Aus dem Institut für Experimentelle Virologie des TWINCORE Zentrum für Experimentelle und Klinische Infektionsforschung
Characterization of the Antiviral Activity of Silvestrol against
Hepatitis E Virus Infection
DISSERTATION
Zur Erlangung des Doktorgrades der Medizin in der M edizinischen H ochschule H annover
vorgelegt von
N ora M aria Proske, geb. M öller geb. in Hannover
Hannover 2019
Angenommen vom Senat am: 12.04.2021
Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover
Präsident: Prof. Dr. med. Michael P. Manns Betreuer der Arbeiter: Prof. Dr. rer. nat. Eike Steinmann
1. Referent: Prof. Dr. Abel Viejo-Borbolla, PhD 2. Referent: Prof. Dr. rer. nat. Florian Kühnel
Tag der mündlichen Prüfung: 12.04.2021 Prüfungsausschuss
Vorsitz: Prof. Dr. med. Hans-Heinrich Kreipe 1. Prüfer: PD Dr. med. Albert Heim
2. Prüfer: Prof. Dr. med. Andreas Klos
TABLE OF CONTENT 1
TA BLE OF CON TEN T
1 Abbreviations ... 2
2 Zusammenfassung ... 3
3 Introduction ... 5
3.1 Hepatitis E: Epidemiology ... 5
3.2 Hepatitis E: Classification and Molecular Virology ... 6
3.3 Hepatitis E: Pathophysiology and Clinical Manifestation ... 9
3.4 Hepatitis E: Diagnostics ... 11
3.5 Hepatitis E: Treatment ... 12
4 Aim of the thesis... 14
5 Manuscript ... 15
6 Discussion and Outlook ... 24
7 References ... 29
8 Danksagungen ... 38
9 Lebenslauf ... 39
10 Eidesstaatliche Erklärung nach § 2 Abs. 2 Nrn. 6 und 7 ... 41
11 Appendix ... 42
Abbreviations 2
1 A bbreviations
ALAT alanine aminotransferase CC50 cytotoxic concentration 50 EBOV Ebola virus
eIF4A eukaryotic initiation factor 4A ELISA enzyme-linked immunosorbent assays ER endoplasmic reticulum
gt genotype
GTP guanosine triphosphate HCV Hepatitis C Virus
Hel RNA helicase
HEV Hepatitis E Virus
HEVcc cell culture-derived infectious HEV HLC hepatocyte-like cells
IC50 inhibitory concentration 50
IgM/IgG Immunoglobulin M/ Immunoglobulin G
kb kilobyte
MT methyltransferase ORF open reading frame PCR polymerase chain reaction
Pol RNA-dependent RNA polymerase PPR polyproline region
Pro cysteine protease RBV ribavirin
RNA ribonucleic acid
sg subgenomic
SOT solid organ transplantation UTR untranslated region
Zusammenfassung 3
2 Zusam m enfassung
Weltweit infiziert das Hepatitis-E-Virus (HEV) mehr als 3 Millionen Menschen pro Jahr. Die WHO beschreibt die HEV-Infektion als Hauptursache für akute virale Hepatitiden. Schätzungen gehen von jährlichen 70 000 Toten durch HEV aus. Dennoch gilt HEV als bisher wenig erforscht und die Behandlungsmöglichkeiten sind äußerst begrenzt. Zurzeit stehen ausschließlich die noch nicht zugelassenen Medikamente Ribavirin (RBV), pegiliertes Inteferon-alpha und Sofosbuvir für die Therapie von HEV-Infektionen zur Verfügung, welche allerdings schwere Nebenwirkungen zur Folge haben können.
Viele Jahre lang waren HEV-Infektionen vor allem aus endemischen Gebieten wie Indien, Südostasien und Subsahara-Afrika bekannt. Doch mit steigender Inzidenz werden HEV- Erkrankungen auch in Europa und anderen Industrieländern verzeichnet. Hier sind besonders immunsupprimierte Patienten betroffen, da diese durch das geschwächte Immunsystem häufiger an den Folgen einer chronischen Hepatitis E mit Leberzirrhose bis hin zum Leberversagen erkranken. Die Infektionswege mit HEV unterscheiden sich hierbei beträchtlich. Während in den Entwicklungsländern Erkrankungen vor allem durch die fäkal-orale Übertragung hervorgerufen werden, besteht die Hauptinfektionsquelle in den industrialisierten Ländern im Verzehr von nicht ausreichend gegartem Schweinefleisch. Damit stellen HEV-Infektionen ein wachsendes Gesundheitsproblem dar. Dieses hat wesentliche Einschränkungen im alltäglichen Leben zur Folge und kann sozioökonomische Konflikte verursachen.
In der veröffentlichten Studie, die Inhalt der vorliegenden Dissertation ist, hat sich unsere Arbeitsgruppe der Aufgabe gewidmet, nach neuen Therapiemöglichkeiten von HEV-Infektionen zu suchen. Dabei konnten wir Silvestrol als potentiellen Wirkstoff identifizieren.
Mahagoniepflanzen aus Indonesien bilden Silvestrol, das sich aus deren Blättern extrahieren lässt.
Silvestrol ist bekannt als spezifischer Inhibitor der DEAD-box RNA-Helikase eIF4A und wurde bereits als möglicher Wirkstoff gegen bestimmte Tumore und einige Viren beschrieben.
