key: cord-0753524-0dsj3226
authors: Lokhande, Kiran Bharat; Banerjee, Tanushree; Swamy, Kakumani Venkateswara; Ghosh, Payel; Deshpande, Manisha
title: An in silico scientific basis for LL‐37 as a therapeutic for Covid‐19
date: 2021-08-09
journal: Proteins
DOI: 10.1002/prot.26198
sha: b0cbc7a1f929fdbdf90b1ae592fbca8997f12773
doc_id: 753524
cord_uid: 0dsj3226

A multi‐pronged approach with help in all forms possible is essential to completely overcome the Covid‐19 pandemic. There is a requirement to research as many new and different types of approaches as possible to cater to the entire world population, complementing the vaccines with promising results. The need is also because SARS‐CoV‐2 has several unknown or variable facets which get revealed from time to time. In this work, in silico scientific findings are presented, which are indicative of the potential for the use of the LL‐37 human anti‐microbial peptide as a therapeutic against SARS‐CoV‐2. This indication is based on the high structural similarity of LL‐37 to the N‐terminal helix, with which the virus interacts, of the receptor for SARS‐CoV‐2, Angiotensin Converting Enzyme 2. Moreover, there is positive prediction of binding of LL‐37 to the receptor‐binding domain of SARS‐CoV‐2; this is the first study to have described this interaction. In silico data on the safety of LL‐37 are also reported. As Vitamin D is known to upregulate the expression of LL‐37, the vitamin is a candidate preventive molecule. This work provides the possible basis for an inverse correlation between Vitamin D levels in the body and the severity of or susceptibility to Covid‐19, as widely reported in literature. With the scientific link put forth herein, Vitamin D could be used at an effective, medically prescribed, safe dose as a preventive. The information in this report would be valuable in bolstering the worldwide efforts to eliminate the pandemic as early as possible.

typical approaches. In this work, a different possibility is presented, in the form of the human anti-microbial peptide LL-37 as a therapeutic, the reasoning for which is collated and elucidated below.

Converting Enzyme 2 (ACE2) by the binding of the Receptor Binding Domain (RBD) of its Spike protein to ACE2. The N-terminal helix (NTH) of the peptidase domain of ACE2 is mainly responsible for the interaction with the RBD. 4 The Receptor Binding Motif (RBM) within the RBD, forms a concavity into which the NTH fits in. Most of the residues of ACE2 that form interactions with the RBD are present in the N-terminal helix. A stretch of ACE2 from Ser19 to Asn53 comprises the NTH. 5 LL-37, a 37 amino acid peptide, produced from the cleavage of an 18 kDa polypeptide, hCAP18, is an anti-microbial peptide (AMP) present in humans. 6 It is produced not only by multiple immune cells like NK cells, B cells, mast cells, and so forth, but also by epithelial cells present in the skin and the respiratory tract. 7 LL-37 is majorly present extracellularly. 8 Within cells, it is present in its proform in granules.

LL-37 can get internalized into human macrophages where it can act in clearance of intracellular pathogens. 9 LL-37 is internalized by human mast cells which it induces to release nucleic acids. 10 Besides the action of LL-37 in destabilizing membranes of pathogens, LL-37 has also been reported to be a cell-penetrating peptide (CPP). 11 The physiological concentration of LL-37 is known to be in the range of 2-5 μg/ml. However, in case of infection its concentration could rise up to 20 μg/ml. 12 LL-37 also exhibits antiviral activity against several viruses by interacting with and destabilizing the viral envelopes. 13 LL-37 has also been found to interfere with the replication of several single-stranded enveloped RNA viruses similar to SARS-CoV-2, such as Respiratory Syncytial Virus, Influenza A hepatitis C virus, Dengue virus, HIV-1 and Vaccinia Virus. 14 Furthermore, LL-37 has been found to inhibit viral binding to host cells. LL-37 inhibits Dengue virus type 2 from entering cells by binding to the E protein of the virus. 15 Binding of Respiratory Syncytial Virus to epithelial cells is diminished by LL-37. 16 LL-37 also had an inhibitory effect on the binding of nonenveloped Enterovirus 71 to U251 cells. 17 A metabolite of Vitamin D3, 1α,25-dihydroxy vitamin D3 (1,25D3), is known for its role in transcriptional regulation of many genes of the immune system. 18 Genes regulated by 1,25D3 also code for anti-microbial peptides.

