Meeting Unmet Medical Needs
• Anticancer therapeutics
• Neuroactive drugs
Advancing strategies in drug discovery and development
• Computational medicinal chemistry
• Discovery of small molecule probes
• New chemical entities from nature
• Redox active targets and compounds
CONFIRMED PLENARY SPEAKERS
We have investigated the structures of eukaryotic ribosomes and their complexes involved in initiation and maturation and complexes involved in regulation of protein synthesis. These results provide insights into the architecture of the eukaryotic ribosome and into various eukaryotic-specific aspects of protein synthesis. Using electron microscopy, we determined the complete molecular structure of the 55S mammalian mitoribosome. The maps that we calculated between 3.4 and 3.6 A resolution allowed de-novo tracing of a large number of mitochondrial specific ribosomal proteins and visualization of interactions between tRNA and mRNA in the decoding centre, the peptidyl transferase center, and the path of the nascent polypeptide through the idiosyncratic tunnel of the mammalian mitoribosome. Furthermore, the structure suggested a mechanism of how mitochondrial ribosomes, specialized for the synthesis of membrane proteins, are attached to membranes.
How well do we understand these properties on the molecular level? Is it only important that a sufficient number of hydrogen bonds are formed and the shape of the molecules fit perfectly together? How much contributes the burial of hydrophobic surface portion and how important are changes of the degrees of freedom upon complex formation? Both binding partners are conformationally flexible species and will adapt to one another upon complex formation. However, who is going to pay for these adaptations? All biological processes occur in water, thus the ubiquitously present water molecules take an important impact on protein-ligand binding, and even rearrangements of water molecules on the surface of a formed protein-ligand complex modulate the affinity of complex formation.
Structural and thermodynamic considerations help to get insight into the driving forces for complex formation. Water takes in manifold ways influence on the structure and energetics of protein-ligand complex formation. The impact of such effects is less apparent in a strong modulation of affinity (ΔG) but, owing to compensatory effects, it strongly shifts the enthalpy/entropy inventory and tunes binding kinetics. Upon ligand binding water molecules are displaced, rearranged or recruited to engage in new contacts between protein and ligand. The newly formed complex is coated by a rearranged water shell. All these processes take influence on the thermodynamic and binding-kinetic signature of the formed complex. Decisive for the characteristics of the involved hydrophobic effect is the state of the water molecules before and after ligand binding. The displacement of ordered and firmly embedded water molecules results in another signature than the displacement of disordered, partly dynamic water molecules. In case binding occurs in empty or partly solvated but structurally stable pockets significantly different energetics are observed compared to transient pockets opened upon binding. The water network created around exposed ligand functional groups in flat solvent-exposed pockets takes strong impact on the thermodynamic signature of the complex and seems to govern binding kinetics. As the parameters affinity and binding kinetics are determinant for the efficacy of binding, they must be optimized individually for each drug in tailored fashion. By closely linking the results of high resolution X-ray and neutron diffraction, microcalorimetry, binding kinetics and computer simulations we want to characterize the determining influence of water on the efficacy of ligand binding.
Results of mutational and photoaffinity ligand studies in GABAA receptors can be analyzed in the light of the model structures. This leads to an assignment of candidate ligands to proposed novel pockets. Candidate binding sites for several ligands are presented. The structural studies can serve as hypotheses generators, and some previously controversial structural interpretations of biochemical data can be resolved in the light of the presented multi-template approach to comparative modeling. Crystal and cryo- EM microscopic structures of the closest homologues that were solved in different conformational states provided important insights into structural rearrangements of binding sites during conformational transitions. The impact of structural variation and conformational motion on the shape of the investigated binding sites can be studied in this way.
Structure guided approaches to identify new binding sites along with specific ligands for these are possible, and lead to a new generation of subtype selective allosteric modulators. The structures can also shed light on the molecular basis that underlies the functional diversity among receptor isoforms. A showcase of ligand development will be presented.
One of our research lines is focused on the generally under-investigated approach: exploitation of intrinsic property of some dyes for aggregation, whereby monomeric and aggregated dye differ strongly not only in target recognition but also in spectroscopic properties1. Thus, one dye molecule could bind with similar affinity to several targets (DNA, RNA, protein) giving different spectroscopic responses for each target: to some polynucleotide sequence dye would bind as monomer, for other sequence as dimer, and protein binding site would induce different spectroscopic response. The ongoing research endeavours to establish for the low molecular weight dyes the structure-activity guidelines for fine tuning of DNA - RNA - protein preferences combined with recognition by set of sensitive and bio-applicable spectrometric methods.
