Discovery of a Novel Class of Norovirus Inhibitors with High Barrier of Resistance.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting in ~219,000 deaths annually and a societal cost of ~USD60 billion. There are no antivirals or vaccines available to treat and/or prevent HuNoV. In this study, we performed a large-scale phenotypical antiviral screening using the mouse norovirus (MNV), which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre (Sapienza University, Rome). Compound 3-((3,5-dimethylphenyl)sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide (compound 1) was identified as an inhibitor of MNV replication with an EC50 of 0.5 ± 0.1 µM. A series of 10 analogs were synthesized of which compound 6 showed an improved potency/selectivity (EC50 0.2 ± 0.1 µM) against MNV; good activity was also observed against the HuNoV GI replicon (EC50 1.2 ± 0.6 µM). Time-of-drug-addition studies revealed that analog 6 acts at a time point that coincides with the onset of viral RNA replication. After six months of selective pressure, two compound 6res variants were independently selected, both harboring one mutation in VPg and three mutations in the RdRp. After reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly compound 6res phenotype. In this study, we present a class of novel norovirus inhibitors with a high barrier to resistance and in vitro antiviral activity.

Ligand Binding in Allosteric Flavoproteins: Part 2. Quantitative Analysis of the Redox-Dependent Interaction of the Apoptosis-Inducing Factor (AIF) with Its Protein Partner.

To perform their action usually flavoproteins interact transiently with a variety of molecular partners, whose binding is reciprocally affected and often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe an approach for the quantitative characterization of the redox-controlled interaction of the mammalian apoptosis inducing factor (AIF) with one of its known protein partners, namely, the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). In particular, we report a protocol for the titration of the flavoprotein in both in its oxidized and reduced states with CHCHD4, using an implementation of the MicroScale Thermophoresis (MST) technique.

Discovery of Zika Virus NS2B/NS3 Inhibitors That Prevent Mice from Life-Threatening Infection and Brain Damage.

Zika virus (ZIKV) infection, which initially was endemic only in Africa and Asia, is rapidly spreading throughout Europe, Oceania, and the Americas. Although there have been enormous efforts, there is still no approved drug to treat ZIKV infection. Herein, we report the synthesis and biological evaluation of agents with noncompetitive mechanism of the ZIKV NS2B/NS3 protease inhibition through the binding to an allosteric site. Compounds 1 and 2 showed potent activity in both enzymatic and cellular assays. Derivative 1 efficiently reduced the ZIKV protein synthesis and the RNA replication and prevented the mice from life-threatening infection and the brain damage caused by ZIKV infection in a ZIKV mouse model.

Detecting the nature and solving the crystal structure of a contaminant protein from an opportunistic pathogen.

The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is presented. The contamination led to the unintended crystallization and structure analysis of a cyanase hydratase from a bacterial strain of the Serratia genus, an opportunistic enterobacterium that grows under conditions similar to those of E. coli and that is found in a variety of habitats, including the laboratory environment. In this context, the procedures that were adopted to identify the contaminant based on crystallographic data only are presented and the crystal structure of Serrata spp. cyanase hydratase is briefly discussed.

Broad spectrum anti-flavivirus pyridobenzothiazolones leading to less infective virions.

We report the design, synthesis, and biological evaluation of a class of 1H-pyrido[2,1-b][1,3]benzothiazol-1-ones originated from compound 1, previously identified as anti-flavivirus agent. Some of the new compounds showed activity in low µM range with reasonable selectivity against Dengue 2, Yellow fever (Bolivia strain), and West Nile viruses. One of the most interesting molecules, compound 16, showed broad antiviral activity against additional flaviviruses such as Dengue 1, 3 and 4, Zika, Japanese encephalitis, several strains of Yellow fever, and tick-borne encephalitis viruses. Compound 16 did not exert any effect on alphaviruses and phleboviruses and its activity was maintained in YFV infected cells from different species. The activity of 16 appears specific for flavivirus with respect to other virus families, suggesting, but not proving, that it might be targeting a viral factor. We demonstrated that the antiviral effect of 16 is not related to reduced viral RNA synthesis or virion release. On the contrary, viral particles grown in the presence of 16 showed reduced infectivity, being unable to perform a second round of infection. The chemical class herein presented thus emerges as suitable to provide pan-flavivirus inhibitors.

Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference.

The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods.

A Single Nucleoside Viral Polymerase Inhibitor Against Norovirus, Rotavirus, and Sapovirus-Induced Diarrhea.

