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Mono- and combinational drug therapies for global viral pandemic preparedness

Summary Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies.


INTRODUCTION
Despite advances in modern medicine, viral diseases consistently pose a substantial economic and public health burden throughout the world. In fact, both the World Health Organization and the United Nations have highlighted the specific need for better management of viral diseases as priorities for future development (World Health Organization, 2018). This burden is likely due to viruses' ability to regularly emerge and re-emerge into the human population from natural reservoirs such as wild and domesticated animals, leading to unpredictable outbreaks and wildly destructive health consequences (Choi, 2021). However, despite this constant threat of viral outbreaks, the landscape of antiviral targets is still underdeveloped, with over 200 human viral diseases that lack approved antiviral treatments.
Because the development of novel antivirals is long, laborious, and often unprofitable, the current strategy for the management of viral outbreaks is heavily reliant on the development of vaccines over antiviral treatments (Monto, 2006). However, while vaccines are an effective public health measure to stop the community spread of a well-characterized virus, it is impossible to develop vaccines against viral diseases that may emerge in the future. Therefore, antiviral development remains a crucial aspect of viral disease management to ensure timely and effective treatment of infected individuals and to reduce virus transmission.
Antiviral drugs are approved medicines that stop viruses from multiplying. Currently, there are 179 approved antiviral drugs, which are derived from 88 unique drug structures. Antiviral drugs currently represent 4.4% of 4,051 approved medicines. However, 10 of 88 have been withdrawn due to side effects (Chaudhuri et al., 2018;Wishart et al., 2018). The most common side effects of many antiviral drugs are nausea, vomiting, allergic reactions, drowsiness, insomnia, heart problems, and dependence (Morris, 1994). Side effects can also be associated with the capacity of the drugs to either enhance or suppress intrinsic immune functions of infected cells or alter the activity of immune cells within the host (Holstein and McCarthy, 2017). Antiviral drugs with immunostimulatory properties could lead to ''cytokine storm,'' which could be associated with an overwhelming systemic inflammation that leads to multiple organ dysfunction and potentially death (Fajgenbaum and June, 2020). By contrast, antivirals with Broad-spectrum antivirals (BSAs) can inhibit the replication of multiple viruses from the same or different viral families (Andersen et al., 2020). One efficient method of BSA development is drug repurposing/repositioning, a strategy for identifying new uses for approved or investigational antiviral drugs that are outside the scope of the original medical indication (Pushpakom et al., 2019). BSAs are cost-effective because the overall development cost can be distributed across many viral indications. Critically, robust BSA development fosters future pandemic preparedness because BSA activity facilitates enhanced coverage of newly emerged viruses.
Ongoing viral replication and prolonged exposure to certain drugs can lead to the selection of drug-resistant viruses through mutations in viral proteins. For example, mutations in HCV proteins confer resistance to NS3-4A, NS5A, and NS5B inhibitors (Ahmed and Felmlee, 2015). To mitigate the development of antiviral drug resistance, researchers developed antivirals that target protein-protein interactions rather than active sites of viral or host enzymes (Massari et al., 2021;Schormann et al., 2011). Another alternative is to combine antivirals . Additive, multiplicative, and synergistic drug combinations are more effective than monotherapies, allowing for successful treatments at lower dosage and reduction of harmful side effects. Indeed, a combination of IFN-a and ribavirin was the ''gold standard'' for the treatment of chronic HCV infection for more than a decade (Ilyas and Vierling, 2014). Furthermore, ribavirin-and IFN-a-containing combinations have been used against other viruses (Li et al., 2011;Tong et al., 2018) (NCT04412863), suggesting that BSA-containing combinations (BCCs) can be used to target a broad range of viruses.
Care needs to be taken when finding the correct BCCs. Drugs with unique mechanisms of action (MoA) are often paired together to minimize side effects and maximize efficacy. Drugs with the same MoA, such as nucleoside/nucleotide analogs, cannot be taken together (Zoulim, 2005) because they compete, rather than produce synergistic or additive effect. Such combinations could also have higher toxicity than monotherapies. In addition, drug antagonism can reduce the effectiveness of treatment and lead to an increased risk of virologic failure (failure to meet a specific drug target). Ideally, one wants the smallest number of drugs in cocktail due to the potential for increased toxicity and additional side effects with each additional drug (Radhakrishnan and Tidor, 2008).
Here we have reviewed the available scientific and clinical information and identified the basic principles behind activities of BSAs and BCCs to predict novel drug cocktails for the treatment of emerging and re-emerging viruses with pandemic potential. The approach described herein could facilitate the development of cost-effective and lifesaving countermeasures to fight new viral outbreaks.

