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Human adenoviruses: new bioassays for antiviral screening and CD46 interaction
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
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 [en]
Adenovirus, CD46, hemagglutination, antiviral, small molecule, screening
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-35733ISBN: 978-91-7459-056-2 (print)OAI: oai:DiVA.org:umu-35733DiVA: diva2:346552
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
List of papers
1. Adenovirus interactions with CD46 on transgenic mouse erythrocytes
Open this publication in new window or tab >>Adenovirus interactions with CD46 on transgenic mouse erythrocytes
2010 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 402, no 1, 20-25 p.Article in journal (Refereed) Published
Abstract [en]

Hemagglutination is an established method but has not been used previously to determine the efficacy of virus binding to a specific cellular receptor. Here we have utilized CD46-expressing erythrocytes from a transgenic mouse to establish whether and to what extent the species B adenoviruses (Ads) as well as Ad37 and Ad49 of species D can interact with CD46. A number of different agglutination patterns, and hence CD46 interactions, could be observed for the different adenovirus types. In this system Ad7p, Ad11a, and Ad14 did not agglutinate mouse erythrocytes at all. Hemagglutination of CD46 expressing erythrocytes with high efficiency was observed for the previously established CD46 users Ad11p and Ad35 as well as for the less investigated Ad34. Ad50 agglutinated with moderate efficiency. Ad16, Ad21 and Ad49 gave incomplete agglutination. Ad16 was the only adenovirus that could be eluted. No specific CD46 interaction could be observed for Ad3p or for Ad37.

Keyword
Adenovirus, CD46, Hemagglutination
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-33397 (URN)10.1016/j.virol.2010.03.004 (DOI)000277967500003 ()20347110 (PubMedID)
Available from: 2010-04-22 Created: 2010-04-22 Last updated: 2010-09-03Bibliographically approved
2. Adenovirus 11p downregulates CD46 early in infection
Open this publication in new window or tab >>Adenovirus 11p downregulates CD46 early in infection
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2010 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 405, no 2, 474-482 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus 11 prototype (Ad11p), belonging to species B, uses CD46 as an attachment receptor. CD46, a complement regulatory molecule, is expressed on all human nucleated cells. We show here that Ad11p virions downregulate CD46 on the surface of K562 cells as early as 5min p.i. Specific binding to CD46 by the Ad11p fiber knob was required to mediate downregulation. The complement regulatory factors CD55 and CD59 were also reduced to a significant extent as a consequence of Ad11p binding to K562 cells. In contrast, binding of Ad7p did not result in downregulation of CD46 early in infection. Thus, the presumed interaction between Ad7p and CD46 did not have the same consequences as the Ad11p-CD46 interaction, the latter virus (Ad11p) being a promising gene therapy vector candidate. These findings may lead to a better understanding of the pathogenesis of species B adenovirus infections.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-35716 (URN)10.1016/j.virol.2010.06.026 (DOI)000281130500023 ()20638094 (PubMedID)
Available from: 2010-09-01 Created: 2010-09-01 Last updated: 2012-02-24Bibliographically approved
3. 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
Open this publication in new window or tab >>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
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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
Keyword
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:nbn:se:umu:diva-35281 (URN)10.1128/AAC.00203-10 (DOI)000281005900048 ()20585112 (PubMedID)
Available from: 2010-08-11 Created: 2010-08-11 Last updated: 2014-04-25Bibliographically approved
4. Synthesis, biological evaluation, and structure-activity relationships of 2-[2-(benzoylamino)benzoylamino]benzoic acid analogues as inhibitors of adenovirus replication
Open this publication in new window or tab >>Synthesis, biological evaluation, and structure-activity relationships of 2-[2-(benzoylamino)benzoylamino]benzoic acid analogues as inhibitors of adenovirus replication
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2012 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 7, 3170-3181 p.Article in journal (Refereed) Published
Abstract [en]

2-[2-Benzoylamino)benzoylamino]benzoic acid (1) was previously identified as a potent and nontoxic antiadenoviral compound ( Antimicrob. Agents Chemother. 2010 , 54 , 3871 ). Here, the potency of 1 was improved over three generations of compounds. We found that the ortho, ortho substituent pattern and the presence of the carboxylic acid of 1 are favorable for this class of compounds and that the direction of the amide bonds (as in 1) is obligatory. Some variability in the N-terminal moiety was tolerated, but benzamides appear to be preferred. The substituents on the middle and C-terminal rings were varied, resulting in two potent inhibitors, 35g and 35j, with EC(50) = 0.6 μM and low cell toxicity.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012
Keyword
stem-cell transplantation; immunocompromised host; formazan assay; infection; pcr; recipients; reduction; cidofovir
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-54029 (URN)10.1021/jm201636v (DOI)000302591100027 ()22369233 (PubMedID)
Available from: 2012-04-12 Created: 2012-04-12 Last updated: 2017-12-07Bibliographically approved

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