Enterovirus 71 VPg Uridylation Uses a Two-Molecular Mechanism of 3D Polymerase

VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3D^pol) and the transfer of UMP by 3D^pol to the hydroxyl group of the third amino acid Tyr of VPg. Previous studies suggested that different picornaviruses employ distinct mechanisms during VPg binding and uridylylation. Here, we report a novel site (Site-311, located at the base of the palm domain of EV71 3D^pol) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and I317) at Site-311 reduced the VPg uridylylation activity of 3D^pol by >90%. None of the Site-311 mutations affected the RNA elongation activity of 3D^pol, which indicates that Site-311 does not directly participate in RNA polymerization. However, mutations that abrogated VPg uridylylation significantly reduced the VPg binding ability of 3D^pol, which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3D^pol. Mutation of a polymerase active site in 3D^pol and Site-311 in 3D^pol remarkably enables trans complementation to restore VPg uridylylation. In contrast, two distinct Site-311 mutants do not cause trans complementation in vitro. These results indicate that Site-311 is a VPg binding site that stabilizes the VPg molecule during the VPg uridylylation process and suggest a two-molecule model for 3D^pol during EV71 VPg uridylylation, such that one 3D^pol presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3D^pol, which in turn catalyzes VPg→VPg-pU conversion. For genome-length RNA, the Site-311 mutations that reduced VPg uridylylation were lethal for EV71 replication, which indicates that Site-311 is a potential antiviral target.     

The crystal structure of coxsackievirus B3 RNA-dependent RNA polymerase in complex with its protein primer VPg confirms the existence of a second VPg binding site on Picornaviridae polymerases

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D^pol) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D^pol at this site excludes its uridylation by the carrier 3D^pol. We suggest that VPg at this position is either uridylated by another 3D^pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D^pol/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.     

Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions

The viral RNA-dependent RNA polymerases show a conserved structure where the fingers domain interacts with the top of the thumb domain to create a tunnel through which nucleotide triphosphates reach the active site. We have solved the crystal structures of poliovirus polymerase (3D^pol) in complex with all four NTPs, showing that they all bind in a common pre-insertion site where the phosphate groups are not yet positioned over the active site. The NTPs interact with both the fingers and palm domains, forming bridging interactions that explain the increased thermal stability of 3D^pol in the presence of NTPs. We have also examined the importance of the fingers-thumb domain interaction for the function and structural stability of 3D^pol. Results from thermal denaturation experiments using circular dichroism and 2-anilino-6-napthaline-sulfonate (ANS) fluorescence show that 3D^pol has a melting temperature of only ∼ 40 °C. NTP binding stabilizes the protein and increases the melting by 5-6 °C while mutations in the fingers-thumb domain interface destabilize the protein and reduce the melting point by as much as 6 °C. In particular, the burial of Phe30 and Phe34 from the tip of the index finger into a pocket at the top of the thumb and the presence of Trp403 on the thumb domain are key interactions required to maintain the structural integrity of the polymerase. The data suggest the fingers domain has significant conformational flexibility and exists in a highly dynamic molten globule state at physiological temperature. The role of the enclosed active site motif as a structural scaffold for constraining the fingers domain and accommodating conformational changes in 3D^pol and other viral polymerases during the catalytic cycle is discussed.     

Chemical compositions and characteristics of organic compounds in propolis from Yemen

Propolis is a gummy material made by honeybees for protecting their hives from bacteria and fungi. The main objective of this study is to determine the chemical compositions and concentrations of organic compounds in the extractable organic matter (EOM) of propolis samples collected from four different regions in Yemen. The propolis samples were extracted with a mixture of dichloromethane and methanol and analyzed by gas chromatography–mass spectrometry (GC–MS). The results showed that the total extract yields ranged from 34% to 67% (mean = 55.5 ± 12.4%). The major compounds were triterpenoids (254 ± 188 mg g⁻¹, mainly α-, β-amyryl and dammaradienyl acetates), n-alkenes (145 ± 89 mg g⁻¹), n-alkanes (65 ± 29 mg g⁻¹), n-alkanoic acids (40 ± 26 mg g⁻¹), long chain wax esters (38 ± 25 mg g⁻¹), n-alkanols (8 ± 3 mg g⁻¹) and methyl n-alkanoates (6 ± 4 mg g⁻¹). The variation in the propolis chemical compositions is apparently related to the different plant sources. The compounds of these propolis samples indicate that they are potential sources of natural bio-active compounds for biological and pharmacological applications.     

Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue

NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM. The induced cultures were then grown for 20 h at 18 °C and cells were harvested by centrifugation at 6000 x g for 15 min at 4 °C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems.     

Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents

Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55 nM and 60 nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells.     

Solution structure of the HIV-1 exon splicing silencer 3

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3′ splice sites by binding 5′-UAG-3′ elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A⁺C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5′-UAGU-3′ element and a proximal 5′-GAU-3′ motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5′-GAU-3′ and 5′-UAGU-3′ motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K_d = 37.8 ± 1.1 nM) complex with ESS3 via site-specific interactions with the loop.     

Design,synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino,N-formamido,pyrrole- and imidazole-containing polyamides

Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K_eq = 2.4 × 10⁸ M^?1) and with comparable sequence selectivity to its cognate sequence 5′-ACGCGT-3′ when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5′-ACGCGT-3′ via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5′-ATGCAT-3′ (K_eq = 7.4 × 10⁶ M^?1) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5′-AAATTT-3′ (K_eq = 4.8 × 10⁷ M^?1), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5′-ATCGAT-3′ as a stacked dimer and it has the lowest affinity among the diamino PAs tested (K_eq <1 × 10⁵ M^?1). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the ‘core rules’ of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.     

The RNA Template Channel of the RNA-Dependent RNA Polymerase as a Target for Development of Antiviral Therapy of Multiple Genera within a Virus Family

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2''-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3D^pol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3D^pol, (ii) had reduced activity against EMCV 3D^pol with the resistance mutations, and (iii) was most efficient in inhibiting 3D^pol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3D^pol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3D^pol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.     

Complete Protein Linkage Map of Poliovirus P3 Proteins: Interaction of Polymerase 3Dpol with VPg and with Genetic Variants of 3AB

Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3D^pol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3D^pol interaction. The viral proteinase 3C^pro was not found to interact with other 3C^pro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CD^pro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.     

An LP1 analogue,selective MOR agonist with a peculiar pharmacological profile,used to scrutiny the ligand binding domain

The hypothesis that central analgesia with reduced side effects is obtainable by occupying an ‘allosteric’ site in the MOR ligand binding domain requires the development of new ligands with peculiar pharmacological profile to be used as tools. New benzomorphan derivatives, analogues of LP1, a multitarget MOR agonist/DOR antagonist, were designed to examine in depth MOR ligand binding domain. Compound 5, bearing a diphenylic N-substituent on the benzomorphan nucleus, showed an affinity (K_i^μ = 0.5 ± 0.2 nM) comparable to that of LP1 and a better selectivity versus DOR and KOR. It elicits antinociceptive effects in ex vivo (GPI) and in vivo. This new compound engages receptor amino acidic residues not reached by LP1 and by other established MOR ligands. Molecular modeling studies, conducted on 5 and on several reference compounds, allowed us to propose possible residues in the MOR ligand binding domain essential for their interactions with ‘orthosteric’ and ‘allosteric’ binding sites.     

Radiosynthesis and evaluation of new PET ligands for peripheral cannabinoid receptor type 1 imaging