Salicylanilides (2-hydroxy-N-phenylbenzamides) have exhibited interesting antimicrobial properties including drug-resistant Mtb. and NTM (MIC ≥0.5 µM), but their potential use is prevented due to limited solubility and comparatively higher toxicity. These obstacles can be overcome, i.a., by employment of DDS. That is why salicylanilides bearing a carbonyl group were selected as model compounds for the evaluation of oligotuftsine carriers.
Oligotuftsin-based carriers ([TKPKG]n, n=1-4) were synthesized by solid-phase synthesis (Fmoc/tBu strategy, rink amide MBHA resin, diisopropylcarbodiimide/HOBt, NMP). N-Terminus and/or side chain lysine ε-amino group(s) were substituted to obtain carriers with various properties. Carboxylic acids (acetic, succinic, palmitic etc.) modify lipophilicity, short peptide spacers (G5, GFLG cleavable by cathepsin B) were used to control the cellular site where the drug is released, whereas fluorescein enables to determine cellular uptake by flow cytometry and fluorescent microscopy. One or more aminooxyacetic acid molecule(s) were coupled with peptides to provide a reactive group for the attachment of active molecules. After these modifications, peptide carriers were cleaved from the resin TFA and purified. Then, carriers were coupled with salicylanilide derivatives to form acid stable oxime bond. Novel conjugates were purified and characterised.
Generally, salicylanilide-oligotuftsin conjugates exhibited a significant extracellular antimyco-bacterial activity including against drug-resistant Mtb. Moreover, they are more effective against intracellular mycobacteria than parent salicylanilides. Cellular uptake was enhanced substantially, too, together with decreased cytostatic and cytotoxic effects on mammalian cells. In conclusion, our tuftsine peptides are perspective carriers for antimycobacterial agents.
This work was supported by the Czech Science Foundation project No. 17-27514Y.
Metalloenzymes are enzyme proteins containing metal ions (metal cofactors), which are directly bound to the protein or to enzyme-bound nonprotein components (prosthetic groups). Approximately one-third of proteins are metalloproteins, which serve to execute a wide array of functions in vivo, including facilitating matrix degradation, modulating DNA transcription, and many others. Given the importance of these functions, metalloenzymes play a significant role in human, virus and parasite physiology. Pathologies for which metalloenzymes are implicated include cancer, heart disease, influenza A and HCV. Given the impact of these diseases on human health, metalloenzyme inhibition offers an appealing approach to disease treatment. Indeed, sales of metalloprotein inhibitors account for billions of dollars in pharmaceutical sales annually.
Hydroxamates act as bidentate ligands and are able to form hydrogen bonds; they can act as potent inhibitors of any enzyme that contains metal ion and residues able to act as hydrogen-bond donors or acceptors. Almost all the enzymes that contain M2+ ion are easily coordinated with any hydroxamic acid derivative. In most of the enzyme proteins containing metal ions, hydroxamates bind bidentately to their catalytic M2+ ion to create a distorted trigonal bipyramidal geometry around the M2+. Thus, the metal-chelating property and multiple hydrogen-bond formation ability of hydroxamates have made them an intriguing family of compounds with a wide spectrum of biological activities, among them antiviral and antiparasitic.
Acknowledgments: Prof. Zoidis would like to thank the National and Kapodistrian University of Athens (ELKE Account) for financial support.
Bis-indole marine alkaloids, have emerged as important lead compounds for the discovery of new biologically active derivatives due to their potent biological activities shown. Among them, nortopsentins A–C having a characteristic 2,4-bis(3’-indolyl)imidazole skeleton, isolated from Spongosorites ruetzleri, exhibited in vitro cytotoxicity against P388 cells (IC50 values: 4.5–20.7 µm) and their synthetic analogs in which the imidazole moiety of nortopsentin was replaced by different 5-membered heterocycles, showed improved biological activity against a wide range of human tumor cell lines with GI50 values reaching sub-micromolar level. In particular, thiazole nortopsentin analogs 1-4, in which one or both indole units of the natural nortopsentins were also manipulated, showed even better activity and inhibition of cyclin-dependent kinase 1 (CDK1). Many derivatives, belonging to this class of compounds, revealed significant biological activity also in STO e MesoII cell lines, derived from human diffuse malignant peritoneal mesothelioma (DMPM), a very aggressive and resistant form of cancer, inducing a caspase-dependent apoptotic response, with a concomitant reduction of the expression of the anti-apoptotic protein survivin. The most active derivatives were also investigated in vivo showing a significant tumor volume inhibition of DMPM xenografts (range, 58%-75%) at well-tolerated doses, and two complete responses in each treatment group.
