with inhibition constant (Ki,app) values ranging from 0.5 to 13 µM. According to the structure and potency some of these molecules could be of great therapeutic potential. The MIF inhibitors emerging from these studies could be divided into four classes: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1 which could be classified into 5 groups; 2) a novel class of catalytic site inhibitors, 3) a class of trimer destabilizing inhibitors. These findings demonstrate the potential of HTS assays as attractive means to identify novel classes of MIF inhibitors as lead drug candidates and/or chemical tools for elucidating the biochemical and structural bases underlying the multifunctionality of MIF's in health and disease. Having these molecules in hand, will advance our understanding of protein-protein interactions mechanism for MIF oligomerization and may help to elucidate the role of MIF enzymatic activity in health and disease.

EPFL2010