Human antimicrobial peptide hCAP-18/LL-37 gene is one such gene which is upregulated by Vitamin D. 19 Moving on to the logic of this study, a large collection of evidence has mounted that there is a correlation between Covid-19 and Vitamin D. In PubMed, there are numerous articles reporting or discussing such correlation. To give an example, in an Israeli population-based analysis involving 7807 people, 10% individuals who were Covid-19 positive had lower 25(OH) Vitamin D levels in the plasma than the individuals who were tested negative and the finding was statistically significant. 20 In another work, which is most relevant to our study, the binding of LL-37 to enveloped viruses and their consequent disablement has been described and it has been postulated that Vitamin D deficiency manifests as aggravated Covid-19 through consequent low expression of cathelicidin. 14 Besides severity of disease, a recent study 21 implicates an increased risk of contracting Covid-19 when there is deficiency of Vitamin D. Also, several studies have found Vitamin D deficiency to be linked with greater risk of viral respiratory tract infections. 22 In our study, the question was asked-Could this association be something that could be tapped for developing a potential therapy? LL-37 is known to have a helical structure 23 as depicted in Figure 1 (Panel I). The actual part of ACE2 that the RBD binds to is the N-terminal helix (NTH) (Figure 1 , Panel I) of ACE2.

Thus, a hypothesis was made that LL-37, could be binding to RBD and if true, this could be one of the most effective way in which LL-37 could interfere with SARS-CoV-2 by binding to the receptorbinding domain (RBD) of the virus. Therefore, in this work, in silico studies were undertaken, mainly to test the hypothesis put forth and to unearth the possibility, if any, of binding of LL-37 to RBD. A great value of this approach to develop a treatment would be that a molecule that is already present in the body's circulation could be used as a therapeutic, as the safety criteria for the therapeutic would be more likely to be met than extraneous peptides or those not already present in the circulation.

The Spike protein RBD is amply glycosylated 24 and the glycan moieties are considered to be steric hindrances for neutralizing antibodies to bind-a mechanism of immune evasion by the virus. The RBD gets exposed when the virus is about to bind to ACE2; this is referred to as the "UP" state of the RBD, as against the unavailable "DOWN" state, these states being alternated by the control of glycans. 25 The development of neutralizing antibodies as a therapeutic depends on the "Open" or available state of the RBD as against its "closed" state. 26 So, in the UP or Open state of the RBD, LL-37 could also bind to it. in complex with its receptor, that is, human ACE2 (hACE2) were retrieved from the PDB database (https://www.rcsb.org/) with PDB ID: 2K6O, 27 6LZG, 28 and 6M0J, 4 respectively. Another three peptides that are structurally similar to LL-37 have been used as "controls."