THE LANDSCAPE OF BROAD-SPECTRUM ANTIVIRAL ACTIVITIES CAN BE EXPANDED
To identify known BSAs we have extensively reviewed published antivirals using PubMed.gov, ClinicalTrials.org and DrugBank.ca (Kim et al., 2021;Ursu et al., 2019;Wishart et al., 2018). Each of the resulting antiviral drug terms in this initial list was queried in combination with the terms ''virus,'' ''antiviral,'' or one of the known human viruses obtained from Virus Pathogen Database and Analysis Resource (Pickett et al., 2012). From this, we have compiled a list of antiviral drugs which we checked in the DrugBank. We desalted the compounds. Metals, mixtures, illicit and exclusively veterinary drugs were excluded. The returned results were examined to determine if antiviral activity has been demonstrated between the drug and two or more viruses from two different viral families. If antiviral activity could be established in more than 2 viral families, then all such drug-virus combinations would be recorded. Altogether, we identified 255 approved, investigational and experimental BSAs that target 104 human viruses from 24 families ( Figure S1; Data S1).
To expand the activity spectrum of BSAs, we analyzed relationships between drug activity and virus phylogeny. For this, we first build a phylogenetic tree using the CLUSTALW2 algorithm and amino acid sequences of viral polymerases (pols) and reverse transcriptases (RTs) extracted from GenBank (Aiewsakun and Simmonds, 2018;Larkin et al., 2007) ( Figure 1A). Notably, some viruses are represented by only small portions of pol and RT sequences (Data S1). Next, we identified the number of BSAs found to have activity against each corresponding virus ( Figure 1B). Although the phylogenetically similar viruses will likely be responsive to the same drug, Figure 1C indicates that most BSAs are only tested against a small subpopulation of related viruses.
From this information, we can identify a wide range of previously untested BSA-virus interactions, which could demonstrate novel antiviral activities. For example, 54 BSAs have been proven effective for SARS-CoV-2, but not other coronaviruses indicating higher likelihood of antiviral activity between those 54 BSAs and several other coronavirus species. Due to the high probability of coronavirus emergence, this inference could further be applied to coronaviruses that may arise in the future. However, it is important to note that our analysis is limited to viruses that encode their own pols or RTs and for which full-or near full-length sequences of these enzymes are available. For viruses that do not encode their own pols and RTs or are thus far poorly characterized, an analysis of virus taxonomy and BSA activity may be required to make similar inferences (Kuhn, 2021).

VIRUS AND HOST TARGETS FOR BROAD-SPECTRUM ANTIVIRALS
We next reviewed the known or suspected primary BSA targets (Data S1). We were able to identify primary targets for a fraction of BSAs. Most virus-directed BSAs work by inhibiting viral nucleic acid synthesis or protein processing ( Figure 3A). Among host-directed BSAs, mechanisms appeared to be more varied and included the inhibition of protein translation, trafficking, modification, or degradation, receptor-mediated signaling, lipid metabolism, etc. ( Figure 3B). However, in contrast to most host-directed BSAs that work The circular dendrogram shows the SAR of BSAs from our database. We also used SAR analysis to identify BSA candidates from the list of 11,834 compounds from DrugBank. Three compound sub-clusters that include two or more know BSAs are shown.

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iScience 25, 104112, April 15, 2022 5 iScience through the inhibition of host factors, several host-directed BSAs also work to activate innate immune responses against viruses. For example, IFNs are natural host-directed activators that bind their receptors to trigger cellular antiviral responses, which attenuate viral replication (Park and Iwasaki, 2020), and ABT-263 (navitoclax) targets the Bcl-xL protein to initiate apoptosis of infected cells without affecting non-infected cells (Ianevski et al., 2020a). Some BSAs, such as suramin, can simultaneously target host and viral factors (Langendries et al., 2021;Yin et al., 2021). Lastly, some BSAs are given in the form of prodrugs such as ganciclovir and gemcitabine which are activated by virus or host factors to achieve their antiviral effect (Walther et al., 2017).
By interrogating the Drug-Gene Interaction database (Fajgenbaum and June, 2020), we found that several host-directed BSAs can target multiple cellular factors involved at several stages of viral replication. These extra drug targets are generally unexplored and are most likely associated with side effects, although they

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may also affect viral replication. Perhaps, the most important of these targets are immunomodulatory. We compiled the BSAs with secondary immunomodulatory targets and showed those in Figure 3C. From this, we found that many BSAs target similar clusters of immunomodulatory genes, indicating some structural and functional similarities between the targets, but no overarching immunomodulatory targets that may suggest a contribution to antiviral activity. Further analysis is needed to elucidate the exact role that these immunomodulatory targets play in host pharmacodynamics or their contribution to antiviral activity. Notably, the potential immunomodulatory side effects of BSAs can be mitigated by lower dosage of drugs in synergistic combinations.

