Structural investigations on the mitochondrial uncouplers niclosamide and FCCP.

There has been renewed interest in using mitochondrial uncoupler compounds such as niclosamide and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) for the treatment of obesity, hepatosteatosis and diseases where oxidative stress plays a role. However, both FCCP and niclosamide have undesirable effects that are not due to mitochondrial uncoupling, such as inhibition of mitochondrial oxygen consumption by FCCP and induction of DNA damage by niclosamide. Through structure-activity analysis, we identified FCCP analogues that do not inhibit mitochondrial oxygen consumption but still provided good, although less potent, uncoupling activity. We also characterized the functional role of the niclosamide 4'-nitro group, the phenolic hydroxy group and the anilide amino group in mediating uncoupling activity. Our structural investigations provide important information that will aid further drug development.

Conjugating uncoupler compounds with hydrophobic hydrocarbon chains to achieve adipose tissue selective drug accumulation.

One potential approach for treating obesity is to increase energy expenditure in brown and white adipose tissue. Here we aimed to achieve this outcome by targeting mitochondrial uncoupler compounds selectively to adipose tissue, thus avoiding side effects from uncoupling in other tissues. Selective drug accumulation in adipose tissue has been observed with many lipophilic compounds and dyes. Hence, we explored the feasibility of conjugating uncoupler compounds with a lipophilic C8-hydrocarbon chain via an ether bond. We found that substituting the trifluoromethoxy group in the uncoupler FCCP with a C8-hydrocarbon chain resulted in potent uncoupling activity. Nonetheless, the compound did not elicit therapeutic effects in mice, likely as a consequence of metabolic instability resulting from rapid ether bond cleavage. A lipophilic analog of the uncoupler compound 2,6-dinitrophenol, in which a C8-hydrocarbon chain was conjugated via an ether bond in the para-position (2,6-dinitro-4-(octyloxy)phenol), exhibited increased uncoupling activity compared to the parent compound. However, in vivo pharmacokinetics studies suggested that 2,6-dinitro-4-(octyloxy)phenol was also metabolically unstable. In conclusion, conjugation of a hydrophobic hydrocarbon chain to uncoupler compounds resulted in sustained or improved uncoupling activity. However, an ether bond linkage led to metabolic instability, indicating the need to conjugate lipophilic groups via other chemical bonds.

Exploring the Fatty Acid Binding Pocket in the SARS-CoV-2 Spike Protein - Confirmed and Potential Ligands.

Severe Acute Respiratory syndrome 2 (SARS-CoV-2) is a respiratory virus responsible for coronavirus disease 19 (COVID-19) and the still ongoing and unprecedented global pandemic. The key viral protein for cell infection is the spike glycoprotein, a surface-exposed fusion protein that both recognizes and mediates entry into host cells. Within the spike glycoprotein, a fatty acid binding pocket (FABP) was confirmed, with the crystallization of linoleic acid (LA) occupying a well-defined site. Importantly, when the pocket is occupied by a fatty acid, an inactive conformation is stabilized, and cell recognition is hindered. In this review, we discuss ligands reported so far for this site, correlating their activity predicted through in silico studies with antispike experimental activity, assessed by either binding assays or cell-infection assays. LA was the first confirmed ligand, cocrystallized in a cryo-EM structure of the spike protein, resulting in increased stability of the inactive conformation of the spike protein. The next identified ligand, lifitegrast, was also experimentally confirmed as a ligand with antiviral activity, suggesting the potential for diverse chemical scaffolds to bind this site. Finally, SPC-14 was also confirmed as a ligand, although no inhibition assays were performed. In this review, we identified 20 studies describing small-molecule compounds predicted to bind the pocket in in silico studies and with confirmed binding or in vitro activity, either inhibitory activity against the spike-ACE2 interaction or antiviral activity in cell-based assays. When considering all ligands confirmed with in vitro assays, a good overall occupation of the pocket should be complemented with the ability to make direct interactions, both hydrophilic and hydrophobic, with key amino acid residues defining the pocket surface. Among the active compounds, long flexible carbon chains are recurrent, with retinoids capable of binding the FABP, although bulkier systems are also capable of affecting viral fitness. Compounds able to bind this site with high affinity have the potential to stabilize the inactive conformation of the SARS-CoV-2 spike protein and therefore reduce the virus's ability to infect new cells. Since this pocket is conserved in highly pathogenic human coronaviruses, including MERS-CoV and SARS-CoV, this effect could be exploited for the development of new antiviral agents, with broad-spectrum anticoronavirus activity.

