Lung Surfactant Protein B Peptide Mimics Interact with the Human ACE2 Receptor

Lung surfactant is a complex mixture of phospholipids and surfactant proteins that is produced in alveolar type 2 cells. It prevents lung collapse by reducing surface tension and is involved in innate immunity. Exogenous animal-derived and, more recently, synthetic lung surfactant has shown clinical efficacy in surfactant-deficient premature infants and in critically ill patients with acute respiratory distress syndrome (ARDS), such as those with severe COVID-19 disease. COVID-19 pneumonia is initiated by the binding of the viral receptor-binding domain (RBD) of SARS-CoV-2 to the cellular receptor angiotensin-converting enzyme 2 (ACE2). Inflammation and tissue damage then lead to loss and dysfunction of surface activity that can be relieved by treatment with an exogenous lung surfactant. Surfactant protein B (SP-B) is pivotal for surfactant activity and has anti-inflammatory effects. Here, we study the binding of two synthetic SP-B peptide mimics, Super Mini-B (SMB) and B-YL, to a recombinant human ACE2 receptor protein construct using molecular docking and surface plasmon resonance (SPR) to evaluate their potential as antiviral drugs. The SPR measurements confirmed that both the SMB and B-YL peptides bind to the rhACE2 receptor with affinities like that of the viral RBD–ACE2 complex. These findings suggest that synthetic lung surfactant peptide mimics can act as competitive inhibitors of the binding of viral RBD to the ACE2 receptor.


Introduction
The COVID-19 pandemic has resulted in hundreds of millions of infected patients, with disease severity ranging from mild flu-like symptoms to severe acute respiratory distress syndrome (ARDS) and death. In humans, infection with the SARS-CoV-2 virus can lead to an acute viral infection with a mean incubation time of around 5 days. Clinical symptoms include fever, cough, fatigue, muscle pain, and dyspnea. A subsequent rapid worsening of respiratory problems may require invasive mechanical ventilation with oxygen supplementation and an administration of antivirals, monoclonal antibody therapy, and anti-coagulants [1,2].
In humans, membrane-bound protein angiotensin-converting Enzyme 2 (ACE2) is expressed in the epithelium of the lungs, small intestines, heart, liver, and kidneys. In the lungs, ACE2 is mainly present on the membranes of alveolar epithelial Type 2 cells. Binding of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike 1 (S1) protein to ACE2 plays an essential role in viral entry and results in damage to the alveoli. Following infection, SARS-CoV-2 replicates in the cells of the respiratory and intestinal epithelium, leading to tissue damage and associated clinical symptoms. The destruction of alveolar Type 2 cells by SARS-CoV-2 results in a loss of active lung surfactant and native immunity because these cells produce and secrete lung surfactant [1,3].
Mammalian lung surfactant consists of about 80% phospholipids, 10% neutral lipids, and 10% proteins. Two of the four surfactant proteins, the hydrophilic surfactant proteins A and D (SP-A and SP-D), are important for native immunity of the lung, whereas the two hydrophobic surfactant proteins, B and C (SP-B and SP-C), are surface active, reduce surface tension at the air-liquid interface in the alveoli, and prevent alveolar collapse. The absence of SP-B in the lung is lethal in humans and mammals. Preterm infants with neonatal respiratory distress syndrome (RDS) due to lung immaturity and surfactant deficiency benefit from treatment with the intratracheal instillation of an exogenous lung surfactant. The clinical surfactant, produced by extracting animal lungs and lavage material, consists of surfactant lipids and SP-B and SP-C and has led to a sharp reduction in morbidity and mortality of preterm infants. In a small clinical study, surfactant treatment reduced mortality and duration of mechanical ventilation in ARDS patients with COVID-19-associated ARDS [4]. The anti-inflammatory activity of SP-B and phospholipids of exogenous surfactant may also provide lung-protective support in these cases [5,6]. Recently, the availability of highly functional SP-B and SP-C peptide mimics has led to the development of advanced synthetic lung surfactants [7]. Two of these surfactant formulations consist of 3% of the SP-B peptide mimics, Super Mini-B (SMB) [8] or B-YL [9] (Figure 1), mixed in three surfactant phospholipids (DPPC, POPC, and POPG 5:3:2 by weight).
ACE2 plays an essential role in viral entry and results in damage to the alveoli. Following infection, SARS-CoV-2 replicates in the cells of the respiratory and intestinal epithelium, leading to tissue damage and associated clinical symptoms. The destruction of alveolar Type 2 cells by SARS-CoV-2 results in a loss of active lung surfactant and native immunity because these cells produce and secrete lung surfactant [1,3].
Mammalian lung surfactant consists of about 80% phospholipids, 10% neutral lipids, and 10% proteins. Two of the four surfactant proteins, the hydrophilic surfactant proteins A and D (SP-A and SP-D), are important for native immunity of the lung, whereas the two hydrophobic surfactant proteins, B and C (SP-B and SP-C), are surface active, reduce surface tension at the air-liquid interface in the alveoli, and prevent alveolar collapse. The absence of SP-B in the lung is lethal in humans and mammals. Preterm infants with neonatal respiratory distress syndrome (RDS) due to lung immaturity and surfactant deficiency benefit from treatment with the intratracheal instillation of an exogenous lung surfactant. The clinical surfactant, produced by extracting animal lungs and lavage material, consists of surfactant lipids and SP-B and SP-C and has led to a sharp reduction in morbidity and mortality of preterm infants. In a small clinical study, surfactant treatment reduced mortality and duration of mechanical ventilation in ARDS patients with COVID-19-associated ARDS [4]. The anti-inflammatory activity of SP-B and phospholipids of exogenous surfactant may also provide lung-protective support in these cases [5,6]. Recently, the availability of highly functional SP-B and SP-C peptide mimics has led to the development of advanced synthetic lung surfactants [7]. Two of these surfactant formulations consist of 3% of the SP-B peptide mimics, Super Mini-B (SMB) [8] or B-YL [9] (Figure 1), mixed in three surfactant phospholipids (DPPC, POPC, and POPG 5:3:2 by weight). Thus, a key element in the infection of host cells by SARS-CoV is the binding of the viral S1 protein to the ACE2 receptor on the host cellular membrane. This initial interaction of the viral RBD with the host membrane receptor is a major determinant of the membrane fusion of the virus with the host lung cells [10,11]. Recently, there have been several peptide-based approaches that target these initial steps involved in the interaction RBD of the viral S1 protein with the ACE2 receptor and aim to prevent viral entry [12][13][14][15][16]. In this study, we characterize the interaction of the synthetic lung SP-B peptide mimics, SMB and B-YL, with the ACE2 receptor. Thus, a key element in the infection of host cells by SARS-CoV is the binding of the viral S1 protein to the ACE2 receptor on the host cellular membrane. This initial interaction of the viral RBD with the host membrane receptor is a major determinant of the membrane fusion of the virus with the host lung cells [10,11]. Recently, there have been several peptidebased approaches that target these initial steps involved in the interaction RBD of the viral S1 protein with the ACE2 receptor and aim to prevent viral entry [12][13][14][15][16]. In this study, we characterize the interaction of the synthetic lung SP-B peptide mimics, SMB and B-YL, with the ACE2 receptor.

Determination of the Potential SMB and B-YL Peptides to Form Complexes with Human ACE2 Receptor Using Molecular Docking
The molecular coordinates of the crystal structure of the ACE2-SARS-CoV-2 spike protein RBD complex (PDB accession code: 6m0j) were used as the initial template for the prediction of the degree of interaction of the ACE2-RBD interfacial domain ( Figure 2). The binding interface contact residue pairs are listed in Table 1. This complex coordinate set was used to determine binding affinity by contacts-based prediction methodology [17]. Both the affinity or binding-free energy (∆G) and dissociation constant (K D ) were predicted from this structural information. The structurally derived binding metrics compare favorably with experimentally determined binding measurements of ACE2-SARS-CoV-2 interactions using SPR (Table 2). Residue pair interactions include both residue side chain ion pairs and hydrophobic interactions of aromatic residue side chains that stabilize the receptor-viral protein complex. The correlations of these in-silico predictions with experimental binding observations suggest that the use of this structure-based approach to the prediction of the potential binding of peptides and proteins to the viral-receptor interface is a reasonable approach for the estimation of possible experimental residue-specific molecular interactions. The potential SMB and B-YL peptide interactions with the ACE2 receptor prot were estimated from the docking of the peptide into the crystal structure of each of molecular complex coordinates (PDB accession code: 6m0j) using integrative modeling the potential SMB-ACE2 and B-YL-ACE2 biomolecular complexes [18]. As shown Figure 3, the SMB and B-YL peptides can be docked into the ACE2 viral binding site the protein constructs. Analysis of the complex structures with contact-based predict methodology indicates that binding affinities are like that of the native biomolecu complex (Table 2), and that the interactive residues of the ACE2 construct are in the sa N-terminal helical domain as observed for the ACE2-RBD crystal structure.  