The basis for selection of these three peptides is explained in the Structural Alignment Results section. The three-dimensional structures of these three peptides viz. Pituitary adenylate cyclaseactivating peptide-38 (PACAP-38) (PDB ID: 2D2P), 29 Amylin endocrine hormone (PDB ID: 2KB8) 30 and Glucagon-like peptide-2 (GLP-2) (PDB ID: 2L63) 31 were retrieved from the PDB database. The sequences of NTH and the peptides are given in Table 1 2.2 | Quantitative assessment of NTH structure similarity with LL-37, PACAP-38, amylin, and GLP-2 Firstly, the four peptide sequences were compared with the NTH sequence using BLASTP (National Center for Biotechnology Information, U.S. National Library of Medicine). To assess the structural similarity between N-terminal Helix (NTH) of hACE2 with LL-37, and the three control peptides, the quantitative assessment of protein structure similarity was carried out using TM-Align online server 33 (https:// zhanglab.ccmb.med.umich.edu/TM-Align/). The TM-Align program produces the optimized residual alignment between two structures depending on their structural similarity followed by structural superimposition and gives the TM-score which is a measure of the structural similarity. TM-score gives the values in between 0 and 1, where 0 indicates the given structures are structurally different while 1 indicates perfect alignment and implies that both the structures are identical to each other. If the TM-score is higher than 0.5, then it signifies that similar structural folds are present in both the structures and a score < 0.3 indicates random structural similarity. Further, a greater value for TM-score signifies stronger structural similarity. 34 CATH (Database based on Class, Architecture, Topology, and Homologous superfamily) and SCOP (Structural classification of Proteins database) are commonly used standards for comparison and classification of protein structures. 35 For CATH, a TM-score of 0.5 means that it is 37% probable that the two structures fall into to the same topology family; when the TM-score is 0.6, this probability elevates to 80%. When TM-score is <0.4, it means that are nearly zero protein pairs in the same SCOP Fold family. When the TM-score is >0.6, the probability of the two proteins being in the same SCOP Fold is raised to >65%. TM-Align also classifies the alignment of amino acid pairs according to a distance cut-off "d," distinguishing the pairs with d < 5.0 Å from those with d > 5.0 Å, the former being the better aligned pairs than the latter.

For the macromolecular docking of given structures, we used the HDOCK online server for blind docking (http://hdock.phys.hust.edu. cn/) to predict their binding complexes with binding affinity. HDOCK with SARS-CoV-2 RBD (from 6LZG). Also, as a different type of control, LL-37 was docked with the Spike protein region from residue 96 to residue 318, which is adjacent (immediately upstream) to RBD by extracting the structure of this region from the full spike protein structure of SARS-CoV-2 (PDB ID: 6Z97), 37 using Maestro software.

All docked complexes were downloaded from the HDOCK server and their intermolecular interaction patterns were analyzed using Maestro.

To analyze the binding strength of LL-37, NTH and control peptides with SARS-CoV-2 RBD, 100 ns molecular dynamic (MD) simulations were implemented with the help of Desmond software. 38 These docked complexes were prepared for MD simulation using the system T A B L E 1 (A) The amino acid sequences of NTH and the peptides used in this study. (B) Quantitative assessment, using TM-Align server, of hACE2 NTH Structural Similarity with peptides from RCSB (https://www.rcsb.org/) selected on the basis as described in the Results. The reference protein was NTH of ACE2. The descriptions are from (https://www.rcsb.org/) builder of a panel of Desmond software. All the complex systems were immersed in a solvated cubic box of size 10 Å using explicit solvent, that is, the TIP3P water model. The suitable counter ions (Na + / Cl À ) were added to the solvated system to neutralize the absolute charge of the complex system. The steepest descent energy minimization method was utilized during 100 ns MD simulation along with the OPLS-2005 force field. 39 Also, all the systems were simulated using NPT (N/number of atoms, P/pressure, T/temperature) ensemble at 300 K temperature and 1 bar pressure. The Martyna-Tobias-Klein barostat method and Noose-Hoover chain thermostat method were used to control the pressure and the temperature during the 100 ns MD simulation. The short-range interactions, that is, columbic interactions were maintained at 9.0 Å cut-off radius, and to integrate the equations of motion leap-frog algorithm was used with 2 fs time step. 

