ebola : possible future cure of ebola
Answer by Kettner Griswold:
A key step in designing antivirals is understanding the life cycle of the virus. Lets start by looking at the ebola virus itself to familiarize ourselves with how it functions. I will propose key therapeutic methods afterwards.
Ebola's genome is made up of linear single stranded RNA about 18-19 thousand base pairs in length. The genome encodes for seven proteins. Let's look at what they do.
The viral RNA polymerase (L) binds the encapsidated genome at the leader region, then sequentially transcribes each genes by recognizing start and stop signals flanking viral genes. This produces mRNAs are capped and polyadenylated by the polymerase during synthesis. Those mRNAs are later translated by the infected cells into proteins.
Not all genes transcribe fully though. The primary product of the unedited transcript of GP gene yields a smaller non-structural glycoprotein sGP which is efficiently secreted from infected cells. This sGP protein acts as a diversion tactic for ebola virus. Instead of generating antibodies directly targeting the full length Glycoprotein (GP) that is presented on the outside of the Ebola virus, your body is more likely to develop antibodies targeting sGP instead. This is seen in a majority of ebola victims. The "Glyco" prefix here refers to the fact that the protein is coated with short chains of sugar molecules. This has the effect of creating a flexible exterior, rendering the production of effectively "sticky" antibodies, incredibly difficult. However this is just one of the ways ebola plays tricks on our immune systems.
We also have so called innate immune systems. This is our body's way of flagging down offending pathogens with broadly applicable signatures. One such signature is that there should never be double stranded RNA (dsRNA) in our cells, or uncapped RNA molecules with triphosphates added on the end. If these types of RNA appear in our cells, it initiates a series of cellular defenses because viruses tend to use these types of RNA for replicating, among other things.
In our cells we have numerous mechanisms to recognize these events and call for help. One such signaling pathway involves a protein called Retinoic acid-inducible gene I, (RIG-I) which is involved in regulation of the innate immune system around viral RNA. RIG-I typically recognizes short 5′ triphosphate uncapped double stranded or single stranded RNA. Normally RIG-I detects these events and then signals to your cells to make lots of interferon, a protein released by host cells that causes nearby cells to ramp up their anti-viral defenses. Not so fast! Ebola virus inhibits this pathway using viral protein 35 (VP35) and viral protein 24 (VP24), disrupting the interaction of RIG-I with important signaling molecule PACT, and others, thereby short circuiting the interferon production process.
A RIG1 Protein Complex
Your cells have other defenses once they are infected. One such defense is RNA interference, a means of using RNA to target other RNA for destruction. The DICER protien is the key enzyme in your cell that recognizes dsRNA and destroys it. In addition to viral protein 35 (VP35), it has been found that VP30 and VP40 independently act as suppressors of RNA interference. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of silencing RNAs. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Collectively these proteins from ebola effectively inhibit RNA interference in your cells.
How DICER binds dsRNA
When Ebola Nucleoprotein (NP) is expressed in mammalian cells, it assembles into helical structures. Recombinantly expressed NP helix can become associated with non-viral RNA, and digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that the RNA is an essential structural component of the helix. Ebola uses NP to form a structural scaffold around its genomic RNA, and to protect the RNA from degrading enzymes of the host cell.
Ebola enters cells by macropinocytosis which is non-specific and non-absorptive.
The vesicle here is called the endosome. The GP protein on the outside of ebola plays a key role in initiating the macropinocytosis process and allows the virus to escape the endosome and enter the cytoplasm. Most endosomes contain proteases, enzymes that breakdown other proteins. Ebola actually requires these proteins to be active in order to shed off excess protein sequences on GP that activate Ebola's ability to escape the endosome and enter the cytoplasm.
The mucin regions here are glycosylated regions on GP
So now that I've laid out how ebola functions, I will go ahead and include the technical summary behind where I would develop therapeutics.
