Natural Bioactive Compounds from Fungi as Potential Candidates for Protease Inhibitors and Immunomodulators to Apply for Coronaviruses.

The inhibition of viral protease is an important target in antiviral drug discovery and development. To date, protease inhibitor drugs, especially HIV-1 protease inhibitors, have been available for human clinical use in the treatment of coronaviruses. However, these drugs can have adverse side effects and they can become ineffective due to eventual drug resistance. Thus, the search for natural bioactive compounds that were obtained from bio-resources that exert inhibitory capabilities against HIV-1 protease activity is of great interest. Fungi are a source of natural bioactive compounds that offer therapeutic potential in the prevention of viral diseases and for the improvement of human immunomodulation. Here, we made a brief review of the current findings on fungi as producers of protease inhibitors and studies on the relevant candidate fungal bioactive compounds that can offer immunomodulatory activities as potential therapeutic agents of coronaviruses in the future.


Introduction
Coronaviruses (CoVs) are a large group of enveloped viruses with non-segmented, single-strand, and positive-sense RNA genomes. CoVs are classified in the family Coronaviridae of the order Nidovirales. Notably, CoVs have been identified as zoonotic viruses that can be transmitted between humans and animals and they are known to cause a wide range of infections. These infections can appear as symptoms that range from those of the common cold to much more fatal diseases, like respiratory syndrome, as well as enteric and central nervous system diseases. Two highly pathogenic microorganisms with approximately 30,000 nucleotides, the Severe Acute Respiratory Syndrome (SARS-CoV, or SARS) and the Middle East Respiratory Syndrome (MERS-CoV, or MERS), have resulted in regional and global outbreaks. In 2002, SARS originated in southern China, while MERS was first known to infect a patient in Saudi Arabia in 2012 [1][2][3]. A novel coronavirus, which was previously designated as SARS-CoV-2, was identified as a causal agent of pneumonia in Wuhan, a city Protease inhibitors play an important role in viral replication by selectively binding to viral proteases and blocking proteolytic cleavage of the protein precursors that are necessary for the production of infectious viral particles [7,8,11]. The papain-like protease (PL pro ) and the 3-chymotrypsin-like protease (3CL pro , also known as the main proteases that are suitable targets for viral inhibitors) have been identified in CoVs, for which both proteases are believed to be essential in the role of viral replication and are considered to be attractive targets for antiviral therapeutics [12,13]. Numerous previous studies have identified compounds and drugs that can inhibit protease activity through docking/molecular dynamic experimentation and their inhibition activity on CoVs replication in the cell cultures of mice and in non-human primate (NHP) models, as has been previously reported [14]. On the other hand, human immunodeficiency virus type 1 (HIV-1) protease inhibitors (tipranavir, saquinavir, ritonavir, nelfinavir, lopinavir, indinavir, darunavir, atazanavir, and amprenavir) that have been approved for clinical applications by the Food and Drug Administration (FDA) are widely reported to be able to deactivate 3CL pro . Hence, they have been identified as potential drugs in the treatment of CoV infections. Lopinavir, atazanavir and indinavir have been identified as potential candidates as 3CL pro inhibitors [15,16]. Furthermore, RNA-dependent RNA polymerase inhibitors, e.g., remdesivir and favilavir, have been used to effectively treat CoVs infections [16,17]. Remdesivir or chloroquine, glucocorticoids and the combined protease inhibitor lopinavir-ritonavir have been used to treat SARS-CoV and MERS-CoV infections [4,[16][17][18].
