EBOV protein functions and availability of structural informationa.
The recent outbreak of Ebola viral disease (EVD) in West Africa reminded us that an effective anti-viral treatment still does not exist, despite the significant progress that has recently been made in understanding biology and pathology of this lethal disease. Currently, there are no approved vaccine and/or prophylactic medication for the treatment of EVD in the market. However, the serious pandemic potential of EVD mobilized research teams in the academy and the pharmaceutical industry in the effort to find an Ebola cure as fast as possible. In this chapter, we are giving the condensed review of different approaches and strategies in search of a drug against Ebola. We have been focusing on the review of the targets that could be used for in silico, in vitro, and/or in vivo drug design of compounds that interact with the targets in different phases of the Ebola virus life cycle.
- small molecule inhibitors
- Ebola virus
- drug design
- protein targets
- structure and action
Ebola virus (EBOV) is a (-)ssRNA filovirus, known for its extreme insidiousness. Case fatality rates of the current 2014 outbreak in West Africa are 50–70% . Transmission of EBOV is predominantly via physical contact with bodily fluids of infected people or corpses and can be limited by a proper combination of early diagnosis, contact tracing, isolation of patients, infection control, and safe burial [2, 3].
The infection is characterized by suppression of the immune system and of the systemic inflammatory response, followed by the collapse of the vascular and immune systems, and multi-organ failure. The patient dies from a combination of dehydration, massive bleeding, and shock. Currently, there are no approved drugs for the hemorrhagic fever caused by EBOV. However, there is some conflicting clinical evidence that antibodies isolated from survived patients may be effective in the treatment of the infection caused by EBOV [4, 5].
In this book chapter, we will review possible targets that are being used or could be used for structure-based design of small molecule inhibitors against EBOV. We will start the chapter with a brief review of the structure and action of EBOV, and then we will describe the targets along with possible hotspots. Additionally, we will present a short review of small molecules that could be used as medicaments against EBOV.
2. Structure and action of Ebola virus
Knowledge about the life cycle of EBOV, supported with structural information, is crucial for the successful design of antivirals. This is the reason why we will start our review with the structural information about EBOV.
The RNA genome of Ebola virus contains information for constructing seven proteins (GP, VP24, VP30, VP35, VP40, L-protein, nucleoprotein), which assemble with the genomic RNA to form one of the most lethal viruses . EBOV's RNA exists in antisense form, which means that it cannot be used for proteins' production directly . For protein building, the complementary copy of the negative RNA is required, which is produced with the help of the viral polymerase (L-protein). Not all genes are transcribed fully through. For example, transcription of GP gene could lead to three different proteins: GP, sGP, and ssGP. A small nonstructural sGP (secretory glycoprotein) is the protein that is efficiently secreted from infected cells. sGP acts as mimic of full GP that is presented at the surface of EBOV, this mimicry is one of the ways of how the Ebola virus deceive the immune system, by urging the body to develop antibodies to sGP instead of full GP [8, 9]. EBOV is enclosed by a membrane hijacked from an infected cell and covered with Ebola glycoproteins. A layer of matrix proteins supports the membrane on the inside and holds a cylindrical nucleocapsid at the center, which stores and delivers the RNA genome.
The main task of Ebola glycoprotein (GP) is binding to receptors located on a host-cell surface and getting the Ebola genome inside. GP is distributed throughout the whole viral membrane surface and the large proportion of oligosaccharides, which are attached to the GP making the virus unrecognizable for the adaptive immune system. GP is a highly dynamic protein that snaps into different shapes when it binds to a cell surface, driving the virus close enough to get fused with the membrane.
The viral matrix is composed of two proteins: VP40 and VP24. The function of VP40, known as the major matrix protein, is to assist in the process of budding. VP40 hexamers form layers that support the nucleocapsid in the middle of the virion. The minor matrix protein VP24 is involved in interferon antagonism.
Another important structural element of EBOV is the nucleocapsid, which is located in the middle of the virion. The nucleocapsid is wound in a regular helix shape 50 nm in diameter. The nucleocapsid is composed of a series of viral proteins, which are attached to EBOV's genome, 19 kb linear, negative-sense RNA. The nucleocapsid is composed from the inner part where RNA is packed with NP protein, and the outer shell is composed of VP24, VP30, VP35, and polymerase (L-protein).