Scalable Production of Monoclonal Antibodies that Target Ebola Virus proteins GP1, GP2
Concept: Construct vectors producing ZMAPP and other Mabs targeting ebola in P pastoris (glycoswitch) strain, and show that the production method is scalable. It is believed that ZMAPP generates an immune response to extracellular Ebola virions and inhibits macropinocytosis. Glycoengineered Pichia pastoris can produce as much as 1g/L of culture. (Potgieter et al. 2009)
Pichia Pastoris Glycoswitch Strain
Inhibiting Endosomal Escape
Concept: The Ebola virus enters cells by a macropinocytosis-related pathway and requires endosomal cysteine proteases, particularly, cathepsin B, and an intact fusion peptide to reach the cytoplasm. A wide array of diseases result from elevated levels of cysteine targeting cathepsins, which causes numerous pathological processes including cell death, inflammation, and production of toxic peptides. Cathepsin inhibitors have been historically pursued in clinical applications for treatment of multiple diseases. In a streptococcus pneumoniae meningitis rodent model, cathepsin inhibitor treatment greatly improved the clinical course of the infection and reduced brain inflammation and inflammatory Interleukin-1beta (IL1-beta) and tumor necrosis factor-alpha (TNFalpha). ( Hoegen et al. 2011)
After transport of the virus to the endosome ebola virus GP is processed by cathepsin B (CatB) and/or L (CatL) proteases. CatB and/or CatL are essential for ebola virus infection. Indeed, inhibition of CatL and CatB by specific protease inhibitors or siRNA reduces entry of Zaire ebola virus and zaire ebola virus GP-pseudotyped viruses alike. (Lee et al. 2009) Misasi and colleagues obtained evidence for virus-specific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae.(Misasi et al. 2012)
E-64 irreversibly binds to the active thiol group of many cysteine proteases such as papain, actinidase, and cathepsins B, H and L to form a thioether linkage. E-64 is a very useful cysteine protease inhibitor for use in in vivo studies because it has a specific inhibition, it is permeable in cells and tissues, it has low toxicity, it is easily synthesized and it is stable. It is active around 10um concentration in tissues. (Joyce et al. 2004) It’s irreversible broad spectrum effects on cysteine proteases are of potential concern for clinical purposes, and therefore the turnover rate of inactivated cysteine proteases will be important to understand in non-human primate models.
An E-64 molecule.
Chemical structures of the small molecules identified by a pseudovirus inhibition assay.
Four small molecules showed inhibition of Ebola virus. The authors found that these novel broad-spectrum small molecules could block cathepsin L-mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from Ebola virus. The small molecules can be further optimized and developed into a potent broad-spectrum antiviral Drug. In general, the small-molecule inhibitors appear to be more specific to CatL than to CatB. The future testing of these inhibitors against live viruses would be important and beneficial in developing them into potent broad-spectrum inhibitor. (Elshabrawy et al. 2014)
N-arylaminonitriles are bioavailable peptidomimetic inhibitors of cathepsin B. (Greenspan et al 2003)
A N-arylaminonitrile, candidate.
Reviving and Enhancing Intracellular Innate Immunity
Concept: Ebola interferes with the innate immune system. Retinoic acid-inducible gene I, (RIG-I) and relatives are involved in regulation of the innate immune system around viral RNA. RIG-I typically recognizes short (< 4000nt) 5′ triphosphate uncapped double stranded or single stranded RNA. Ebola inhibits this pathway using VP35, disrupting the interaction of PACT with RIG-I thereby short circuiting Interferon induction. Interferons (IFNs) are proteins made and released by host cells in response to the presence of pathogens. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to ramp up their anti-viral defenses It has been shown that preactivation of RIG-I reduces Ebola virus titers in cell culture up to ∼1000-fold (Spiropoeulou et al., 2009) Drugs that lead to upregulation of RIG-I/RLR pathways, could therefore be used for those suspected of having contact with ebola victims, during the incubation period.
A RIG1 Protein Complex
Delivery of innocuous 5’ triphosphorylated RNA molecules is generally sufficient to upregulate the expression of RIG-1 leading to enhanced anti-viral response. The virus like particle delivery and vaccination approached discussed later is a viable means of delivering such RNA molecules for RIG-I upregulation. Interferon delivery is also potentially promising for such situations. It should be noted that EBOV replication and subsequent lethal effects in primates can be reduced by treatment with siRNA. However, siRNA treatment is required over time to maintain the efficacy, demonstrating a balance between viral replication and innate immune response by the host and a potential role for EBOV suppression of RNA silencing.
The overall structure of the VP35 IID-dsRNA complex
Concept: We can revive the DICER dsRNA knockdown methods with interfering RNA aptamers. In addition to viral protein 35 (VP35), it has been found that VP30 and VP40 independently act as suppressors of RNA silencing. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). (Fabozzi et al. 2011) Generalized dsRNA interference can be achieved by catalytic complexes acting as artificial nucleases that selectively target dsRNA. Binning and colleagues developed RNA aptamers that have high (10–50 nM) specifity against VP35 proteins. The SELEX approach used for development of aptamers against VP35 is generalizable to creating VP30 and VP40 targeting aptamers.