Currently, there is a lack of an effective treatment or a vaccine to prevent SARS-CoV-2 infection. Parent compounds are being tested to prevent SARS-CoV-2 infection in in vitro or clinical studies-based SARS-CoV and MERS-CoV trials. Remdesivir or chloroquine has been highly effective in the inhibition of SARS-CoV-2 infection in vitro [18]. Lopinavir-ritonavir has been suggested to be one of the therapeutic agents of SARS-CoV-2 [17,19,20]. Hence, presently, there are no approved vaccines or drugs for SARS-CoV-2 infection. Potential candidates as targets for further in vitro and in vivo studies for SARS-CoV-2 prevention and treatment are 3CL pro , Spike, RNA-dependent RNA polymerase (RdRp), PL pro and human angiotensin-converting enzyme 2 (human ACE2). Interestingly, the potential unitality of clinical drugs and natural products for the treatment of SARS-CoV-2 infection was studied while using computational methods. Specifically, 3CL pro is recognized as an important target anti-SARS-CoV-2 drug that was designed and modified to play an important role in the maturation of SARS-CoV-2. Nelfinavir has been predicted as a potential inhibitor of the SARS-CoV-2 main protease, but has been used in treatment application [4]. Nevertheless, favilavir has been approved for use in the investigational therapy for SARS-CoV-2 infected patients in China [21]. The US patient infected with SARS-CoV-2 has been treated by supportive care and remdesivir, for which no adverse reactions were observed during administration [22,23]. In February 2020, the remdesivir treatment of SARS-CoV-2 was initiated in Wuhan and Beijing, China for clinical trials, but the safety and efficacy of remdesivir in the treatment process need to be fully evaluated [24]. Therefore, this study has summarized the current findings on the natural antiviral compounds that were obtained from fungi for the purpose of employing them as protease inhibitors. The findings reveal a noteworthy potential in the development of antiviral agents for CoVs. Moreover, the fungal bioactive compounds that possess immunomodulatory activities reveal themselves to be a potential resource in the treatment of CoVs.

Potential of Fungal Antiviral Bioactive Compounds as Protease Inhibitors to Treat CoVs
Fungi (including filamentous fungi and mushrooms) represent a rich source of various biologically active compounds that can serve as a major source of new compounds in the development of small-molecule drugs. This development process could involve direct or semi-synthetic methods, while the findings of this research could serve as a source of inspiration in the investigation of chemical scaffolds. Bioactive compounds that were obtained from fungi with potent antiviral activity are presently under investigation, and the number of studies is continually increasing [25][26][27][28][29][30]. Fungal bioactive compounds can be divided into two major groups of molecules; small organic molecules (secondary metabolites) produced by filamentous fungi, especially endophytic fungi, and high molecular weight compounds in the extracts or products that were obtained from the fruiting bodies of edible or medicinal mushrooms [26,[31][32][33]. Fungal small organic molecules are low molecular weight compounds that are produced by filamentous fungi. These compounds were synthesized by fungal hyphae and later secreted. They are commonly studied through the cultivation of fungal hyphae in culture media. Small molecular weight compounds with antiviral capabilities have been classified as indole alkaloids, non-ribosomal peptides, polyketides, and hybrids of non-ribosomal peptides and polyketides, and terpenoids [24,25,30,31,[33][34][35][36][37][38]. The antiviral activity of high molecular weight compounds that are extracted from fruiting bodies and fungal mycelia have been reported and classified as lignin derivatives, polysaccharides (e.g. chitin, glucan, lentinan, and mannan), proteins, and polysaccharide-protein/amino acid complexes [28][29][30][39][40][41][42]. Drugs or compounds with special effects on viral protease inhibitors, like HIV-1 protease inhibitors and hepatitis C virus (HCV) NS3/4A protease inhibitors, have been considered as potential drugs against CoVs, according to the findings of previous studies. Therefore, fungal compounds that have the potential to be candidates as protease inhibitors have been the focus of numerous present studies.
The major viral protease classes have been identified based on the relevant catalytic types including serine, cysteine, aspartic, threonine, glutamic acid, and metalloproteases [43,44]. Most viral proteases can recognize the specific sequences of amino acids in their substrates and cleave the peptide bond via a nucleophilic attack on the side chain of catalytic site [43][44][45]. HIV-1 protease is formed by two identical monomers as shown in Figure 2. Each monomer contributes on catalytic aspartyl residues (Asp25 and Asp25 ) in the active site which lie on the bottom of the cavity that plays a crucial role in substrate binding [46]. Additionally, HCV polyprotein is processed proteolytically upon translation by both host cells and viral proteases to at least 10 individual proteins [47,48]. These include four structural proteins and non-structural (NS) proteins. NS3/4A serine protease is further involved in the proteolytic processing of NS proteins and is also considered necessary for the direct-inhibition of HCV. NS3 is comprised of protease and helicase domains and forms a heterodimer with NS4A. Additionally, NS4A binds to the N-terminal region of NS3 and acts as a cofactor of the protease to enhance cleavage ( Figure 2). The catalytic triad of the NS3/4A protease is formed by His57, Asp81, and Ser139 [47][48][49]. Currently, the NS3 protein has emerged as an important target for anti-HCV drug discovery and development. Notably, a 3CL pro of CoVs is comprised of three-domain cysteine proteases. Furthermore, domain I and II contain β-barrels of the chymotrypsin structure, but domain III consists mainly of α-helices ( Figure 2). Moreover, 3CL pro contains a catalytic dyad defined by His41 and Cys145 [50,51]. This main protease is responsible for maturation of functional proteins and currently represents as a key target for antiviral drugs. Therefore, most of antiviral agents are peptidomimetics and macrocyclic compounds that interact with the active site of a targeted viral protease [52,53]. According to the HIV-1 protease and HCV protease exhibited a similar function as CoVs protease, so protease inhibitors are hypothesized to have the preventive and therapeutic potential against CoVs infection.

