Radim Nencka

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.

Phosphatidylinositol 4-kinases: Function, structure, and inhibition.

The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

The crystal structure of the phosphatidylinositol 4‐kinase IIα

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4‐kinase type IIα (PI4K IIα), in complex with ATP solved by X‐ray crystallography at 2.8 Å resolution. The structure revealed a non‐typical kinase fold that could be divided into N‐ and C‐lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C‐lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.

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.

Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein.

Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.

The high-resolution crystal structure of phosphatidylinositol 4-kinase IIβ and the crystal structure of phosphatidylinositol 4-kinase IIα containing a nucleoside analogue provide a structural basis for isoform-specific inhibitor design.

Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIβ and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIβ and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9 Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.

Structural basis of Zika virus methyltransferase inhibition by sinefungin

Zika virus is considered a major global threat to human kind. Here, we present a crystal structure of one of its essential enzymes, the methyltransferase, with the inhibitor sinefungin. This structure, together with previously solved structures with bound substrates, will provide the information needed for rational inhibitor design. Based on the structural data we suggest the modification of the adenine moiety of sinefungin to increase selectivity and to covalently link it to a GTP analogue, to increase the affinity of the synthesized compounds.

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.