In der Studie wurden die antiviralen Effekte der Substanz Silvestrol auf die Replikation von HEV in vitro wie auch in vivo untersucht. Zunächst wurden verschiedene subgenomische HEV- Reporterviren in Zellkulturen mit Silvestrol behandelt und die Wirkung im Vergleich zu einer Ribavirin Behandlung analysiert. Dabei zeigte sich, dass Silvestrol die HEV-Replikation konzentrationsabhängig inhibiert und sogar weitaus effizienter wirkt als Ribavirin. Eine Kombinationsbehandlung aus Silvestrol mit Ribavirin führte zu deutlich additiven Effekten.
Zusammenfassung 4 Ferner reduzierte Silvestrol die Replikation des volle Länge Konstrukts HEVp6 und führte zu einer verminderten Produktion von infektiösen HEV-Partikeln. In einem weiteren Schritt wurde der Infektionsverlauf von HEV mit und ohne Silvestrol in Stammzellen untersucht, die zu Leberzellen ausdifferenziert waren. Zur Infektion nutzten wir hierbei Hepatitis-E-Viren vier unterschiedlicher Genotypen, die alle aus Patienten stammten und aufgereinigt worden waren.
Auch hier hemmte Silvestrol die HEV-Replikation in den infizierten Zellen. Um zu untersuchen, ob Silvestrol auch in vivo die Virusreplikation inhibiert, wurden chronisch mit HEV infizierte Mäuse für jeweils eine Dauer von 6 oder 10 Tagen mit Silvestrol behandelt. Nach Beginn der Behandlung reduzierte sich die Viruslast in den mit HEV infizierten Mäusen deutlich, was einen antiviralen Effekt von Silvestrol ebenfalls in vivo bestätigte.
Zusammenfassend lässt sich sagen, dass die Ergebnisse unserer Studie die Hoffnung wecken, dass Silvestrol ein wirksames Mittel gegen Hepatitis E sein könnte. Sowohl in den in vitro wie in vivo Experimenten hemmte Silvestrol die HEV-Replikation effizient und zeigte dabei keine starke zytotoxische Wirkung. Damit legen unsere Untersuchungen einen Grundstein, um in weiteren Studien das klinische Potenzial von Silvestrol gegen HEV ausloten zu können.
Introduction 5
3 Introduction
The hepatitis E virus (HEV) infects worldwide approximately 20 million people and leads to more than 3 million cases of acute hepatitis every year (WHO, 2017). Although the virus is known for more than 3 decades, HEV research has been neglected for a long time (Dalton et al., 2018). In the past, hepatitis E virus infections were generally known from endemic countries such as the Indian subcontinent, South-East Asia and Sub-Saharan Africa but not considered as a clinical problem in developed countries. However, with cases of chronic hepatitis emerging in immunocompromised patients in the developed world, HEV has been studied more intensively (Dalton et al., 2008; Kamar et al., 2012; Wedemeyer et al., 2012).
3.1 H epatitis E: Epidem iology
HEV is mainly transmitted through the food chain via contaminated drinking water or infected animals. There are at least 5 human-pathogenic HEV genotypes (gt 1–4 and 7), of which genotypes 1 and 2 infect only human individuals whereas genotypes 3, 4 and 7 are zoonotic and can cross the species barrier (Doceul et al., 2016; Kamar, Izopet et al., 2017). The global presence of the HEV genotypes is illustrated in Figure 1. The distribution of the genotypes shows a specific pattern:
HEV gt1 and gt2 are highly endemic in the developing world, including Africa, South-East Asia and Mexico, causing large epidemics of acute hepatitis (Aggarwal, 2011). The outbreaks usually occur by contamination of the drinking water due to poor sanitary infrastructure. After heavy rainfall and flooding, the water supplies get polluted with human feces and the virus is transmitted via the fecal-oral route among humans (Viswanathan, 1957; Naik et al., 1992; Khuroo and Khuroo, 2016).
In contrast, the reservoir of HEV genotypes 3 and 4 has mainly been described in swine, wild boars and deer. The newly discovered gt7 has been identified in dromedaries. HEV genotypes 3, 4 and 7 are common in developed countries as Europe, USA and China and spread by consumption of undercooked meat or direct contact with infected animals (Van der Poel, W. H., 2014; Sayed et al., 2015; Rasche et al., 2016). Genotype 3 is the main HEV genotype circulating in Europe.
Whereas in China and other developed Asian regions, Hepatitis E is primarily caused by genotype 4. Nevertheless, sporadic cases of genotype 4 infections have also been described for Europe in the meantime (Doceul et al., 2016). The most relevant host of HEV worldwide are domestic swine,
Introduction 6 which do not show any symptoms upon infection, but are highly contagious. Thus, individuals who consume infected meat or are in direct contact with animals, most notably veterinarians and farmers have a higher risk to develop a HEV infection (Dalton et al., 2008; Kamar et al., 2012;
Kamar, Izopet et al., 2017). But not only in products of mammals HEV-RNA has been detected, in addition the virus has been found in shellfish, soft fruits and salads probably caused by irrigation with contaminated water (Dalton et al., 2018).