HIV-1 Protease Inhibitors Isolated from Fungi
Several HIV-1 protease inhibitor drugs have been made available in the human clinical use of CoVs [4,7,8,[16][17][18]. Nelfinavir was found to strongly inhibit the replication of SARS-CoV [54,55]. Antiviral drugs (ribavirin, lopinavir, and ritonavir), steroids, proteins that are known as immunoglobulins, type I interferon, and convalescent plasma have been used in the clinical treatment of SARS and MERS patients [56][57][58][59][60]. A diagnostic test for early SARS and MERS illnesses has not been validated; therefore, treatment could only be initiated once patients have met the criteria of a clinical and epidemiological case definition. Patient characteristics, such as age and the presence of diabetes, have been associated with severe diseases and they can confound treatment effects. Certain potential drugs that have been approved by the FDA and identified as potential inhibitors of 3CL pro of SARS-CoV-2 have been reported by Hosseini and Amanlou [11] while using a virtual screening and the molecular docking procedure. The ten potential drugs include paclitaxel, simeprevir, docetaxel, palbociclib, cabazitaxel, alctinib, imatinib, plerixafor, azelastine, and dasabuvir. Paclitaxel and simeprevir HCV NS3/4A protease inhibitors) revealed a strong degree of interaction with the SARS-CoV-2 protease binding pocket and it has been placed well into the pocket when compared to the antiviral drugs. Interestingly, virtual screening has confirmed that indinavir was selected as the SARS-CoV-2 main protease (PDB code 6LU7).
Ryang et al. [74] reported that 20 µg/mL of paclitaxel could inhibit HIV-1 protease activity in a similar manner to the positive control pepstatin A (80 µg/mL) in the in vitro experiment. A combination of paclitaxel and protease inhibitors (indinavir, nelfinavir, or combinations of these agents) at recommended dosages and schedules was used to treat patients of HIV-associated Kaposi's Sarcoma without enhancing toxicity [75]. In the virtual screening procedure, paclitaxel has been suggested as a therapeutic agent of SARS-CoV-2 based on its higher binding energy (-11.33 kcal/mol) to the active site of SARS-CoV-2 protease than that of lopinavir (-5.36 kcal/mol) and ritonavir (-5.04 kcal/mol) [21]. However, patient conditions for paclitaxel applications should be considered because of its side effects on bone marrow suppression. In addition, two semicochliodinols (semicochliodinol A and B) and didemethylasterriquinone D that were isolated from a microfungus, Chrysosporium merdarium, displayed an inhibitory effect on HIV-1 protease activity [76,77].