The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

Rational design of novel nucleoside analogues reveals potent antiviral agents for EV71.

Different viruses belonging to distinct viral families, such as enterovirus 71, rely on the host methyltransferase METTL3 for the completion of fundamental cytoplasmic stages of their life cycle. Modulation of the activity of this enzyme could therefore provide a broad-spectrum approach to interfere with viral infections caused by viruses that depend on its activity for the completion of their viral cycle. With the aim to identify antiviral therapeutics with this effect, a series of new nucleoside analogues was rationally designed to act as inhibitors of human METTL3, as a novel approach to interfere with a range of viral infections. Guided by molecular docking studies on the SAM binding pocket of the enzyme, 24 compounds were prepared following multiple-step synthetic protocols, and evaluated for their ability to interfere with the replication of different viruses in cell-based systems, and to directly inhibit the activity of METTL3. While different molecules displayed moderate inhibition of the human methyltransferase in vitro, multiple novel, potent and selective inhibitors of enterovirus 71 were identified.

Identification of Small Molecular Chaperones Binding P23H Mutant Opsin through an In Silico Structure-Based Approach.

N-terminal P23H opsin mutation accounts for most of retinitis pigmentosa (RP) cases. P23H functions and folding can be rescued by small chaperone ligands, which contributes to validate mutant opsin as a suitable target for pharmacological treatment of RP. However, the lack of structural details on P23H mutant opsin strongly impairs drug design, and new chemotypes of effective chaperones of P23H opsin are in high demand. Here, a computational-boosted workflow combining homology modeling with molecular dynamics (MD) simulations and virtual screening was used to select putative P23H opsin chaperones among different libraries through a structure-based approach. In vitro studies corroborated the reliability of the structural model generated in this work and identified a number of novel chemotypes of safe and effective chaperones able to promote P23H opsin trafficking to the outer cell membrane.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2 )2 (tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) "RuRuPhen" blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

CHIKV strains Brazil (wt) and Ross (lab-adapted) differ with regard to cell host range and antiviral sensitivity and show CPE in human glioblastoma cell lines U138 and U251.

Chikungunya virus (CHIKV), a (re)emerging arbovirus, is the causative agent of chikungunya fever. To date, no approved vaccine or specific antiviral therapy are available. CHIKV has repeatedly been responsible for serious economic and public health impacts in countries where CHIKV epidemics occurred. Antiviral tests in vitro are generally performed in Vero-B4 cells, a well characterised cell line derived from the kidney of an African green monkey. In this work we characterised a CHIKV patient isolate from Brazil (CHIKVBrazil) with regard to cell affinity, infectivity, propagation and cell damage and compared it with a high-passage lab strain (CHIKVRoss). Infecting various cell lines (Vero-B4, A549, Huh-7, DBTRG, U251, and U138) with both virus strains, we found distinct differences between the two viruses. CHIKVBrazil does not cause cytopathic effects (CPE) in the human hepatocarcinoma cell line Huh-7. Neither CHIKVBrazil nor CHIKVRoss caused CPE on A549 human lung epithelial cells. The human astrocyte derived glioblastoma cell lines U138 and U251 were found to be effective models for lytic infection with both virus strains and we discuss their predictive potential for neurogenic CHIKV disease. We also detected significant differences in antiviral efficacies regarding the two CHIKV strains. Generally, the antivirals ribavirin, hydroxychloroquine (HCQ) and T-1105 seem to work better against CHIKVBrazil in glioblastoma cells than in Vero-B4. Finally, full genome analyses of the CHIKV isolates were done in order to determine their lineage and possibly explain differences in tissue range and antiviral compound efficacies.

Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase.

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure-activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2)2(tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) "RuRuPhen" blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

SARS-CoV-2 Virus-Host Interaction: Currently Available Structures and Implications of Variant Emergence on Infectivity and Immune Response.