In the developed countries the human-to-human infection is no less relevant than the environmental transmission. HEV can further be transmitted via blood transfusion or organ transplants from infected donors. This transmission route is particularly dangerous, given that immunosuppressed patients are prone to develop a chronic hepatitis upon infection. (Schlosser et al., 2012; Hewitt et al., 2014).
Figure 1: Worldwide distribution of the different hepatitis E virus (HEV) genotypes. (Kamar, Izopet et al., 2017) (Reprint with permission granted by Springer Nature)
3.2 H epatitis E: Classification and M olecular V irology
The Hepatitis E Virus is a member of the family of Hepeviridae which is divided into two different genera, Orthohepevirus and Piscihepevirus. The Piscihepevirus genus consists of only the cutthroat trout virus, whereas the Orthohepevirus genus includes further four species (A to D). All HEV strains infecting humans (HEV gt 1-4 and 7) belong to the Orthohepevirus species A. Other isolates
Introduction 7 detected in animals as chicken, rats, ferrets or bats are classified into the species Orthohepevirus A–D and cannot be transmitted to humans (Smith et al., 2014; Meng, 2016).
HEV is a small, icosahedral shaped RNA virus with a single-stranded, positive-sense genome of approximately 7.2 kb. The virion measures 27–32 nm in diameter and is called ‘quasi-enveloped’
since the virus exists in an enveloped and non-enveloped form (Yin et al., 2016). Recent data imply that virions secreted into the bile are non-enveloped whereas virions found in the bloodstream are wrapped in cellular membranes (Debing, Y. et al., 2016).
The viral genome consists of three open reading frames (ORFs), which are additionally framed by untranslated regions as depicted in Figure 2. The 5’-non-coding region is capped with a 7- methylguanosine and the 3’ end is polyadenylated. ORF1 encodes the non-structural proteins that are essential for the HEV RNA replication including a methyltransferase (MT), cysteine protease (Pro), RNA helicase (Hel) and RNA-dependent RNA polymerase (Pol). The function of the Y- domain, polyproline region (PPR) and the X-domain are mainly unknown so far. An additional ORF4 in the HEV genome of genotype 1 increases the virus polymerase activity (Kamar, Izopet et al., 2017; Nimgaonkar et al., 2018). The ORF2 is translated into the 660-amino-acid virus capsid protein which is important for particle assembly and host cell binding. Montpellier et al.
reported that during infection at least 3 different forms of the capsid protein are processed of which only one is associated with infectious particles (Montpellier et al., 2018). Finally, the short ORF3 encodes for a viroporin which is involved in the release of infectious particles (Ding et al., 2017). Furthermore, the ORF3 protein interacts with a variety of other host proteins which are assumed to create a host environment that facilitates the viral life cycle within the cell (Chandra et al., 2008).
Figure 2: Genetic organization of hepatitis E virus. (Kamar, Izopet et al., 2017) (Reprint with permission granted by Springer Nature)
Introduction 8 Although HEV is a primary hepatotropic and non-cytopathic virus, several studies have shown that HEV is also able to replicate in other tissues such as neuronal cells, placental cells, kidney cells and cells of the gastrointestinal tract. (Bose et al., 2014; Drave et al., 2016; Geng et al., 2016;
Kamar et al., 2016).
The viral life cycle of HEV is only partially understood. The key steps of infection, replication, assembly and release in the hepatocytes are depicted in Figure 3. Initially the virus attaches to heparan sulfate proteoglycans on the surface and enters the cell through a receptor-dependent clathrin-mediated endocytosis (Kalia et al., 2009; Kapur et al., 2012). After uncoating, the viral RNA is translated by host proteins into the polyprotein of ORF1. Then the viral RNA polymerase is able to transcribe the positive-sense (+) RNA into a full-length negative-sense (-) transcript.
This serves as a template to produce more full-length (+) RNA as well as subgenomic (sg) RNA that comprises ORF2 and ORF3 (Varma et al., 2011). During this step the RNA helicase plays an important role in unwinding the two RNA strands (Karpe and Lole, 2010). The capsid and ORF3 protein are translated from the sgRNA and then processed within the endoplasmic reticulum (ER). Finally, the viral RNA is packaged to allow formation and assembly of new virions. It is assumed that HEV utilizes the ORF3 protein in connection with the cellular secretory machinery to establish an interaction with the endosomal sorting complexes required for transport (ESCRT) for viral release from the host cell (Nagashima et al., 2011; Nimgaonkar et al., 2018).
The virions get secreted with an additional envelope of ORF3 proteins and lipids. The envelope is destroyed by bile salts when released into the biliary canaliculi. In contrast, virions secreted into the bloodstream maintain the envelope (Okamoto, 2013; Debing, Y. et al., 2016).
Introduction 10 inflammation or injury. During the following icteric phase, the bilirubin level is elevated and patients might suffer from vomiting, diarrhea and itching accompanied by jaundice and dark- colored urine. These symptoms are normally self-limiting. The viral load and the elevated liver enzymes decrease without the requirement of antiviral therapy (Wedemeyer et al., 2012; Dalton et al., 2018). Nevertheless, in some cases infected individuals progress to a fulminate hepatitis with acute liver failure, which leads to mortality rates of 0,5–4% in the population without preexisting liver diseases (Blasco Perrin et al., 2015).
