**3. Drug repurposing in viral diseases**

Over the last decades the world has seen multiple severe viral outbreaks resulting in millions of deaths. Among the deadliest were the Influenza pandemics such as H1N1 (1918), H2N2 (1957), H3N2 (1968) and H1N1 (2009). The HIV/AIDS epidemic that was first recognized in the 1980s and went global has also caused up to an estimated amount of 36 million deaths and is still ongoing. Besides the large, deadly pandemics there have been smaller but very impactful localized epidemics such as Dengue virus (DENV), Zika virus (ZIKV), Ebola virus (EBOV) and Middle East respiratory-syndrome corona virus (MERS-CoV) which pose serious challenges to public health. Most recently in 2019 there was an outbreak of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which caused the COVID-19 pandemic, affecting nearly every country in the world with over 226 million reported cases to date [72]. Despite the advances in controlling viral pathogens that come with the widespread mass vaccination, there are no approved specific (effective) therapies for the treatment of most viral infections.

By exploring new targets and mechanisms, drug repurposing provides new indications for old drugs. The major time advantage of repurposing is that this approach allows repurposed drugs to quickly enter clinical trials, which is of significant importance in reacting to disease outbreaks, especially in the case of worldwide pandemics.

#### **3.1 Drug repurposing for COVID-19**

The outbreak of COVID-19, caused by SARS-CoV-2, has spread across the world. There is, as yet, no specific treatment for COVID-19 approved. Drug repurposing provides a fast and economical option for the identification of medications targeting SARS-CoV-2.

#### *Drug Repurposing Techniques in Viral Diseases DOI: http://dx.doi.org/10.5772/intechopen.101443*

SARS-CoV-2 is a member of of the betacoronaviruses family. It is a singlestranded RNA virus, characterized by large crown-like spikes protruding on the viral surface and an unusually large RNA genome which encodes four main structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) [62, 73, 74]. Like other coronaviruses, SARS-CoV-2 cell entry is mediated by the spike glycoprotein. The spike glycoprotein is composed of two subunits, S1 and S2, which mediate viral-host attachment and viral-host membrane fusion cascade, respectively [75]. SARS-CoV-2 spike recognizes and binds to the human ACE2 (hACE2) receptor through its receptor-binding domain (RBD) and is primed and activated by proteolytic cleavage by enzymes such as Furin and transmembrane serine protease 2 (TMPRSS2) [76, 77]. Spike, ACE2, Furin and TMPRSS2 have been shown to play a key role in mediating viral-host fusion attachment and fusion, and the Furin cleavage site on the Spike protein has been indicated as one of major reasons SARS-CoV-2 is so infectious [78, 79]. This makes these potentially promising drug targets for COVID-19 treatment [80, 81].

#### *3.1.1 Drug repurposing targeting viral fusion*

Inhibition of the Spike-Ace2 interaction is a primary target for drug repurposing as it is crucial to viral entry. *In silico* approaches have been performed and identified Simeprevir, an HCV NSP3A/4 protease inhibitor, as a potential blocker of Spike-Ace2 interaction by binding the RBD [82]. However, other *in vitro* studies showed that Simeprevir is not necessarily active against the RBD but is targeting the viral replication [83].

Another promising target is TMPRSS2, a serine protease. TMPRSS2 is associated to the host endothelial cell surface and cleaves the viral spike glycoprotein after binding to ACE2, activating it. The activation of spike protein then facilitates viral entry [84]. Camostat mesilate (a serine protease inhibitor) has been approved for the treatment of chronic pancreatitis, postoperative reflux esophagitis and kidney or liver disease fibrosis [81, 84, 85]. Since it is an established serine protease inhibitor it is a prime candidate to inhibit the TMPRSS2. An *in vitro* study indeed showed that camostat mesilate can suppress viral replication by halting the fusion of virus-cell membranes through the inhibition of TMPRSS2 [84]. Clinical trials using this drug are currently ongoing [86]. However, recently the results of a small double-blind randomized clinical trial using camostat mesilate performed in patients hospitalized with COVID-19. The trial determined no adverse effects of treatment with camostat but also no increase in positive clinical outcomes [87].

