Multifaceted roles for BCL3 in cancer: a proto-oncogene comes of age.

In the early 1990's a group of unrelated genes were identified from the sites of recurring translocations in B-cell lymphomas. Despite sharing the nomenclature 'Bcl', and an association with blood-borne cancer, these genes have unrelated functions. Of these genes, BCL2 is best known as a key cancer target involved in the regulation of caspases and other cell viability mechanisms. BCL3 on the other hand was originally identified as a non-canonical regulator of NF-kB transcription factor pathways - a signaling mechanism associated with important cell outcomes including many of the hallmarks of cancer. Most of the early investigations into BCL3 function have since focused on its role in NF-kB mediated cell proliferation, inflammation/immunity and cancer. However, recent evidence is coming to light that this protein directly interacts with and modulates a number of other signaling pathways including DNA damage repair, WNT/ß-catenin, AKT, TGFß/SMAD3 and STAT3 - all of which have key roles in cancer development, metastatic progression and treatment of solid tumours. Here we review the direct evidence demonstrating BCL3's central role in a transcriptional network of signaling pathways that modulate cancer biology and treatment response in a range of solid tumour types and propose common mechanisms of action of BCL3 which may be exploited in the future to target its oncogenic effects for patient benefit.

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.

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.

Design, synthesis, and biological investigation of selective human carbonic anhydrase II, IX, and XII inhibitors using 7-aryl/heteroaryl triazolopyrimidines bearing a sulfanilamide scaffold.

A novel library of human carbonic anhydrase (hCA) inhibitors based on the 2-sulfanilamido[1,2,4]triazolo[1,5-a]pyrimidine skeleton modified at its 7-position was prepared by an efficient convergent procedure. These derivatives were evaluated in vitro for their inhibition properties against a representative panel of hCA isoforms (hCA I, II, IV, IX, and XII). The target tumour-associated isoforms hCA IX and XII were potently inhibited with KIs in the low nanomolar range of 5-96 nM and 4-72 nM, respectively. Compounds 1d, 1j, 1v, and 1x were the most potent hCA IX inhibitors with KIs of 5.1, 8.6, 4.7, and 5.1 nM, respectively. Along with derivatives 1d and 1j, compounds 1r and 1ab potently inhibited hCA XII isoform with KIs in a single-digit nanomolar range of 8.8, 5.4, 4.3, and 9.0 nM, respectively. Compounds 1e, 1m, and 1p exhibited the best selectivity against hCA IX and hCA XII isoforms over off-target hCA II, with selectivity indexes ranging from 5 to 14.

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.

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.

A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle.

Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the ß-ladder domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 "Wing" domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 -NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a trans-complementation assay, we show that both cis and trans acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.

Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]thiophene derivatives as potent apoptosis-inducing agents.

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure-activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

A Computer-Based Methodology to Design Non-Standard Peptides Potentially Able to Prevent HOX-PBX1-Associated Cancer Diseases.

In the last decades, HOX proteins have been extensively studied due to their pivotal role in transcriptional events. HOX proteins execute their activity by exploiting a cooperative binding to PBX proteins and DNA. Therefore, an increase or decrease in HOX activity has been associated with both solid and haematological cancer diseases. Thus, inhibiting HOX-PBX interaction represents a potential strategy to prevent these malignancies, as demonstrated by the patented peptide HTL001 that is being studied in clinical trials. In this work, a computational study is described to identify novel potential peptides designed by employing a database of non-natural amino acids. For this purpose, residue scanning of the HOX minimal active sequence was performed to select the mutations to be further processed. According to these results, the peptides were point-mutated and used for Molecular Dynamics (MD) simulations in complex with PBX1 protein and DNA to evaluate complex binding stability. MM-GBSA calculations of the resulting MD trajectories were exploited to guide the selection of the most promising mutations that were exploited to generate twelve combinatorial peptides. Finally, the latter peptides in complex with PBX1 protein and DNA were exploited to run MD simulations and the ΔGbinding average values of the complexes were calculated. Thus, the analysis of the results highlighted eleven combinatorial peptides that will be considered for further assays.

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.

Characterizing the original anti-C5 function-blocking antibody, BB5.1, for species specificity, mode of action and interactions with C5.