The clinical manifestation of HEV is dependent on the HEV genotype besides the individual host factors. Infections with the tropical genotypes 1 and 2 affect more frequently younger people (15–
35 years) (Purcell and Emerson, 2008). These infections are symptomatic in 15–20% of the cases.
Additionally, gt1 and 2 seem to show a higher manifestation rate with often a more severe progression of the disease (Rein et al., 2012). Especially pregnant women in the second or third trimester are at high risk to develop a life-threating hepatitis caused by an infection with HEV genotype 1. Besides acute liver failure, hemorrhages and stillbirth, the infection in pregnant women results in more than a quarter of the cases in mortality. The underlying pathomechanisms are still mainly unknown (Pérez‐Gracia et al., 2017). Individuals with pre-existing liver diseases more regularly experience a fulminant course of the infection and display higher mortality (Kumar and Saraswat, 2013). In high-income countries elderly males tend to get infected and develop symptoms of acute hepatitis. For the most part, individuals infected with genotypes 3 or 4 develop no symptoms and progression to acute liver failure is uncommon (Hartl et al., 2016).
In immunocompromised patients HEV replication can persist longer than 3–6 months. Only for genotype 3, 4 and 7 chronic infections have been described leading to liver fibrosis, cirrhosis and eventually chronic liver failure (Kamar et al., 2013). As the clinical course of HEV infection is dependent on the strength of the host’s immune system, most chronic hepatitis occur in solid- organ-transplanted (SOT) patients receiving a immunosuppressive therapy (Kamar et al., 2008).
Chronic infection is further associated with any immunosuppression in the context of hematological disorders, rheumatic diseases, stem cell transplantation and HIV-infected individuals.
Although HEV mainly replicates in the liver, extrahepatic manifestations are increasingly diagnosed in association with HEV infection. Most cases are reported for neurological disorders including Guillain-Barré-Syndrome, neuralgic amyotrophy and encephalitis. Furthermore, renal
Introduction 11 injuries, pancreatitis, thyroiditis, myocarditis and hematological disorders have been described as extrahepatic manifestations of HEV (Kamar et al., 2016; Pischke et al., 2017).
Figure 4: HEV infection and hepatitis E. (Kamar, Izopet et al., 2017) (Reprint with permission granted by Springer Nature)
3.4 H epatitis E: Diagnostics
HEV infections can be diagnosed either indirectly by the detection of anti-HEV antibodies (IgM and IgG) in the serum via enzyme-linked immunosorbent assays (ELISA), or directly by the detection of HEV capsid antigen or HEV RNA in the blood or stool through quantitative PCR.
To diagnose HEV-specific antibodies, several ELISA-based assays exist which vary considerably in their specificity and sensitivity, so that they must be evaluated with greatest care (Norder et al., 2016). An acute infection is marked by the detection of anti-HEV IgM in the serum. Within 2–4 weeks following virus transmission, an IgM-response can be detected in immunocompetent individuals. The antibody increase is generally accompanied by elevated liver enzymes and lasts 6–9 months. A positive anti-HEV IgG test indicates a previous HEV infection. The IgG antibodies persist for many years but do not completely protect against re-infection (Huang et al., 2010).
However, immunocompromised patients might have delayed antibody responses or remain antibody negative caused by the immunosuppression. Therefore, immunosuppressed individuals should always be tested via direct methods. The direct detection and quantification of viral RNA via viral nucleic acid testing represents the gold standard of the diagnostics of a current HEV infection. To improve comparability of interlaboratory results, the World Health Organization introduced international standards for HEV PCR techniques in 2013 (Baylis et al., 2013).
Introduction 12 3.5 H epatitis E: Treatm ent
Most hepatitis E infections are self-limited and do not require any antiviral therapy. However, some infected patients are at high risk to progress from acute to chronic hepatitis.
Immunosuppressed individuals represent the majority of patients who do not achieve a viral clearance without treatment. Viral infection is defined as chronic after 3 months of persistent infection (Kamar et al., 2008). A reduction of the immunosuppressive drugs is the first therapeutic option for patients under immunosuppressive therapy. Especially calcineurin and mTOR inhibitors were found to elevate the risk of developing chronic HEV (Wang et al., 2014). Previous studies show that approximately one-third of patients spontaneously clear the virus after the reduction of immunosuppression (Kamar, Abravanel, Selves et al., 2010; Kamar et al., 2011). The second line of therapy is an off-label use of ribavirin (RBV) for chronic as well as acute severe hepatitis.
Kamar et al. published in 2014 a large multicentric study involving 59 chronically infected patients who received SOT. They were treated for 3 months at a median dose of 600 mg RBV per day.
After the treatment period 78% of the patients had achieved a sustained virological response, meaning undetectable HEV RNA levels in the blood 6 months after the end of therapy. Today RBV monotherapy is the only efficient treatment for chronic hepatitis. Nevertheless, treatment failure occurred in some case reports, mainly caused by a RBV dose reduction required because of side-effects as anemia (Kamar et al., 2014).
