toremifene and Hemorrhagic-Fever--Ebola

Niemann-Pick C1 (NPC1) receptor is an intracellular protein located in late endosomes and lysosomes whose main function is to regulate intracellular cholesterol trafficking. Besides being postulated as necessary for the infection of highly pathogenic viruses in which the integrity of cholesterol transport is required, this protein also allows the entry of the Ebola virus (EBOV) into the host cells acting as an intracellular receptor. EBOV glycoprotein (EBOV-GP) interaction with NPC1 at the endosomal membrane triggers the release of the viral material into the host cell, starting the infective cycle. Disruption of the NPC1/EBOV-GP interaction could represent an attractive strategy for the development of drugs aimed at inhibiting viral entry and thus infection. Some of the today available EBOV inhibitors were proposed to interrupt this interaction, but molecular and structural details about their mode of action are still preliminary thus more efforts are needed to properly address these points. Here, we provide a critical discussion of the potential of NPC1 and its interaction with EBOV-GP as a therapeutic target for viral infections.

Topics: Antibodies; Ebolavirus; Glycoproteins; Hemorrhagic Fever, Ebola; Humans; Molecular Docking Simulation; Niemann-Pick C1 Protein; Protein Binding; Small Molecule Libraries; Virus Internalization

Ebola virus (EBOV), one member of the family Filoviridae, can causes hemorrhagic fever and other severe diseases in humans with a high mortality rate (25-90%). Until recently, there were no approved drugs and very limited treatment method for Ebola virus disease. In this study, we discovered a series of potent Ebola entry inhibitors with the (3S,4aS,8aS)-2-(3-amino-2-hydroxypropyl)decahydroisoquinoline-3-carboxamide scaffold from high-throughput screening in reported pseudotyped virus system. Further optimization resulted a most potent compound 28 (IC

Topics: Antiviral Agents; Ebolavirus; Hemorrhagic Fever, Ebola; HIV Fusion Inhibitors; Humans; Molecular Docking Simulation; Virus Internalization

The recent Ebola epidemics in West Africa underscore the great need for effective and practical therapies for future Ebola virus outbreaks. We have discovered a new series of remarkably potent small molecule inhibitors of Ebola virus entry. These 4-(aminomethyl)benzamide-based inhibitors are also effective against Marburg virus. Synthetic routes to these compounds allowed for the preparation of a wide variety of structures, including a conformationally restrained subset of indolines (compounds

Topics: A549 Cells; Animals; Antiviral Agents; Benzamides; Chlorocebus aethiops; Cytochrome P-450 CYP3A Inhibitors; Drug Evaluation, Preclinical; Hemorrhagic Fever, Ebola; Humans; Marburg Virus Disease; Microsomes, Liver; Molecular Docking Simulation; Structure-Activity Relationship; Toremifene; Vero Cells; Viral Envelope Proteins; Virus Internalization

Ebola virus (EBOV) causes a deadly hemorrhagic fever in humans and non-human primates. There is currently no FDA-approved vaccine or medication to counter this disease. Here, we report on the design, synthesis and anti-viral activities of two classes of compounds which show high potency against EBOV in both in vitro cell culture assays and in vivo mouse models Ebola viral disease. These compounds incorporate the structural features of cationic amphiphilic drugs (CAD), i.e they possess both a hydrophobic domain and a hydrophilic domain consisting of an ionizable amine functional group. These structural features enable easily diffusion into cells but once inside an acidic compartment their amine groups became protonated, ionized and remain trapped inside the acidic compartments such as late endosomes and lysosomes. These compounds, by virtue of their lysomotrophic functions, blocked EBOV entry. However, unlike other drugs containing a CAD moiety including chloroquine and amodiaquine, compounds reported in this study display faster kinetics of accumulation in the lysosomes, robust expansion of late endosome/lysosomes, relatively more potent suppression of lysosome fusion with other vesicular compartments and inhibition of cathepsins activities, all of which play a vital role in anti-EBOV activity. Furthermore, the diazachrysene 2 (ZSML08) that showed most potent activity against EBOV in in vitro cell culture assays also showed significant survival benefit with 100% protection in mouse models of Ebola virus disease, at a low dose of 10 mg/kg/day. Lastly, toxicity studies in vivo using zebrafish models suggest no developmental defects or toxicity associated with these compounds. Overall, these studies describe two new pharmacophores that by virtue of being potent lysosomotrophs, display potent anti-EBOV activities both in vitro and in vivo animal models of EBOV disease.

Topics: Animals; Antiviral Agents; Chrysenes; Ebolavirus; Hemorrhagic Fever, Ebola; Lysosomes; Mice; Surface-Active Agents; Virus Internalization; Zebrafish

Here, we show that four chemically divergent approved drugs reported to inhibit Ebolavirus infection, benztropine, bepridil, paroxetine and sertraline, directly interact with the Ebolavirus glycoprotein. Binding of these drugs destabilizes the protein, suggesting that this may be the mechanism of inhibition, as reported for the anticancer drug toremifene and the painkiller ibuprofen, which bind in the same large cavity on the glycoprotein. Crystal structures show that the position of binding and the mode of interaction within the pocket vary significantly between these compounds. The binding constants (K

Topics: Animals; Antiviral Agents; Chlorocebus aethiops; Ebolavirus; Glycoproteins; Hemorrhagic Fever, Ebola; Models, Molecular; Protein Conformation; Thermodynamics; Vero Cells; Virus Internalization

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug's observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.

Topics: Animals; Antiviral Agents; Biological Transport; Cell Line; Chlorocebus aethiops; Ebolavirus; Hemorrhagic Fever, Ebola; Humans; Hydrazones; Lysosomes; Macrophages; Marburgvirus; Morpholines; Nocodazole; Phosphoinositide-3 Kinase Inhibitors; Pyrimidines; Toremifene; Triazines; Vero Cells; Virus Internalization

Although a group of FDA-approved drugs were previously identified with activity against Ebola virus (EBOV), most of them are not clinically useful because their human blood concentrations are not high enough to inhibit EBOV infection. We screened 795 unique three-drug combinations in an EBOV entry assay. Two sets of three-drug combinations, toremifene-mefloquine-posaconazole and toremifene-clarithromycin-posaconazole, were identified that effectively blocked EBOV entry and were further validated for inhibition of live EBOV infection. The individual drug concentrations in the combinations were reduced to clinically relevant levels. We identified mechanisms of action of these drugs: functional inhibitions of Niemann-Pick C1, acid sphingomyelinase, and lysosomal calcium release. Our findings identify the drug combinations with potential to treat EBOV infection.

Topics: Animals; Antiviral Agents; Cell Line; Chlorocebus aethiops; Clarithromycin; Drug Combinations; Drug Synergism; Ebolavirus; Hemorrhagic Fever, Ebola; High-Throughput Screening Assays; Humans; Mefloquine; Sphingomyelin Phosphodiesterase; Toremifene; Triazoles; Vero Cells; Virus Internalization

Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)- and ex-US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.

Topics: Animals; Cathepsins; Chlorocebus aethiops; Clomiphene; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Approval; Ebolavirus; Endosomes; Hemorrhagic Fever, Ebola; Hep G2 Cells; Humans; Hydrogen-Ion Concentration; Mice; Mice, Inbred C57BL; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Survival Analysis; Toremifene; United States; United States Food and Drug Administration; Vero Cells; Virion; Virus Internalization