Repurposing of Approved Drugs with Potential to Block SARS-CoV-2 Surface Glycoprotein Interaction with Host Receptor

Background: Respiratory transmission is the primary route of SARS-CoV-2 infection. Angiotensin I converting enzyme 2 (ACE2) is the known receptor of SARS-CoV-2 spike glycoprotein for entry into human cells. A recent study reported absent to low ACE2 promoter activity in a variety of human lung epithelial cell samples. Three bioprojects (PRJEB4337, PRJNA270632 and PRJNA280600) invariably found abundant expression of ACE in human lungs compared to very low expression of ACE2. Methods: In silico tools were applied to assess potential interaction of SARS-CoV-2 surface spike protein with human ACE as well as predict the drugs that may block SARS-CoV-2 interaction with host receptor. Results: Although it is not obvious from the primary sequence alignment of ACE2 and its homolog ACE (also known as ACE1), comparison of X-ray crystallographic structures show striking similarity in the regions of these proteins which is known (for ACE2) to interact with the receptor binding domain (RBD) of SARS-CoV-2 spike protein. Critical amino acids that mediate interaction with the viral spike protein in ACE2 are organized in the same order in ACE. In silico analyses predicts comparable interaction of SARS-CoV-2 spike protein with ACE2 and ACE. In addition, this study predicts and selects already approved drugs from a list of 1263, which may interfere with the binding of SARS-CoV-2 spike glycoprotein to ACE2 and/or ACE.

SARS-CoV (caused an outbreak in 2003) in making its way to the host cell [3,4]. Angiotensin I converting enzyme 2 (ACE2) is the known cellular receptor for both SARS-CoV and SARS-CoV-2 in human [3,5]. The receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of these viruses interact with the extracellular peptidase domain (PD) of ACE2 using electrostatic as well as van der Waals forces [3,6]. Despite the overall similarity in structure, SARS-COV-2 spike protein has evolved with a number of sequence variations and conformational deviations from that of SARS-CoV in the RBD at the interface with ACE2 [3,5].
Structural analyses have revealed key atomic-level interactions between the SARS-CoV-2 spike protein RBD and ACE2 [3,5]. Binding ability of SARS-CoV-2 has evolved [5]. SARS-CoV-2 is assumed to bind human ACE2 more efficiently using its modified spike protein than the SARS-CoV [5]. Binding affinity of the spike protein to ACE2 is one of the most important determinants of SARS-CoV-2 infectivity [5]. SARS-CoV-2 might have gained its high capability to infect and transmit in humans through enhanced binding.
The primary physiological role of ACE2 is in the maturation of angiotensin, which controls vasoconstriction and blood pressure [7]. ACE2 is a homolog of angiotensin converting enzyme (ACE) with subtle differences in the active site [8,9]. Whereas ACE2 act as a carboxypeptidase by removing a single amino acid from the C-terminus of susceptible substrates, ACE acts as a carboxy-dipeptidase (or, peptidyl-dipeptidase) and removes a C-terminal dipeptide [10]. A recent study reported absent to low ACE2 promoter activity in a variety of human lung epithelial cell samples [11]. Three bioprojects (PRJEB4337, PRJNA270632 and PRJNA280600) invariably found very low expression of ACE2 in human lungs, whereas ACE was found to be much highly expressed (Supplementary figure 1-3) [12]. Although it is not obvious from the primary sequence alignment, ACE has striking similarity in the PD region with ACE2 that interact with the SARS-CoV-2 spike protein.
Till April 13, 2020 COVID-19 has spread in 213 countries and regions on earth with over 1,775,000 confirmed cases of infection and more than 112,000 deaths. Despite an urgent need to find options to help tens of thousands of patients and preclude potential death, there is no proven therapy to treat COVID-19 [13]. Repurposing of already approved drugs, if available, may be an immediate and promising option to tackle COVIDd-19. It is unlikely for the virus to mutate and evolve to bind an entirely different receptor within days or even months as such functional relationships are established by evolution over a long period of time [13]. Therefore, one strategy might be the use of an agent that binds to the receptor region recognized by the RBD of SARS-CoV-2 spike protein.
Since SARS-CoV-2 has evolved with increased affinity of the surface spike protein for its known receptor ACE2, this study explored the possibility of interaction of this spike protein with its ACE-a homolog of ACE2, which is more abundant in human lungs. This study also investigated the potential of 1263 already approved drugs to bind and interfere at the interface of ACE and ACE2 with the SARS-CoV-2 S protein.