BROAD-SPECTRUM ANTIVIRAL-CONTAINING DRUG COMBINATIONS FOR THE TREATMENT OF VIRAL INFECTIONS
Despite demonstrated efficacy at the early stages of drug development, many antiviral monotherapies are often found to be ineffective in clinical settings (Consortium et al., 2021). Because of this, antiviral cocktails have increasingly become the focus of drug developers. Antiviral combinations have several benefits over monotherapies. Namely, they can prevent the development of drug-resistant strains by completely halting viral replication, an advantage rarely achieved with monotherapies. Further, drugs administered together as cocktails may achieve an expanded antiviral activity, allowing for the treatment of multiple types of viral infections at once (Shyr et al., 2021). Because of this, BCCs are favorable candidates for front-line therapy against poorly characterized emerging viruses, re-emerging drug-resistant viral variants, and viral co-infections.
To underscore the potential benefits and provide an organized summary of known dual antiviral drug combinations, we manually reviewed scientific literature and patent applications and constructed a BCC database (Data S1). The database comprises 538 drug cocktails. It includes 612 unique drugs and covers 68 viruses. We were able to identify primary targets for 415 drugs (Figure 4). Of these, we found that 211 BCCs have components that both primarily target viral factors, 74 have components that both primarily target host factors, and 130 BCCs in which one drug primarily targets the virus while the other primarily targets the host. We were not able to identify specific targets for 160 BCCs due to one or more BSA in the BCC having an unknown mechanism of action. We suspect that the overrepresentation of virus-virus and virus-host targeting BCCs is as drugs that were developed to specifically target virus factors may be more successful in achieving a direct antiviral effect while minimizing severe side effects. Thus, virus-virus and virus-host targeting BCCs are superior to host-host BCCs in many ways, including the leveraging of antiviral synergism, reduction of toxicity. However, host-host targeting BCCs have lower risk of drug resistance and an expanded spectrum of antiviral activity.

ASSOCIATION BETWEEN INFECTED ORGAN SYSTEMS AND ROUTES OF BROAD-SPECTRUM ANTIVIRALS/BCCS ADMINISTRATION
Viruses often preferentially infect hosts in one or more specific organ systems of the human body ( Figure 5A). In theory, BSAs and BCCs must be rapidly delivered to the infected organs using an amenable route of administration (RoA) to preserve the drug structure, maximize antiviral effect, and reduce drug toxicity or other adverse events. For example, if a virus infects and replicates in the respiratory system, medications administered by inhalation may be preferable. Likewise, if the virus infects the cardiovascular system, intravenous drug administration could be considered, etc. However, intravenous administration prevents widespread use of the BCCs because use is restricted to specialized care centers such as hospitals. In cases of advanced or systemic virus infections that affect multiple organ systems, antivirals intravenous administration may be preferable. However, most of the BSAs and BCCs reviewed here are delivered orally, most likely due to the preferential development of orally bioavailable drugs by pharmaceutical companies because of their increased marketability and potential for global distribution ( Figures 5B and 5C).

