Candidates to drug repurposing
The drugs herein reported (either approved or investigational) are excellent examples of possible candidates for repositioning as SARS-CoV-2 Mpro inhibitors. As an evidence of the reliability of our results, several of the proposed candidates are viral protease inhibitors, which in some cases are already under study as COVID-19 therapeutic agents.
Saquinavir (DB01232) is an effective anti-retroviral drug used for AIDS treatment, targeting HIV-1 protease18. As discussed below, two major saquinavir metabolites (M2 and M10) were predicted to bind to the SARS-CoV-2 Mpro with a higher affinity with respect to the parent compound.
The hydroxymethyl ketones EXPT02467 (cruz-2, DB02128) and EXPT02989 (cruz-1, DB01871) are two reversible inhibitors of Cruzipain, a cysteine-type endopeptidase of Tripanosoma cruzi19. The structures of their complexes with the Cruzipain target are available with PDB IDs: 1ME3 and 1ME4, respectively. Another interesting Cruzipain inhibitor emerging from our screening is WRR-204 (EXPT03235, DB04502), which is an irreversible inhibitor (PDB ID: 1EWO).
MMI-175 (DB02378) is an experimental drug that inhibits β-secretase (BACE-1)20, one of the two aspartic proteases responsible for the generation of amyloid-β peptides in the neurons. As such, drugs blocking this enzyme may help slowing down Alzheimer’s disease progression21. According to the predicted pose (Figure 2, panel a) and the binding affinities, this compound is expected to efficiently bind to the SARS-CoV-2 enzyme. The ability of this compound to cross the blood brain barrier would be of high interest for COVID-19 treatment22. In fact, previous studies have reported the presence of coronavirus particles in the CNS and their potential association with neurologic manifestations in patients22,23.
JE-2147 (EXPT01956, DB02668) is an HIV-1 protease inhibitor, whose X-ray crystal structure was deposited (PDB ID: 1KZK). It is remarkably potent against several common resistant strands24.
EH58 (EXPT01332, DB03063) is a potent inhibitor of Plasmepsin 2, an aspartic protease in the food vacuole of Plasmodium falciparum (PDB ID: 1LF3), provided with antimalarial activity (Ki=100 nM)25. Another Plasmepsin 2 inhibitor worth of consideration, even if predicted scores are lower than those of EH58, is RS370 (DrugBank ID: DB04378, PDB ID: 1LF2), with a reported Ki of 30 nM26.
EXPT00713 (DB03648) is a P. falciparum formylmethionine deformylase inhibitor with a reported activity of 130 nM27. As already mentioned, other antimalarial drugs, such as chloroquine and hydroxychloroquine, are under the spotlight of COVID-19 researches11.
QF34 (EXPT02729, DB04353) is a pseudopeptide inhibitor of several variants of HIV-1 and HIV-2 proteases28, and even of some highly resistant mutants. The compound was also crystallized with the HIV-1 protease (PDB IDs: 1IZH, 1IZI).
Compounds I2 (DB04692), N1 (DB04710) and N3 (PRD_002214, DB04595), three experimental inhibitors of the SARS-CoV Mpro reported in 2005, could also be valuable candidate inhibitors of the SARS-CoV-2 Mpro. Compounds I2, N1, and N3 were co-crystallized in complex with SARS-CoV Mpro (PDB IDs: 2D2D, 1WOF, and 2AMQ, respectively). Notably, compound N3 has been very recently confirmed to bind and inhibit the SARS-CoV-2 Mpro, the crystal structure of its complex being the starting point of our virtual screening (PDB ID: 6LU7)29.
Rupintrivir (AG-7088, DB05102) is a potent irreversible rhinovirus 3C and 3C-Like protease inhibitor in development for use against human rhinoviral (HRV) infections that has been recently co-crystallized with its target (PDB ID: 6KU8)30. Rupintrivir has subsequently shown to be a broad-spectrum antiviral agent, acting against picornavirus, norovirus and coronavirus proteases31. Although the drug failed to meet the requirements of the Phase II clinical trials for the treatment of common cold, it was reconsidered in 2003 as a good candidate for the inhibition of SARS-CoV Mpro, as it shares homology with the HRV-C 3C protease32.Therefore, rupintrivir is a very interesting candidate for testing against COVID-19, as recently suggested also by Liu et al33.
Larazotide (DB05645) is a peptidic inhibitor of paracellular permeability, investigated for the treatment of autoimmune diseases, such as diabetes mellitus type I, and gastrointestinal diseases and disorders34. Larazotide acetate is known to prevent immunologic changes induced by gluten consumption in patients with celiac disease. We predict that larazotide might bind to the SARS-CoV-2 Mpro protease very efficiently.
Compound WRR-183 (DB08732) is a α,β-epoxyketone that irreversibly inhibits the SARS-CoV Mpro (PDB code 2OP9)35. This compound is predicted to bind the SARS-CoV-2 Mpro tightly and with a similar pose, the epoxide being in close proximity to the Cys residue. Moreover, WRR-183 and especially its C-2 (R) epoxide isomer WRR-182 are highly active against SARS-CoV spike-mediated entry36. Therefore, they show the potential to block SARS-CoV at two different steps of the replication cycle, i.e. viral entry and particle assembly36.
Birinapant (DB11782) is an inhibitor of apoptosis proteins as XIAP, currently under investigation against solid tumors37. Birinapant was crystallized in complex with XIAP (PDB ID: 4KMP). XIAP stops apoptotic cell death induced either by viral infection or by overproduction of caspases38. In a recent study, birinapant treatment resulted in a rapid clearance of detectable Hepatitis B Virus (HBV) genetic material from serum39. Combination treatment with birinapant and entecavir (a reverse transcriptase inhibitor) is non-toxic and leads to a quicker clearance with respect to treatment with either drugs alone39. Therefore, birinapant modulates a host cell protein involved in viral persistence and may be useful to eliminate rather than merely control the SARS-CoV-2 infection.
Difelikefalin (formerly known as CR-845, DB11938) is a highly selective agonist of the κ-opioid receptor40. It is an analgesic opioid peptide that acts peripherally, under investigation for the treatment of acute and post-operative pain and, more recently, chronic pruritus40. Difelikefalin is currently in two Phase II clinical trials for the treatment of pruritus in atopic dermatitis and biliary cholangitis (Clinical Trials Identifiers: NCT04018027 and NCT03995212). The peripheral analgesic activity of the compound, together with the potential SARS-CoV-2 Mpro activity, may prove beneficial to COVID-19 patients experiencing peripheral neurologic symptoms and pain.
Ipamorelin (DB12370) is a selective agonist of the growth hormone (GH) secretagogue receptor increasing GH levels in plasma41. It was investigated for the treatment of gastrointestinal hypomotility disorders (Clinical Trials Identifiers: NCT01280344, NCT00672074).
Candidates to drug repurposing with beneficial polypharmacology
Polypharmacological ligands are extremely interesting in drug repurposing, because they offer the potential for higher efficacy and a combination of synergistic effects13. Therefore, for each top-ranking compound we investigated whether a possible beneficial polypharmacological effect may arise thanks to the reported biological activities and original therapeutic indications.
Caspofungin (DB00520) is an antifungal echinocandine possessing a cyclic peptide core. The reported mechanism of action involves inhibition of β-glucane synthase, which affects the fungal cell wall42. The in silico results returned a promising pose in complex with the SARS-CoV-2 protease, suggesting a possible antiviral activity. It is worth to note that caspofungin is the first-choice treatment for Pneumocystis pneumonia, one of the most serious secondary opportunistic mycotic infections that many severely ill COVID-19 patients tend to develop43.
Enalkiren (DB03395) belongs to the class of direct renin inhibitors. By mimicking the transition state of angiotensin, enalkiren is able to block the first step of the renin-angiotensin system44. It has been recently reported that SARS-CoV-2 binds to the widespread angiotensin-converting enzyme 2 (ACE2) to enter target cells45, and that levels of serum angiotensin II are considerably increased in COVID-19 patients46. Therefore, the modulation of the renin-angiotensin system by enalkiren, coupled with inhibition of the SARS-CoV-2 Mpro, might exhibit beneficial effects to treat COVID-19. According to our analyses, enalkiren is well accommodated within the SARS-CoV-2 Mpro binding site (Figure 2, panel b). Interestingly, another very recent computational study based on a different workflow also identified enalkiren as a potential candidate for the SARS-CoV-2 Mpro33, further supporting its selection as a promising candidate for COVID-19 treatment.
The Calpain inhibitor IV (ZLLYCH2F, DB04653), a covalent inhibitor of the Calpain-1 cysteine protease (PDB ID: 1ZCM)47, is a top ranking candidate with peculiar characteristics. In our docked structure, the reactive methylene group of the compound is in close proximity to the Cys residue of the SARS-CoV-2 Mpro active site. This means that a covalent bond providing specificity and higher affinity over other proteases can potentially be formed. Interestingly, in 2004 calpain inhibitor IV resulted as an active agent against SARS-CoV48. Calpain regulates the activity of proteins that are part of processes influencing neuronal plasticity, cognition and neurodegeneration49. In particular, Calpain-1 is a calcium-activated cysteine protease that plays an important role in neutrophil motility and is a potential target for intervention in inflammatory diseases50. Interestingly, sarilumab and tocilizumab IL-6 antagonists and anakinra (IL-1 antagonist), which were approved for inflammatory diseases, are now under evaluation for their effectiveness against COVID-19 (Clinical Trials Identifiers: NCT04315298, NCT04317092, NCT04306705, NCT04315480, NCT04339712).
Ethylsulfonamide-D-Trp-Gln-p-aminobenzamidine (DB04758) was designed to potently inhibit factor VIIa (FVIIa), whose complex with tissue factor (TF) starts the extrinsic coagulation cascade51. Compared with other anti-thrombotic agents, the specific targeting of the extrinsic coagulation provides lesser risks of bleedings. SARS-CoV-2 infection often causes dramatic consequences to the circulatory system52,53. Preliminary reports include thrombocytopenia, elevated d-dimer levels, prolonged prothrombin time, and disseminated intravascular coagulation54. Our in silico findings suggested that DB04758 could also bind with high affinity to the SARS-CoV-2 Mpro. Therefore, this molecule might exhibit a dual activity against two crucial aspects of the virus infection.
