Contemporary Anti-Ebola Drug Discovery Approaches and Platforms

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Contemporary anti-Ebola drug discovery approaches and platforms Elena K. Schneider-Futschik, Daniel Hoyer, Alex Khromykh, Jonathan B. Baell, Glenn Marsh, Mark A. Baker, Jian Li, and Tony Velkov ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00285 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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ACS Infectious Diseases

Contemporary anti-Ebola drug discovery approaches and platforms

Elena K. Schneider-Futschik1, Daniel Hoyer1,2,3,, Alexander A. Khromykh4, Jonathan B. Baell5,6, Glenn A. Marsh7, Mark A. Baker8, Jian Li9, Tony Velkov1*

1Department

of Pharmacology & Therapeutics, School of Biomedical Sciences, Faculty of

Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia; 2The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, VIC, 3052, Australia. 3Department of Molecular Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA Microbiology and Parasitology, 4Australian Infectious Diseases Research Centre, School of Chemistry & Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072. 5School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China; 6Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia.

7CSIRO

Livestock Industries, Australian Animal Health

Laboratory, Geelong, Australia. 8Priority Research Centre in Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia.9Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.

* Corresponding author: Tony Velkov Phone: + 61 3 83449846 E-mail: [email protected]

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Email address (all authors) Elena K. Schneider-Futschik: [email protected] Daniel Hoyer: [email protected] Alexander A. Khromykh: [email protected] Jonathan B. Baell: [email protected] Glenn A. Marsh: [email protected] Mark A. Baker: [email protected] Jian Li: [email protected] Tony Velkov:[email protected]

Keywords: Ebola virus, drug development, FDA approved drugs, discovery platforms.


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The Ebola virus has a grave potential to destabilise civil society as we know it. The past few deadly Ebola outbreaks were unprecedented in size: The 2014-15 Ebola West Africa outbreak saw the virus spread from the epicentre through to Guinea, Sierra Leone, Nigeria, Congo and Liberia. The 2014-15 Ebola West Africa outbreak was associated with almost 30,000 suspected or confirmed cases and over 11,000 documented deaths. The more recent 2018 outbreak in the Democratic Republic of Congo has so far resulted in 216 suspected or confirmed cases and 139 deaths. There is a general acceptance within the World Health Organisation (WHO) and the Ebola outbreak response community that future outbreaks will become increasingly more frequent and more likely to involve inter-continental transmission. The magnitude of the recent outbreaks demonstrated in dramatic fashion the shortcomings of our mass casualty disease response capabilities and lack of therapeutic modalities for supporting Ebola outbreak prevention and control. Currently, there are no approved drugs although vaccines for human Ebola virus infection are in the trial phases and some potential treatment have been field tested most recently in the Congo Ebola outbreak. Treatment is limited to pain management and supportive care to counter dehydration and lack of oxygen. This underscores the critical need for effective anti-viral drugs that specifically target this deadly disease. This review examines the current approaches for the discovery of anti-Ebola small molecule or biological therapeutics, their viral targets, mode of action and contemporary platforms, which collectively form the backbone of the anti-Ebola drug discovery pipeline.



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An in-depth understanding of the biology of a disease is often a key aspect that underlies any successful drug discovery program. Ebola virus (EBOV) infection is a highly infectious zoonotic disease with a very high fatality rate in humans (up to 90%).1-2 Human EBOV infection is characterised by fever, myalgia, headache, gastrointestinal symptoms, and a maculopapular rash.3 Fatal outcomes correlate with viremia, convulsions, and intravascular haemorrhage. Worryingly, fruit bats, which are common in many countries, have been identified as a reservoir host for Ebola,4 although non-human primates may also be a source of contamination. EBOV is an encapsidated single-stranded negative RNA (ssRNA) virus belonging to the family Filoviridae.2 Due to their genome organisation, filoviruses belong together with the paramyxo-, rhabdo- and borna-viruses to the order Mononegavirales. Members of the family Filoviridae include three genera, one of which is Ebolavirus. Members of Ebola virus genera include Ebola virus (EBOV, species Zaire ebolavirus), Sudan virus, Tai Forest virus, Bundibugyo virus, Reston virus, and Bombali virus, with only the first 4 viruses known to cause disease in humans EBOV, Marburg virus, Sudan virus and Bundibugyo virus; all of which cause severe viral haemorrhagic fevers in humans.2 Electron microscopy studies show that the EBOV has a filamentous appearance, hence its name sake (Figure 1). EBOV viral particles in infected individuals are found in skin, mucus membranes, bodily fluids, and nasal secretions.5 EBOV infection begins when an individual comes into contact with the viral particles at an endothelial or epithelial site through mucus membranes or small skin abrasions; these can include mucus membranes like the nose, mouth, eyes and gastrointestinal tract. Luckily, aerosol transmission of EBOV has not yet been conclusively demonstrated. The high virulence, rapid onset and high mortality of EBOV infection in humans, together with the sparseness of effective therapeutic modalities has led to its categorisation by the National Institute of Allergy and Infectious Diseases (US) and the Centre for Disease Control and Prevention (CDC) as an urgent unmet medical challenge. Moreover, EBOV is classified as a Critical Biological Agents Category C Biosafety Level 4 pathogen (BSL4), which restricts live virus research to a very limited number of high-level bio-containment facilities. 4 ACS Paragon Plus Environment