The information about the function of EBOV's proteins about its structural data along with potential targets is collected in Table 1.
for the protein
|Nucleocapsid and inclusion of body
formation; encapsulation of RNA genome,
replication and transcription of viral genome.
Binds VP35, and associates with VP30
4z9p, 4ypi, 4zta
|target interactions with
or binding sites on VP35,
VP30 and L
|Associates in RNP complexes,
binds RNA. Together with L, it forms replicase–
trancriptase holoenzyme, initiates transcription. EBOV VP35 suppresses
Type-I IFN production.
|Target basic patch of VP35
around the Arg312 to prevent
VP35 binding to double stranded
DNA (dsRNA) and promote
|Viral matrix protein. Accumulates
at cellular membrane proliferation sites,
responsible for viral budding. Assocites with microtubules and
outer cell membranes.
1h2c, 1h2d, 2kq0,
4eje, 4ldb, 4ldd,
|Compound 5539-0062 inhibits interaction of VP40 with
Tsg101 a factor involved in
endosomal protein sorting.
|Transmembrane protein that mediates
viral attachment to the cell membrane
and promote viral entry. Some domains of this protein may have
2ebo, 2rlj, 3csy,5f18,5f1b
|Disruption of interaction
between primed EBOV-GP and
NPC1. Interaction of EBOV-GP
with neutralizing antibodies
|Secondary nucleoprotein, binds NP
and ssRNA, part of ribonucleoprote-in complex.
VP30 in oligomeric form is neded for
the activation of transcription.
2i8b, 3v7o, 5dvw
|Target basic patch around
LYS 180 to inhibit the activation
|Secondary matrix protein, it colocalizes
with VP40 and has important function in
nucleocapsid formation. VP24 inhibits cellular
responses to IFN.
3vne, 3vnf, 4d9o, 4m0q, 4or8, 4u2x
|Inhibition of VP24 binding to karyopherin.|
|RNA dependent RNA polymerase;
binds VP35 to form replicase
|Only sequence||Poly-adenylation, profreading function or interaction
3. Collection of targets for small molecule inhibitor design
In this section, we describe different targets and strategies for structure-based design of EBOV's small molecule inhibitors. Based on the knowledge of EBOV action, different strategies for curing Ebola viral disease are proposed: prevention of EBOV adhesion to host cells (monoclonal antibodies; inhibitors of host-cell receptors, …) ; inhibition of viral escape from endosome (inhibition of NPC1, protease inhibitors) ; reviving and enhancing intracellular innate immunity; orthogonal RNA destruction mechanism targeting essential Ebola genes; inhibiting viral RNA processing (inhibition of RNA polymerase) ; disruption of viral assembly (nucleoprotein, VP40, VP24, VP35), etc.
Some of these mechanisms will be described in this section along with proposed targets. The ideogram where the connection between Ebola virus life cycle and possible therapeutic targets is shown in Figure 2.
The main function of nucleoprotein (NP) is encapsulation of the viral genome. EBOV's RNA located in the cage assembled mainly from nucleoprotein, which serves also as a scaffold for additional viral proteins forming nucleocapsid, is protected from the action of nucleases. The model of cross-section of RNA's cage composed from NP and VP35, based on recent X-ray diffraction structure (PDB-ID:4ypi) is shown in Figure 3 . A possible strategy to fight against EBOV is to prevent assembly of the RNA's cage. Binning
3.2. VP35 as a target for EVD therapeutics
EBOV's VP35 protein is a multifunctional protein, together with nucleoprotein and RNA, which is the main building block for the assembly of the nucleocapsid. Another important function of this protein is inhibition of interferon IFN-α/β production . Additionally, VP35 is also a cofactor of the viral RNA polymerase. In this subsection, we will introduce the strategy for inhibition of VP35's polymerase activity with small molecules.