Several mechanisms of action have been described for RBV. The depletion of intracellular guanosine triphosphate (GTP) pools has been found crucial as antiviral mechanism of RBV in vitro. The inhibition of the inosine monophosphate dehydrogenase by RBV 5’monophosphat leads to a reduced production of GTP which is necessary for viral RNA synthesis (Debing, Yannick et al., 2014). Furthermore, it has been shown that RBV has a mutagenic effect on the HEV genome by inducing mismatches and nucleotide substitutions. On the one hand the increase of mutations can cause the extinction of the virus population. On the other hand, due to the higher mutation rates RBV treatment can result in the selection of viral variants with enhanced viral replication fitness (Todt, Walter et al., 2016). Todt et al., Debing et al. identified several novel single nucleotide variation sites in the viral genome emerging during RBV treatment in patients (Debing, Y., Ramiere et al., 2016; Todt, Gisa et al., 2016). Both studies describe the previously discovered G1634R mutation in the polymerase domain of HEV ORF1 protein which results in a higher replication fitness of the virus in vitro (Debing, Y. et al., 2014). The research groups correlate
Introduction 13 RBV treatment failure in patients with the mutated viral intra-host populations that might have acquired variants leading to an apparent resistance to ribavirin.
As treatment failures occur under RBV, another therapy option is the administration of PEGylated Interferon-α for 3 months. In a small patient cohort, a number of chronically infected individuals successfully cleared HEV. However, interferon therapy has to be evaluated carefully because it stimulates the immune system and can lead to acute rejection in SOT recipients (Kamar, Abravanel, Garrouste et al., 2010; Kamar, Rostaing et al., 2010; Haagsma et al., 2010).
Only recently, a study of Dao Thi et al. showed that sofosbuvir, normally used as potent inhibitor of the hepatitis C virus in clinics, has also some antiviral activity against HEV in vitro (Dao Thi et al., 2016). The results were promising, indicating that sofosbuvir might also act against chronic HEV infection in patients. So far only few case reports exist and in those sofosbuvir did not show such efficacy (Kamar, Wang et al., 2017; Van der Valk et al., 2017; Donnelly et al., 2017). Thus, further studies are needed to evaluate the potential benefit of sofosbuvir for the therapy of chronic HEV in vivo.
An effective vaccine against HEV, labelled ‘Hecolin’ or HEV 293 is licensed only for China, where it is available since 2012. While a preventive strategy for endemic regions and high-risk groups as pregnant women is urgently needed, the WHO has reviewed the HEV 293 vaccine as not sufficiently evaluated concerning its safety in pediatric, elderly and other subpopulations and recommends a phase IV trial despite a “reassuring phase I, II and III” (WHO, 2014; Zhang et al., 2015).
Aim of the thesis 14
4 A im of the thesis
Infections with the Hepatitis E virus present an increasing health problem worldwide. They cause high rates of morbidity and mortality among vulnerable subpopulations such as immunocompromised individuals, pregnant women and persons with pre-existing liver injuries. No direct-acting and non-teratogenic therapies against HEV infections are currently available. As described above, only ribavirin and type I interferon represent effective treatment options although they have severe side effects and are contra-indicated in pregnant women.
Throughout our project we aimed to address this problem and screened for new HEV inhibitors.
In the process, the natural compound silvestrol was identified. Silvestrol belongs to the rocaglate derivates and can be isolated from the plant species Aglaia foveolata of the Mahogany family (Kim et al., 2007). The compound bears a unique cyclo-penta[b]tetrahydrobenzofuran skeleton and is described as an efficient and specific inhibitor of the DEAD-box RNA helicase eIF4A (Bordeleau et al., 2008). The helicase is part of the eIF4F complex that is able to initiate cap-dependent translation in eukaryotes.
The aim of our study was to evaluate the anti-HEV activity of silvestrol in HEV experimental model systems in vitro as well as in vivo. We wanted to analyze how efficient silvestrol inhibits the replication of different HEV gts in cell culture. The Hepatitis C virus (HCV) was used as reference because the virus drives the translation initiation cap-independent and should be less susceptible to silvestrol. Additionally, we performed parallel experiments with RBV in order to compare the inhibitory effects and characteristics. We further wanted to investigate the antiviral effect of silvestrol on cell culture derived infectious viral particles and on primary HEV isolates.
Therefore, we assessed the infectious virus production and HEV RNA copy number respectively under silvestrol treatment. In close collaboration with the research group of Philip Meuleman from the Ghent university we aimed to proof the antiviral effect of silvestrol also in an HEV animal model system. Using human liver chimeric mice which can be chronically infected with HEV, we wanted to study if a treatment with silvestrol can also lead to a decline of HEV infection in vivo.
In the last part we planned to investigate the combinatory activity of silvestrol with RBV. Since so far RBV is the most effective treatment option against HEV, we hoped to find synergistic antiviral effects in combining RBV with silvestrol.
Manuscript 15
5 M anuscript
TH E N A TU R A L COM POU N D SILV ESTR OL IN H IBITS H EPA TITIS E V IU R S (H EV ) R EPLICA TION IN V ITR O A N D IN V IV O
Todt, D.§; Moeller, N.§; Praditya, D.; Kinast V.; Friesland M.; Engelmann M.; Verhoye L.;
Sayed I.M.; Behrendt, P.; Dao Thi, V.L.; Meuleman, P.; Steinmann E. Antiviral Research. 157, 151-158 (2018) https://doi.org/10.1016/j.antiviral.2018.07.010.
