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The discovery of antiviral compounds targeting adenovirus and herpes simplex virus: assessment of synthetic compounds and natural products
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. (Wadell, G.)
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 [en]
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: urn:nbn:se:umu:diva-88186ISBN: 978-91-7601-043-3 (print)OAI: oai:DiVA.org:umu-88186DiVA: diva2:713928
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
List of papers
1. 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: 2017-12-12Bibliographically approved
2. 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
3. 2-[4,5-Difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic acid, an antiviral compound with activity against acyclovir-resistant isolates of herpes simplex virus type 1 and 2
Open this publication in new window or tab >>2-[4,5-Difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic acid, an antiviral compound with activity against acyclovir-resistant isolates of herpes simplex virus type 1 and 2
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2012 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 56, no 11, 5735-5743 p.Article in journal (Refereed) Published
Abstract [en]

Herpes simplex viruses (HSV-1 and HSV-2) are responsible for life-long latent infections in humans, with periods of viral reactivation associated with recurring ulcerations in the orofacial and genital tract. In immunosuppressed patients and neonates, HSV infections are associated with severe morbidity, and in some cases even mortality. Today, acyclovir is the standard therapy for management of HSV infections. However, the need for novel antiviral agents is apparent since HSV isolates resistant to acyclovir therapy are frequently isolated in immunosuppressed patients. In this study, we assessed the anti-HSV activity of the anti-adenoviral compounds 2-[2-(2-benzoylamino)-benzoylamino]benzoic acid, (Benzavir-1) and 2-[4,5-difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic acid, (Benzavir-2) on HSV-1 and HSV-2. Both compounds were active against both viruses. Importantly, Benzavir-2 had similar potency to acyclovir against both HSV types and it was active against clinical acyclovir-resistant HSV isolates.

Place, publisher, year, edition, pages
American Society Microbiology, 2012
Keyword
Herpes simplex virus, HSV, inhibitor, 2-[2-(benzoylamino)benzoylamino]benzoic acid, antiviral, Benzavir
National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-60354 (URN)10.1128/AAC.01072-12 (DOI)000310055800039 ()22908173 (PubMedID)
Available from: 2012-10-09 Created: 2012-10-09 Last updated: 2017-12-07Bibliographically approved
4. Isolation and characterization of anti-adenoviral secondary metabolites from marine actinobacteria
Open this publication in new window or tab >>Isolation and characterization of anti-adenoviral secondary metabolites from marine actinobacteria
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2014 (English)In: Marine Drugs, ISSN 1660-3397, E-ISSN 1660-3397, Vol. 12, no 2, 799-821 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus infections in immunocompromised patients are associated with high mortality rates. Currently, there are no effective anti-adenoviral therapies available. It is well known that actinobacteria can produce secondary metabolites that are attractive in drug discovery due to their structural diversity and their evolved interaction with biomolecules. Here, we have established an extract library derived from actinobacteria isolated from Vestfjorden, Norway, and performed a screening campaign to discover anti-adenoviral compounds. One extract with anti-adenoviral activity was found to contain a diastereomeric 1:1 mixture of the butenolide secondary alcohols 1a and 1b. By further cultivation and analysis, we could isolate 1a and 1b in different diastereomeric ratio. In addition, three more anti-adenoviral butenolides 2, 3 and 4 with differences in their side-chains were isolated. In this study, the anti-adenoviral activity of these compounds was characterized and substantial differences in the cytotoxic potential between the butenolide analogs were observed. The most potent butenolide analog 3 displayed an EC50 value of 91 μM and no prominent cytotoxicity at 2 mM. Furthermore, we propose a biosynthetic pathway for these compounds based on their relative time of appearance and structure.

Place, publisher, year, edition, pages
MDPI, 2014
Keyword
adenovirus; antiviral; natural products; secondary metabolites; marine actinobacteria; extract screening; butenolides
National Category
Basic Medicine
Identifiers
urn:nbn:se:umu:diva-86525 (URN)10.3390/md12020799 (DOI)000335745100011 ()24477283 (PubMedID)
Available from: 2014-03-03 Created: 2014-02-28 Last updated: 2017-12-05Bibliographically approved

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