A safe and highly efficient antiviral is needed for the prophylaxis and/or treatment of viral diarrhea. We here demonstrate the in vitro antiviral activity of four 2'-C-methyl nucleoside analogues against noro-, rota-, and sapoviruses. The most potent nucleoside analogue, 7-deaza-2'-C-methyladenosine, inhibits replication of these viruses with a 50% effective concentration < 5 µM. Mechanistically, we demonstrate that the 2'-C-methyl nucleoside analogues act by inhibiting transcription of the rotavirus genome. This provides the first evidence that a single viral-diarrhea-targeted treatment can be developed through a viral-polymerase-targeting small molecule.

Functionalized 2,1-benzothiazine 2,2-dioxides as new inhibitors of Dengue NS5 RNA-dependent RNA polymerase.

Over recent years, many RNA viruses have been "re-discovered", including life-threatening flaviviruses, such as Dengue, Zika, and several encephalitis viruses. Since no specific inhibitors are currently available to treat these infections, there is a pressing need for new therapeutics. Among the flaviviral proteins, NS5 RNA-dependent RNA polymerase (RdRp) represents a validated target being essential for viral replication and it has no human analog. To date, few NS5 RdRp inhibitor chemotypes have been reported and no inhibitors are currently in clinical development. In this context, after an in vitro screening against Dengue 3 NS5 RdRp of our in-house HCV NS5B inhibitors focused library, we found that 2,1-benzothiazine 2,2-dioxides are promising non-nucleoside inhibitors of flaviviral RdRp with compounds 8 and 10 showing IC50 of 0.6 and 0.9 µM, respectively. Preliminary structure-activity relationships indicated a key role for the C-4 benzoyl group and the importance of a properly functionalized C-6 phenoxy moiety to modulate potency. Compound 8 acts as non-competitive inhibitor and its proposed pose in the so-called N pocket of the RdRp thumb domain allowed to explain the key contribution of the benzoyl and the phenoxy moieties for the ligand binding.

Targeting flavivirus RNA dependent RNA polymerase through a pyridobenzothiazole inhibitor.

RNA dependent RNA polymerases (RdRp) are essential enzymes for flavivirus replication. Starting from an in silico docking analysis we identified a pyridobenzothiazole compound, HeE1-2Tyr, able to inhibit West Nile and Dengue RdRps activity in vitro, which proved effective against different flaviviruses in cell culture. Crystallographic data show that HeE1-2Tyr binds between the fingers domain and the priming loop of Dengue virus RdRp (Site 1). Conversely, enzyme kinetics, binding studies and mutational analyses suggest that, during the catalytic cycle and assembly of the RdRp-RNA complex, HeE1-2Tyr might be hosted in a distinct binding site (Site 2). RdRp mutational studies, driven by in silico docking analysis, allowed us to locate the inhibition Site 2 in the thumb domain. Taken together, our results provide innovative concepts for optimization of a new class of anti-flavivirus compounds.

PPNDS inhibits murine Norovirus RNA-dependent RNA-polymerase mimicking two RNA stacking bases.

Norovirus (NV) is a major cause of gastroenteritis worldwide. Antivirals against such important pathogens are on demand. Among the viral proteins that orchestrate viral replication, RNA-dependent-RNA-polymerase (RdRp) is a promising drug development target. From an in silico-docking search focused on the RdRp active site, we selected the compound PPNDS, which showed low micromolar IC50vs. murine NV-RdRp in vitro. We report the crystal structure of the murine NV-RdRp/PPNDS complex showing that two molecules of the inhibitor bind in antiparallel stacking interaction, properly oriented to block exit of the newly synthesized RNA. Such inhibitor-binding mode mimics two stacked nucleotide-bases of the RdRp/ssRNA complex.

Structural bases of norovirus RNA dependent RNA polymerase inhibition by novel suramin-related compounds.

Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM); the compound inhibits also murine NV (mNV) RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3-7) hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability.

Naphthalene-sulfonate inhibitors of human norovirus RNA-dependent RNA-polymerase.

Noroviruses are members of the Caliciviridae family of positive sense RNA viruses. In humans Noroviruses cause rapid onset diarrhea and vomiting. Currently Norovirus infection is responsible for 21 million gastroenteritis yearly cases in the USA. Nevertheless, despite the obvious public health and socio-economic relevance, no effective vaccines/antivirals are yet available to treat Norovirus infection. Since the activity of RNA-dependent RNA polymerase (RdRp) plays a key role in genome replication and in the synthesis/amplification of subgenomic RNA, the enzyme is considered a promising target for antiviral drug development. In this context, following the identification of suramin and NF023 as Norovirus RdRp inhibitors, we analyzed the potential inhibitory role of naphthalene di-sulfonate (NAF2), a fragment derived from these two molecules. Although NAF2, tested in enzymatic polymerase inhibition assays, displayed low activity against RdRp (IC50=14µM), the crystal structure of human Norovirus RdRp revealed a thumb domain NAF2 binding site that differs from that characterized for NF023/suramin. To further map the new potential inhibitory site, we focused on the structurally related molecule pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) tetrasodium salt (PPNDS). PPNDS displayed below-micromolar inhibitory activity versus human Norovirus RdRp (IC50=0.45µM), similarly to suramin and NF023. Inspection of the crystal structure of the RdRp/PPNDS complex showed that the inhibitor bound to the NAF2 thumb domain site, highlighting the relevance of such new binding site for exploiting Norovirus RdRp inhibitors.