Coronavirus disease 19, or COVID-19, is an infection associated with an unprecedented worldwide pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which has led to more than 215 million infected people and more than 4.5 million deaths worldwide. SARS-CoV-2 cell infection is initiated by a densely glycosylated spike (S) protein, a fusion protein, binding human angiotensin converting enzyme 2 (hACE2), that acts as the functional receptor through the receptor binding domain (RBD). In this article, the interaction of hACE2 with the RBD and how fusion is initiated after recognition are explored, as well as how mutations influence infectivity and immune response. Thus, we focused on all structures available in the Protein Data Bank for the interaction between SARS-CoV-2 S protein and hACE2. Specifically, the Delta variant carries particular mutations associated with increased viral fitness through decreased antibody binding, increased RBD affinity and altered protein dynamics. Combining both existing mutations and mutagenesis studies, new potential SARS-CoV-2 variants, harboring advantageous S protein mutations, may be predicted. These include mutations S13I and W152C, decreasing antibody binding, N460K, increasing RDB affinity, or Q498R, positively affecting both properties.

Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells.

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on ß-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.

Structure-Activity Relationship Studies on Novel Antiviral Agents for Norovirus Infections.

Human norovirus is the leading cause of acute gastroenteritis worldwide, affecting every year 685 million people. Norovirus outbreaks are associated with very significant economic losses, with an estimated societal cost of 60 billion USD per year. Despite this, no therapeutic options or vaccines are currently available to treat or prevent this infection. An antiviral therapy that can be used as treatment and as a prophylactic measure in the case of outbreaks is urgently needed. We previously described the computer-aided design and synthesis of novel small-molecule agents able to inhibit the replication of human norovirus in cell-based systems. These compounds are non-nucleoside inhibitors of the viral polymerase and are characterized by a terminal para-substituted phenyl group connected to a central phenyl ring by an amide-thioamide linker, and a terminal thiophene ring. Here we describe new modifications of these scaffolds focused on exploring the role of the substituent at the para position of the terminal phenyl ring and on removing the thioamide portion of the amide-thioamide linker, to further explore structure-activity relationships (SARs) and improve antiviral properties. According to three to four-step synthetic routes, we prepared thirty novel compounds, which were then evaluated against the replication of both murine (MNV) and human (HuNoV) norovirus in cells. Derivatives in which the terminal phenyl group has been replaced by an unsubstituted benzoxazole or indole, and the thioamide component of the amide-thioamide linker has been removed, showed promising results in inhibiting HuNoV replication at low micromolar concentrations. Particularly, compound 28 was found to have an EC50 against HuNoV of 0.9 µM. Although the most active novel derivatives were also associated with an increased cytotoxicity in the human cell line, these compounds represent a very promising starting point for the development of new analogues with reduced cytotoxicity and improved selectivity indexes. In addition, the experimental biological data have been used to create an initial 3D quantitative structure-activity relationship model, which could be used to guide the future design of novel potential anti-norovirus agents.

Anti-schistosomal activities of quinoxaline-containing compounds: From hit identification to lead optimisation.

Schistosomiasis is a neglected disease of poverty that is caused by infection with blood fluke species contained within the genus Schistosoma. For the last 40 years, control of schistosomiasis in endemic regions has predominantly been facilitated by administration of a single drug, praziquantel. Due to limitations in this mono-chemotherapeutic approach for sustaining schistosomiasis control into the future, alternative anti-schistosomal compounds are increasingly being sought by the drug discovery community. Herein, we describe a multi-pronged, integrated strategy that led to the identification and further exploration of the quinoxaline core as a promising anti-schistosomal scaffold. Firstly, phenotypic screening of commercially available small molecules resulted in the identification of a moderately active hit compound against Schistosoma mansoni (1, EC50 = 4.59 µM on schistosomula). Secondary exploration of the chemical space around compound 1 led to the identification of a quinoxaline-core containing, non-genotoxic lead (compound 22). Compound 22 demonstrated substantially improved activities on both intra-mammalian (EC50 = 0.44 µM, 0.20 µM and 84.7 nM, on schistosomula, juvenile and adult worms, respectively) and intra-molluscan (sporocyst) S. mansoni lifecycle stages. Further medicinal chemistry optimisation of compound 22, resulting in the generation of 20 additional analogues, improved our understanding of the structure-activity relationship and resulted in considerable improvements in both anti-schistosome potency and selectivity (e.g. compound 30; EC50 = 2.59 nM on adult worms; selectivity index compared to the HepG2 cell line = 348). Some derivatives of compound 22 (e.g. 31 and 33) also demonstrated significant activity against the two other medically important species, Schistosoma haematobium and Schistosoma japonicum. Further optimisation of this class of anti-schistosomal is ongoing and could lead to the development of an urgently needed alternative to praziquantel for assisting in schistosomiasis elimination strategies.

Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin.

Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA.

Targeting the Complement Serine Protease MASP-2 as a Therapeutic Strategy for Coronavirus Infections.

MASP-2, mannose-binding protein-associated serine protease 2, is a key enzyme in the lectin pathway of complement activation. Hyperactivation of this protein by human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 has been found to contribute to aberrant complement activation in patients, leading to aggravated lung injury with potentially fatal consequences. This hyperactivation is triggered in the lungs through a conserved, direct interaction between MASP-2 and coronavirus nucleocapsid (N) proteins. Blocking this interaction with monoclonal antibodies and interfering directly with the catalytic activity of MASP-2, have been found to alleviate coronavirus-induced lung injury both in vitro and in vivo. In this study, a virtual library of 8736 licensed drugs and clinical agents has been screened in silico according to two parallel strategies. The first strategy aims at identifying direct inhibitors of MASP-2 catalytic activity, while the second strategy focusses on finding protein-protein interaction inhibitors (PPIs) of MASP-2 and coronaviral N proteins. Such agents could represent promising support treatment options to prevent lung injury and reduce mortality rates of infections caused by both present and future-emerging coronaviruses. Forty-six drug repurposing candidates were purchased and, for the ones selected as potential direct inhibitors of MASP-2, a preliminary in vitro assay was conducted to assess their interference with the lectin pathway of complement activation. Some of the tested agents displayed a dose-response inhibitory activity of the lectin pathway, potentially providing the basis for a viable support strategy to prevent the severe complications of coronavirus infections.

Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C.

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine-which also targets 2C-but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

Novel Nucleoside Analogues as Effective Antiviral Agents for Zika Virus Infections.

Previously considered a neglected flavivirus, Zika virus has recently emerged as a public health concern due to its ability to spread rapidly and cause severe neurological disorders, such as microcephaly in newborn babies from infected mothers, and Guillain-Barré syndrome in adults. Despite extensive efforts towards the identification of effective therapies, specific antivirals are still not available. As part of ongoing medicinal chemistry studies to identify new antiviral agents, we screened against Zika virus replication in vitro in a targeted internal library of small-molecule agents, comprising both nucleoside and non-nucleoside agents. Among the compounds evaluated, novel aryloxyphosphoramidate prodrugs of the nucleosides 2'-C-methyl-adenosine, 2-CMA, and 7-deaza-2'C-methyl-adenosine, 7-DMA, were found to significantly inhibit the virus-induced cytopathic effect in multiple relevant cell lines. In addition, one of these prodrugs exhibits a synergistic antiviral effect against Zika virus when applied in combination with an indirect antiviral agent, a l-dideoxy bicyclic pyrimidine nucleoside analogue, which potently inhibits vaccinia and measles viruses in vitro by targeting a host pathway. Our findings provide a solid basis for further development of an antiviral therapy for Zika virus infections, possibly exploiting a dual approach combining two different agents, one targeting the viral polymerase (direct-acting antiviral), the second targeting a host-directed autophagy mechanism.

Synthesis and biological evaluation of novel flexible nucleoside analogues that inhibit flavivirus replication in vitro.

Flaviviruses, such as Dengue (DENV) and Zika (ZIKV) viruses, represent a severe health burden. There are currently no FDA-approved treatments, and vaccines against most flaviviruses are still lacking. We have developed several flexible analogues ("fleximers") of the FDA-approved nucleoside Acyclovir that exhibit activity against various RNA viruses, demonstrating their broad-spectrum potential. The current study reports activity against DENV and Yellow Fever Virus (YFV), particularly for compound 1. Studies to elucidate the mechanism of action suggest the flex-analogue triphosphates, especially 1-TP, inhibit DENV and ZIKV methyltransferases, and a secondary, albeit weak, effect on the DENV RNA-dependent RNA polymerase was observed at high concentrations. The results of these studies are reported herein.

Computational Studies towards the Identification of Novel Rhodopsin-Binding Compounds as Chemical Chaperones for Misfolded Opsins.

Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber's congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber's congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases.