key: cord-0854929-gtpmx20t
authors: Fernandes, Leonardo Antônio; Gomes, Anderson Albino; Magalhães, Maria de Lourdes Borba; Ray, Partha; da Silva, Gustavo Felippe
title: Engineering Defensin α-helix to produce high-affinity SARS-CoV-2 Spike protein binding ligands
date: 2022-02-10
journal: bioRxiv
DOI: 10.1101/2022.02.09.479781
sha: 0b5f8a36e638c5bc9a298ba6de0880fba8df86cb
doc_id: 854929
cord_uid: gtpmx20t

The binding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike protein to the Angiotensin-Converting Enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high-affinity and cost-effective ACE2 peptidomimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small and highly stable proteins, and engineered the amino acid residues on its conformationally constrained alpha-helices to mimic the critical residues on the ACE2 helix 1 that interacts with the Spike-protein. The engineered proteins were soluble and purified to homogeneity with high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability, high-affinity binding to the Spike protein with dissociation constants in the low nanomolar range, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical peptidomimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing high-affinity ligands. Broad audience statement The engineered proteins developed in this study are cost-effective and highly stable reagents for SARS-CoV-2 detection. These features may allow large-scale and cost-effective production of diagnostic tests to assist COVID-19 diagnostic and prevention.

Coronavirus 2 (SARS-CoV-2) emerged as a pandemic in March 2020 (1, 2) , resulting in over 300 million infections and more than 5.5 million deaths worldwide as of January 2022 (3) . The emergence of highly transmissible variants and the possible occurrence of other pandemics calls for innovative protein engineering strategies to develop improved viral detection probes for rapid diagnostic followed by proper therapeutic interventions.

The viral entry into human cells occurs when SARS-CoV-2 Spike glycoprotein (S) binds the human Angiotensin-Converting Enzyme 2 (ACE2) followed by its proteolytic cleavage by the cellular Trans-Membrane Protease Serine 2 (TMPRSS2) (4) . Therefore, binding to the human ACE2 receptor is a critical initial step for SARS-CoV-2 infection, and targeting such Protein-Protein Interactions (PPIs) via competitive-binding might be a promising strategy for blocking viral entry into human cells (5) . Accordingly, studies have shown that soluble ACE2 inhibits SARS-CoV-2 infection by blocking the interaction between the human receptor and the viral protein (6) (7).

Biochemical and structural studies have demonstrated that interactions with the Receptor-Binding Domain (RBD) of Spike SARS-CoV-2 mainly involve ACE2 N-terminal -helix 1 (8) , and such α-helical peptide may effectively mimic ACE2 and bind to the Spike protein. However, once a short peptide is isolated from the original protein, much of its binding ability is lost, as the peptide tends to adopt random-coil conformations, reducing the number of optimal binding conformations (9) (10) (11) (12) .

Therefore, the development of stabilized α-helical mimetic ligands is crucial for Spike protein binding, allowing SARS-CoV-2 detection, COVID-19 neutralizing antibody analysis, and therapeutic intervention design. For that reason, several attempts to produce stable and potent -helical ACE2 peptidomimetics are underway (13) (14) (15) (16) .

The proteolytic instability of peptides is an additional factor that limits their utility as reagents in diagnostic and therapy since flexible peptides are also easy targets for proteases (17) . In this regard, reduced conformational heterogeneity of stabilized helical peptides has been demonstrated to increase protease resistance (12) .

Defensins are small (45-54 amino acid residues), cysteine-rich, cationic proteins found in animals, plants, and fungi that play crucial roles in the innate immune system by protecting against pathogens (18, 19) . These proteins usually enclose three to five disulfide bonds that confer protease resistance (20) , extreme temperature tolerance (21) , and wide pH-range stability (22) .

In plants, all defensins share a common topology that consists of one -helix and three antiparallel -strands stabilized by at least four disulfide bonds (23) . However, in vertebrates, -helices are present only in a subset of defensins called beta-type that contain a -helix restricted to the carboxy-terminal region of these proteins (18, 24) .

In this study, we hypothesized that the design of ACE2 -helix peptidomimetics using defensin scaffolds could be advantageous given their stability and helix-constrained conformation. Therefore, considering the published crystal structure of the SARS-CoV-2 RBD-ACE2 complex (25), we selected the main residues located on the -helix 1 of ACE2, contacting RBD, and introduced them into the -helix of plant and human defensins to investigate the ability of the engineered proteins to bind the RBD of Spike protein from SARS-CoV-2.

The use of prokaryotic expressed proteins presents several advantages, including production cost and time efficiency compared to antibodies or eukaryotic proteins. The engineered proteins developed in this study were highly expressed in Escherichia coli, binding SARS-CoV-2 Spike protein with low nanomolar Kd values and presenting exceptional thermal stability. In addition, proteins were able to detect HEK-293/SARS-5 CoV-2 Spike expressing cell lines and detected the presence of SARS-CoV-2 neutralizing antibodies via competitive ELISA tests. In summary, we report a novel strategy to generate constrained -helix SARS-CoV-2 binders stabilized by defensin scaffolds that are promising candidates for COVID-19 diagnostics and viral neutralization.

To produce a potentially low-immunogenic and stable ACE2 mimic we initially chose a human scaffold (defensin PDB1IRH). Analysis of the complex ACE2-RBD structure using the Pymol software reveals that most RBD contacts involve the surface located ACE2 helix 1 residues L29, D30, K31, N33, H34, E35, E37, D38, and L39. Therefore, we inserted residues D30, K31, H34, and E35 into the human defensin -helix to produce the engineered protein named h-deface2 ( Figure 1 , Supplementary Data S1). The chosen defensin scaffold contains a -helix stabilized by two disulfide bonds; therefore, we hypothesized they would provide a constrained and stable framework to display ACE2 residues responsible for Spike RBD binding.

Nonetheless, the human defensin contains a short -helix that accommodates the insertion of only four -helix ACE2 residues ( Figure 1B) . Therefore, we further selected a defensin from the tobacco plant (PDB 6MRY) that contained a more extended -helical structure, which accommodated five additional ACE2 residues, intending to produce higher affinity binders ( Figure 1C ). Therefore, residues L29, D30, K31, N33, H34, E35, E37, D38, and L39 were introduced into the plant defensin -helix, and the resulting protein was named p-deface2 ( Figure 1 ). In addition, based on recent studies claiming increased binding upon ACE2 mutations E35K and K31N (26) , we introduced such mutations on p-deface2 to produce a new version of the protein named p-deface2-MUT.

Engineered proteins were initially expressed as Trx-fusion peptides (Supplementary data S1) to assist disulfide bond formation, which led to soluble protein expression of pronounced yield (>50mg/L). To rule out the possibility of Trx-tag interference into the binding, Trx-free proteins were also expressed, purified, and tested accordingly. Both the Trx tagged and untagged proteins exhibited similar binding to the ECD of S-protein, demonstrating that the tag did not interfere with the binding (Supplementary data S2).

However, heterologous expression of engineered peptides in the absence of Trx-tag resulted in a substantial yield decrease (< 5 mg/L).

Biolayer Interferometry (BLI) experiments were initially performed using biotinylated ACE2-peptidomimetics and SARS-CoV-2 S-protein to investigate the molecular interactions. Biotinylated h-deface2 and p-deface2 were immobilized on streptavidinconjugated biosensors, and experiments were performed using a gradient of SARS-CoV-2 S-protein. Measured data indicates association and dissociation phases (Figure 2A and 2B), but curves did not follow a single exponential kinetic behavior. Therefore, kinetic analysis using either 1:1 or 1:2 binding models was not possible using this method, and so were the Kd calculations. We, therefore, calculated the apparent dissociation constants using dose-response curves from ELISA assays and the apparent Kd values were determined using nonlinear regression Michaelis-Menten curve fits ( Figure 2C ). Accordingly, calculated apparent Kd values were 54.4±11.3, 33.5±8.2, and 14.4±3.5 nM for h-deface2, p-deface2, and p-deface2-MUT, respectively (Table 1) .

To test the binding specificity of the engineered ACE2-peptidomimetics, the amino-acid residues D30, K31, E35, D38, and L39 were mutated to alanine to produce p-deface2 ala mutant, and binding analysis was performed. We found that the alanine mutation 7 disrupted binding to the SARS-CoV-2 S protein, thus reconfirming the importance of these residues for the binding interaction ( Figure 3 ).

We also tested the thermostability of the engineered proteins. For this, we pre-incubated the purified proteins at specific temperatures and followed the binding using standard ELISA assay. We observed that all three proteins retained binding activity after heating ( Figure 4) . Additionally, the proteins retained their total activity upon lyophilization, followed by reconstitution (Supplementary data S3).

Next, we investigated the potential application of ACE2-peptidomimetics in a diagnostic platform for detecting SARS-CoV2 neutralizing antibodies. For this, we used a Brazilian Health Regulatory Agency (ANVISA)-approved neutralizing antibody test. The assay is based on the principle of competitive ELISA. Briefly, the biotinylated-ACE2 protein is immobilized on the streptavidin-coated microplates that can bind HRP-coupled SARS-CoV-2 Spike protein and generate a signal in the presence of HRP-substrate. However, in the presence of SARS-CoV2 anti-S-protein binding antibodies (neutralizing sera), the availability of HRP-coupled SARS-CoV-2 Spike protein to bind the ACE2 protein decreases, thus resulting in a decrease of the measured signal. We modified this assay and replaced the biotinylated-ACE2 with the biotinylated p-deface2 protein ( Figure 5 ). This was followed by adding HRP-coupled SARS-CoV-2 Spike protein and the test (neutralizing) or control (non-neutralizing) human serum provided with the kit. The presence of neutralizing antibodies disrupted the interaction between p-deface2 and HRPcoupled SARS-CoV-2 Spike protein, thus decreasing the measured signal compared to the control non-neutralizing sera, indicating that biotinylated p-deface2 could be applied in a diagnostic assay platform to screen between positive and negative SARS-CoV-2 neutralizing serum ( Figure 5 ).

Finally, we wanted to determine if h-deface2 and p-deface2 could bind the S-protein expressed on the cell surface that might mimic the architecture of the trimeric-S-protein displayed on the SARS-CoV2 surface. Transgenic HEK293 cell lines stably transfected with (1) Wild-type (Wuhan-Hu-1) or (2) Interestingly, at higher concentrations, h-deface2 and p-deface2 displayed loss of binding to the cells, as evident from the "bell-shaped curve" (Supplementary data S5), suggesting that oligomerization of the proteins at high concentrations might be responsible for removing the engineered proteins from the solution phase. Therefore, based on the results from titration experiments, we selected the protein concentration of 0.1 g/L for both h-deface2 and p-deface2 to perform our subsequent assay to test the specificity of the proteins. We observed higher binding of both the h-deface2 and p-deface2 proteins to the Spike protein (Wuhan and Delta) expressing HEK293 cells than the control HEK293 cells (Supplementary data S6, S7, and Figure 6 ). However, a noticeable background binding of h-deface2 and p-deface2 to the control, HEK293 cells was also observed. Defensins are highly basic proteins, and the interaction of their positively charged amino-acid 9 residues with negatively charged phosphatidyl-lipid moieties on the cell plasma membrane is most likely responsible for this background binding.