§ equal contribution
Reprint with permission granted by Elsevier in accordance with the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license;
https://creativecommons.org/licenses/by-nc-nd/4.0/.
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Discussion and Outlook 24
6 Discussion and Outlook
In our study, we characterized the anti-HEV effect of the natural compound silvestrol as part of the screening approach for novel HEV inhibitors. Silvestrol is already known from literature as potent antitumor as well as antiviral agent and was originally found in a screen for eukaryotic initiation factor 4A (eIF4A) activity inhibitors by Bordeleau et al. (Bordeleau et al., 2008). They showed that silvestrol inhibits translation initiation by modulating the eIF4A helicase and thereby alters the drug sensitivity of cancer cells. The compound seems to induce dimerization and it increases the binding affinity between eIF4A helicase and RNA. This results in a dysregulation of the ribosome recruitment and in an inability to bind the translations preinitiation complex (Bordeleau et al., 2008; Cencic et al., 2009). Subsequently, different research groups demonstrated a significant therapeutic activity of silvestrol on mouse tumor models targeting the translation machinery in cancer (Kogure, Takayuki et al., 2013; Patton et al., 2015). From that it became clear, that the eIF4A helicase represents an essential drug target that is involved in the host cellular respectively oncogenic protein synthesis.
Similar to cancer cells, most viruses including HEV rely on the host cellular machinery using the translation mechanisms to synthesize their own viral proteins. The HEV mRNA is capped at the 5’ end and contains a short 5’ untranslated region (UTR). This indicates that the virus is dependent on cap-recognizing proteins for an efficient RNA replication. In accordance with that, Zhou et al. previously showed that HEV indeed requires the host cellular eIF4F complex for RNA translation (Zhou et al., 2015). As a heterotrimeric protein complex, eIF4F is composed of the DEAD-box helicase eIF4A, a 5ʹ mRNA cap-binding subunit eIF4E and the large scaffolding protein eIF4G. The function of the complex is to recruit the mRNA to the ribosomes and to facilitate the scanning of the 5’ UTRs (Bhat et al., 2015). This process is referred to as ‘translation initiation’ of the eIF4F complex and is the first and most rate-limiting step of translation. During the translation initiation the eIF4A RNA helicase unwinds RNA secondary structures in the 5’- UTRs of mRNAs. By this mechanism, silvestrol interferes with the translation. Sadlish et al.
identified eIF4A mutants that are resistant to silvestrol, which further proved that eIF4A has to be the molecular target of silvestrol (Sadlish et al., 2013).
As HEV is expected to require the cellular translation machinery including the eIF4F complex in order to initiate the translation of the viral proteins, we investigated the effects of silvestrol on the HEV life cycle. First, we explored the inhibitory effects of silvestrol in a HEV replicon system
Discussion and Outlook 25 with the human hepatoma cell line Huh7.5. Our following observations that silvestrol inhibited the replication of different subgenomic HEV replicons in a dose-dependent manner were consistent with the earlier findings (Bordeleau et al., 2008; Cencic et al., 2009). The different HEV replicons included a gt1 construct, a gt3-based wild board isolate, the most used gt3 construct p6 and a variant of p6 with a mutation in the polymerase region (G1634R), that leads to a higher replication fitness. For all constructs we observed an inhibitory effect at low nanomolar concentrations of silvestrol. Whereas the inhibitory concentrations 50 (IC50) of silvestrol ranged between 4 and 6.6 nM, the IC50 for RBV were 1000-fold higher. It is remarkable that for the same inhibitory effect we needed far less silvestrol than RBV since RBV is the mostly used HEV inhibitor. Then the inhibition efficiency of silvestrol on HEV replication was compared to a replicon construct based on HCV. HCV translation requires an internal ribosomal entry side (IRES) and is therefore independent of the eIF4A helicase cap-driven translation initiation. That explains the modest inhibition of HCV replication in our experiments and supports the expected mode of action of silvestrol to modulate the eIF4A helicase. As silvestrol targets host factors, it was important to monitor the cell viability under treatment. Throughout the whole study we assessed silvestrol toxicity using a MTT assay and determined viable cells via visual cell counting to exclude severe antiproliferative properties of silvestrol. After 24h treatment we observed only small toxic effects and the cytotoxic concentration 50 (CC50) was never reached. Longer incubation periods caused a decreasing cell metabolic activity although after 42h the combined values of IC50 and CC50 still resulted in a selectivity index of about 8.6 for the p6 strain. These findings lead to the prediction that within a therapeutic window silvestrol can be applied to efficiently inhibit HEV replication at non-toxic concentrations.
To further confirm our results, we performed viral infection experiments. In two different assay setups simulating the whole HEV viral life cycle, we observed inhibitory effects of silvestrol on infectious viral particle production and HEV RNA copy numbers. In the first HEV infection assay HepG2/C3A cells were infected with cell culture-derived infectious HEV (HEVcc) and the impact of silvestrol at different concentrations and incubation periods was tested. The most efficient inhibition was reached with 20 nM and 50 nM silvestrol at a late time point. Surprisingly the overall antiviral activity of silvestrol was less prominent in that assay than in the before described experiments with Huh7.5 cells. Therefore, a direct comparison of the antiviral effect was performed on HEVp6 replication in HepG2/C3A and Huh7.5 cells. The results confirmed our observations and showed a shift of the dose-response curves with a higher effectivity of silvestrol in transfected Huh7.5. Although the reasons for these differences are not immediately apparent, they may be
Discussion and Outlook 26 related to the complex mode of action of silvestrol. On the one hand the translation of the viral RNA is inhibited and on the other hand the translation of host cellular mRNAs. Because the host cellular mRNAs vary from cell line to cell line, the differences of silvestrol’s effectivity might be explained.
