Molecular proling of Neprilysin expression and its interactions with SARS-CoV-2 spike proteins to develop evidence base pharmacological approaches for therapeutic intervention

Neprilysin due to its peptidase activity is involved in several physiological and pathological processes. Recently our group has reported the association of neprilysin with angiotensin-converting enzyme 2 (ACE2) network proteins which facilitate the entry of SARS-COV2 virus. The potential role of neprilysin beyond its peptidase activity is not known. Using the established sequence analysis and molecular docking tools, this study evaluated the molecular prole of neprilysin interaction with SARS-COV2 virus proteins. Human neprilysin protein showed a signicant sequence similarity with SARS-COV2 spike protein, which was further conrmed by observation of considerable interaction in the molecular docking. Human neprilysin protein was also found to additionally interact with SARS-COV2 proteins facilitating virus replication. The potential of neprilysin inhibitors (Sacubitril and Sacubitrilat) to interfere with neprilysin and SARS-COV2 proteins interactions was assessed. The neprilysin inhibitors showed binding ecacy within therapeutically feasible concentration range (1 to 150 uM). This study while reporting a novel role of neprilysin as potential receptor for SARS-COV2 virus, highlights the merit in assessing clinical ecacy of neprilysin inhibitors for the management of SARS-COV2 infection.


Introduction
Neprilysin is a widely expressed peptidase located on the cell surface and has very diverse substrate speci city (Esser and Zraika, 2019;Karoor et al., 2013;Nalivaeva et al., 2020). Due to its diverse substrate speci city it is involved in regulating variety of physiological (electrolyte balance, blood pressure regulation, analgesia) and pathological (T cell lymphoma, Alzheimer's, diabetes, diarrhoea) process. The biochemical function of neprilysin is to hydrolyse peptides by cleaving them at their N-terminal side of hydrophobic amino acid residues (Acanfora et al., 2020;Esser and Zraika, 2019;Karoor et al., 2013;Mangia co et al., 2013;Nalivaeva et al., 2020;Rice et al., 2004). The peptidase activity of the neprilysin is seen as its major physiological role which is achieved by modulating signalling of a variety of peptides in various organ systems (Mangia co et al., 2013). Neprilysin is also reported to cleave several mitogenic peptides and hence can curtail development of tumours (Mangia co et al., 2013;Nalivaeva et al., 2020).
The major expression of neprilysin in mammals is reported in kidneys, lungs, GI tract and neuronal cells (Mangia co et al., 2013;Nalivaeva et al., 2020;Pavo et al., 2019b;Shipp et al., 1991). Neprilysin is also located on the neutrophils and in lung, wherein respiratory irritants are reported to downregulate its expression and trigger in ammatory response (Borson et al., 1989;Dempsey et al., 2009). In contrast plasma neprilysin activity was reported to be increased in preclinical models associated with systemic in ammation, such as obesity and insulin resistance (Borson et al., 1989;Dempsey et al., 2009;Esser and Zraika, 2019;Karoor et al., 2013). Neprilysin is also expressed on common lymphoid progenitors, which give rise to cells (T, B and NK cells) having signi cant role in tissue in ammation and immune response (Pavo et al., 2019b;Rice et al., 2004;Shipp et al., 1991;Song et al., 2016). However it is not clear if changes in the activity of neprilysin is a cause or consequence of in ammation.
Neprilysin inhibitors are approved for clinical use in human patients with heart failure (Acanfora et al., 2020; El Tabaa and El Tabaa, 2020; Srivastava and Fonarow, 2019). In the past neprilysin inhibitors have shown e cacy as therapeutics for several diseases associated with systemic in ammation (Acanfora et al., 2020;Esser and Zraika, 2019;Liczek et al., 2018). Although the role of neprilysin with airway in ammatory cascade is previously reported (Acanfora et al., 2020;El Tabaa and El Tabaa, 2020;Esser and Zraika, 2019;Liczek et al., 2018), its role in in ammatory cascade triggered by viruses in human patients is not clear. Our group recently reported the interaction of SARS-COV2 virus with several angiotensin-converting enzyme 2 (ACE2) network proteins (DPP4 and Meprin A alpha) with superior e cacy and neprilysin was observed as one of the ACE2 network proteins in this study (Goothy and Kumar, 2020). A low lymphocyte count among patients with SARS-COV2 infection is reported to be signi cantly associated with fatal outcome. Considering the expression of neprilysin on lymphocytes and its role in regulation of peptides interacting with ACE2, this study tested the hypothesis that SARS-COV2 virus can directly interact with neprilysin. Hence molecular interactions of neprilysin and its inhibitors with SARS-COV-2 proteins were assessed to establish their potential therapeutic merit.