Early studies revealed that soluble ACE2 inhibits SARS-CoV-2 infection, showing that blocking the PPIs between ACE2 and Spike RBD might be a promising strategy for COVID-19 diagnosis and treatment (6) . Since then, many trials using soluble ACE2 variants have been underway. However, although soluble ACE2 is a promising approach, it will be both cost and time prohibitive to mass-produce the protein in a eukaryotic expression system for deploying it in the production of COVID19 diagnostics or therapeutics. In this respect, peptide-based therapeutics present advantages, including cost and time efficiency in synthesis compared to eukaryotic proteins. Therefore, researchers are focusing on developing minimalist and efficient -helical mimetic structures suitable for large-scale and cost-effective production (27) . Most rationally designed peptidomimetics are based on ACE2 N-terminal helix 1 since this is the secondary structure that encloses most RBD interactions.

Unconstrained -helical peptide inhibitors based on ACE2-helix 1 were able to block the interaction between Spike protein and ACE2 (27) with binding affinities at the millimolar range. Linear -helices are flexible and less structured, frequently leading to lower affinity binders. Therefore, conformationally restricted ACE2 helix1-stapled peptides were produced, but affinity increased only to the micromolar range (28) . Other stapled peptides were created and presented no evidence of viral neutralization (11) , indicating that enhanced binding interactions are required to outcompete all the interactions of membrane-bound ACE2 and the virus Spike-protein to prevent viral infection effectively.

In this regard, SARS-CoV-2 mini protein inhibitors containing residues 23 to 46 from helix 1 were also developed presenting dissociation constant at a low nanomolar range 10 (29). Mini proteins are a diverse group of protein scaffolds characterized by small (1-10 kDa) size, stability, and versatility (30, 31) , being currently used as scaffolds for protein engineering. These SARS-CoV-2 mini protein inhibitors were computer-generated and effectively targeted the ACE2-RBD interface, however, as non-natural scaffolds, little is known about their stability or cellular toxicity (29) .

Expanding the portfolio of nontoxic, stable, and inexpensive ACE2 peptidomimetics is crucial for producing SARS-CoV-2 detection probes, detecting neutralizing antibodies, and generating new therapeutic strategies. Therefore, our study intended to create conformationally constrained helices structured on a prokaryotically expressed and highly stable tertiary structure to generate potent and stable binders of SARS-CoV-2 Spike RBD. We used defensins as scaffolds, which are small and highly stable proteins found in nature. Defensins are efficiently expressed in E. coli, nontoxic for eukaryotes, present exceptional thermostability, and often enclose a disulfide-stabilized -helix. We reasoned that defensins would provide a well-structured framework to display critical ACE-2 residues on a constrained -helical structure.

Defensins exhibit antimicrobial properties against pathogenic organisms, including bacteria, fungi and viruses (32) . Recently, a human beta-defensin expressed in the human respiratory epithelium was shown to bind to the Spike RDB and inhibit SARS-CoV-2 pseudovirus infection on ACE2 expressing HEK 293Tcells (33) . Earlier studies also demonstrated the binding of a fungal defensin into the Spike RBD from SARS-CoV-2 (34) . However, unlike our study, in both these cases, the defensin alpha-helix was not engineered to mimic the ACE2 alpha helix1, and perhaps for that reason, their binding affinities for SARS-CoV-2 Spike protein were observed at micromolar ranges.

The engineered proteins were highly expressed in a bacterial expression system, and purified to homogeneity, demonstrating exceptional thermostability and potent binding 11 against SARS-CoV-2 Spike protein. We observed that the apparent dissociation constant for h-deface2 is higher than p-deface2. The reason is likely due to the shortened length of the human defensin -helix. The use of plant defensin provided a more extended -helix that accommodated ACE2 residues 29 to 39, improving binding as observed by a lower apparent Kd value for p-deface2. Based on the three-dimensional structure reported earlier, we noticed that ACE2 displays a palindromic region where Histidine 34 is centered and the pairs L29/D30 and D38/L39 are equidistant located from H34. We reasoned that this palindromic positioning could allow spike docking in both orientations, and therefore we introduced these additional residues into p-deface2.

