Michal Šála

Nucleoside Inhibitors of Tick-Borne Encephalitis Virus

ABSTRACT Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2′-C-methyladenosine (7-deaza-2′-CMA), 2′-C-methyladenosine (2′-CMA), and 2′-C-methylcytidine (2′-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 μM for 7-deaza-2′-CMA, 7.1 ± 1.2 μM for 2′-CMA, and 14.2 ± 1.9 μM for 2′-CMC) and viral antigen production. Notably, 2′-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 μM). The anti-TBEV effect of 2′-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2′-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2′-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2′-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.

Design, synthesis, and biological evaluation of novel coxsackievirus B3 inhibitors

The synthesis and SAR study of a novel class of coxsackievirus B3 (CVB3) inhibitors are reported. These compounds could be considered as the 6-chloropurines substituted at position 9 with variously substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene-norbornane or norbornene). The synthesis and biological evaluation of 31 target compounds are described. Several of the analogues inhibited CVB3 in the low micromolar range (0.66-2muM). Minimal or no cytotoxicity was observed.

Highly Selective Phosphatidylinositol 4-Kinase IIIβ Inhibitors and Structural Insight into Their Mode of Action

Phosphatidylinositol 4-kinase IIIβ is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional replication machinery. Selective inhibitors of this enzyme are potential broad-spectrum antiviral agents, as inhibition of this enzyme results in the arrest of replication of PI4K IIIβ-dependent viruses. Herein, we report a detailed study of novel selective inhibitors of PI4K IIIβ, which exert antiviral activity against a panel of single-stranded positive-sense RNA viruses. Our crystallographic data show that the inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent the phosphorylation reaction.

SAR studies of 9-norbornylpurines as Coxsackievirus B3 inhibitors

Coxsackievirus and related enteroviruses are important human pathogens that cause various diseases with clinical manifestations ranging from trivial flu-like syndromes to dangerous or even fatal diseases such as myocarditis, meningitis and encephalitis. Here, we report on our continuous SAR study focused on 9-(bicyclo[2.2.1]hept-2-yl)-9H-purines as anti-enteroviral inhibitors. The purine moiety was modified at positions 2, 6 and 8. Several analogues inhibited Coxsackievirus B3 as well as other enteroviruses at low-micromolar concentrations. The 6-chloropurine derivative was confirmed as the most active compound in this series.

Semi-organic salts of aniline with inorganic acids: prospective materials for the second harmonic generation

Three novel inorganic salts of aniline with sulfuric and selenic acids were prepared and characterized by X-ray structural analysis. Anilinium(1+) selenate, (C6H5NH3+)2SeO42−, and anilinium sulfate, (C6H5NH3+)2SO42−, crystallize in the monoclinic space groupC2. The crystal structures are based on hydrogen bonded layers of alternating anilinium cations and inorganic anions. Anilinium(1+) selenate dihydrate, (C6H5NH3+)2SeO42−·2H2O, crystallizes in the monoclinic space groupC2/c. The crystal structure is formed by a network of alternating anilinium cations, selenate anions and water molecules connected by a system of intermolecular hydrogen bonds. The FTIR and Raman spectra of all the compounds have been recorded and discussed as well as their crystal structures. According to the DSC curves and temperature dependence of lattice parameters, anilinium sulfate exhibits phase transitions at 217 and 182 K. The appropriate changes of vibrational spectra were also recorded during cooling of the sample especially in the N–H stretching and sulfate antisymmetric stretching (ν3SO42−) spectral regions. The quantitative measurements of the second harmonic generation at 1064 nm were performed using powdered samples of anilinium sulfate, anilinium chloride and anilinium selenate, and the relative efficiencies deff = 0.05dKDP, deff = 2.33dKDP and deff = 0.05dKDP (KDP; i.e.KH2PO4) have been observed, respectively.

9-Norbornyl-6-chloropurine (NCP) induces cell death through GSH depletion-associated ER stress and mitochondrial dysfunction