Orthogonal RNA Destuction Mechanisms Targeting Essential Ebola Genes
Concept: Ebola has unique proteins that are essential for replication. Antisense oligonucleotides can allow for the targeting of ssRNAs in a sequence specific fashion. They act by hybridizing to specific ssRNAs and inhibit expression of particular proteins by RNase H-mediated destruction. This is a promising orthogonal RNA interference route as it is is not inhibited by ebola interference mechanisms. The RNA:DNA hybrid duplex that is formed between the antisense oligonucleotide and a target mRNA interferes directly with protein synthesis, resulting in reduced expression of the coded protein. RNase H destroys the ssRNA strand, and both the RNAse H enzyme and the antisense oligonucleotide remain intact at the end of the cycle: the antisense effect is therefore catalytic. A single antisense oligonucleotide can partake in the destruction of many mRNA molecules by this mechanism. The enzyme RNase H occurs in normal cells where it plays an important role in removing short pieces of RNA from the lagging strand of DNA replication. DNAase resistant oligos are ideally suited to this application and can be delivered by multiple means. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogues that silence expression of specific genes. They have high potency as sequence specific antivirals inhibiting replication and translation mRNA of necessary viral proteins. Targets include NP, VP35, VP40, GP.
Antiviral Morpholino Conjugates
Inhibiting Viral RNA processing
Concept: Certain genes in Ebola virus require RNA editing for expression. A novel synthetic adenosine analogue, has been shown to inhibit infection of distinct filoviruses in human cells. Biochemical, reporter-based and primer-extension assays indicate that analog inhibits viral RNA polymerase (L gene) function, acting as a non-obligate RNA chain terminator. Post-exposure intramuscular administration of the analog protected against Ebola virus and Marburg virus disease in rodent models. Most importantly, the analog completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection.
Attacking intracellular structural assembly
Concept: Ebola uses a series of structural proteins to construct the virus within a human cell. They are: NP (Nucleoprotein) which binds the dsRNA along its length. VP40 (Matrix protein) virus assembly and budding at the plasma membrane of infected cells. VP24 (Membrane associated protien) Plays a role in assembly of viral nucleocapsid and virion budding, blocks interferon-alpha/beta (IFN-alpha/beta) and IFN-gamma signaling pathways. SELEX produced RNA aptamers with high affinity against these proteins are desirable to inhibit formation of necessary structural complex formation key to viron production. We could deliver aptamers as TAT protein conjugates. other methods include liposomal delivery.
Aptamer affinity verified by Isothermal Titration Calorimetry against NP (Binning et al. 2013)
The aptamers were shown to compete with dsRNA for binding to VP35 and disrupt the eVP35–nucleoprotein (NP) interaction. We believe the approach will be effective provided that the delivery of the aptamer is successful and efficient.
Ebola Virus Like Particle Delivery: A Trojan Horse Approach
Concept: Use Ebola VLPs as delivery vectors of therapeutic nucleic acid molecules. They are plausibly immunogenic for vaccination purpose and Ebola VLPs can serve as means of siRNA and DNA delivery to ebola targeted cell. Coexpression of the Ebola virus glycoprotein (GP) and matrix protein (VP40) in mammalian cells results in spontaneous production and release of virus-like particles that resemble the distinctively filamentous infectious virions. (figure below.) Warfield and colleagues showed that mice vaccinated with eVLPs were 100% protected from an otherwise lethal Ebola virus inoculation. (Warfield et al. 2003)
In 2006 they demonstated that 100% of 5 vaccinated non-human primates with eVLPs containing EBOV glycoprotein (GP), nucleoprotein (NP), and VP40 matrix protein and challenged the macaques with 1000 pfu of EBOV. The Control animals died. (Warfield et al. 2006)
Expanding on Warfield’s method, propose using alternative VLP production platforms to package arbitrary siRNA, interfering morpholinos, and linearized plasmid DNA sequences into the Ebola Virus like particles using Pichia Pastoris Glycoswitch and cell free platforms.
Conceptually, these goals can be achieved through addition of expression of Ebola virus nucleoprotein (NP). When NP is expressed in mammalian cells, it assembles into helical structures. Recombinantly expressed NP helix can become associated with non-viral RNA, and digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that the RNA is an essential structural component of the helix. (Noda et al. 2010) Our proposed strategy is to pretreat cell free lysates to remove undesirable RNA and DNA molecules. Thereafter, The VLP expression plasmid and huge excess of siRNA are added to the expression system. We expect that this will efficiently pack and deliver siRNA. Depending on the backbone flexibility, we believe that ssDNA and possibly morpholinos should become packaged as well for antisense therapy.
Binning, J. M., Wang, T., Luthra, P., Shabman, R. S., Borek, D. M., Liu, G., … Amarasinghe, G. K. (2013). Development of RNA aptamers targeting Ebola virus VP35. Biochemistry, 52(47), 8406–8419.