Mitochondrial ferritin is a functional iron-storage protein in cucumber (Cucumis sativus) roots.

In plants, intracellular Fe trafficking must satisfy chloroplasts' and mitochondrial demands for Fe without allowing its accumulation in the organelles in dangerous redox-active forms. Protein ferritin is involved in such homeostatic control, however its functional role in mitochondria, differently from its role in chloroplasts, is still matter of debate. To test ferritin functionality as a 24-mer Fe-storage complex in mitochondria, cucumber seedlings were grown under different conditions of Fe supply (excess, control, deficiency) and mitochondria were purified from the roots. A ferritin monomer of around 25 KDa was detected by SDS-PAGE in Fe-excess root mitochondria, corresponding to the annotated Csa5M215130/XP_004163524 protein: such a monomer is barely detectable in the control mitochondria and not at all in the Fe-deficient ones. Correspondingly, the ferritin 24-mer complex is abundant in root mitochondria from Fe-excess plants and it stores Fe as Fe(III): such a complex is also detectable, though to a much smaller extent, in control mitochondria, but not in Fe-deficient ones. Cucumber ferritin Csa5M215130/XP_004163524 is therefore a functional Fe(III)-store in root mitochondria and its abundance is dependent on the Fe nutritional status of the plant.

Structure-based inhibition of Norovirus RNA-dependent RNA polymerases.

Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.

Biofortification for combating 'hidden hunger' for iron.

Micronutrient deficiencies are responsible for so-called 'hidden undernutrition'. In particular, iron (Fe) deficiency adversely affects growth, immune function and can cause anaemia. However, supplementation of iron can exacerbate infectious diseases and current policies of iron therapy carefully evaluate the risks and benefits of these interventions. Here we review the approaches of biofortification of valuable crops for reducing 'hidden undernutrition' of iron in the light of the latest nutritional and medical advances. The increase of iron and prebiotics in edible parts of plants is expected to improve health, whereas the reduction of phytic acid concentration, in crops valuable for human diet, might be less beneficial for the developed countries, or for the developing countries exposed to endemic infections.

Identification of an Arabidopsis mitoferrinlike carrier protein involved in Fe metabolism.

Iron has a major role in mitochondrial as well as in chloroplast metabolism, however the processes involved in organelle iron transport in plants are only partially understood. To identify mitochondrial iron transporters in Arabidopsis, we searched for proteins homologous to the Danio rerio (zebrafish) Mitoferrin2 MFRN2, a mitochondrial iron importer in non-erythroid cells. Among the identified putative Arabidopsis mitoferrinlike proteins, we focused on that one encoded by At5g42130, which we named AtMfl1 (MitoFerrinLike1). AtMfl1 expression strongly correlates with genes coding for proteins involved in chloroplast metabolism. Such an unexpected result is supported by the identification by different research groups, of the protein encoded by At5g42130 and of its homologs from various plant species in the inner chloroplastic envelope membrane proteome. Notably, neither the protein encoded by At5g42130 nor its homologs from other plant species have been identified in the mitochondrial proteome. AtMfl1 gene expression is dependent on Fe supply: AtMfl1 transcript strongly accumulates under Fe excess, moderately under Fe sufficiency and weakly under Fe deficiency. In order to understand the physiological role of AtMfl1, we isolated and characterized two independent AtMfl1 KO mutants, atmfl1-1 and atmfl1-2: both show reduced vegetative growth. When grown under conditions of Fe excess, atmfl1-1 and atmfl1-2 mutants (seedlings, rosette leaves) contain less total Fe than wt and also reduced expression of the iron storage ferritin AtFer1. Taken together, these results suggest that Arabidopsis mitoferrinlike gene AtMfl1 is involved in Fe transport into chloroplasts, under different conditions of Fe supply and that suppression of its expression alters plant Fe accumulation in various developmental stages.

Arabidopsis CYP82C4 expression is dependent on Fe availability and circadian rhythm, and correlates with genes involved in the early Fe deficiency response.