UNLABELLED 9-Norbornyl-6-chloropurine (NCP) is a representative of a series of antienteroviral bicycle derivatives with selective cytotoxicity towards leukemia cell lines. In this work we explored the mechanism of the antileukemic activity of NCP in T-cell lymphoblast cells (CCRF-CEM). Specifically, we searched for a potential link between its ability to induce cell death on the one hand and to modulate intracellular glutathione (GSH) that is necessary to its metabolic transformation via glutathione-S-transferase on the other hand. We have observed that GSH levels decreased rapidly in NCP-treated cells. Despite a complete regeneration following 24h of incubation with NCP, this profound drop in cellular GSH content triggered ER stress, ROS production and lipid peroxidation leading to the loss of mitochondrial membrane potential (MMP). These events induced concentration-dependent cell cycle arrest in G2/M phase and apoptosis. Both MMP loss and apoptosis were reversed by sulfhydryl-containing compounds (GSH, N-acetyl-l-cysteine). Furthermore, we have also shown that NCP-induced GSH decrease activated the Nrf2 pathway and its downstream targets NAD(P)H quinone oxidoreductase (NQO-1) and glutamate cysteine ligase modifier subunit (GCLm), thus explaining the fast restoration of GSH pool and ROS decrease. Importantly, we confirmed that the cell death-inducing properties of the compounds were co-dependent on their ability to diminish cellular GSH level by analyzing the relationships between the GSH-depleting potency and cytotoxicity in a series of other norbornylpurine analogs. Altogether, the results demonstrated that in CCRF-CEM cells NCP triggered apoptosis through GSH depletion-associated oxidative and ER stress and mitochondrial depolarization.

Norbornane-based nucleoside and nucleotide analogues locked in North conformation

We report on the synthesis of novel conformationally locked nucleoside and nucleotide derivatives, which are structurally closely related to clinically used antivirals such as didanosine and abacavir. As a suitable conformationally rigid substitute of the sugar/pseudosugar ring allowing a permanent stabilization of the nucleoside in North conformation we employed bicyclo[2.2.1]heptane (norbornane) substituted in the bridgehead position with a hydroxymethyl group and in the C-3 position with a nucleobase. Prepared nucleoside derivatives were also converted into appropriate phosphoramidate prodrugs (ProTides) in order to increase delivery of the compounds in the cells. All target compounds were evaluated in a broad antiviral and cytostatic assay panel.

Discovery of Dual Death-Associated Protein Related Apoptosis Inducing Protein Kinase 1 and 2 Inhibitors by a Scaffold Hopping Approach

DRAK2 emerged as a promising drug target for the treatment of autoimmune diseases and to prevent graft rejection after organ transplantation. Screening of a compound library in a DRAK2 binding assay led to the identification of an isothiazolo[5,4-b]pyridine derivative as a novel ligand for DRAK2, displaying a Kd value of 1.6 μM. Subsequent medicinal chemistry work led to the discovery of a thieno[2,3-b]pyridine derivative with strong DRAK2 binding affinity (Kd = 9 nM). Moreover, this compound also behaves as a functional inhibitor of DRAK2 enzymatic activity, displaying an IC50 value of 0.82 μM, although lacking selectivity, when tested against DRAK1. This paper describes for the first time functionally active dual DRAK1 and DRAK2 inhibitors that can be used as starting point for the synthesis of chemical tool compounds to study DRAK1 and DRAK2 biology, or they can be considered as hit compounds for hit-to-lead optimization campaigns in drug discovery programs.

One-pot build-up procedure for the synthesis of variously substituted purine derivatives

In this article, we report a one-pot build-up procedure leading to 6-chloro- or 2-amino-6-chloropurines bearing various alkyl or aryl substituents in position N-9. This reaction is simple, fast and effective with up to 96% yields depending on the starting amine. This reaction may be easily combined with further nucleophilic displacement of the C-6 chlorine atom using various reagents, making this procedure very attractive in the field of medicinal chemistry pertaining to compounds based on a purine scaffold.

Dynamics of water molecules and sodium ions in solid hydrates of nucleotides

Nuclear magnetic resonance experiments, together with molecular dynamics simulations and NMR calculations, are used to investigate mobility of water molecules and sodium ions in solid hydrates of two nucleotides. The structure of guanosine monophosphate system (GMP) is relatively rigid, with a well-ordered solvation shell of the nucleotide, while the water molecules in the uridine monophosphate system (UMP) are shown to be remarkably mobile, even at −80 °C. The disorder of water molecules is observed in the 13C, 31P, and 23Na solid-state NMR experiments as multiple signals for equivalent sites of the nucleotide corresponding to different local arrangements of the solvation shell. Deuterium NMR spectra of the samples recrystallized from D2O also confirm differences in water mobility between the two systems. The experiments were complemented with NMR calculations on an ensemble of structures obtained from DFT molecular dynamics (MD) simulations. The MD simulations confirmed higher water mobility in the UMP system and the calculated chemical shifts and quadrupolar couplings were consistent with the experimental data. The disordered solvation shell in UMP is likely to be a good model for solvated nucleotides in general, with fast reorientation of water molecules and fluctuations in the hydrogen-bond network.