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The genome of the EBOV consists of a 19 kb non-segmented negative-sense ssRNA which encodes seven structural proteins namely NP, VP35, VP40 (major matrix protein), GP (glycoprotein), VP30, VP24 (minor matrix protein) and L (syn. RNA-dependant-RNA polymerase, RdRp; Figures 1 and 2).2,

6

EBOV only has a single promoter at the 3′ end of its genome for the RdRp to bind,

consequently, as the RdRp moves down the genome it tends to fall off (and start over at the 3′ end of the genome) as it hits the bumps of the translation start and stop signals of the seven individual viral genes. Thus, genes located near the promoter (i.e. 3′ end of the genome) are expressed at much higher levels than genes toward the 5′ end of the genome. Therefore, the NP protein whose gene is situated at the 3′ end is expressed at the highest levels, with VP40 at intermediate levels, while the L protein is usually expressed at the lowest levels. The EBOV viron surface is made up of glycoprotein (GP) spikes and a lipid bilayer derived from infected cell membranes, as the virus buds from the cell. This helps EBOV evade the immune system, because most of the antigenic components besides GP are cloaked by the host derived lipid bilayer. Beneath the lipid bilayer, there are the matrix proteins VP40 and VP24, which play a structural role in maintaining the shape of the viral particle. The nucleocapsid forms the core of the viron, consisting of a complex of NP, VP35, VP30, and L proteins and the ssRNA viral genome (Figure 2). The fact that all of these viral components are very distinct from any known host factors and, there are no human homologs represents an opportunity and makes the discovery of EBOV specific drugs highly feasible.

In the first stage of its replication cycle, the infecting EBOV binds to attachment factors and receptors on the cell surface through the viral GP protein (Figure 3, Step 1).7 This in turn triggers a process called macropinocytosis (Step 2),8 and the cell imports the virus inside the endosomes.9 EBOV then employs a fusion interaction of its GP with the endosome to make its way out of the late endosomes (Step 3).10 Basically, the virus containing endosome is processed, and eventually becomes a late endosome where the pH drops leading to an acidification of its viral payload. This in 5 ACS Paragon Plus Environment


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turn triggers the activity of the endosomal enzymes Cathepsin B and L that sequentially cleave GP 1,2 generating a primed 19-kDa form of GP coined sGP that initiates fusion between the viral and endosomal membranes resulting in practice, the release of viral nucleocapsids pop out of the cellular trash can and into the cytoplasm (Step 4).11 Then, viral genome replication ensues whereby the RdRP initiates (with the aid of the VP35 and VP30 viral proteins) at the extreme 3′ promoter end of the -ssRNA genome to synthesise a full-length complementary antigenome (+ssRNA). This antigenome then serves as the master template for the synthesis of full-length progeny genomes. The progeny genomes can then be utilised as templates for secondary transcription, or assembled into infectious particles. Finally, viral proteins and genomic RNA are assembled into complete virus particles and the virus exits the cell by budding through the plasma membrane (Step 5). Intuitively, the steps into the EBOV life cycle that can be exploited for antiviral therapeutic development are the endosomal fusion processes and the RdRp machinery.

The current status of small molecules, antibodies and nucleic acids with antiviral activity against EBOV has recently been covered in detail across a series of excellent reviews,12-28 hence we will focus our discussions in overview on the mode of action, viral targets of these leads and finally in greater detail the developmental platforms.

The EBOV RdRp, syn. large (L) polymerase protein is the heart of the viral replication machinery and has no human homologues as such it is a highly attractive drug target. In fact RdRp inhibitors already have a proven track record as therapies for human viral infections.29-30 RdRp is responsible for catalysing several enzymatic activities that drive viral RNA transcription and replication.31 Sequence comparisons of Mononegavirales L proteins have revealed six highly conserved domains I-VI (Figure 1); and there is a general assent that the enzymatic activities of RdRp are located in these conserved regions.32 In line with this putative domain-like architecture, RdRp domains have been expressed independently in Escherichia coli and shown to retain their enzymatic function.32 A 6 ACS Paragon Plus Environment

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developmental nucleoside drug (BCX4430) that inhibits the EBOV RdRp was reported in Nature by BioCryst Pharmaceuticals.15 Unfortunately, the compound displayed rather poor pharmacokinetic (PK) properties with a very short plasma half-life in rodents and macaques (

Contemporary Anti-Ebola Drug Discovery Approaches and Platforms

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