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The pandemic threat from newly emerging viral diseases constitutes a major unsolved issue for global health. Antiviral therapy can play an important role in treating and preventing the spread of unprecedented viral infections. A repository of compounds exhibiting broad-spectrum antiviral activity against a series of different viral families would be an invaluable asset to be prepared for future pandemic threats. Utilizing an open innovation crowd-sourcing paradigm, we were able to identify a compound class of diphenylureas that exhibits in vitro antiviral activity against multiple viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), adenovirus, dengue virus, herpes, and influenza viruses. Compound 4 among the series exhibits strong activity against dengue virus, a growing global health problem with high medical need and no approved antiviral drug. The compounds are active against SARS-CoV-2 in a primary human stem cell-based mucociliary airway epithelium model and also active in vivo, as shown in a murine SARS-CoV-2 infection model. These results demonstrate the potential of the chemical class as antivirals on the one hand and the power of open innovation, crowd-sourcing, and repurposing on the other hand.

The Rift Valley fever virus (RVFV) is an emerging high-priority pathogen endemic in Africa with pandemic potential. There is no specific treatment or approved antiviral drugs for the RVFV. We previously developed a cell-based high-throughput assay to screen small molecules targeting the RVFV and identified a potential effective antiviral compound (1-N-(2-(biphenyl-4-yloxy)ethyl)propane-1,3-diamine) as a lead compound. Here, we investigated how structural modifications of the lead compound affected the biological properties and the antiviral effect against the RVFV. We found that the length of the 2-(3-aminopropylamino)ethyl chain of the compound was important for the compound to retain its antiviral activity. The antiviral activity was similar when the 2-(3-aminopropylamino)ethyl chain was replaced with a butyl piperazine chain. However, we could improve the cytotoxicity profile of the lead compound by changing the phenyl piperazine linker from the para-position (compound 9a) to the meta-position (compound 13a). Results from time-of-addition studies suggested that compound 13a might be active during virus post-entry and/or the replication phase of the virus life cycle and seemed to affect the K+ channel. The modifications improved the properties of our lead compound, and our data suggest that 13a is a promising candidate to evaluate further as a therapeutic agent for RVFV infection.

Most flaviviruses are arthropod-borne viruses, transmitted by either ticks or mosquitoes, and cause morbidity and mortality worldwide. They are endemic in many countries and have recently emerged in new regions, such as the Zika virus (ZIKV) in South-and Central America, the West Nile virus (WNV) in North America, and the Yellow fever virus (YFV) in Brazil and many African countries, highlighting the need for preparedness. Currently, there are no antiviral drugs available to treat flavivirus infections. We have previously discovered a broad-spectrum antiviral compound, benzavir-2, with potent antiviral activity against both DNA- and RNA-viruses. Our purpose was to investigate the inhibitory activity of benzavir-2 against flaviviruses. We used a ZIKV ZsGreen-expressing vector, two lineages of wild-type ZIKV, and other medically important flaviviruses. Benzavir-2 inhibited ZIKV derived reporter gene expression with an EC₅₀ value of 0.8 +/- 0.1 µM. Furthermore, ZIKV plaque formation, progeny virus production, and viral RNA expression were strongly inhibited. In addition, 2.5 µM of benzavir-2 reduced infection in vitro in three to five orders of magnitude for five other flaviviruses: WNV, YFV, the tick-borne encephalitis virus, Japanese encephalitis virus, and dengue virus. In conclusion, benzavir-2 was a potent inhibitor of flavivirus infection, which supported the broad-spectrum antiviral activity of benzavir-2.

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.

According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-inhuman antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.

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.

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. 

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.

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.

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.