3. Viral Protease Inhibitors

3.1. Lopinavir/Ritonavir (Kaletra)
Lopinavir (Figure 6) is an orally bioavailable, small peptidomimetic antiretroviral agent that acts as an HIV-1 aspartate competitive inhibitor [83]. The drug inhibits the cleavage of viral Gag-Pol polyprotein precursors into individual functional proteins required for infectious HIV. The inhibition eventually results in the formation of immature, noninfectious viral particles. The drug was approved by the U.S. FDA in combination with ritonavir (Figure 6), which is another antiretroviral aspartate protease inhibitor. Ritonavir does not only provide an additive effective, but it is also a pharmacokinetic booster, i.e., it inhibits the CYP3A-mediated metabolism of lopinavir and thus increases its plasma level [83,84]. Currently, lopinavir/ritonavir is being evaluated for the treatment of SARS-CoV-2 patients in more than 35 interventional trials alone or in conjugation with hydroxychloroquine, inhaled INF-α, INF-β 1b and hydroxychloroquine, or oseltamivir (an inhibitor of neuraminidase in influenza virus) (for details refer to clinicaltrials.gov).
The rationale for using lopinavir is attributed to multiple studies. Lopinavir exhibited an antiviral activity against SARS-CoV-2 virus in Vero E6 cells with an estimated EC50 value of 26.63 μM [85]. Computational studies have also suggested that lopinavir may inhibit the viral main protease Mpro, perhaps by targeting its active site [86,87]. Earlier, lopinavir exhibited in vitro activity against SARS-CoV-1 and MERS-CoV [88,89,90]. It also showed beneficial effects in animal studies for the treatment of MERS-CoV [91,92]. Furthermore, there is an evidence of some clinical benefit for lopinavir/ritonavir when used with ribavirin and/or INFs against MERS-CoV and SARS-CoV [88,93,94]. Yet, coronavirus proteases, including Mpro, do not have a C2-symmetric protein architecture which is the target of lopinavir and all HIV-1 protease inhibitors. This subsequently sheds doubts on the prospect of HIV-1 aspartate protease inhibitors in treating COVID-19.
In this direction, a randomized, open-label trial in China in COVID-19 patients (n = 199), who were hospitalized with severe illness, compared lopinavir/ritonavir (400 mg/100 mg twice a day for 14 days) along with the standard care to the standard care alone [94]. The trial found that the time to achieve clinical improvement was similar in the two groups and that no statistically significant improvement, with respect to the viral load, oxygen therapy duration, hospitalization duration, or time to death, was achieved by the use of the drug combination [95]. Furthermore, a retrospective cohort study in China evaluated the use of lopinavir/ritonavir with or without umifenovir in COVID-19 patients (n = 16). On the seventh day, SARS-CoV-2 was not detected in the nasopharyngeal specimens of 35% of lopinavir/ritonavir-treated patients compared to 75% of lopinavir/ritonavir/umifenovir-treated patients. Chest computerized tomography scans were also better in the latter group (29% versus 69%) [96]. Moreover, a randomized, open-label trial in COVID-19 adults (n = 127) with mild to moderate symptoms in Hong Kong suggested that adding ribavirin and INF-β-1b to lopinavir/ritonavir increased the efficacy of the treatment when it was initiated within 7 days of the symptoms onset [59]. Other studies also suggested a limited benefit of lopinavir/ritonavir with or without INFs in patients with COVID-19 [97,98,99]. Recently, a small, randomized study in hospitalized COVID-19 adults (n = 22) in China compared lopinavir/ritonavir (lopinavir 400 mg/ritonavir 100 mg twice daily for 10 days) against chloroquine (500 mg twice daily for 10 days) and found that chloroquine was linked to a shorter time to RT-PCR conversion and a faster recovery [100].