Some HIV-1 protease inhibitors have been isolated from certain mushrooms, especially some edible and medicinal mushrooms. Lingzhi mushrooms (Ganoderma species) have been generally acknowledged as a nutritional supplement across the world due to their association with long-term safety and the fact that they possess a vast array of medicinal properties. Various compounds that have exhibited inhibitory effects against HIV-1 protease activity have been identified from Ganoderma lucidum including ganolucidic acid A, 3β-5α-dihydroxy-6β-methoxyergosta-7,22-diene, ganoderic acid A-C, ganoderic acid β, ganodermanondiol, ganodermanontriol and lucidumol B [78][79][80]. Six colossolactones, ganomycin I, and ganomycin B isolated from G. colosum have displayed anti-HIV-1 protease activity [81,82]. Twenty-five metabolites were isolated from the fruiting body of G. sinnense, and it was found that ganoderic acid GS-2, 20-hydroxylucidenic acid N, 20(21)-dehydrolucidenic acid N and ganoderiol F exhibited a high potential to inhibit HIV-1 protease activity [83]. Notably, crude extracts of tiger milk mushroom (Lignosus rhinocerus) displayed inhibitory activity against HIV-1 protease activity on infected cells, while in silico analysis showed that heliantriol F displayed significant binding energy at -12.57 kcal/mol on the active site of HIV-1 protease [84]. Hexane extract fractions obtained from a jelly fungus (Auricularia polytricha) could effectively inhibit HIV-1 protease activity in vitro, while four major compounds, ergosterol, linoleic acid and two triacylglycerols were found to be present [85]. Moreover, adenosine and iso-sinensetin isolated from golden cordycep (Cordycep militaris), and 4.5 kDa protein isolated from Russula paludosa, have been reported as anti-HIV-1 replications by inhibition of HIV-1 protease activity [86,87].

Potential of Fungal Bioactive Compounds for Immunomodulators
Inflammasome is a cytosolic multiprotein oligomer of the innate immune system that is responsible for the activation of inflammatory responses. Inflammasome induction by coronavirus was first reported in porcine reproductive and respiratory syndrome virus [105]. Currently, the transport of Ca 2+ by SARS-CoV has been reported to trigger inflammasome activation. It has been suggested that the cytokine storm is associated with cases of pneumonia that were infected by SARS-CoV-2 [106]. Cytokines and chemokines have been recognized for playing an important role in immunity and immunopathology in the body during virus infection. They are an important part of the first barrier of innate immunity that serves as a defense against the viruses. The massive infiltrated inflammatory cells and the elevated proinflammatory cytokines/chemokines can lead to fatal acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [107,108]. A clinical study of 41 patients infected with SARS-CoV-2 in Wuhan, China showed that 63% of the patients had lymphopenia, 12% had ARDS, all patients had pneumonia, and the intensive care patients reported higher plasma levels of IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1A, and TNF-α than the non-intensive care patients [108]. Researchers also noted that patients with high concentrations of IL-1β, IFN-γ, IP10, and MCP1 were likely associated with activated T-helper-1 (Th1) cell responses.
Immunomodulators are the bioactive substances that can play a role or affect the regulating of the immune system, which is the first barrier against infectious diseases [109]. Clinically, immunomodulators are usually classified into three categories based on their relevant activities including: (1) reducing the stimulation of the immune system or reducing the effectiveness of the immune system (immunosuppressants), (2) promoting the response of the innate immune system (immunostimulants), and (3) enhancing the efficacy of vaccines to stimulate immunity (immunoadjuvants) [109,110]. Many drugs are known to be immunomodulatory substances because they have significant clinical efficacy for altering host responses in the therapy of viral and bacterial infections [111][112][113][114]. Various edible mushrooms have been studied for many years in terms of the effects of their metabolites in boosting immune responses and treating infectious [115][116][117][118]. The principal immunomodulatory effect of active substances derived from mushrooms is to stimulate immune effector cells such as T cells, cytotoxic T lymphocytes (CTL), dendritic cells (DCs), lymphocytes, macrophages and natural killer (NK) cells, resulting in cytokine expression and secretion including interleukins (ILs), tumor necrosis factor-alpha (TNF)-α, and interferon-gamma (INF)-γ [119,120].
Immunomodulators derived from mushrooms are classified into four groups, including lectins, proteins, polysaccharides, and terpenoids [109]. Lectins are carbohydrate-binding proteins that can be found in many organisms and are extracted from mushrooms. They have specific immune cell functions such as antiproliferative, and antitumor activities [108].