In order to confirm the therapeutic efficacy of this series of compounds, further studies were performed against cancer stem cells (CSCs) subpopulation freshly isolated from surgical resections of patients with the aim to identify new molecules potentially useful in therapy. Preliminary data showed that the treatment of colorectal CSCs, bearing different mutational background, with thiazole nortopsentin analogs, induced reduction of cells viability at 24 hours, potently induced the exit of CSCs from dormancy state. This phenomenon rendered the highly resistant CSCs sensitive to conventional chemotherapy drugs, oxaliplatin and 5FU. These results supported the use of these compounds as an innovative differentiation therapy that could be used in combination with standard chemotherapy as additive effect.
Although the relationship between their activity and the chemical composition has been extensively investigated over the past three decades, there are some conflicting reports on this topic. Polysaccharides with strong immunomodulatory activity include neutral and acidic ones; some of them are bound to protein or peptide residues. In addition to the primary structure of polysaccharides, higher structure, such as chain conformation, also plays an important role in their activities.
Although structure-activity relationship (SAR) among higher sugars is not clear, it is assumed that determinants of their immunomodulatory activity are: the monosaccharide composition, water solubility, molecular weight, branching degrees (DBs) and triple helical conformation. The next issue to be solved is the mechanism of immunomodulating activity of mushroom polysaccharides. One possible pathway of host-mediated actions from polysaccharide-drug Lentinan is binding of β-glucans to iC3b-receptors (CR3, CD11b/CD18) of phagocytic cells and natural killer (NK) cells and stimulation of phagocytosis and/or cytotoxic degranulation, as suggested by Chihara (1992) and later modified by Wasser (1999).
The polysaccharide fractions isolated from the natural sources (bacteria, fungi, plants) or obtained by biotechnological methods are often subjected to structural modifications in order to improve their pharmacological activity and/or pharmacokinetic properties. Uncertainty of the structural data on the atomic level considerably hinders this task.
An example of structural modifications of polysaccharides that lead to changes in their biological activity is incorporation of selenium atoms performed in our laboratory by biotechnological methods. We have isolated a Se-containing polysaccharide-protein fraction from selenium-enriched mycelial culture of Lentinula edodes. The results of preliminary tests of immunomodulatory activity of these fractions were surprising: the effect of Se-polysaccharides on the proliferation of human blood lymphocytes demonstrated their selective immunosuppresive activity and a very low toxicity at the same time. This activity is opposite to immunostimulating activity of L. edodes-derived polysaccharides, described in the literature. We speculate that incorporation of selenium into exopolysaccharide molecules changes their spatial structure which affects the biological activity.
Acknowledgement: Investigation of structure-activity relationship in Se-enriched polysaccharides is supported by grant from the Polish National Science Centre DEC-2013/09/B/NZ7/03978.
Acknowledgement: We are thankful for support to Biomagi, Slovakia and VEGA 1/0634/13.
In the first step, we developed a selective and highly potent noncovalent BChE inhibitor. In the next step, this compound was used as a starting point for the synthesis of a comprehensive series of close structural analogues in order to study the structure-activity relationships and to obtain compounds with improved on-target activities. More than 100 compounds were synthesized resulting in improved inhibitors with nano to picomolar inhibition constants. The most promising inhibitor improved memory, cognitive functions, and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic side effects. This inhibitor therefore provides a promising advanced lead compound for the alleviation of symptoms caused by cholinergic hypofunction in AD.
Limited clinical efficacy of current symptomatic treatment and minute effect on the progression of AD has shifted the research focus from targeting single enzyme or receptor towards multi-target-directed ligands (MTDLs). These ligands are able to interact with multiple pathological processes of AD, and have the unmet potential to halt the progression or even to cure the disease. Therefore, our potent BChE inhibitors were used as starting points to develop a new series of multifunctional ligands. The derivatives designed and synthesized displayed balanced BChE inhibition, antioxidant activity and inhibition of amyloid β aggregation. The co-crystal structure of typical inhibitor in complex with BChE explained the molecular basis for its low nanomolar inhibition of the enzyme. The most promising compounds also showed metal chelating properties, inhibited redox activity of chelated Cu2+ ions, were non-cytotoxic, and were not substrates of active efflux transport system as determined in Caco2 cells, thereby providing promising lead multifunctional ligands for AD treatment. A series of dual BChE/monoamine oxidase-B (MAO-B) inhibitors was also developed based on the selective BChE inhibitors. In addition, nanomolar MAO-A and MAO-B inhibitors with piperidine scaffold were derived thereof, and they showed promising neuroprotective activity in 6-hydroxydopamine cell model of Parkinson’s disease.