Cannabinoid receptor type 1 (CB1) is mainly expressed in the brain, as well as being expressed in functional relevant concentrations in various peripheral tissues. 1-(4-Chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 1) was developed as a potent allosteric antagonist for CB1 and its oral administration led to reductions in the appetite and body weight of rats. Several analogs of 1 (compounds 2 and 3) were recently identified through a series of structure-activity relationship studies. Herein, we report the synthesis of radiolabeled analogs of these compounds using [¹¹C]COCl₂ and an evaluation of their potential as PET ligands for CB1 imaging using in vitro and in vivo techniques. [¹¹C]2 and [¹¹C]3 were successfully synthesized in two steps using [¹¹C]COCl₂. The radiochemical yields of [¹¹C]2 and [¹¹C]3 were 17 ± 8% and 20 ± 9% (decay-corrected to the end of bombardment, based on [¹¹C]CO₂). The specific activities of [¹¹C]2 and [¹¹C]3 were 42 ± 36 and 37 ± 13 GBq/μmol, respectively. The results of an in vitro binding assay using brown adipose tissue (BAT) homogenate showed that the binding affinity of 2 for CB1 (K_D = 15.3 µM) was much higher than that of 3 (K_D = 26.0 µM). PET studies with [¹¹C]2 showed a high uptake of radioactivity in BAT, which decreased in animals pretreated with AM281 (a selective antagonist for CB1). In conclusion, [¹¹C]2 may be a useful PET ligand for imaging peripheral CB1 in BAT.     

3D-QSAR CoMFA study of benzoxazepine derivatives as mGluR5 positive allosteric modulators

Positive allosteric modulation of the metabotropic glutamate receptor subtype 5 was studied by conducting a comparative molecular field analysis on 118 benzoxazepine derivatives. The model with the best predictive ability retained significant cross-validated correlation coefficients of q² = 0.58 (r² = 0.81) yielding a standard error of 0.20 in pEC₅₀ for this class of compounds. The subsequent contour maps highlight the structural features pertinent to the bioactivity values of benzoxazepines.     

Brevenal,a brevetoxin antagonist from Karenia brevis,binds to a previously unreported site on mammalian sodium channels

Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize the effects of brevenal, a radioactive analog ([³H]-brevenol) was produced by reducing the terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A K_D of 67 nM and B_max of 7.1 pmol/mg protein were obtained for [³H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [³H]-brevenol binding with K_i values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol inhibited brevetoxin binding, brevetoxin was completely ineffective at competition for [³H]-brevenol binding. After examining other site-specific compounds, it was determined that [³H]-brevenol binds to a site that is distinct from the other known sites on the sodium channel, including the brevetoxin site, (site 5) although some interaction with site 5 is apparent.     

RNA Signals in Entero- and Rhinovirus Genome Replication

The VPg-linked, plus-stranded RNA genomes of entero- and rhinoviruses contain very different 5′ and 3′ terminal regions which harbor signals for RNA replication. The terminal cloverleaf-like structure of the 5′-nontranslated region (5′NTR) is known to be required for plus-strand RNA synthesis. Genetic evidence suggest that two stem-loop structures and the poly(A) tail of the 3′NTR have a function in minus-strand synthesis. All of the nonstructural viral proteins, and possibly also some cellular polypeptides, are believed to be involved in RNA replication. RNA synthesis is initiated on a poly(A) template and involves uridylylation of VPg to yield VPgpU(pU). This precursor is likely to serve as primer for the RNA polymerase 3D^polduring both minus- and plus-strand RNA synthesis.     

Development of thioquinazolinones,allosteric Chk1 kinase inhibitors

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K_m for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site ～13 Å from the ATP binding site. Preliminary data is presented for several of these compounds.     

Liquid phase combinatorial synthesis of 1,2,5-trisubstituted benzimidazole derivatives as human DHODH inhibitors

The synthesis of 1,2,5-trisubstituted benzimidazole derivatives was carried out using liquid phase combinatorial approach using soluble polymer assisted support (PEG5000). Synthesised compounds were characterised by FTIR, ESI-MS, ¹H NMR and ¹³C NMR. The purity of compounds was confirmed with HPLC analysis. Compounds were also docked into the binding site of human dihydroorotate dehydrogenase (hDHODH). The synthesised compounds were screened for hDHODH enzyme inhibition assay using brequinar as standard compound. The synthesised compounds demonstrated comparative biological activity. Synthesised compounds 8d and 8e demonstrated IC₅₀ value of 81 ± 2 nM and 97 ± 2 nM, respectively.