Interaction between ACE and SARS-CoV-2 surface spike glycoprotein
Based on the sequence similarities to SARS-CoV spike proteins, it has been suggested that SARS-CoV-2 also exploits ACE2 to mediate infection in human cells [5]. Alignment of X-ray crystallographic structures of ACE and ACE2 reveals striking similarities in the tertiary structures of the PD regions that interact with the RBD of SARS-CoV-2 spike protein ( Figure   1A). Critical amino acids in this region of ACE2 [3,5] that interact with the spike protein occupy similar positions in ACE ( Figure 1B and C). Lys31 and Lys 353 in ACE2 are particularly considered as critical in the PD of ACE2 for interaction with the viral spike protein [5]. Although it is not obvious in the primary sequence alignment, these important amino acid residues in the PD of ACE and ACE2 are present in the same order ( Figure 1B).
Receptor-ligand interaction analysis using molecular docking technique could predict the amino acids at the interface of ACE and ACE2 PD regions with RBD of the spike protein ( Figure 2).
Although amino acid residues at the interface of ACE2 and spike proteins are already known from X-ray crystallographic analysis, this in silico prediction was performed as control to compare with the predicted analysis between ACE and S protein. The amino acid residues in ACE2 at the interface with the SARS-CoV-2 spike protein matched to the previous reports [3,5].
Similar interactions were observed in the predicted interactions between ACE and the spike protein. Predicted interactions of ACE and ACE2 with the spike protein involve similar forces and z-scores (Supplementary table 2). As in SARS-CoV/SARS-CoV-2 and ACE2 [5], the predicted interface between SARS-CoV-2 and ACE maintains a highly polar environment ( Figure 2). In fact, the predicted interaction model suggests (Supplementary table 2) the ACEspike protein complex to be electrostatically more stable than the ACE2-spike protein complex.
As SARS-CoV-2 spike protein has evolved to bind ACE2 with higher affinity than does the SARS-CoV [4] and gained more power to transmit and infect humans, mere speculation based on sequence comparison with SARS-CoV might not be enough to define its receptor.

In silico assessment of drugs with potentials to block SARS-CoV-2 spike protein interaction with ACE and ACE2
A total of 1263 approved drugs (Supplementary table 1) were assessed for potential interactions with ACE and ACE2 at regions that overlap with the predicted and known binding regions of RBD of the SARS-COV-2 spike protein, respectively. Angiotensin II is a substrate of ACE2 [10]. Molecular docking with AutoDock Vina predicted an interaction of angiotensin II with the PD of ACE2 with a binding energy of -6.0 kcal/mol. Drugs that bind to overlapping regions in the PD of ACE and ACE2 and, therefore, may perturb interaction with the SARS-CoV-2 spike protein and has more stable binding than the native substrate (i.e., predicted to release energy > 6.0 kcal/mol) are listed in table 1. Several of these predicted interactions are shown in figure 3 and 4. Table 1 also provides brief description of the drugs along with their current approval status. Some drugs have multiple statuses as these have been approved for certain condition(s), but are currently on clinical trials for one or more different indications.
There are differences between the compositions and structures of ACE and ACE2. Based on in silico analysis, among the 1263 analyzed drugs in this study, only 10 may bind to regions in both ACE and ACE2 that overlap with the binding sites of the key interacting spike protein amino acid residues. Pibrentasvir is one such drug which is used to treat infection mediated by Hepatitis C Virus (HCV)-a positive-strand RNA virus [28]. In silico analysis could predict a few more antiviral drugs (Indinavir, Baloxavir marboxil, Maraviroc, Doravirine, and Nelfinavir), which may interfere with the binding of SARS-CoV-2 spike protein with ACE2 only. Several of the drugs may play dual roles by blocking the binding of virus to the receptor as well as fight other associated infections. For example, Azithromycin, Cefoperazone, Natamycin, Nystatin, Rifapentine, etc may be used to manage infection as well as interfere with SARS-CoV-2 binding.
These may serve as a two edged sword by blocking the binding to the receptor as well as inhibiting secondary infections [29]. Two angiotensin II analogs (Azilsartan kamedoxomil and Saralasin) were predicted to bind with higher affinity to ACE2 than angiotensin II. These two drugs bind to regions that overlap with the binding site of SARS-CoV spike protein. Mefloquinean anti-malarial drug may compete with spike protein for ACE2 rather than Hydroxychloroquine, which binds to other region of ACE2 (Table 1 and supplementary table 1).

Conclusion
Although there has been discussion on whether it would be safe to use angiotensin receptor blockers (ARB) in the treatment of COVID-19, the Council on Hypertension of the European Society of Cardiology, the American Heart Association, the Heart Failure Society of America, and the American College of Cardiology recommended that the physicians and patients should continue treatment with their usual anti-hypertensive therapy because there is no clinical or scientific evidence to suggest that treatment with angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) should be discontinued because of the COVID-19 infection [30,31]. In an acid lung injury model of mice SARS-CoV worsened lung injury by down-regulation of ACE2, which was improved by treatment with angiotensin receptor blocker (ARB) [32].
No specific therapeutics for COVIDD-19 is yet available. A better understanding of the underlying pathobiology will be useful in finding a cure [33]. Till then, already available potential options might be explored to bring comfort to the world.

Conflict of interests:
There is no known conflict of interest.     Approved Long-acting synthetic antidiarrheals, which has no effect on the adrenergic system or central nervous system, but may antagonize histamine and interfere with acetylcholine release locally. Shows a high order of in vitro activity against many species of fungi and without effect on bacteria, rickettsiae, and viruses.

NYSTATIN -6.8 _ Approved
A polyene antifungal drug that has broad-spectrum fungicidal and fungistatic activity against a number of yeasts and fungi, most notably Candida species