The implication of complement in multiple diseases over the last 20 years has fuelled interest in developing anti-complement drugs. To date, the focus has been on C5; blocking cleavage of C5 prevents formation of two pro-inflammatory activities, C5a anaphylatoxin and membrane attack complex. The concept of C5 blockade to inhibit inflammation dates back 30 years to the description of BB5.1, an anti-C5 blocking monoclonal antibody raised in C5-deficient mice. This antibody proved an invaluable tool to demonstrate complement involvement in mouse disease models and catalysed enthusiasm for anti-complement drug development, culminating in the anti-human C5 monoclonal antibody eculizumab, the most successful anti-complement drug to date, already in clinical use for several rare diseases. Despite its key role in providing proof-of-concept for C5 blockade, the mechanism of BB5.1 inhibition remains poorly understood. Here, we characterized BB5.1 cross-species inhibition, C5 binding affinity and chain specificity. BB5.1 efficiently inhibited C5 in mouse serum but not in human or other rodent sera; it prevented C5 cleavage and C5a generation. BB5.1 bound the C5 α-chain with high affinity and slow off-rate. BB5.1 complementarity-determining regions were obtained and docking algorithms were used to predict the likely binding interface on mouse C5.

Enhanced efficacy of endonuclease inhibitor baloxavir acid against orthobunyaviruses when used in combination with ribavirin.

OBJECTIVES: Baloxavir acid is an endonuclease inhibitor approved for use against influenza. We evaluated whether this compound also targets the endonuclease domain of orthobunyaviruses and therefore could potentially be used against orthobunyavirus infections. METHODS: We performed a thermal shift assay and a fluorescence resonance energy transfer (FRET)-based nuclease monitoring assay using the La Crosse virus (LACV) endonuclease and baloxavir acid to prove their interaction and identify an inhibitory effect. Their interaction was further studied in a docking simulation using Glide SP. We show that baloxavir acid inhibits the viral replication of Bunyamwera virus (BUNV)-mCherry in vitro using high-content imaging and virus yield assay. Lastly, we investigated the use of baloxavir acid in combination with ribavirin in vitro by implementing the Zero Interaction Potency response surface model. RESULTS: We show that baloxavir acid augments LACV enzyme's melting temperature with ΔTm 9.5 ± 0.4°C and inhibited substrate cleavage with IC50 0.39 ± 0.03 µM. Moreover, our docking simulation suggests that baloxavir acid is able to establish an efficient binding with the LACV endonuclease. In the cell-based assay, we observed that baloxavir acid and ribavirin inhibited BUNV-mCherry with an EC50 of 0.7 ± 0.2 µM and 26.6 ± 8.9 µM, respectively. When used in combination, we found a maximum synergistic effect of 8.64. CONCLUSIONS: The influenza endonuclease inhibitor baloxavir acid is able to bind to and interfere with the endonuclease domain of orthobunyaviruses and yields a more potent antiviral effect than ribavirin against BUNV-mCherry. The combination of both compounds results in a more potent antiviral effect, suggesting that these molecules could potentially be combined to treat orthobunyavirus-infected patients.

In Vitro Topical Delivery of Chlorhexidine to the Cornea: Enhancement Using Drug-Loaded Contact Lenses and ß-Cyclodextrin Complexation, and the Importance of Simulating Tear Irrigation.

Microbial keratitis is a severe, sight-threatening condition caused by various pathogens. Eyedrops are the standard delivery modality for treating these disorders; however, blinking reflex, elevated tear production, and nasolacrimal drainage eliminate much of the instilled dose within a few seconds. Therefore, eyedrops must be applied repeatedly for prolonged periods. The present study aimed to probe more effective ocular delivery of chlorhexidine based upon drug-loaded hydrogel contact lenses and ß-cyclodextrin (ß-CD), while also determining the effect of constant irrigation with simulated tear fluid (STF) in in vitro experiments. Chlorhexidine digluconate (as 0.2 and 2% solutions, ß-CD inclusion complexes, and loaded hydrogel contact lenses) were applied to enucleated porcine eyes as single or multiple 10 µL doses, or as drug-loaded contact lenses, with and without ß-CD. The corneas were then excised and drug-extracted quantified by high-performance liquid chromatography (HPLC). The effect of constant irrigation by STF was evaluated to test the effect of increased tear production on corneal delivery. Potential antimicrobial activity of the delivered drug was also assessed. Results showed that drug-loaded contact lenses delivered the greatest amount of chlorhexidine into the cornea over a 24 h period, while the eyedrop solution comparator delivered the least. The ß-CD significantly enhanced chlorhexidine delivery to the cornea from eyedrop solution, although contact lenses loaded with chlorhexidine-ß-CD failed to enhance delivery. ß-CD within the hydrogel matrix impeded drug release. Constant irrigation with STF significantly reduced the amount of drug delivered to the cornea in all cases. Chlorhexidine retained antimicrobial activity in all delivery methods. Hydrogel contact lenses loaded with chlorhexidine delivered significantly higher levels to the cornea compared to eyedrops, either multiple hourly doses or a single dose. They also offer reduced application, in particular, to a nonulcerated corneal infection. Finally, the importance of fully accounting for tear production in in vitro ocular delivery experiments was highlighted.