CONFIRMED KEYNOTE SPEAKERS
In recent years, bioorthogonal bond cleavage (or bioorthogonal elimination) reactions have emerged as a thriving area of chemical research, with examples such as the palladium-mediated cleavage of allyl and propargyl carbamates or the elimination of a carbamate-functionalized trans-cyclooctene (release-TCO, rTCO) upon reaction with a 1,2,4,5-tetrazine (Tz). The latter reaction has recently led to novel therapeutic concepts, and applications in chemical biology.
We have designed and prepared several prodrugs using carbamates and rTCO as protective triggers also in combination with self-immolative linkers. In general, we have observed that these prodrugs are significantly less toxic to tumor cells than the respective parent drugs, but can be activated by co-treatment with a palladium catalyst or tetrazines, respectively. For example, a Tz-moiety was attached to a highly selective covalent inhibitor affording a bioorthogonal probe that was successfully applied for pretargeted activation of rTCO-modified prodrugs. In this talk, an overview will be given of these studies and our recent activities regarding prodrug activation through bioorthogonal elimination.
On the basis of QM calculations and EVB QM/MM simulations, we have proposed a new two-step hydride mechanism for the MAO catalysis which is gaining some affirmation in the literature, and is fully corroborated by a very recent 13C kinetic isotope effect measurements. Calculations of the pKa values of three tyrosine residues revealed that MAO active site is hydrophilic, but turns hydrophobic upon the substrate entrance that binds in the monocationic form. MAO selectivity has been investigated in the case of histamine, which is not a physiological MAO substrate, yet is efficiently metabolized by MAO upon the N-methylation of the imidazole ring by histamine N-methyltransferase. This fact raises a very important and intriguing question: for a promiscuous enzyme such as MAO, what is the origin of its unexpected selectivity towards two very similar compounds, yet completely identical in their reactive ethylamine chain parts? We utilized a combination of MD simulations, MM-PBSA binding free energy evaluations, and QM calculations to address the MAO specificity with two substrates, histamine and N-methylhistamine, differing only in a single methyl group distant from the reactive centre. The insight gained through all these results led us to propose several promising strategies of preventing neurodegeneration.
Among various synthetic procedures, amidoxime route and 1,3 dipolar cycloaddition are the most commonly used, however 1,2,4-oxadiazole derivatives have been identified in nature as well, e.g. antibacterial metabolite of a fish pathogen Vibrio parahaemolyticus. As 3-pyrazinyl-1,2,4-oxadiazoles proved to inhibit growth of clinical strains of Mycobacterium, we tried to synthesize 3-pyrazinyl-5-substituted-1,2,4-oxadiazoles combining amidoximes and reactive carbonyl compounds. All identified compounds have been tested on antifungal and antibacterial including antimycobacterial effects.
In addition, the synthesis of conjugates combining a cytotoxic compound and azasteroidal lactams will be analyzed. These conjugates improve the physicochemical properties (lipophilicity and solubility) of known classical alkylators, such as POPAM ((bis(2-chloroethyl)amino)phenoxy) propanoic acid). The synthesized homo-azasteroidal alkylators showed relatively lower acute toxicity, very promising and prominent antileukemic activity both in vitro and in vivo.
In our research program aimed at obtaining modulators of the ECS, we focused our attention on compounds of general structure A, B and C as selective CB2R agonists, selective MAGL inhibitors and multi-target ECS modulators, respectively.
Bearing in mind the assumptions of aforementioned strategy, and with the aim of obtaining more efficacious antiepileptic drugs (AEDs) that will suppress different types of human seizures, the new hybrid anticonvulsants based on the pyrrolidine-2,5-dione scaffold have been obtained. These hybrid molecules join on the one chemical template the structural fragments of clinically relevant AEDs such as ethosuximide, levetiracetam, and lacosamide. As a result, the hybridization process yielded substances effective in three the most important animal seizure models, namely the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (scPTZ) test, and notably the six-Hertz (6 Hz) model of pharmacoresistant limbic seizures in mice. These substances displayed wider spectrum of protection, more potent efficacy or/and better safety profile than respective AEDs creating hybrid structure (ethosuximide, levetiracetam, and lacosamide). Additionally, several compounds diminished the pain responses in the formalin model of tonic pain and notably in the neurogenic pain models (capsaicin-induced nociception and oxaliplatin-induced neuropathy) in mice.