Material And Methods
Protein network and sequence analysis: The neprilysin protein network was analysed using the STRING database (https://string-db.org/cgi/info.pl) (Sharma et al., 2015). The STRING database was searched using the neprilysin as protein name and Homo sapiens as organism. The Basic Local Alignment Search Tool (BLAST) was used to identify the regions of similarity between sequences of the neprilysin network proteins identi ed in the string database. Subsequent to initial alignment with all network proteins, similarities were further assessed between neprilysin, DPP4 and ACE2. To know the potential interaction of neprilysin with SARS-CoV-2 spike proteins the similarity between their sequences was compared (Goothy and Kumar, 2020;Hruz et al., 2008;Sharma et al., 2015).
Simulation of dose response curves: Dose-response curves were modelled based on nonlinear regression analysis as reported before (Kumar, 2020;Sagar and Kumar, 2020).
Interaction analysis using Ligplot: The protein-protein or protein-ligand interactions was evaluated using the LigPlot software. The PDB output les from molecular docking were inputted into the LigPlot and the intermolecular interactions and their features (hydrogen bonds, hydrophobic contacts, and atom accessibilities) were assessed. Hydrogen bonds are represented by dashed lines. The amino acid residues of the protein involved in the molecular interactions are represented by an arc with spokes emerging towards the ligand atoms in contact (Bharatham et al., 2008;Mishra and Dey, 2019).

Results
The network analysis of human neprilysin protein in the string database showed 10 proteins in its primary network ( Figure 1A). The Basic Local Alignment Search Tool (BLAST) analysis of the sequence of the neprilysin network proteins, showed a very weak identify (0.082%) (Table 1, Figure 1B). We and others have reported the role of DPP4 and ACE2 proteins as cell surface receptors for entry of coronaviruses. (Goothy and Kumar, 2020;Stower, 2020) As both DPP4 and ACE2 were observed in the neprilysin protein work, BLAST was use to assess the similarities between these three proteins. Compared to the general neprilysin network proteins, both DPP4 and ACE2 showed signi cantly better identity (2.205%, 112 similar positions) in their sequence (Table 1, Figure 1B). Which suggested neprilysin may be a potential receptor for coronaviruses. Hence the SARS-COV2 Post fusion core of S2 subunit (PDB ID: 6LXT), which is reported to be an important component of spike protein interacting with ACE2 receptor was compared to neprilysin. The sequence similarities (235 similar positions) between neprilysin and 6LXT was signi cantly better (9.058 Vs 2.205%) than that between neprilysin and DPP4/ACE2 (Table 1, Figure 1B). The expression pro le of receptors is essential to effectively correlate the associated pathology and as well for designing rationale therapeutic measures. Hence the relevant database were analysed to pro le the gene, transcript and protein expression pattern of neprilysin in various human tissues ( Figure 2). Differences between the relative expression of neprilysin RNA, gene and protein were observed across various human tissues/organs, which although not surprising was nevertheless interesting (Figure 2).