Inspired by the success of Brown
Ebola virus specifically inhibits the dsRNA (double stranded RNA) within cells via a sequestration process. The molecular basis of such sequestration is shown in Figure 5, where the complex between VP35 and model of dsRNA is presented. Mutagenesis studies have shown that critical residues for binding dsRNA are Phe 239, Arg 312, Arg 322, and Lys 339. Mutation of these residues to Ala results in VP35 complete loss of its ability to bind dsRNA, and thus were also unable to suppress IFN-β promoter . Compounds like Ampligen, a immunomodulatory double stranded RNA, may be able to overcome this deficiency in host response [24, 25].
3.3. VP24 is vital for budding and acts also as interferon antagonist
VP24 is a secondary matrix protein that is colocalized with VP40 in virions and it is important for intracellular nucleocapsid assembly. It is also a type I interferon antagonist. This protein plays an important role in the budding. VP24 by itself has features that are common to the viral matrix protein (VP40), such as hydrophobicity, membrane binding, and self-oligomerisation . An important role of VP24 in the process of nucleocapsid formation has been demonstrated using electron microscopy .
Secondary matrix protein VP24 is one of the virion proteins that play a crucial role in Ebola virus disease pathology . Recently, VP24 was recognized as a major virulent factor. The virulent action of VP24 is initiated by binding of interferon (IFN) to the IFN receptors located on the host cells' surface. The activation of interferon leads to activation of STAT1 protein via Janus tyrosine kinase (JAK1). Activated by phosphorylation, STAT1 forms a dimer that subsequently interacts with KPNA5 (karyopherin 5, also known as importin). KPNA5 is vital for translocation of STAT1 dimer into the nucleus where STAT1 acts as a transcription activator for the expression of IFN stimulated genes. Recent studies have also shown that VP24 competes with STAT1 to bind KPNA5. These studies have shown that KPNA5 binds to VP24 more than 100 times tighter than to the STAT1:STAT1 dimer . The sequestration of free KPNA5 finally results in prevention of STAT1:STAT1 entrance into the nucleus and blocks the subsequent activation of numerous genes that are involved in antiviral activity. The mechanism of VP24 interferon signal path inhibitory action is shown in Figure 6.
Another successful approach of protection against EBOV infection is to use phosphorodiamidate morpholino oligomers (PMOs), which are able to bind mRNA in a sequence-specific fashion. The study of Warren
Molecules that bind to the VP24 surface where KPNA5 interacts could be inhibitors of viral action of VP24. Pleško
This study by Pleško
3.4. Secondary nucleoprotein VP30
The Ebola protein VP30 is known as a secondary nucleoprotein, the protein is colocalized with the nucleoprotein (NP) in inclusion bodies when both proteins are coexpressed
The study of Modrof
3.5. The viral matrix protein VP40
The viral matrix protein VP40 is the most abundant among Ebola virus' proteins, and it is encoded by the most conserved filovirus gene. VP40 is responsible for numerous functions of Ebola virus by rearranging into different oligomeric structures . Each of the structure has different role in the EV life cycle. VP40 dimer traffics to the cellular membrane, where the electrostatic interactions induce its rearrangement into a linear hexamer. VP40 hexamers form a multilayered matrix filament that is critical for budding of virion. Another structural rearrangement of VP40 is in the form of an octameric ring. The RNA-binding VP40 octameric ring is important for the regulation of viral transcription . Different assemblies of VP40 are shown in Figure 10.
The VP40 dimer with some of the potential sites for inhibition of viral budding is shown in Figure 11 . The area inside the magenta sphere represents the N-terminal domain interface that is responsible for the dimer formation. This interface domain involves two alpha helices 52-65 and 108-117, it is mainly composed of hydrophobic residues, thus the hydrophobic effect could be the driving force in the dimerization. It has been shown that mutation of Leu 117 disrupts the formation of VP40 dimers and inhibits viral budding. The C-terminal domain hexameric interface is shown in red. Met 241 and Ile 307 are residues that are mainly involved in the formation of hexamers. Disruption of the hexameric domain by a set of mutations reduces viral budding from plasma membranes. The cationic patch (blue area) consists of numerous positively charged residues: Lys 224, Lys 225, Lys 274, and Lys 275, which may interact via electrostatic interactions with the anionic leaflet of the plasma membrane. Mutation of these Lys residues reduces the viral budding. The area within the green sphere represents a hydrophobic loop, which penetrates into the plasma membrane—this step is necessary for viral budding.