Fungal immunomodulatory proteins (FIPs) are small molecular weight proteins, ∼13 kDa and 110-114 amino acids, displaying immunomodulatory activity. They are a type of bioactive substance that can be derived from some edible mushrooms. Meanwhile, mushrooms are an essential source of immunomodulatory polysaccharides, which are long chains of carbohydrate molecules, particularly polymeric carbohydrates, that are composed of monosaccharides linked together by glycosidic bonds [108]. Polysaccharides are responsible for immuno-modulating activities that include stimulating phagocytic activity, acting as inflammatory mediators and in cytokine production [121][122][123]. Terpenes and terpenoids are a large and diverse class of hydrocarbon compounds and typically consist of five-carbon isoprene units [109,124]. Many terpenoids are biologically active and have been widely used for the treatment of many diseases. Simultaneously, they play a diverse role in the fields of cosmetic and food production and have been associated with hormones, medicines, vitamins, etc. [124]. Triterpenoids such as lanostane are the highly oxidized substances that can be isolated from wood-decaying mushrooms, Ganoderma sp. These compounds display immunomodulating and anti-infective effects [125][126][127]. Many species of mushrooms have been found to produce immunomodulators, such as Agaricus bisporus, Agaricus blazei, Amanita pantherina, Boletus satanas, Coprinus cinereus, Cordyceps sinensis, Ga. lucidum, Grifola frondosa, Flammulina velutipes, Ischnoderma resinosum, Lactarius deterrimus, Laetiporus sulphureus, Lentinus tigrinus, Trametes versicolor, and Volvariella volvacea [115,[128][129][130], as is detailed in Figure 5 and Table 3.   Inducing synthesis of IFN-γ [144] β-glucan Grifola frondosa Activating macrophages, NK cells, lymphokines and cytokines [145,146] Polysaccharides Galactomannan Morchella esculenta, Morchella conica Activating macrophages and cytokines [147,148] Grifolan Grifola frondosa Activating macrophages and lymphokines [149] Lentinan Lentinus edodes T-cell-oriented adjuvant [149] PS-G Ganoderma lucidum Activating macrophages and T lymphocytes [135,136] Schizophyllan Schizophyllum commune Activating T cell, increasing interleukin and TNF-α production [150] Terpenoids Exobiopolymers Ganoderma applanatum Activating NK cell [128] Ganolucidoid A and B Ganoderma lucidum NO production, anti-inflammatory activities [130] Lanostane Hypholoma fasciculare NO production, anti-inflammatory activities [151] The fungal immunomodulatory protein FIP-fve that was obtained from Flammulina velutipes has been employed to suppress the respiratory syncytial virus (RSV), which is known to cause bronchiolitis. FIP-fve effectively decreased RSV replication, IL-6 expression, and inflammation via inhibition of NF-κB translocation and respiratory pathogenesis in RSV-challenged mice. Interestingly, FIP-fve maymight be seen as a safe substance for viral prevention and disease therapy [133]. Immunomodulators have become useful agents in relieving the pathology that is associated with viral infections going forward [152]. The immunomodulatory mechanisms of mushroom products involve stimulating innate and adaptive immune responses through the activation of macrophages, T lymphocytes, DCs, NK cells, and cytokines. A study of the relationship between the structure and activity of immunomodulators will encourage the development of new therapeutic agents for the treatment of viral infection diseases.

Conclusions
The discovery and production of antiviral metabolites from fungi have emerged as part of an exciting field in viral therapeutic and antiviral drug development. Although, CoVs vaccines have been continually developed to alter the occurrence of virally associated diseases, viral protease inhibitors and immunomodulators have become extremely useful agents in this process. The results of the current studies indicate that fungi are an important source of the natural bioactive compounds that have potential as protease inhibitors and immunomodulations. Fungal protease inhibitors reveal strong potential as future candidates in the development of antiviral drugs or alternative and complementary medicals prevention and treatment of CoVs. However, it is of particular interest and concern that fungal protease inhibitors and fungal extracts could have both poisonous and curative effects against CoVs. Presently, there has been a lack of clinical tests that can validate these determinations. Consequently, these circumstances may result in consumers delaying or stopping their pursuit of appropriate medical treatment, which may lead to serious and life-threatening harm to those individuals. Therefore, laboratory assays and clinical tests are needed to fully understand the level of toxicity and pharmacokinetic profile of these viral protease inhibitors and immunomodulators. The important research must be done before the application of these fungal compounds can be used for the prevention and treatment of CoVs in the future, particularly with regard to SARS-CoV-2.
Author Contributions: The project approach was conceptually designed by N.S., J.K., C.S., S.L.; writing and original draft preparation, N.S., J.K., K.S., T.P., C.S.; chemical structure drawing, K.S.; the research was supervised by N.S., S.L.; All authors have read and agreed to the published version of the manuscript.
Funding: This research work was partially supported by Chiang Mai University.