To underline the specificity of silvestrol on HEV replication, pluripotent stem cell-derived hepatocyte-like cells (HLCs) were used. These cells are not cancer-derived cell lines and do not have alterations in metabolic, innate immune and apoptotic responses. Therefore, HLCs offer a more physiologically relevant cell culture system (Wu et al., 2018). The cells were infected with patient-derived HEV from genotypes 1–4 and treated with silvestrol post-infection. No cytotoxicity due to treatment was observed but a clear reduction of HEV RNA copy numbers in infected HLCs for all four different genotypes. This assay proved a pangenotypic effect of the compound and an even more pronounced inhibition of HEV infection in these cells.
The results raised the question whether treatment with silvestrol would also inhibit HEV infection in vivo. As mentioned above silvestrol was originally discovered as anti-cancer compound and in the beginning mainly characterized in anti-cancer studies. Several research groups showed that silvestrol effectively inhibits tumor growth in vivo without evidence of toxicity (Cencic et al., 2009; Lucas et al., 2009; Alinari et al., 2012; Kogure, T. et al., 2013). The studies demonstrated how crucial the translational control in cancer development and progression is (Silvera et al., 2010). The treatment with silvestrol seemed to affect translation initiation in the tumor cells, leading to a reduced progression of tumor spread in the different animal models.
The study of HEV infection was hampered for a long time because of the absence of a small animal model. Ordinary mice cannot be infected with HEV and only lately researchers are able to infect immune-deficient human liver chimeric mice with HEV. This animal model allows the preclinical in vivo evaluation of novel antiviral compounds (Sayed et al., 2017). Based on the antitumor in vivo studies, the chronically infected animals were treated intraperitoneal with silvestrol for either six or ten days. The administration of silvestrol was effective within the first days, resulting in a rapid decline of HEV RNA levels in the feces compared to the control group. Although there was only a small number of animals tested, this first successful in vivo study validated the promising therapeutic potential of silvestrol and can lead to further silvestrol-based assays to improve established antiviral strategies.
Discussion and Outlook 27 Previously to our study, Biedenkopf et al. published a paper on the inhibitory effects of silvestrol on Ebola virus (EBOV) replication. Like HEV and all other viruses, EBOV protein synthesis depends on the cellular translation machinery. Biedenkopf et al. showed for the first time that specifically targeting the host factor eIF4A with silvestrol leads to a sufficient inhibition of the viral propagation before any effects on cellular functions occur (Biedenkopf et al., 2017). Further studies also reported antiviral effects of silvestrol on other RNA viruses, including corona- and picornaviruses, Zika and Chikungunya (Müller, C. et al., 2018; Elgner et al., 2018; Henss et al., 2018).
In contrast to direct-acting antivirals that target viral proteins, molecules like silvestrol interfere with cellular factors involved in the viral life cycle. On the one hand, targeting host factors bears the risk to inhibit essential metabolic processes required for cellular homeostasis. On the other hand, agents that inhibit host factors have the advantage that they are less susceptible to escape mutations by viruses. That feature can hamper the evolution of viral evasion mechanisms and prevent viral resistance (Müller, K. et al., 2012; Zeisel et al., 2015). Especially HEV is a virus where escape mutations play a major role whether a treatment leads to viral clearance or not.
Deep sequencing results revealed that several HEV RNA mutations were the reason for ribavirin resistance and treatment failure in patients (Debing, Y., Ramiere et al., 2016; Lhomme et al., 2016; Todt, Gisa et al., 2016). Generally, RNA viruses like HEV consist of so-called mutant clouds.
In HEV infected individuals the viral population contains a repertoire of HEV variants that comprises genomes that are similar but not-identical. High replication rates and high error-rates of the RdRP are the source of those mutant clouds. The mutations facilitate virus adaptation and evasion of selective constraints like antiviral agents. (Domingo et al., 2012). Using a combination of drugs with different targets and mechanisms of action, may prevent viral resistance in the course of HEV infection. Moreover, possible synergistic antiviral effects of two compounds have the advantage of a reduction in the necessary doses, minimizing the risk of toxicity and side effects.
Therefore, we evaluated a combinatory treatment of silvestrol and the known HEV-inhibitor ribavirin. To analyze the interactions between the two compounds, two different models were used. The calculated combination indices as well as the three-dimensional surface plot indicated that the combination of silvestrol with RBV has additive antiviral effects in the HEV replication system. Although clear synergistic effects could not be observed, the combination may offer an option in case of treatment failure as soon as an optimal ratio of the drugs in vivo is identified.
