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Identification and evaluation of antiviral compounds targeting Rift Valley fever virus
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases. (Clas Ahlm)
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Rift Valley fever virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease Rift Valley fever (RVF). RVFV causes significant morbidity and mortality in humans and livestock throughout Africa and the Arabian Peninsula. RVFV is an emerging virus and is capable of infecting a broad range of mosquito species distributed around the world, so it poses a potential threat globally. A wide range of livestock animals (e.g. sheep, goats, cows, and camels) and some wild animals become highly affected by RVFV. In humans, RVFV infection presents as an acute self-limiting febrile illness that may lead to more severe hemorrhagic fever and encephalitis. The severity of the disease is mostly dependent on age and the species of mammal, but other factors are also important.

There are no licensed RVFV vaccines for humans, and there is a lack of effective antiviral drugs. Moreover, due to the severe pathogenicity, higher-level facilities are needed―biosafety level 3 (BSL-3) or more―to work with RVFV, which makes antiviral drug development more challenging. Because RVFV causes severe disease in Africa and the Arabian Peninsula, and has the potential to spread globally, it is essential that safe, efficient antiviral drugs against this virus are developed.

The previously reported antiviral compound benzavir-2 inhibits the replication of several DNA viruses, i.e. human adenoviruses, herpes simplex virus (HSV) type 1, and HSV type 2, indicating a broadranging activity. We wanted to evaluate whether benzavir-2 had an effect against the RNA virus RVFV. For these and subsequent studies, we used a recombinant, modified RVFV strain with a deleted NSs gene, which was replaced by a reporter gene (rRVFVΔNSs::Katushka), enabling the studies to be conducted under BSL-2 conditions. The NSs gene is the main virulence factor for RVFV and without it, RVFV become less pathogenic. The reporter gene made it possible for us to quantify infection with the help of the red fluorescent protein. We found that benzavir-2 effectively inhibited RVFV infection in cell culture at an effective concentration showing 50% inhibition (EC50) of 0.6 μM. Benzavir-2 also inhibited the production of progeny virus. When we studied the pharmacokinetic properties, we found that benzavir-2 had good in vitro solubility, permeability, and metabolic stability. When we investigated the oral bioavailability in mice by administering benzavir-2 in peanut butter pellets, high systemic distribution was observed without any adverse toxic effects. Benzavir-2 thus inhibited RVFV infection in cell culture and showed excellent pharmacokinetic properties, suggesting the possibility of evaluating its effectiveness in an animal model. Since benzavir-2 has a broad effect against both RNA and DNA viruses, we speculated that the antiviral mechanism affects cellular targets.

We also wanted to explore a large number of small chemical compounds with unknown properties and identify any anti-RVFV activities. Thus, we developed a whole-cell-based high-throughput reporter-based assay, and screened 28,437 small chemical compounds. The assay was established after optimization of several parameters. After primary and secondary screening, we identified 63 compounds that inhibited RVFV infection by 60% at a concentration of 3.12 μM and showed ≥ 50% cell viability at 25 μM. After a dose-dependent screening of these 63 compounds, several compounds were identified with highly efficient anti-RVFV properties. Finally, N1-(2-(biphenyl-4-yloxy)ethyl)propane-1,3-diamine (compound 1) was selected as the lead compound. We performed a structure-activity relationship (SAR) analysis of compound 1 by replacing and changing component after component of the chemical compound to see how this affected the antiviral activity. After the SAR analysis, the antiviral activity did not change, but we could improve the cytotoxicity profile. Our studies suggested that the improved compound, 13a, might be targeting the early phase of the RVFV lifecycle.

In conclusion, we developed an efficient and reliable screening method that creates possibilities for discovering and developing antivirals against RVFV under BSL-2 conditions. We also identified several chemical compounds with anti-RVFV activities, which might lead to development of therapies for RVFV infection.

Abstract [sv]

Rift Valley fever virus (RVFV) är ett vektorburet RNA-virus som orsakar Rift Valley fever (RVF). RVF virus orsakar betydande sjukdom och död hos människor och boskap i hela Afrika och på den Arabiska halvön. Viruset kan orsaka stora, plötsliga utbrott och eftersom det kan infektera många olika myggarter som finns runt om i världen så utgör RVF ett potentiellt globalt hot. Ett stort antal olika boskapsdjur och några vilda djur drabbas hårt under RVF utbrott. Människor får en sjukdom som kan vara mild, men kan utvecklas till allvarligare symptom som blödarfeber och hjärninflammation (encefalit) och under RVF-utbrott är antalet döda högt. Hur allvarlig sjukdomens blir är mest beroende av ålder, bakomliggande symptom och för djur, vilken djurart som infekteras.

Frånvaron av licensierade vacciner för människor och brist på effektiva antivirala läkemedel gör behandling och prevention av denna sjukdom utmanande. Dessutom, på grund av att den kan orsaka allvarlig sjukdom och död behövs höga nivåer av biosäkerhet (biosäkerhetsnivå-3, BSL-3) för att arbeta med RVFV vilket försvårar utvecklingen av läkemedel. Eftersom RVFV orsakar allvarlig sjukdom i Afrika och Arabiska halvön och har potential att spridas globalt är det viktigt att utveckla säkra effektiva antivirala medel för detta virus.