Modeling Epac1 interactions with the allosteric inhibitor AM-001 by co-solvent molecular dynamics.

The exchange proteins activated by cAMP (EPAC) are implicated in a large variety of physiological processes and they are considered as promising targets for a wide range of therapeutic applications. Several recent reports provided evidence for the therapeutic effectiveness of the inhibiting EPAC1 activity cardiac diseases. In that context, we recently characterized a selective EPAC1 antagonist named AM-001. This compound was featured by a non-competitive mechanism of action but the localization of its allosteric site to EPAC1 structure has yet to be investigated. Therefore, we performed cosolvent molecular dynamics with the aim to identify a suitable allosteric binding site. Then, the docking and molecular dynamics were used to determine the binding of the AM-001 to the regions highlighted by cosolvent molecular dynamics for EPAC1. These analyses led us to the identification of a suitable allosteric AM-001 binding pocket at EPAC1. As a model validation, we also evaluated the binding poses of the available AM-001 analogues, with a different biological potency. Finally, the complex EPAC1 with AM-001 bound at the putative allosteric site was further refined by molecular dynamics. The principal component analysis led us to identify the protein motion that resulted in an inactive like conformation upon the allosteric inhibitor binding.

Rational modifications, synthesis and biological evaluation of new potential antivirals for RSV designed to target the M2-1 protein.

Respiratory syncytial virus (RSV) is the main cause of lower respiratory tract diseases in infants and young children, with potentially serious and fatal consequences associated with severe infections. Despite extensive research efforts invested in the identification of therapeutic measures, no vaccine is currently available, while treatment options are limited to ribavirin and palivizumab, which both present significant limitations. While clinical and pre-clinical candidates mainly target the viral fusion protein, the nucleocapsid protein or the viral polymerase, our focus has been the identification of new antiviral compounds targeting the viral M2-1 protein, thanks to the presence of a zinc-ejecting group in their chemical structure. Starting from an anti-RSV hit we had previously identified with an in silico structure-based approach, we have designed, synthesised and evaluated a new series of dithiocarbamate analogues, with which we have explored the antiviral activity of this scaffold. The findings presented in this work may provide the basis for the identification of a new antiviral lead to treat RSV infections.

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.

Design, synthesis and biological evaluation of 2-alkoxycarbonyl-3-anilinoindoles as a new class of potent inhibitors of tubulin polymerization.

A new class of inhibitors of tubulin polymerization based on the 2-alkoxycarbonyl-3-(3',4',5'-trimethoxyanilino)indole molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. The results presented show that the methoxy substitution and location on the indole nucleus plays an important role in inhibition of cell growth, and the most favorable position for the substituent was at C-6. In addition, a small-size ester function (methoxy/ethoxycarbonyl) at the 2-position of the indole core was desirable. Also, analogues that were alkylated with methyl, ethyl or n-propyl groups or had a benzyl moiety on the N-1 indolic nitrogen retained activity equivalent to those observed in the parent N-1H analogues. The most promising compounds of the series were 2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-5-methoxyindole 3f and 1-methyl-2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-6-methoxy-indole 3w, both of which target tubulin at the colchicine site with antitubulin activities comparable to that of the reference compound combretastatin A-4.

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.

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.

Synthesis and Biological Evaluation of 2-Substituted Benzyl-/Phenylethylamino-4-amino-5-aroylthiazoles as Apoptosis-Inducing Anticancer Agents.

Induction of apoptosis is a common chemotherapeutic mechanism to kill cancer cells The thiazole system has been reported over the past decades as a building block for the preparation of anticancer agents. A novel series of 2-arylalkylamino-4-amino-5-(3',4',5'-trimethoxybenzoyl)-thiazole derivatives designed as dual inhibitors of tubulin and cyclin-dependent kinases (CDKs) were synthesized and evaluated for their antiproliferative activity in vitro against two cancer cell lines and, for selected highly active compounds, for interactions with tubulin and cyclin-dependent kinases and for cell cycle and apoptosis effects. Structure-activity relationships were elucidated for various substituents at the 2-position of the thiazole skeleton. Among the synthesized compounds, the most active analogues were found to be the p-chlorobenzylamino derivative 8e as well as the p-chloro and p-methoxyphenethylamino analogues 8f and 8k, respectively, which inhibited the growth of U-937 and SK-MEL-1 cancer cell lines with IC50 values ranging from 5.7 to 12.2 µM. On U-937 cells, the tested compounds 8f and 8k induced apoptosis in a time and concentration dependent manner. These two latter molecules did not affect tubulin polymerization (IC50 > 20 µM) nor CDK activity at a single concentration of 10 µM, suggesting alternative targets than tubulin and CDK for the compounds.