Highest expression of neprilysin RNA was observed in adipocytes, whole blood, olfactory bulb and lymphoma ( Figure 2A). In contrast highest expression of neprilysin gene was observed in glomerulus, jejunum and nephrons ( Figure 2B). The RNA and gene expression pattern of neprilysin in human tissue differed from its protein expression pattern, although gene and protein expression pattern was similar ( Figure 2B and C). Highest expression of neprilysin protein was observed in small intestines, followed by that in duodenum, colon, kidneys, and granulocytes ( Figure 2C). Hence considering this expression pattern of neprilysin, it is likely that gastrointestinal, renal and immune physiology are predominantly in uenced by factors interfering with neprilysin function. The symptoms of nausea, diarrhoea, generalised in ammation observed with SARS-COV2 infection does correlate with the expression pattern of neprilysin in human tissues. Hence to assess if SARS-COV2 virus proteins can interact with neprilysin, molecular docking of the selected combination of protein (Table 2) was performed. SARS-COV2 virus proteins (6LXT, 6Y2E, 6W6Y, 6ZB5) were observed to signi cantly interact with neprilysin through formation of hydrogen bonds in su cient numbers for the interaction to be biochemically feasible ( Figure  3A, table 2). The interaction of neprilysin with 6LXT was observed at Glu646, His587, His583, Asp950 regions with 44 hydrogen bonds, suggesting the possibility of neprilysin serving as a receptor for SARS-COV2 spike protein ( Figure 3B, table 2). Of considerable interest was the superior interaction (more number of hydrogen bonds) of neprilysin with SARS-COV2 proteins (6Y2E, 6W6Y) involved in its replication (Table 2). Sacubitril and Sacubitrilat are neprilysin inhibitors, which are approved for clinical use in humans. Hence the binding a nity of these two drugs against SARS-COV2 proteins was assessed. The binding a nity ( Figure 4A) and the IC 50 (Table 3) of Sacubitril and Sacubitrilat against the SARS-COV2 proteins (6LXT, 6Y2E and 6W6Y) were observed to be within therapeutically feasible concentration ( Figure 4A, table 3).
Based on the IC 50 values, simulated dose response curves for Sacubitril and Sacubitrilat were generated for optimal estimation of therapeutic concentration range (1 to 150 uM) ( Figure 4B). Sacubitril showed superior e cacy than sacubitrilat in interacting with SARS-COV2 targets ( Figure 4B). Sacubitril was observed to form 26 hydrogen bonds with 6LXT (selectively at Asp950, Gln949 and Gln1180) suggesting signi cant binding a nity ( Figure 4C, Table 2) and its potential to block interaction of neprilysin with the SARS-COV2 spike protein.

Discussion
Several cell surface receptors are known to facilitate the entry of viruses, which can be target for therapeutic intervention (Goothy and Kumar, 2020;Stower, 2020). This study reports an unexpected observation from network protein analysis, which resulted in identi cation of neprilysin as a potential receptor for the key proteins of recently reported SARS-COV2 virus. The molecular interaction of neprilysin was observed with SARS-COV2 virus post fusion core of S2 subunit (6LXT), spike protein open con rmation (6ZB5), ADP ribose phosphatase of NSP3 (6W6Y) and its main protease (6Y2E) suggesting neprilysin may facilitate both viral attachment, entry and its replication. This novel role of neprilysin as a receptor for viruses is not reported before.
Viruses which spread at a pandemic scale are unlikely to depend on a single receptor type for it attachment and entry into the host cell. Dependency on multiple receptor types has been shown for many strains of coronaviruses reported previously (Goothy and Kumar, 2020;Stower, 2020). Most of these strains of coronaviruses have caused infections in large scale, both in humans and animals despite the endemic nature of the infections (Renu et al., 2020). SARS-COV2 is the recently reported strain of coronaviruses which has spread to a pandemic scale, (Goothy and Kumar, 2020;Renu et al., 2020;Stower, 2020) and paraphs it does utilize multiple receptor types at least in non-experimental settings for attachment and entry into the host cells. We have recently reported the role of ACE2 network proteins in facilitating SARS-COV2 virus attachment and entry into the host cells, with some of the network proteins (DPP4, Meprin A and XPNPEP2) showing superior molecular interactions with SARS-COV2 virus spike proteins compared to ACE2 (Goothy and Kumar, 2020). Coincidental observation of neprilysin association with the ACE2 network protein and its sequence similarities with the SARS-COV2 spike protein observed in this study suggest the possibility of neprilysin being a receptor for coronaviruses similar to DPP4 and ACE2 (Goothy and Kumar, 2020;Renu et al., 2020). Additionally superior molecular interaction of neprilysin with SARS-COV2 proteins (6W6Y, 6Y2E) regulating its replication was also observed. Although neprilysin is predominantly expressed on cell membrane, a few studies have reported its subcellular localisation, (Gregoriou et al., 2020;Nalivaeva et al., 2020) suggesting the potential role of neprilysin beyond its peptidase activity against natriuretic peptides. Facilitating attachment, entry and replication of virus in the host cell may be one alternative role of neprilysin, which merits further investigation.