BROAD-SPECTRUM ANTIVIRAL AND BCC SCORING SYSTEMS
To identify the most promising monotherapies we developed a six-component  iScience Perspective To calculate the final BSA score, we sum the points across all six components using the following formula: BSA score = CSAR + CDDS + CTR + CIC + CRoA + CPhyl (Equation 1) For example, the BSA score of favipiravir in relation to its activity against Ebola virus (EBOV) is 5.57. Favipiravir is an orally available nucleoside analog, which blocks viral RNA synthesis by inhibiting viral RdRP activity. Its immunomodulatory properties were not reported, and it is in phase 3 clinical trials against EBOV (NCT02329054). Therefore, the component values are as follows: C SAR = 1, C DDS = 0.75, C DTR = 1, C IC = 1, C RoA = 1, and C Phyl = 1 (Data S1).
Another example is merimepodib. Its BSA score in relation to its EBOV activity is 4.25. Merimepodib is an orally available inhibitor of host inosine monophosphate dehydrogenase (IMPDH), which controls the intracellular guanine nucleotide levels that are required for viral RNA synthesis. It possesses anti-EBOV activity in vitro and suppresses host immunity (Jain et al., 2001;Tong et al., 2018). Therefore, the component values are as follows: C SAR = 1, C DDS = 0.25, C DTR = 1, C IC = 0, C RoA = 1, and C Phyl = 1 (Data S1).
To identify the most promising combinational therapies, we invented a four-coefficient BCC scoring system. It utilizes the following BCC coefficients: if both BSA components can be administrated by the same route, but the RoA cannot be used for targeted delivery to the diseased system, k RoA = 1; if the two BSA components cannot be administrated via the same route, k RoA = 0.8; From these we calculate a BCC score using the following formula: BCC score = kDI Ã kDTI Ã kDRS Ã kRoA Ã ðBSA score drug 1 + BSA score drug 2Þ (Equation 2) If the BCC score exceeds the sum of the individual BSA scores by 5, we consider this combination to be effective (Data S1). For example, for favipiravir-merimepodib targeting EBOV, the k DI is 1.0 because the MoAs of the drugs are different; the k DTI is 1.1, because the drugs target viral RdRP and host IMPDH; the k DRS is 1.2, because both drugs reduce the synthesis of viral RNA, and k RoA 1.2, because both drugs can be taken orally, which allows delivery of the combination to multiple infected organs. Therefore, the BCC score of favipiravir-merimepodib is 15.8, whereas the combined BSA score of the combination is 10 (Table 1). Because the BCC score is greater than the combined BSA score by over 5 points, this combination would be considered to have high potential based on our scoring system. Indeed, by reviewing the literature, we found that this combination has been independently tested against EBOV in vitro and was shown to be effective (Tong et al., 2018). In contrast, the BCC score for favipiravir-ribavirin against EBOV is 7.3. This is lower than the sum of individual BSA scores of favipiravir and ribavirin, which is 8.5, which predicts suboptimal performance for this combination (Table 1). Literature review shows that this prediction is consistent with efficacy studies in monkeys (Madelain et al., 2020). Thus, we demonstrated that the results from our scoring system are consistent with real-life experimental evidence.

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Next, we used our scoring system for the identification of novel potential BCCs (Data S1). As mentioned above, we focused on novel combinations for which BCC scores exceed the sum of the individual BSA scores by > 5. In this way, we have identified several unexplored drug combinations that may be prioritized for development in preparation for future resurgent outbreaks or the appearance of newly emerging viruses (Table 1).
Interestingly, many predicted BCCs contain nucleotide/nucleoside analogs along with inhibitors of pyrimidine/purine biosynthesis, cap analogs, or IFNs, which also target viral RNA synthesis via IFN-induced RNases. Indeed, such combinations showed synergy in experiments performed in our and other laboratories (Bellobuono et al., 1997;Byrn et al., 2015;Falloon et al., 2000;Herring et al., 2021;Ianevski et al., 2021aIanevski et al., , 2021bLi et al., 2020Li et al., , 2022Phillips et al., 2015;Pires de Mello et al., 2018;Schultz et al., 2022;Tong et al., 2018;Wedemeyer et al., 2013). Thus, our preliminary results suggest that scores could correlate with the antiviral efficacy of BCCs and that some of these combinations could be used as pan-and even cross-virus family .

Limitations of the study
The MoA of many BSAs remains elusive. In addition, many BSAs have been tested only in vitro. These lowered the final scores of BSAs and BCCs and thus affected the prediction capacity of our approach. Therefore, the MoAs of BSAs and BCCs should be studied in vitro and their efficacy and toxicity should be evaluated in vivo. In addition, immunological properties and RoA of mono-and combinational therapies should be evaluated. Finally, the prospects for clinical trials of the most effective and least toxic drug combinations should be assessed.

CONCLUSIONS AND FUTURE PERSPECTIVES
New life-threatening viruses emerge and pose a serious threat to public health. Thereby, broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after a new virus started to spread from human to human (Andersen et al., 2020). To identify novel pan-and cross-virus family treatments, we established a scoring system, which is based on analysis of conserved druggable virus-host interactions, MoAs, immunomodulatory properties of BSAs, RoAs, and BSA interactions with other antivirals. The system prioritizes the development of the most promising few of the thousands of potentially viable BSAs and BCCs. However, the effectiveness of the predicted BSAs and BCCs needs to be confirmed in vitro and in vivo to prepare them for clinical trials . Therefore, we will invite researchers to validate our proposed BSAs and BCCs and optimize our approach further using mathematical modeling, machine-learning, and other tools. If handled correctly, the development of the right BSAs and BCCs can have a global impact by enhancing preparedness for future viral outbreaks, filling the void between virus identification and vaccine development with life-saving countermeasures and improving the protection of the general population against emerging viral threats.