3.6. Ebola virus surface's glycoprotein
The EBOV glycoprotein decorates the surface of virions, and it is responsible for the attachment of virus to the receptor cells and subsequent entry into the cells. Glycoprotein (GP1,2) is a transmembrane protein composed of two subunits, GP1 and GP2, linked by a disulfide bond. N- and O-linked glycosylation accounts for about one-third of the molecular mass . The posttranslational cleavage of the precursor GP into GP1 (~160 kDa) and GP2 (~38-45 kDa) is required for successful infection. Both subunits have an important role in the mechanism of viral infection: heavily glycosylated GP1 is responsible for the attachment to host cells, while GP2 is responsible for fusion of the viral and host-cell membrane once the virion has entered the endosome (lysosome). Both subunits have functional domains that are reported to have activities not connected with binding and entry. GP1, for example, has a domain at the N-terminus, which suppresses lymphocyte blastogenesis
An innovative approach to prevent adhesion of EBOV to host cell is blocking receptors (DC-SIGN, L-SIGN) located at the cell surface with glycodendritic structures. DC-SIGN (dendritic cell specific intercellular adhesion molecule-3-grabbing non-integrin) is one of the most important pathogen recognition receptors . This lectin is specific for the recognition of highly mannosylated branched oligosaccharides of EBOV's glycoprotein. Since the affinity between the single copy of the oligosaccharide and its respective receptor is often weak, researchers used the approach of multivalency to enhance the activity. Researchers have developed a series of dendritic glycoconjugates such as Boltorn-type glycodendrimers, glycodendrofullerenes, and virus-like gycodendronanoparticles [44–46]. Some representatives of dendrimeric structures that could successfully bind to the receptors at the host cell surface with high affinity are depicted in Figure 12.
In the most recent approach, researchers have synthesized globular multivalent glycofullerenes that act as potent inhibitors in a model of EBOV infection . They recognize hexakis adducts of 60-fullerene as useful building blocks since the obtained products maintain globular shape and with the aspect that it is relatively easy to control the size and multivalency.
Targeting GPs exposed at the surface of EBOV with neutralizing antibodies is one of the most often used strategies in the fight against Ebola. The GP structure represented in Figure 13 includes neutralizing antibodies from a person who survived infection by the virus. The antibodies bind to the bottom side of the glycoprotein, the portion of protein essential for the process of fusion, which is not usually decorated by oligosaccharidic chains.
3.7. The human Niemann-Pick disease type C1 protein
The human Niemann-Pick disease type C1 (NPC1) is a membrane protein that is predominantly required for intracellular transport of cholesterol and lipids in mammals. A deficiency of this protein leads to abnormal accumulation of lipids and cholesterol within cells. Recent studies of Côté
The proposed model for Ebola virus entry via binding of Ebola virus to NPC1 is presented in Figure 14. Binding of EBOV to the receptors (DC-SIGN [48, 49], TIM-1 , …) at the cell surface is the first and essential step in the viral infection. After successful attachment, the viruses undergo endocytosis and enter the cell internalized in late endosomes. In the next phase, cysteine proteases, primarily cathepsin B, cleave EBOV-GP to a 19-kD fragment. The cleaved EBOV-GP serves as a ligand for NPC1, a multimembrane spanning cholesterol transport protein. After binding of EBOV-GP to NPC1, EBOV nucleocapsid is released into the cell cytosol.
We are expecting further development of antivirals for blocking endosomal escape since the structure of the EBOV's GP2 to its endosomal receptor Niemann-Pick C1 has been recently solved .