Loss of smell and taste is reported to an early symptom of SARS-COV2 infection. While the exact mechanisms responsible for loss of this physiology is not known (Kilroy and Kumar, 2020), it is interesting to note that higher expression of neprilysin is observed in olfactory bulb. In contrast expression of neprilysin was least in the tongue. The differential loss of smell and taste sensation in SARS-COV2 infection are perhaps mediated by different pathways, with a potential role of neprilysin in regulating the sense of smell. The predominant expression of neprilysin in GI tract, kidneys and granulocytes, does correlate with the symptoms of nausea, diarrhoea, thrombosis and systemic in ammation observed in patients with SARS-COV2 infection (Kilroy and Kumar, 2020).
Respiratory irritants and pathogens (parain uenza virus and rat coronavirus) are reported to interact with neprilysin on neutrophils and in lung epithelium to trigger in ammatory responses (Borson et al., 1989;Dempsey et al., 2009;Karoor et al., 2013). These prior studies are in concurrence with this study reporting the potential of neprilysin as a receptor for SARS-COV2 virus. Besides these a soluble circulating form of neprilysin is also reported in several body uids (Pavo et al., 2019a), which together with high expression of neprilysin in whole blood may substantiate the systemic nature of in ammation observed in SARS COV2 infection. Neprilysin expression was also highest in the adipose tissue, which paraphs support the incidence of higher mortality rate in obese patients with SARS COV2 infection (Nalivaeva et al., 2020;Shipp et al., 1991;Song et al., 2016). With brinogen as its substrate, neprilysin can regulate brin formation by thrombin (Burrell et al., 2016). Hence factors inhibiting neprilysin can enhance brinogen levels and lead to intravascular coagulation. The molecular interactions observed in this study does indicate the potential of SARS COV2 spike proteins to interact and inhibit neprilysin activity, weather this is the potential mechanism of disseminated intravascular coagulation observed in SARS COV2 infections remains to be validated. Several studies have associated neprilysin activity with negative remodelling of pulmonary and vascular structures, including increased microvascular permeability (Dempsey et al., 2009;Rice et al., 2004;Shipp et al., 1991;Steiner, 2009;Wick et al., 2011). The correlation of these features with symptoms observed in SARS COV2 infections together with the molecular interactions between SARS COV2 proteins and neprilysin reported in this study does support the notion for neprilysin being a potential receptor for interaction with SARS COV2 virus. Further the higher catalytic activity of neprilysin then ACE2 (El Tabaa and El Tabaa, 2020; Srivastava and Fonarow, 2019) may be more favourable for the enveloped virus to enter host cells.
Neprilysin inhibitors (Sacubitril and Sacubitrilat) are currently approved for clinical use and have shown e cacy in the treatment of acute diarrhoea and heart failure (El Tabaa and El Tabaa, 2020; Srivastava and Fonarow, 2019). The binding e cacy of both sacubitril and sacubitrilat against SARS-COV2 proteins (6LXT, 6W6Y and 6Y2E) were within therapeutically feasible range, indicating their potential in not only preventing virus attachment and entry into host cell but also the potential to prevent virus replication. This ability of neprilysin inhibitors to target full cycle of virus entry and replication can lead to synergistic outcomes and improved e cacy. Further the synergistic e cacy of neprilysin inhibitors could be a consequence of targeting both neprilysin as well as SARS-COV2 proteins independently. This dual targeting of both host cell and virus proteins in addition to curtailing the pathogenesis of the virus can also be helpful to harness the collateral bene ts from neprilysin inhibition. Recent studies have supported the bene ts from neprilysin inhibition by reducing the pro-in ammatory cytokines and neutrophil count in patients with SASR-COV2 infections (Acanfora et al., 2020;El Tabaa and El Tabaa, 2020;Srivastava and Fonarow, 2019). Sacubitril in combination with valsartan was reported to increase NO bioavailability and reduce high sensitivity C-reactive protein, which can be additionally bene cial by improving microvascular function and reducing systemic in ammation.
In conclusion the ndings from this study provides evidence for the potential novel role of neprilysin as a receptor for SASR-COV2 virus, which can be effectively targeted by currently approved neprilysin inhibitors.

Declarations
Con ict of interest: none