3.2. Darunavir/Cobicistat (Prezcobix)
Darunavir (Figure 6) is another antiretroviral drug that competitively inhibits the HIV-1 aspartate protease [101,102]. In addition to the active site, it has been reported that the flexible darunavir binds to another site on the surface of the enzyme, which accounts for its resilience against potential mutations in the targeted protease [103]. Darunavir was approved in 2015 by the U.S. FDA and usually prescribed in combination with the pharmacokinetic booster cobicistat. Although structurally similar to ritonavir, cobicistat lacks antiviral activity due to the lack of the central phenyl-propanol moiety, a key structural feature of HIV protease inhibitors.
Darunavir/cobicistat is under clinical investigation for the treatment of SARS-CoV-2 infection. This is potentially attributed to darunavir ability to in vitro inhibit SARS-CoV-2 in Vero E6 cells, albeit at high concentrations (EC50 = 46.41 µM) [104]. Mechanistically, this could be because darunavir potentially inhibits 3CLpro and/or PLpro of SARS-CoV-2. The two enzymes are important for the viral glycoprotein processing. However, in another in vitro study, darunavir/cobicistat demonstrated no activity against SARS-CoV-2 at clinically relevant concentrations in Caco-2 cells [105]. Furthermore, the results from a randomized controlled trial in China showed that darunavir/cobicistat was not effective in treating COVID-19 patients [106]. Regardless, darunavir is being tried in about three trials in combination with cobicistat (NCT04252274; n = 30), ritonavir/hydroxychloroquine (NCT04435587; n = 80), ritonavir/oseltamivir, ritonavir/oseltamivir/hydroxychloroquine, or ritonavir/favipiravir/hydroxychloroquine (NCT04303299; n = 320).
TMC310911 (also known as ASC-09) (Figure 6) is structurally similar to darunavir. It is HIV-1 aspartate protease competitive inhibitor with improved antiviral activity. TMC310911 has potent activity against the wild-type HIV-1 and against an extended spectrum of recombinant HIV-1 clinical isolates, including multiple protease inhibitors-resistant strains [107]. Similar to darunavir, it was evaluated with the pharmacokinetic booster ritonavir [108]. Currently, it is being tested in two clinical trials in China in patients infected with SARS-CoV-2. It is being tested in combination with ritonavir (NCT04261907; n = 160) or oseltamivir (NCT04261270; n = 60). In a recent computational exercise, TMC-310911 was reported as a potential inhibitor of Mpro of SARS-CoV-2 [66], yet this potential is to be experimentally confirmed.

3.3. Atazanavir (Reyataz)
Atazanavir (Figure 6) is another antiretroviral drug that competitively inhibits the HIV-1 aspartate protease. Atazanavir was approved in 2003 by the U.S. FDA and usually prescribed in combination with the pharmacokinetic booster cobicistat (combination is being marketed under the name Evotaz; 2015). Currently, it is being evaluated alone (NCT04468087; n = 189) or in combination with NA-831 (neuroprotective agent; traneurocin) or dexamethasone for the treatment of COVID-19 infection (NCT04452565; n = 525) or with nitazoxanide/ritonavir (NCT04459286; n = 98). The drug alone or in combination with ritonavir demonstrated in vitro activity against SARS-CoV-2 in Vero E6 cells, human epithelial pulmonary cells (A549), and human monocytes [109,110]. In these studies, atazanavir has been identified as inhibitor of Mpro. The drug and its combination have been projected to be 10-fold more potent than lopinavir and its combination with ritonavir. The drug also inhibited the virus-induced enhancement of IL-6 and TNF-α levels [109]. In a separate computational study, atazanavir was reported as a potential inhibitor of SARS-CoV-2 helicase, a viral enzyme that unwinds nucleic acids [111].

3.4. Danoprevir/Ritonavir
Danoprevir (Ganovo) is an orally bioavailable 15-membered macrocyclic peptidomimetic antiviral drug (Figure S4). It is an inhibitor of NS3/4A HCV protease, an important processing enzyme complex. It inhibits the protease with an IC50 value of 0.29 nM [112]. It was approved in China in 2018 to treat chronic HCV patients. At higher concentrations, it also appears to inhibit the aspartate protease of HIV. The NS3/4A protease of HCV is claimed to share a certain level of structural and/or functional similarity to the protease(s) of SARS-CoV-2. Thus, HCV protease inhibitors, including danoprevir, have been proposed as potential therapeutics for COVID-19. This has also been supported by computational work which indicated that HCV protease inhibitors have high binding affinity to 3CLpro of SARS-CoV-2 [113] and by in vitro and clinical studies which showed that patients with SARS-CoV or MERS-CoV may benefit from HCV protease inhibitors [88,90,114]. Currently, danoprevir in combination with ritonavir, is being evaluated in two clinical trials (NCT04345276; n = 10) and (with nebulized INF; NCT04291729; n = 11) for the treatment of COVID-19 patients. As mentioned earlier, ritonavir is an antiviral and pharmacokinetic booster that extends the systemic exposure of patients to potential therapeutic concentration of danoprevir. In fact, a recent clinical study results under review has indicated that danoprevir/ritonavir combination alleviated the symptoms in COVID-patients and accelerated their recovery in 4–12 days [115].

3.5. Maraviroc (Selzentry)
It is a small, synthetic, azabicyclic molecule (Figure S4) that exhibits antiretroviral activity by blocking the interaction between HIV-1 glycoprotein 120 and chemokine receptor 5 (C-C motif receptor 5), on human CD4-presenting cells, that is necessary for HIV-1 to enter cells [116]. The drug was approved by the U.S. FDA in 2007 as an oral treatment for HIV-1. The drug is currently being evaluated in three clinical trials (NCT04441385, NCT04435522, and NCT04475991) for COVID-19 treatment. Recently, it was shown that maraviroc may act as a potential inhibitor of Mpro [117]. However, it appears that it is more realistic to assume that the drug is a viral entry inhibitor and potentially acts by blocking the interaction between the viral spike S protein and the host ACE2 receptor [118].