Den tidigare rapporterade antivirala föreningen benzavir-2 hämmar flera DNA-virus, dvs humant adenovirus, herpes simplexvirus typ-1 och typ-2 infektion, vilket indikerar en bredverkande aktivitet. Vi ville utvärdera om benzavir-2 också hade en effekt mot RNA-viruset RVFV. För dessa och följande studier använde vi en rekombinant, modifierad RVFV med en borttagen NSs-gen, ersatt av en reportergen, som gjorde det möjligt att utföra studierna i BSL-2-förhållanden. NSsgenen är den huvudsakliga virulensfaktorn för RVFV och med den borttagen är RVFV mindre sjukdomsframkallande. Reportergenen gjorde det möjligt att detektera rött fluoroscerande ljus efter virusinfektion och använda det som ett mått på infektion.

Vi observerade att benzavir-2 effektivt inhiberade RVFVinfektion i cellkultur med 50% inhibering vid en koncentration av 0,6μM. Benzavir-2 hämmade också produktion av nya viruspartiklar. När vi studerade dess farmakokinetiska egenskaper fann vi att benzavir-2 hade bra löslighet, permeabilitet och metabolisk stabilitet i cellkultur. När vi undersökte hur bra möss kunde ta upp benzavir-2 genom att äta föreningen blandad med jordnötssmör (den orala biotillgängligheten) observerades en hög serumkoncentration av benzavir-2 utan skadliga effekter. Benzavir-2 hämmade alltså RVFV-infektion i cellkultur och visade på utmärkta farmakokinetiska egenskaper med potential att senare kunna utvärdera effektiviteten i en djurmodell. Eftersom benzavir-2 har en bredverkande effekt mot både RNA och DNA-virus, spekulerar vi att den antivirala mekanismen påverkar cellulära mål.

I den andra studien ville vi undersöka ett stort antal små kemiska föreningar med okända egenskaper och identifiera anti-RVFV aktivitet hos dessa. För att genomföra detta utvecklade vi en helcellsbaserad ”high-throughput-screening” analys då vi använde den rekombinanta, modifierade RVFV beskriven ovan och screenade 28,437 små kemiska föreningar. Till att börja med optimerades analysmetoden med avseende på flera olika parametrar. Därefter utfördes primär och sekundär screening och 63 föreningar som inhiberade RVFV-infektion identifierades. De inhiberade RVFV-infektion med 60% vid 3,12μM och uppvisade ≥50% cellöverlevnad vid 25μM. Efter en dosberoende analys av dessa 63 föreningar kunde vi fokusera på några av föreningarna med mycket effektiva anti-RVFV egenskaper. Slutligen valdes N1-(2-(biphenyl-4-yloxy)ethyl)propane-1,3-diamine (förening 1) som vår ledande förening. Vi utförde därefter en strukturaktivitetsrelationsanalys (SAR) för förening 1. SAR-analys innebär att man byter ut och förändrar komponent efter komponent i den kemiska föreningen för att se hur det påverkar den antivirala aktiviteten. Efter SAR-analysen förändrades inte den antivirala egenskapen, men vi kunde förbättra cytotoxicitetsprofilen så att föreningen hade en låg toxisk effekt på värdcellen. Den förbättrade föreningen benämns 13a, och vår studie föreslog också att förening 13a kan vara aktiv under den tidiga fasen av RVFV-livscykel.

Sammanfattningsvis har vi identifierat flera kemiska föreningar med anti-RVFV aktiviteter som kan leda till utvecklande av terapi mot RVFV. Denna studie öppnar också möjligheter att upptäcka och utveckla antivirala medel mot RVFV i biosäkerhets-2 nivå.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2018. , p. 75
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1980
Keywords [en]
Rift Valley fever virus, antiviral, assay development, screening, drug discovery
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:umu:diva-151843ISBN: 978-91-7601-940-5 (print)OAI: oai:DiVA.org:umu-151843DiVA, id: diva2:1248080
Public defence
2018-10-11, Hörsal 933 Unod B9, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-09-18 Created: 2018-09-13 Last updated: 2018-10-22Bibliographically approved
List of papers
1. Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound
Open this publication in new window or tab >>Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 1925Article in journal (Refereed) Published
Abstract [en]

Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-144950 (URN)10.1038/s41598-018-20362-9 (DOI)000423663100004 ()29386590 (PubMedID)
Available from: 2018-02-22 Created: 2018-02-22 Last updated: 2018-09-14Bibliographically approved
2. High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection
Open this publication in new window or tab >>High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection
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2016 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 21, no 4, p. 354-362Article in journal (Refereed) Published
Abstract [en]

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by 80%, with 50% cell viability at 50 mu M concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with 60% inhibition of RVFV infection at 3.12 mu M compound concentration and 50% cell viability at 25 mu M were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection.

Place, publisher, year, edition, pages
Sage Publications, 2016
Keywords
high-throughput screening, antiviral, cell-based assay, recombinant virus, Rift Valley fever
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-119642 (URN)10.1177/1087057115625184 (DOI)000372883200004 ()26762502 (PubMedID)
Available from: 2016-05-18 Created: 2016-04-25 Last updated: 2018-09-14Bibliographically approved
3. Structural modifications and biological evaluations of Rift Valleyfever virus inhibitors identified from chemical library screening
Open this publication in new window or tab >>Structural modifications and biological evaluations of Rift Valleyfever virus inhibitors identified from chemical library screening
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(English)Manuscript (preprint) (Other academic)
National Category
Dermatology and Venereal Diseases
Identifiers
urn:nbn:se:umu:diva-151875 (URN)
Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2018-09-14

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