Discussion and Outlook 28 In the course of our study the new compound silvestrol was identified which inhibits HEV efficiently in vitro as well as in vivo. Perhaps the still limited treatment options of HEV infections reflect the long-lasting ignorance of HEV as a global pathogen. Currently, ribavirin is the only effective antiviral drug against HEV. Particularly for pregnant women there is a great need for alternative treatments. Unfortunately, specifically ribavirin is contraindicated in pregnant women, who disproportionately suffer from the symptoms of HEV infection. By investigating the anti- HEV properties of silvestrol and additive effects with RBV, our study tried to make a valuable contribution to the field of drug research and future treatment strategies of hepatitis E. Targeting the host factors eIF4A during cap-dependent translation initiation is a promising broad-spectrum antiviral strategy. Both, in our HEV cell culture systems as well as in the animal model system, viral replication of different HEV genotypes was blocked without major toxic side effects. However, many aspects about possible adverse effects in humans are so far completely unknown and there is still a long way to go until silvestrol might be used as proper drug against Hepatitis E, Ebola or other viruses, where antiviral effects are observed.
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Danksagungen 38
8 Danksagungen
Mein besonderer Dank gilt Herrn Professor Eike Steinmann für die Überlassung des interessanten Themas und die Bereitstellung aller notwenigen Ressourcen für die Bearbeitung des Projekts. Ich verdanke ihm die Chance, Teil eines offenherzigen, großartigen und sehr motivierten Teams zu sein und unendlich viel über die Molekularvirologie gelernt zu haben. Er weckte schnell meine Begeisterung an der Forschung und trug erheblich zur erfolgreichen Fertigstellung des Projektes bei.
Ein riesiger Dank gilt ebenfalls Dr. rer. nat. Daniel Todt für die zahlreichen Erklärungen, Hilfestellungen und Gespräche. Seine Tipps, Anregungen und schlussendlich die gemeinsame Bearbeitung des Projekts trugen maßgeblich zur Erstellung der Arbeit bei.
Während meiner gesamten Laborzeit stand mir Martina Friesland mit Rat und Tat zur Seite und führte mich in alle erdenklichen Labortätigkeiten ein. Die hilfreichen Tipps, kreativen Ideen und vielen Stunden, die wir gemeinsam vor allem in der Zellkultur verbrachten haben sind unersetzlich.
Ich bin Martina sehr dankbar, dass sie immer für mich Zeit hatte und mich wieder aufgebaut hat, wenn ein Experiment nicht so lief wie geplant.
Ebenso bedanken möchte ich mich bei Patrick Behrendt, der stets gutgelaunt mir viel klinischen Hintergrund vermittelte und Einblicke in den klinischen Alltag gab. Bedanken möchte ich mich auch bei Prof. Dr. Thomas von Hahn für die Gespräche und seine Mitbetreuung des Projektes im Rahmen der Strukturierten Doktorandenausbildung StrucMed und bei Prof. Dr. Thomas Pietschmann für sein stetiges Interesse und ohne den meine Promotion nicht möglich gewesen wäre.
Ein weiterer großer Dank gilt allen Mitarbeitern des Labors für die Unterstützung und unvergessliche Zeit. Insbesondere hier zu erwähnen sind aus der AG Steinmann Volker Kinast, Dimas Praditya, Michael Engelmann und Birthe Tegtmeyer, mit denen ich viel gelacht und erlebt habe.
Schlussendlich möchte ich Basti, meinen Eltern und all meinen Freundinnen und Freunden danken, die mich auf vielfältigste Weise unterstützt haben. Vielen Dank für das Zuhören, Erörtern, Erklären, Korrekturlesen und das immer offene Ohr, wenn es mal nicht so lief. Ihr seid großartig und ich bin sehr glücklich, Euch in meinem Leben zu haben!
Eidesstaatliche Erklärung nach § 2 Abs. 2 Nrn. 6 und 7 41
10 Eidesstaatliche Erklärung nach § 2 A bs. 2 N rn. 6 und 7
Ich erkläre, dass ich die der Medizinischen Hochschule Hannover zur Promotion eingereichte Dissertation mit dem Titel
“Characterization of the A ntiviral A ctivity of Silvestrol against H epatitis E V irus Infection”
im Institut für Experimentelle Virologie des TWINCORE, Zentrum für Experimentelle und Klinische Infektionsforschung
unter Betreuung von Professor Dr. rer. nat. Eike Steinmann mit der Unterstützung durch das StrucM ed Programm der MHH
ohne sonstige Hilfe durchgeführt und bei der Abfassung der Dissertation keine anderen als die dort aufgeführten Hilfsmittel benutzt habe.
Die Gelegenheit zum vorliegenden Promotionsverfahren ist mir nicht kommerziell vermittelt worden. Insbesondere habe ich keine Organisation eingeschaltet, die gegen Entgelt Betreuerinnen und Betreuer für die Anfertigung von Dissertationen sucht oder die mir obliegenden Pflichten hinsichtlich der Prüfungsleistungen für mich ganz oder teilweise erledigt.
Ich habe diese Dissertation bisher an keiner in- oder ausländischen Hochschule zur Promotion eingereicht.
Weiterhin versichere ich, dass ich den beantragten Titel bisher noch nicht erworben habe.
Ergebnisse der Dissertation wurden im Journal Antiviral Research im Juli 2018 veröffentlicht.
Hannover, den 14.04.2021
Nora Maria Proske
Appendix 42
11 A ppendix
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