Elshabrawy, H. A., Fan, J., Haddad, C. S., Ratia, K., Broder, C. C., Caffrey, M., & Prabhakar, B. S. (2014). Identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and Ebola, Hendra, and Nipah viruses by using a novel high-throughput screening assay. Journal of Virology, 88(8), 4353–65. doi:10.1128/JVI.03050-13
Fabozzi, G., Nabel, C. S., Dolan, M. a, & Sullivan, N. J. (2011). Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. Journal of Virology, 85(6), 2512–23. doi:10.1128/JVI.01160-10
Greenspan, P. D., Clark, K. L., Cowen, S. D., McQuire, L. W., Tommasi, R. A., Farley, D. L., … Zhou, S. (2003). N-Arylaminonitriles as bioavailable peptidomimetic inhibitors of cathepsin B. Bioorganic and Medicinal Chemistry Letters, 13(22), 4121–4124.
Hoegen, T., Tremel, N., Klein, M., Angele, B., Wagner, H., Kirschning, C., … Koedel, U. (2011). The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release. Journal of Immunology (Baltimore, Md. : 1950), 187(10), 5440–51. doi:10.4049/jimmunol.1100790
Iversen, P. L., Warren, T. K., Wells, J. B., Garza, N. L., Mourich, D. V, Welch, L. S., … Bavari, S. (2012). Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infections. Viruses, 4(11), 2806–30. doi:10.3390/v4112806
Joyce, J. a, Baruch, A., Chehade, K., Meyer-Morse, N., Giraudo, E., Tsai, F.-Y., … Hanahan, D. (2004). Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell, 5(5), 443–453. doi:10.1016/S1535-6108(04)00111-4
Lee, J. E., & Saphire, E. O. (2009). glycoprotein structure and mechanism of entry. Future Virology.
Lennemann, N. J., Rhein, B. A., & Ndungo, E. (2014). Comprehensive Functional Analysis of N-Linked Glycans on Ebola. doi:10.1128/mBio.00862-13.Editor
Misasi, J., Chandran, K., Yang, J.-Y., Considine, B., Filone, C. M., Côté, M., … Cunningham, J. (2012). Filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences. Journal of Virology, 86(6), 3284–92. doi:10.1128/JVI.06346-11
Noda, T., Hagiwara, K., Sagara, H., & Kawaoka, Y. (2010). Characterization of the Ebola virus nucleoprotein-RNA complex. The Journal of General Virology, 91(Pt 6), 1478–83. doi:10.1099/vir.0.019794-0
Olinger, G. G., Pettitt, J., Kim, D., Working, C., Bohorov, O., Bratcher, B., … Zeitlin, L. (2012). Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proceedings of the National Academy of Sciences of the United States of America, 109(44), 18030–5. doi:10.1073/pnas.1213709109
Pettitt, J., Zeitlin, L., Kim, D. H., Working, C., Johnson, J. C., Bohorov, O., … Olinger, G. G. (2013). Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Science Translational Medicine, 5(199), 199ra113. doi:10.1126/scitranslmed.3006608
Potgieter, T. I., Cukan, M., Drummond, J. E., Houston-Cummings, N. R., Jiang, Y., Li, F., … d’Anjou, M. (2009). Production of monoclonal antibodies by glycoengineered Pichia pastoris. Journal of Biotechnology, 139(4), 318–25. doi:10.1016/j.jbiotec.2008.12.015
Ritchie, G., Harvey, D. J., Stroeher, U., Feldmann, F., Wahl-jensen, V., Royle, L., … Rudd, M. (2012). NIH Public Access, 1–30. doi:10.1002/rcm.4410.Identification
Spiropoulou, C. F., Ranjan, P., Pearce, M. B., Sealy, T. K., Albariño, C. G., Gangappa, S., … Sambhara, S. (2009). RIG-I activation inhibits ebolavirus replication. Virology, 392(1), 11–5. doi:10.1016/j.virol.2009.06.032
Warfield, K. L., Bosio, C. M., Welcher, B. C., Deal, E. M., Mohamadzadeh, M., Schmaljohn, A., … Bavari, S. (2003). Ebola virus-like particles protect from lethal Ebola virus infection. Proceedings of the National Academy of Sciences of the United States of America, 100(26), 15889–94. doi:10.1073/pnas.2237038100
Warfield, K. L., Swenson, D. L., Olinger, G. G., Kalina, W. V, Aman, M. J., & Bavari, S. (2007). Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge. The Journal of Infectious Diseases, 196 Suppl (Suppl 2), S430–7. doi:10.1086/520583
Warren, T. K., Wells, J., Panchal, R. G., Stuthman, K. S., Garza, N. L., Van Tongeren, S. a, … Bavari, S. (2014). Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430. Nature, 508(7496), 402–5. Retrieved from