The potential SMB and B-YL peptide interactions with the ACE2 receptor protein were estimated from the docking of the peptide into the crystal structure of each of the molecular complex coordinates (PDB accession code: 6m0j) using integrative modeling of the potential SMB-ACE2 and B-YL-ACE2 biomolecular complexes [18]. As shown in Figure 3, the SMB and B-YL peptides can be docked into the ACE2 viral binding site of the protein constructs. Analysis of the complex structures with contact-based prediction methodology indicates that binding affinities are like that of the native biomolecular complex (Table 2), and that the interactive residues of the ACE2 construct are in the same N-terminal helical domain as observed for the ACE2-RBD crystal structure.  A structural analysis of the interface between the ACE2 receptor and the SMB and B-YL peptides indicates that the ACE2 N-terminal helix interacts with the N-terminal helix of the SMB and B-YL peptides. The interactive residues of the helical domains are listed in Table 1 and illustrated in Figures 3 and 4. These amino acid side chain interactions include charged residues that form a salt bridge between glutamic acid residues of an ACE2 helix with an arginine residue of the N-terminal helix of the SMB and B-YL peptides. There are also hydrophobic residue side chain interactions that stabilize the A structural analysis of the interface between the ACE2 receptor and the SMB and B-YL peptides indicates that the ACE2 N-terminal helix interacts with the N-terminal helix of the SMB and B-YL peptides. The interactive residues of the helical domains are listed in Table 1 and illustrated in Figures 3 and 4. These amino acid side chain interactions include charged residues that form a salt bridge between glutamic acid residues of an ACE2 helix with an arginine residue of the N-terminal helix of the SMB and B-YL peptides. There are also hydrophobic residue side chain interactions that stabilize the protein-peptide complex that include the aromatic side chains of tyrosine residue pairs, as well as the leucine and alanine of the ACE2 N-terminal helix interacting with the tyrosine side chains of the SMB and B-YL peptide helical domains.

Surface Plasmon Resonance (SPR) Measurements of SMB and B-YL Peptide Binding to ACE-2 Domain
To better characterize the potential degree and type of interaction of the lung ACE2 receptor protein and lung surfactant peptides SMB and B-YL, we used SPR to test the binding of these peptides to the recombinant human ACE2 receptor construct (Supplementary Materials File S3). The SPR binding study was accomplished by immobilizing the ligand (ACE2) on the SPR sensor surface and flowing the solute containing the SMB or B-YL peptide past the sensor-linked molecule. The binding of the solute to the sensor surface ligand results in an evanescent sensor response and is measured in response units (RU) that are proportional to the bound mass. This technique has been shown to be very useful in the study of ligand binding in protein-protein interactions [19], such as those in the current study.
Representative sensor grams for the interaction of the lung surfactant SMB and B-YL peptides tested in the present study with the ACE-2 receptor protein are shown in Figure  5. The kinetic analysis of these traces to determine relative binding (affinity) of the peptides to the expressed protein construct is summarized in Tables 2 and 3. The ACE2

Surface Plasmon Resonance (SPR) Measurements of SMB and B-YL Peptide Binding to ACE-2 Domain
To better characterize the potential degree and type of interaction of the lung ACE2 receptor protein and lung surfactant peptides SMB and B-YL, we used SPR to test the binding of these peptides to the recombinant human ACE2 receptor construct (Supplementary Materials File S3). The SPR binding study was accomplished by immobilizing the ligand (ACE2) on the SPR sensor surface and flowing the solute containing the SMB or B-YL peptide past the sensor-linked molecule. The binding of the solute to the sensor surface ligand results in an evanescent sensor response and is measured in response units (RU) that are proportional to the bound mass. This technique has been shown to be very useful in the study of ligand binding in protein-protein interactions [19], such as those in the current study.