Z-LY-CMK (DB07571) is a covalent inhibitor of the ATP-dependent Clp protease proteolytic subunit (ClpP)55. Interestingly, the ClpP enzyme has recently gained attention as a promising drug target for antibiotics development56. If confirmed, the possibility of this compound to act both as an antimicrobial agent and SARS-CoV-2 inhibitor would be particularly useful to treat secondary bacterial infections, potentially affecting COVID-19 patients.
The thrombin inhibitor BM51.1011 (DB07934) is particularly interesting, as thrombotic complications appear to be an important issue in patients affected by COVID-1954. As the pandemic is spreading, the reported coagulation disorders in COVID-19 patients and in previous SARS and MERS patients should be carefully addressed. The molecule is reported to bind to the original target (thrombin, PDB ID: 1UVS).
Cobicistat (DB09065) is a CYP3A blocker, co-administered with HIV-1 protease inhibitors (e.g. elvitegravir, darunavir, and atazanavir), which has no activity on the HIV-1 enzyme, but protects protease modulators against liver degradation57. However, we predicted a high binding affinity to the SARS-CoV-2 Main Protease (Figure 2, panel c). Such dual CYP3A/SARS-CoV-2 Mpro inhibition, if demonstrated, would provide a particularly effective antiviral compound against SARS-CoV-2.
Delparantag (formerly known as PMX-60056, DB12955) is a top scoring candidate for all the scoring functions. This molecule reverses the anticoagulation effects of heparin by binding to the pentasaccharide group of unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH)58. Like protamine sulphate, which shares the same mechanism, delparantag should be administered in the minimal quantity required to antagonize heparin-associated bleeding. Heparin has raised increasing attention for its ability to prevent blood coagulation in COVID-19 patients affected by severe pneumonia and concomitant anti-inflammatory effects due to reduction of IL-652. However, a recent study showed that among a cohort of COVID-19 patients at high risk of venous thromboembolism, 11% of them also had a high risk of bleeding59. Patients with COVID-19 can rapidly develop severe or critical vascular diseases, which can affect both venous thromboembolism and bleeding status60. Therefore, controlling venous thromboembolism and bleeding risks regularly is essential. Deparantag may be an effective tool to mitigate bleeding risks while eliciting antiviral activity due to inhibition of the SARS-CoV-2 Mpro.
Remdesivir (DB14761) is a nucleotide prodrug acting on the Ebola virus RNA dependent RNA polymerase and Replicase polyprotein 1ab61. The drug was subsequently found to be able to inhibit MERS and SARS viruses10. It is currently under clinical trials to treat COVID-19. The prodrug itself is well suited to dock and bind to the protease (Figure 2, panel d). Therefore, it may block viral maturation outside of the nucleus. Another nucleotide prodrug of this kind is tenofovir alafenamide (DB09299), a reverse transcriptase inhibitor (HIV-1) and DNA polymerase inhibitor (HSV2)62.
Candidates to drug repurposing based on active drug metabolites
A unique feature of our repurposing strategy regards the inclusion of metabolites among screened compounds. Although they are usually discarded in drug repurposing studies, major metabolites can provide extremely interesting results. For this reason, here we describe some of the top scoring metabolites in our screening. Remarkably, many of these metabolites were shown to bind to the active site of the SARS-Cov-2 Mpro with higher affinity with respect to their parent drug.
Ritonavir and lopinavir, two HIV-1 protease inhibitors that are administered in combination63, are now clinically investigated against COVID-1964. Based on our in silico analyses, neither of them seems to interact favourably with the SARS-CoV-2 Mpro, although the N-desmethyl metabolite M7 of ritonavir (DBMET00084) was demonstrated to better interact with the protein thanks to its free urea group that establishes favourable hydrogen bonds with the SARS-CoV-2 Mpro.
Saquinavir decahydroisoquinoline metabolites M2 (DBMET01550 and DBMET01549), and t-butyl hydroxyl M10 (DBMET01548) were predicted to strongly bind to the SARS-CoV-2 Mpro active site according to the docking scores. Interestingly, while saquinavir metabolites are inactive against HIV-1 protease18, they seem to be more potent than saquinavir itself against the SARS-CoV-2 Mpro. This finding may be relevant for the pharmacokinetics and dosing of this antiviral for the treatment of COVID-19.
The glucuronide metabolite DBMET02115 (major active metabolite) of ezetimibe (DB00973) was predicted to establish favourable interactions with the protease active site. Interestingly, the metabolite is predicted to bind the protease with an affinity higher than that of the drug itself. Considering that this cholesterol-lowering drug is rapidly and extensively metabolized to its active glucuronate form in the liver and intestine65, the metabolite would be readily available to inhibit the viral target.