Furthermore, the addition of K31N and E35K mutations in p-deface2-MUT culminated with improved binding (Kd 14.4 nM). Therefore, the defensin structure was not only able to incorporate the function of the native ACE2 residues for binding, but remarkably it was also able to achieve a gain of function with the same specific mutations discovered in the evolution of ACE2 derivatives (26) , demonstrating that they are versatile scaffolds.

Kd calculations using the BLI assay were not possible since curves did not follow a single exponential behavior, indicating that multiple binding events were likely occurring.

Defensins tend to dimerize through their -sheet face (35) . Additionally, the SARS-CoV Spike glycoprotein undergoes oligomerization (36) . It is possible that binding to multimeric structured defensins and Spike protein produces a more complex interaction that did not allow proper kinetic analysis through BLI assays.

In addition, the engineered proteins distinguished HEK293/SARS-CoV-2 cell lines from the parental HEK293 control cells at the conditions tested. However, a high background binding was observed, which is likely due to the defensins mode of action that involves an electrostatic interaction with the pathogen cell membrane due to the highly cationic character of defensins. Studies have demonstrated that the binding of defensins to cell 12 membrane phosphatidic acids is part of their mechanism of action (37, 38) . Although it might sound contradictory, cell surface stickiness might be a wanted feature of such scaffolds since their local use into the respiratory tract may be favored by a diminished local clearance. Mutations of the engineered proteins are being studied to evaluate this hypothesis by our group.

In addition to the observed thermostability, the proteins retained total biological activity upon lyophilization and reconstitution, important features considering their potential use as point of care diagnostic reagents.

The initial focus of our study was directed against the SARS-CoV-2 that emerged in 2019, but the current emergence of genetic variants introduces new challenges that need to be addressed. Furthermore, recent data demonstrated that therapeutic intervention based on ACE2 hotspot residues is a promising strategy to hit emerging variants and similar SARS viruses (13) . Accordingly, the engineered defensin-ACE2 peptides demonstrated binding towards the Wuhan and Delta variant SARS-CoV-2.

-Helices play a significant role in mediating protein-protein interactions in nature, being stabilized via noncovalent interaction within proteins' tertiary structures. Although chemical biologists frequently use synthetic strategies to stabilize the α-helical conformation of peptides, other strategies involve using mini-proteins as scaffolds to display stable α-helical domains (12) . To our knowledge, defensins have not yet been explored as mini protein scaffolds, and our strategy demonstrated that they might represent a superior strategy for developing constrained alpha-helices mimetic and highaffinity binders for additional targets. Finally, the hypothetical three-dimensional structure of defensin-based peptides were created using The Phyre2 web portal for protein modeling, prediction, and analysis (39) .

All genes were purchased from Biomatik Corporation (Ontario, CA) and cloned into pET-32a (+) between the Nco I and Xho I restriction sites. Therefore, all proteins contained a Trx-Tag, followed by a His-tag and an S-tag (Supplementary data S1). Trx-tag was included in the construct to assist disulfide bond formation. Control experiments using 

The reaction was quenched with 50 μL Stop Solution, and absorbance at 450 nm was measured using an ELISA plate reader. Thermostability assay. Defensin-based ACE-2 peptidomimetics (1g) were preincubated at 37°C, 50°C, 65°C, 80°C or 95°C for five minutes, cooled on ice for additional 5 minutes and mixed to 90 L of PBST containing 1% of BSA and incubated for 1 hour against 50 ng of ECD previously immobilized on polystyrene ELISA plates.

The wells were washed three times with PBST, followed by the addition of streptavidin- Measurements were performed in triplicates and errors correspond to standard deviation. A B 28 28 Figure 6 

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This work received financial support from SENAI Green Chemistry-ABDI inside the SGF project nº 329254 and Grant PAP UDESC FAPESC 2021TR889. 

The authors intend to file a provisional patent application regarding the engineered proteins.