Representative sensor grams for the interaction of the lung surfactant SMB and B-YL peptides tested in the present study with the ACE-2 receptor protein are shown in Figure 5. The kinetic analysis of these traces to determine relative binding (affinity) of the peptides to the expressed protein construct is summarized in Tables 2 and 3. The ACE2 construct ( Figure 5) had the highest binding affinity for the SMB peptide and lowest dissociation constant (K D ). B-YL peptide had a lower affinity with corresponding higher K D values (Table 3).  (Table 3).     SPR metrics derived from the time course of binding of the ACE2 to the SMB and B-YL peptides. Association and dissociation kinetic rate constants (k on , k off ) and equilibrium dissociation constants (K D ) calculated from SPR kinetic measurements for the hACE2 were attached to the Biacore sensor chip, while SMB and B-YL peptides were flowed in the anylate. Peptide was dissolved in HBS-EP buffer and flowed past the recombinant protein constructs on the CSM sensor chip with a Biacore system (Methods). Kinetic rate constants and equilibrium dissociation constants were determined from curve-fitting analysis of SPR traces. Ka = Association constant; kd = Dissociation constant; RI = Bulk refractive index contribution; Rmax = Maximum binding capacity; KA = Equilibrium association constant; KD = Equilibrium dissociation constant/affinity; Req = Response at equilibrium; Kobs = "Observed constant" (kobs = ka × concentration+ kd).

Discussion
Although the development of modern vaccines has been an important step in helping control the spread of the COVID-19 virus [20], an alternative therapeutic approach to viral infection intervention can be found in the development of peptides that bind the host ACE-2 receptor COVID-19-binding domain interface in the lungs, thereby blocking viral entry into the host [12,21]. The peptide-based ACE-2 interaction blockers fall into two categories. One class consists of short peptides that are six amino acid residues in length. Typically, these short peptide amino acid sequences have hydrophobic residues that are dominated by tyrosine interspersed with polar cationic residues of lysine and arginine [13,14]. These short amino acid sequences have little secondary conformation, even when bonded in a complex with the receptor protein. The second class of peptide viral fusion blockers have amino acid sequences that span from approximately 20-60 residues in length [12,16,22]. These blocking inhibitors have considerable alpha helical propensity and assume a tertiary fold of a helix-hairpin-like structure. The amino acids in the longer peptide sequences have a sequential distribution that resembles an amphipathic helix. Polar-charged residues include arginine and lysine, and hydrophobic residues include aromatic amino acids such as tyrosine, phenylalanine, and tryptophan. These amino acid sequences also contain residues such as asparagine and glutamine, which provide additional hydrogen bonding between the peptide and the target receptor sequence and help stabilize the potential receptor-viral protein complex.
In the present study we examined the potential interaction of two hydrophobic lung surfactant peptide mimics of surfactant protein B, named SMB and B-YL, with the ACE-2-SARS-CoV-2 Receptor-Binding Domain. The rationale for the selection of these specific peptide mimics was in part based on the amino acid sequence of the peptides (Figure 1), which are composed of multiple tyrosine residues and cationic polar amino acid residues arginine and lysine. Additionally, both the SMB and B-YL sequences have a distribution of polar and non-polar residues that help enhance the amphipathic alpha helical secondary structure by providing the peptide a helix hairpin character and a tertiary Saposin fold [23,24]. These primary and secondary features of the SMB and B-YL peptides suggest that they resemble many of the known peptide inhibitors of the ACE-2-COVID-19 spike protein complex. Docking of the SMB and B-YL peptides into the ACE-2 RBD binding interface allows for the prediction of a high propensity for interactions between the peptide and the receptor-viral protein interface. Experimental in-vitro SPR measurements of the binding of the SMB and B-YL peptides to the recombinant human ACE-2 receptor protein confirm the binding of the peptides to this target domain. These results suggest that lung surfactant protein B peptide mimics have potential for use in the therapeutic intervention of COVID-19 infection in the lung. We speculate that the observed experimental difference binding affinity of the SMB peptide compared to the B-YL construct for the ACE-1 receptor may be related to the greater rigidity of the disulfide-linked structure of the helix hairpin mimic conformation [23,24].
This study was performed in an aqueous solution, but, since SP-B is a lipid-associated protein, it is expected that these peptides require binding to lipid membranes to adopt a functional structure. This raises the question whether the interaction of SMB and B-YL with ACE2 can be affected by the presence of surfactant membranes. A recent paper by Rozak et al. [25] suggests that lipids do not interfere with viral binding to ACE2, and the same could be true for surfactant lipids and peptide binding.