Under conditions of reduced iron availability, most frequent in calcareous soils, plants induce the "Fe Deficiency Response" to improve root Fe uptake. The transcription factor FIT is essential for such a response in strategy I plants, such as Arabidopsis thaliana. From microarray analysis of Arabidopsis roots, it is known that three different cytochrome P450 genes, CYP82C4, CYP82C3 and CYP71B5 are up-regulated under Fe deficiency through a FIT-dependent pathway. We show that, out of these three P450 genes, only CYP82C4 strongly correlates with genes involved in metal uptake/transport. The CYP82C4 promoter, unlike those of CYP82C3 and CYP71B5, contains several IDE1-like sequences (iron deficiency-responsive element) as well as an RY element. While confirming that the CYP82C4 transcript accumulates in Fe-deficient Arabidopsis seedlings, with circadian fluctuations in a light-dependent way, we also demonstrate that such accumulation is suppressed under Fe excess. Full suppression of CYP82C4 expression, as observed in the atc82c4-1 KO mutant, is associated with longer roots at the seedling stage. We propose that CYP82C4 is involved in the early Fe deficiency response, possibly through an IDE1-like mediated pathway.

AtFer4 ferritin is a determinant of iron homeostasis in Arabidopsis thaliana heterotrophic cells.

In plants, the iron storage protein ferritin can be targeted to both chloroplasts and mitochondria. To investigate the role of Arabidopsis ATFER4 ferritin in mitochondrial iron trafficking, atfer4-1 and atfer4-2 mutant knock-outs for the AtFer4 gene were grown in heterotrophic suspension cultures. Both mutants showed altered cell size and morphology, reduced viability, higher H2O2 content and reduced O2 consumption rates when compared to wt. Although no reduction in total ferritin or in mitochondrial ferritin was observed in atfer4 mutants, total iron content increased in atfer4 cells and in atfer4 mitochondria. Transcript correlation analysis highlighted a partial inverse relationship between the transcript levels of the mitochondrial ferric reductase oxidase FRO3, putatively involved in mitochondrial iron import/export, and AtFer4. Consistent with this, FRO3 transcript levels were higher in atfer4 cells. We propose that the complex molecular network maintaining Fe cellular homeostasis requires, in Arabidopsis heterotrophic cells, a proper balance of the different ferritin isoforms, and that alteration of this equilibrium, such as that occurring in atfer4 mutants, is responsible for an altered Fe homeostasis resulting in a change of intraorganellar Fe trafficking.

Knocking out of the mitochondrial AtFer4 ferritin does not alter response of Arabidopsis plants to abiotic stresses.

Ferritins are iron-storage proteins which, in Arabidopsis, have a clear role in protection against oxidative stress. Plant ferritins are localized mainly in chloroplasts, but they can also be targeted to mitochondria; the ATFER4 ferritin isoform, according to bioinformatic subcellular predictors, has the highest scores for such localization in Arabidopsis. We isolated atfer4-2 mutant KO in the AtFer4 gene and we characterized it together with a second, independent mutant atfer4-1. We show that ATFER4 is indeed localized in mitochondria of Fe-treated Arabidopsis plants; when grown under Fe excess, atfer4 plants manifest, however, the same toxicity symptoms and O(2) consumption rates as wt plants. No enhanced sensitivity to oxidative conditions was observed in atfer4 seedlings exposed to salinity, osmotic stress, cold stress or oxidative stress elicited by paraquat. The growth response of roots and aerial parts in atfer4 plants under different light conditions was the same as wt. Also, the process of natural senescence, in which AtFer1 takes active part, was not perturbed in atfer4 plants. We conclude that the ATFER4 ferritin role in counteracting the environmental or developmental oxidative conditions in Arabidopsis plants is ancillary to that of the other isoforms, regardless of its mitochondrial localization.

Knock-out of ferritin AtFer1 causes earlier onset of age-dependent leaf senescence in Arabidopsis.

Ferritins are iron-storage proteins involved in the regulation of free iron levels in the cells. Arabidopsis thaliana AtFer1 ferritin, one of the best characterized plant ferritin isoforms to date, strongly accumulates upon treatment with excess iron, via a nitric oxide-mediated pathway. However other environmental factors, such as exposure to oxidative stress or to pathogen attack, as well as developmental factors regulate AtFer1 transcript levels. In particular, recent findings have highlighted an accumulation of the ferritin transcript during senescence. To investigate the physiological relevance of AtFer1 ferritin during senescence we isolated an Arabidopsis mutant knock-out in the AtFer1 gene, which we named atfer1-2. We analyzed it together with a second, independent AtFer1 KO mutant, the atfer1-1 mutant. Interestingly, both atfer1-1 and atfer1-2 mutants show symptoms of accelerated natural senescence; the precocious leaf yellowing is accompanied by accelerated decrease of maximal photochemical efficiency and chlorophyll degradation. However, no accelerated senescence upon dark treatment was observed in the atfer1 mutants with respect to their wt. These results suggest that AtFer1 ferritin isoform is functionally involved in events leading to the onset of age-dependent senescence in Arabidopsis and that its iron-detoxification function during senescence is required when reactive oxygen species accumulate.