## *3.1.2 Drug repurposing targeting endocytosis of SARS-CoV-2*

Besides direct membrane fusion, SARS-CoV-2 can also invade cells via endocytosis [88, 89]. This route involves several proteins that play an important role in endosome formation, such as two-pore channel 2 (TPC2), Cathepsin L (CTSL) and Vacuolar-type ATPase (V-ATPase) [90]. These proteins are indicated to be potentially interesting therapeutic targets for COVID-19 treatment.

Tetrandrine is a bisbenzylisoquinoline and calcium channel blocker, known for its anti-inflammatory, immunosuppressive, oncological, and cardiovascular bioactivity [48–49]. The compound has been shown to be effective in the treatment of silicosis [90–92]. According to an *in vitro* study, tetrandrine is a low micromolar inhibitor of viral replication that functions by blocking the two-pore channel 2 (TPC2), which impedes Ca2+ release which in turn prevents acidification of the endosome [92].

The now infamous anti-malarial drugs chloroquine (CQ ) and hydroxychloroquine (HCQ ) were also posited to inhibit endocytosis of SARS-CoV-2 [93, 94]. CQ and HCQ are potentially involved in blocking cleavage of spike by raising the pH of the endosomes, preventing cathepsin L-mediated proteolysis, which is a key element in membrane fusion after binding ACE2. Whilst many potential mechanisms of action have been suggested, none have been rigorously demonstrated. In addition, a recent large meta-data analysis has shown that there is no evidence that treatment with CQ or HCQ reduces COVID-19 mortality in patients [95]. To the contrary, evidence is available that shows HCQ is responsible for a small increase in mortality outcomes. These compounds garnered lots of attention when the presidents of prominent countries started promoting CQ and HCQ as wonder drugs that could combat COVID-19 [96]. However, as mentioned before most evidence points towards the contrary and the WHO recommends against the treatment with these drugs [97]. These cases have shown an important risk in the use of drug repurposing: in the age of hyper connectivity and social media echo chambers dangerous, unfounded ideas can avoid scrutiny and rigorous investigation, leading to large groups of people self-medicating with alternative treatments that have no scientific basis. This poses a problem in general drug development but even more so in drug repurposing cases where these compounds tend to be far more easily obtainable by the general public as they are approved and often available for purchase in pharmacies.

#### *3.1.3 Drug repurposing targeting viral replication*

After invading host cells, the coronavirus comes into the next stage of its life cycle: translation, replication, transcription and Assembly. This process mainly involves five different proteins: Mpro, RdRp, nsp14, MTHFD1 and Plpro, which have different functions [74, 98]. Mpro is also known as 3C-likeprotease (3CLpro) and proteolytically processes the majority of the polyprotein into functional polypeptides [99]. Similar to 3CLpro, Plpro is a viral protease that is responsible for cleaving polyproteins to generate a function replicase complex [100]. RNA synthesis, critical for viral replication, is performed by RdRP and its cofactors nsp7 and nsp8. Nsp14 has an exonuclease activity that supports RNA synthesis with an unusual RNA proofreading function. A study performed by Tinghua University showed that knockdown of MTHFD1, a key enzyme in cellular production of purine, dTMP and methyl groups, significantly inhibits viral replication [101]. As a result of their key functions in the viral life cycle these proteins are promising potential targets for antiviral drugs development. Clofazimine is an anti multibacillary leprosy drug which was approved for medical use in 1986. A recent study performed by The University of Hong Kong showed that clofazimine inhibits both viral spike glycoprotein mediated cell fusion and replication of SARS-CoV-2 *in vitro* [102].