Acknowledgement: The studies were supported by the Polish National Science Centre grant 2015/18/E/NZ7/00509.
We turned our attention to multicomponent reactions (MCRs), in which three or more starting materials react to form a single product, usually in a technically simple, one-pot procedure and in an atom-efficient manner. Among the variety of MRCs, Ugi reaction has been known as a rich source of precursors for heterocyclic ‘drug-like’ scaffolds.
In this lecture development of an Ugi → amide N-de-tertbutylation → intramolecular cyclocondensation synthetic strategy is presented, that leads to anticonvulsant 2,6-DKPs. The SAR information obtained through in vivo studies in animal models of epilepsy is discussed.
Concurrently, we started to build up the systematic database of compounds with indole scaffold, whose properties have been studied by a virtual screening method to find optimal pharmacokinetic and pharmacodynamics properties. The compounds proposed by this study are to be synthesized and tested for their activities as aldose reductase inhibitors and the modulators of other proteins occurring in the development of chronic diseases together with aldose reductase.
Acknowledgement: This work was supported by VEGA 2/0041/15 and VEGA 2/0033/14. We also thank the Slovak Research and Development Agency under the contract No. APVV-15-0455 and SAS – TÜBİTAK Joint Project No. JRP 2015/7 for funding.
The compounds of interest were designed by advanced in silico drug design approaches. Structure- and ligand-based virtual screening was performed on the basis of crystallographic structural data for selected target. Targeting the TLR4–MD-2 interface enabled the discovery of three hit compounds with promising TLR4 antagonist activity. The most potent hit 1-(4-fluorophenyl)-2-(5-(2-hydroxy-5-methoxybenzoyl)pyrimidin-2-yl)guanidine also suppressed cytokine secretion by human PBMC. In a follow-up study, three-step synthetic route was designed and optimized to obtain a series of N-aryl-N’-(5-(2-hydroxybenzoyl)pyrimidin-2-yl)guanidine derivatives as TLR4 antagonists with EC50 values in the low micromolar range. Furthermore, 3D similarity-based virtual screening using ROCS software lead to the discovery of 1-isobutylchromeno[3,4-d]imidazol-4(1H)-one. Additionally, sixteen novel chromeno[3,4-d]imidazol-4(1H)-one derivatives were synthesized in the optimized straightforward synthetic pathway and evaluated for TLR7 modulatory activity in a HEK-Blue™ TLR7 reporter assay. The most promising compound exhibited potent TLR7 agonist activity (IC50 of 1.8 µM) which was in the same range as a marketed drug imiquimod.
Recently, the ligand-based virtual screening using IDO1 inhibitor epacadostat as a query was performed with our software LiSiCA. Moreover, application of structure-based screening protocol on the human form of IDO1 enzyme using our newly developed ProBiS plugin software enabled the discovery of structurally diverse hits which were further evaluated for IDO1 inhibitory activity in an optimized highly sensitive fluorescence-based end-point assay.
Altogether, we obtained useful information about SAR of chromeno[3,4-d]imidazol-4(1H)-one TLR7 agonists, which represent an important starting point for further studies of small-molecule agents targeting TLRs. Furthermore, novel IDO1 inhibitors were also discovered.
Using structure-based design and starting from marine alkaloids clathrodin and oroidin, we have discovered a new, N-phenylpyrrolamide structural class of GyrB and ParE inhibitors. We have determined the crystal structure of the N-phenyl-4,5-dibromopyrrolamide inhibitor-DNA gyrase B complex. Based on this structural information we have designed and prepared novel series of N-phenyl-4,5-dibromopyrrolamides and N-phenyl-3,4-dichloro-5-methylpyrrolamides and evaluated them against DNA gyrase from Escherichia coli (E. coli). The most potent inhibitors had low nanomolar IC50 values against E. coli gyrase. A selected set of compounds was evaluated against DNA gyrase from Staphylococcus aureus (S. aureus) and against topoisomerase IV from E. coli and S. aureus. The binding affinities of selected compounds to E. coli gyrase were studied using surface plasmon resonance (SPR). The structure-activity relationship (SAR) was examined and the results were rationalised with molecular docking. Antibacterial activities of the most promising compounds were evaluated against two Gram-positive and two Gram-negative bacterial strains. The most active compound had a low micromolar minimum inhibitory concentration (MIC90) against Enterococcus faecalis.
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BOOK OF ABSTRACTS
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