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Small molecule screening using a whole cell viral replication reporter gene assay identifies 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid as a novel anti-adenoviral compound
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
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2010 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 54, no 9, 3871-3877 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their anti-adenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential anti-adenoviral compounds. The assay is unique in that it is based on a replication competent adenovirus type 11p GFP-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression in vitro by >/= 80% but only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid, turned out to be a potent inhibitor of adenovirus replication.

Place, publisher, year, edition, pages
American society for microbiology , 2010. Vol. 54, no 9, 3871-3877 p.
Keyword [en]
rapid colorimetric assay; adenovirus infection; transplant recipients; in-vitro; immunocompromised host; formazan assay; renal-failure; cidofovir; growth; proliferation
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-35281DOI: 10.1128/AAC.00203-10ISI: 000281005900048PubMedID: 20585112OAI: oai:DiVA.org:umu-35281DiVA: diva2:342873
Available from: 2010-08-11 Created: 2010-08-11 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Human adenoviruses: new bioassays for antiviral screening and CD46 interaction
Open this publication in new window or tab >>Human adenoviruses: new bioassays for antiviral screening and CD46 interaction
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adenoviruses are common pathogens all over the world. The majority of the population has at some point been infected with an adenovirus. Although severe disease can occur in otherwise healthy individuals an adenovirus infection is most commonly self limited in these cases. For immunocompromised individuals however, adenoviruses can be life-threatening pathogens capable of causing disseminated disease and multiple organ failure. Still there is no approved drug specific for treatment of adenovirus infections. We have addressed this using a unique whole cell viral replication reporter gene assay to screen small organic molecules for anti-adenoviral effect. This RCAd11pGFP-vector based assay allowed screening without any preconceived idea of the mechanism for adenovirus inhibition. As a result of the screening campaign 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid turned out to be a potent inhibitor of adenoviral replication. To establish a structure-activity relationship a number of analogs were synthesized and evaluated for their anti-adenoviral effect. The carboxylic acid moiety of the molecule was important for efficient inhibition of adenovirus replication.

There are 54 adenovirus types characterized today and these are divided into seven species, A-G. The receptors used by species B and other adenoviruses are not fully characterized. CD46 is a complement regulatory molecule suggested to be used by all species B types and some species D types but this is not established. We have designed a new bioassay for assessment of the interaction between adenoviruses and CD46 and investigated the CD46-binding capacity of adenovirus types indicated to interact with CD46. We concluded that Ad11p, Ad34, Ad35, and Ad50 clearly bind CD46 specifically, whereas Ad3p, Ad7p, Ad14, and Ad37 do not.

CD46 is expressed on all human nucleated cells and serves as a receptor for a number of different bacteria and viruses. Downregulation of CD46 on the cell surface occurs upon binding by some of these pathogens. We show that early in infection Ad11p virions downregulate CD46 upon binding to a much higher extent than the complement regulatory molecules CD55 and CD59.

These findings may lead to a better understanding of the pathogenesis of adenoviruses in general and species B adenoviruses in particular and hopefully we have discovered a molecule that can be the basis for development of new anti-adenoviral drugs.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2010. 81 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1366
Keyword
Adenovirus, CD46, hemagglutination, antiviral, small molecule, screening
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-35733 (URN)978-91-7459-056-2 (ISBN)
Public defence
2010-09-24, Sal 914, 9tr NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2010-09-03 Created: 2010-09-01 Last updated: 2014-04-25Bibliographically approved
2. The discovery of antiviral compounds targeting adenovirus and herpes simplex virus: assessment of synthetic compounds and natural products
Open this publication in new window or tab >>The discovery of antiviral compounds targeting adenovirus and herpes simplex virus: assessment of synthetic compounds and natural products
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease.

Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types.

To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity.

This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use. 

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2014. 104 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1647
Keyword
virology, antiviral, adenovirus, herpes simplex virus, small molecule, inhibitor, hsv, drug discovery
National Category
Microbiology in the medical area Pharmacology and Toxicology Microbiology
Research subject
Medical Virology; läkemedelskemi; Microbiology
Identifiers
urn:nbn:se:umu:diva-88186 (URN)978-91-7601-043-3 (ISBN)
Public defence
2014-05-16, Betula, by 6M, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council, 621-2010-4746Swedish Cancer Society, 100356Swedish Research Council, K2007-56X-05688-28-3
Available from: 2014-04-25 Created: 2014-04-24 Last updated: 2014-04-25Bibliographically approved

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Andersson, Emma KStrand, MårtenEdlund, KarinLindman, KristinaEnquist, Per-AndersSpjut, SaraAllard, AnnikaElofsson, MikaelMei, Ya-FangWadell, Göran

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