Genome analysis has demonstrated that SARS-CoV-2 and SARS-CoV genes globally share >80% nucleotide identity and >89% similarity [73, 103, 104]. As a result, the key steps in the CoV family viral life cycle within the host cell are likely to be highly conserved. A key feature of this process is the expression of non-structural proteins (nsps). Subsequent to cell entry, two extended polypeptides (pp1a and pp1ab) from the CoV viral genome are generated by the host cell translation machinery [105, 106]. These two polypeptides then self-cleave into 37 distinct nonstructural (nsp) proteins [107] and anaylsis has demonstrated that the CoV family possesses several proteases involved in this essential self-cleavage process: the papain-like protease (PLpro), and the 3C-like proteinases (3CLpro or Mpro) [108]. CoV generally encode two PLpros within nsp3, with the exception of gamma-CoV,

#### *Drug Repurposing Techniques in Viral Diseases DOI: http://dx.doi.org/10.5772/intechopen.101443*

SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 [100]. Thus, 3d unlike structural/accessory protein-encoding genes, which can show significant sequence variation in order to select between different potential host cell receptors, CLpro plays a central and critical role in CoV replication in host cells and the similarity in essential function leads to a high sequence similarity between the proteases of the CoV family, in particular beta-CoV [105, 106]. This increased similarity in the structure of the 3CLpro has the consequence of high structural between 3CLpro of different CoV family members—and the concomitant increased likelihood of cross-species function of CoV 3CLpro inhibitors and the potential to repurpose these inhibitors. The high sequence homology within Cov 3CLpros also provided high quality model templates—subsequently supported by the availability of high-resolution diffracting crystals—to perform both computational docking experiments, as well as molecular validation by X-ray crystallography [109]. There also exists the potential for the discovery and the development of a pan-anti-CoV inhibitor [110, 111].

This potential has been partially realized in not only the discovery of entirely novel SARS-CoV2 3CLpro inhibitors [99, 112, 113] (refs), but also in a number of reports describing successful identification of potential repurposing candidates [109].

For example, we and others have previously reported the results of a molecular docking experiment that indicated a class of well tolerated compounds (gliptins) as potential SARS-CoV2 inhibitors. For example, Anagliptin, a DPP4 inhibitor, is a well-established treatment for diabetes that is used by millions of patients. It has an excellent safety profile [114]. Computational docking demonstrated an efficient binding, with predicted H-bonds made to the backbone atoms of Gly163, Gly271, and Tyr268 and the side chain of Tyr273. Docking experiments also proposed that α-ketoamide inhibitors of hepatitis C virus (HCV) protease would be potential inhibitors of SARS-CoV-2 3CLpro. Efforts were concentrated on broceprevir and telaprevir as the docking poses were supported by experimental structure analyses (**Figure 1**). Subsequent biochemical assays demonstrated that broceprivir indeed displays strong binding to isolated 3CLpro of SARS-CoV2 and inhibits viral replication in cellular assays.

Similarly to CQ and HCQ, ivermectin, originally an anthelmintic, also gained widespread attention as a potential treatment for COVID-19 and some *in vitro* evidence of SARS-CoV-2 replication inhibition in cell cultures has been provided [115]. The suggested mechanism of action is the inhibition of importin alpha/ beta-1 nuclear transport proteins which the virus uses to enter the nucleus and is an important part of the replication cycle as it suppresses host-immune response [116]. Despite the in vitro effect no clinical data has supported the therapeutic use of ivermectin at concentrations approved for use in humans. However, it gained attention in the media and people started buying ivermectin meant for use in large animals to self-medicate against the virus [117]. Even though ivermectin is approved for human use and is generally safe, the doses used in the treatment of animals are several times larger than recommended for humans and can cause side-effects ranging from mild diarrhea to seizures and coma. This once again shows the care that needs to be taken when repurposing drugs in how scientific results are communicated to the general public, as it can lead to potential dangerous situations [118].