While the present findings, demonstrating the interaction of synthetic surfactant B peptides with the human ACE2 receptor, are very encouraging regarding the possible blocking of the binding site to the COVID-19 spike protein, more detailed studies of the peptides in various synthetic lipid formulations will be required to optimize this system for therapeutic applications.

Protein-Peptide Docking and Prediction of Binding Affinities
The potential interactions of the lung SP-B peptide mimics and the components of the S1 Protein-ACE2 receptor protein complex were initially determined by downloading the molecular coordinates of the SMB and B-YL peptides from the ModelArchive website (https://www.modelachive.org accessed on 25 May 2023; ID: ma-scodz for SMB and ma-vilb7 for B-YL) and the S/RBD-ACE2 crystal structure from the Protein Data Bank (https://www.rcsb.org accessed on 25 May 2023; ID: 6M0J). Protein-peptide docking and analysis were carried out using the HADDOCK 2.4 server (V 2.4-2021.05; https://www. bonvinlab.org/education/HADDOCK24/ accessed on 25 May 2023) [18,26]. The docked structures were then analyzed with Prodigy (https://wenmr.science.uu.nl/prodigy/ accessed on 25 May 2023) [17,27] to estimate the binding-free energy and the affinity constants for the residue-specific interactions of the peptide-protein complex.

Protein and Peptide Constructs
HBS-EP buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.4) was obtained from Biacore (Uppsala, Sweden). Organic solvents used for sample synthesis and purification were HPLC grade or better (Fisher Scientific, Waltham, MA, USA). The synthetic SMB and B-YL lung surfactant peptides, of which amino acid sequences are shown in Figure 1, were synthesized using a standard Fmoc protocol and HPLC purified as described previously [8,23].
SMB and B-YL peptides were assembled using H-Ser(OtBu)-HMPB NovaPEG serine resin (Novabiochem, MilliporeSigma, Burlington, MA, USA) possessing a substitution of 0.62 mmole/g. Peptide synthesis was carried out at a 0.25 mmol scale on a Liberty Microwave Peptide (CEM Corporation, Matthews, NC, USA) synthesizer employing the standard Fmoc protocol [24]. Both SMB and B-YL peptides were double-coupled from the C-terminus to Residue 29, followed by triple-coupling for all remaining amino acid residues to the N-terminal phenylalanine to insure optimal yield. Peptides were cleaved from the resin for one hour using the phenol:thioanisole:ethanedithiol:water:trifluoracetic acid (0.75:0.25:0.5:0.5: 10, v:v) cleavage-deprotection mixture. The crude peptide was then purified to greater than 95% by a Jasco preparative HPLC (Easton, MD, USA) using a VYDAC diphenyl or C8 (1" by 12" width by length) column at 20 mL/min. The peptides were eluted from the column with a 0 to 100% ACN (water to acetonitrile) with 0.1% TFA as an ion-pairing agent added to both aqueous and organic phases with a linear gradient in 1 h. The purified product solution was then lyophilized, and the mass confirmed the resulting peptide powder by Maldi TOF mass spectrometry.
In the case of SMB, the Saposin protein-like peptide folded [24] and directed disulfide formation between Cys-8 and Cys-40, as well as between Cys-11 and Cys-34, and was facilitated by dissolution in TFE with the oxidant dimethylsulfoxide (DMSO) and 50 mM of PBS pH 7.5 (TFE:DMSO:PBS, 3:1.5:5.5, v:v) to mediate oxidation of the thiols to form disulfides [28]. The peptide-aqueous-organic solvent solution was stirred for 48 h at 25 • C to ensure complete disulfide formation before the concentration of the peptide by Speed-Vac ® . Purification of the oxidized SMB by HPLC was performed as described above for the crude product and the oxidized peptides disulfide-linked molecular mass confirmed by Maldi-TOF mass spectrometry. Peptide concentrations were determined by the UV spectrometry method of Anthis and Clore [29].
Expressed viral spike and human ACE2 receptor protein constructs were supplied by R&D Systems (https://www.bio-techne.com accessed on 25 May 2023). The recombinant human ACE2 receptor construct included residues Glutamine 18 to Serine 740 of the full-length protein with a C-terminal 10 Histidine tag.