4. Recent status of EBOV drugs
The most recent outbreak of Ebola viral disease in West Africa 2014 initiated a worldwide activity of searching for an effective cure against one of the most threatening diseases. Numerous bio/med/pharm researches are devoted to the investigation of the action of EBOV at the molecular level as a way to find optimal strategies to combat the virus. In this section, we will describe some small molecule inhibitors that have proven
4.1. Viral transcription modulators
An example of small molecules that alter the process of transcription of EBOV's genome is favipiravir (Figure 15), which was developed as a selective inhibitor of influenza virus replication (inhibits the viral RNA-dependent RNA polymerase). The antiviral potential against EBOV of favipiravir has been recently tested in a small animal model. Total prevention of mortality of the small animals subjected to the EBOV infection was achieved in these experiments . The nucleoside analog BCX4430 (Figure 15) is another compound from the class of viral transcription modulators. BCX4430 is active in vitro against negative-sense RNA-viruses including EBOV. Laboratory tests of BCX4430’s anti-EBOV activity have shown a survival rate between 90% and 100 % of experimentally infected mice. Further experiments reported success with a protective action of BCX4430 in EBOV infection of non human primates . C-c3Ado and c3Nep, which were first reported as S-adenosylhomocysteine hydrolase (SAH) inhibitors, are additional examples of compounds with protective action against small animal experimental EBOV infection .
4.2. Viral entry and fusion modulators
The fusion of EBOV and host cell membrane represents the first phase of EBOV infection . It has been shown that proteolysis of EBOV's glycoprotein GP1,2 represents an obligatory step in virus' life cycle. Proteolytic degradation of GP1,2 was successfully blocked using inhibitors of cysteine proteases, among which E-64d (unselective protease inhibitor), CA-074 (selective cathepsin B inhibitor), FY-DMK (Cathepsin B/L inhibitor) and Z-FY-(t-Bu)-DMK (Cathepsin L inhibitor), which are shown in Figure 16 [60, 61]. It was shown that Leupeptin (inhibitor of serine protease) and CIS23631927 – Cat L inhibitor were able to reduce EBOV infection in macrophages and human embryonic cells .
After fusion, the entry of viral particles is followed by endocytosis, which is dependent on a functional cytoskeleton. The research of Yonezawa
After fusion with the membrane and subsequent macropinocytosis, the Ebola virus is catched in late endosomes. Carette
EBOV-GP, as the key recognition element, represents a crucial mediator of viral budding; therefore, suppression of protein glycosylation is another option to decrease EBOV infection. An example of such treatment is the use of tunicamycin, a N-glycosylation suppressor, which decreases EBOV infection of HeLa cells by more than 90%. Another way to reduce EBOV infectivity is to use imino sugars (IHVR11029, IHVR17028, and IHVR19029) as inhibitors of -glucosidase I, a glycosyl hydrolase from endoplasmic reticulum, which is responsible for proper folding and maturation of nascent proteins .
4.3. Signal pathway modulators
Perturbation of cell signaling pathways involved in EBOV infection is another option to inhibit the devastating action of the virus. Here we will describe a few examples where existing kinase and protease inhibitors were used as a cure for EVD.
siRNA screening of human kinome identified mitogen-activated kinase (MAPK), phosphoinositide 3 kinase (PI3K) , and calcium/calmodulin kinases (CAMK2) as novel cellular targets for therapeutic intervention against EBOV infection . Garcia
By contrast, only dephosphorylated form of VP30, part of EBOV nucleocapsid complex, is required for viral transcription. Studies of Modrof
The use of ion channel blockers was also used to affect the complex process of viral entry, an example of such approach is to use multiple ion channel blockers amiodarone, dronedarone, and the calcium channel blocker verapamil for inhibition of Ebola virus GP1,2-mediated cell entry .
Structures of representative signal pathway modulators are presented in Figure 19.
4.4. Small molecule Ebola virus modulators
in vitro and in vivo
The review of Picazo
Recent outbreak of extremely lethal Ebola hemorrhagic fever in West Africa motivated scientists from all over the world to research EBOV life cycle and the pathology of EVD. The interest to find a successful treatment of Ebola hemorrhagic fever is currently one of the hottest topic in the academy and industry. Our review presents different strategies that could be used for the design of anti-EBOV medications. We have collected what is known about EBOV life cycle, structural information about EBOV protein targets, and some interesting inhibitors in one place. We believe that better knowledge of EBOV life cycle, supported with high quality structural information, could be a deciding factor in accelerating the task of finding a suitable cure against Ebola. Our review has shown that despite a huge collection of data (PDB structures, genome analysis,
This work was supported by Slovenian Research Agency (ARRS) through the research program P1-0201. We are also grateful for the support of dr. Edvard Kobal from Slovenian Science Foundation. And at the end, the authors express their gratitude to Mirzet Čuskić for his continuous moral support of our project.
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