where G complex represents the protein-ligand complex energy, G protein represents the unbound (free) protein energy and G ligand symbolizes the free ligand energy. for all peptides. The validation of the TM-Align server was done by giving the same hACE2 NTH structure as structure one and structure two inputs to the server. The resulting TM-score of this alignment was 1, with root mean square deviation (RMSD) 0 Å and this con- The structural alignments generated from the TM-Align server are shown in Figure 1 (Panel II). It can be seen that LL-37 even shares a similar bend in the helix as NTH, and this could help in fitting of LL-37 into the concavity of RBD. Also, although the peptide Amylin has aligned with NTH with a score a little greater than 0.5, it can be seen from the figure that there is a clear bifurcation of the two structures toward the N-terminus. This study suggests that the reported human anti-microbial peptide LL-37 is structurally very close to hACE2 NTH and this feature could facilitate its binding to RBD. For each peptide, the amino acids that aligned with those of NTH are shown in Figure 2 (Panel I). It can be seen that LL-37 and PACAP-38 have a greater number of aligned residues with d < 5.0 Å, and hence, better alignment than Amylin and GLP-2. Now, those structures with alignment RMSD > 2.0 Å and/or those that are not found as free peptides in the circulation (which includes 2LAT), were eliminated. Thus, 2D2P, 2KB8, and 2L63 were selected as control peptides for docking studies. in each case are shown diagrammatically in Figure 3 was analyzed, and its docking score was À224.14. The detailed interactions are given in Table 2 (B), from which it is seen that the interactions are mostly not common with the experimentally determined interactions. This would be due to the NTH alone docking in a similar but not perfectly the same position as when in the entire ACE2, due to the surrounding influences of ACE2.

The docked complex of 2D2P-RBD 6LZG gives a docking score of Figure 4 (Panel I). There was no commonality between RBD residues interacted with by NTH and by any of the Test or Control peptides. Figure 4 (Panel II) diagrammatically shows the alignments of the regions of RBD spanned by the control peptides. We also explored the binding stability of LL-37 peptide toward the region which is immediately adjacent and upstream to SARS-CoV-2 RBD.

This docking result gives a docking score of À209.74 by forming two interactions at the interface of this complex (Table 3( Table 4 .

Remarkably, the peptide LL37 used in this study gives a stronger binding affinity toward the RBD 6LZG of SARS-CoV-2. The complexes LL37_RBD 6LZG and LL37_RBD 6M0J exhibited À144.50 kcal/mol and À118.98 kcal/mol binding free energies, respectively. Also, NTH extracted from the ACE2 of 6LZG complexed with RBD 6LZG shows À112.26 kcal/mol binding free energy, which is also stronger when compared with the other peptides used as a control in this study.

The control peptides binding to RBD, viz., 2D2P_RBD 6LZG , 2 KB8_RBD 6LZG , and 2 L63_RBD 6LZG give À91.80 kcal/mol, 

For analyzing the variations in the sequence of SARS-CoV-2 isolated from different geographical regions we used the "Latest Global Analysis" tool of the "nextstrain.org" database, 43 

It is recognized that peptides, even including LL-37, could have adverse effects when used as therapeutics. 48 For possible delivery of LL-37 through the inhalatory route, LL-37 was assessed for allergenicity. Tak- ing an example of a human polypeptide which is used by inhalation in clinical practice, namely insulin, it has been reported that allergic reactions can occur although insulin is an innate protein. 49, 50 The algorithm

AllergenFP v.1.0 51 predicted LL-37 to be a "probable non-allergen."

Using the server AllerCatPro 52 LL-37 as a query gave the result "No

Evidence" for allergenicity. AllergenFP v.1.0 predicted human Insulin (UniProt sequence ID P01308) to be a "probable allergen" and AllerCatPro gave the result "Strong Evidence" for insulin as an allergen.

The software program ToxinPred 53 predicted LL-37 to be a Non-Toxin by all Support Vector Machine methods available on the server. For prediction of hemolytic activity, the tool HAPPENN, which employs neural networks method was used. 54 LL-37 was predicted to have very low hemolytic scores of 0.073, 0.089, and 0.09 by the three methods available in the tool, degree of hemolytic activity increasing from 0 to 1.

In summary, based on our in silico findings, we propose that LL-37 could be used as a therapeutic for Covid-19. Besides the known There is also approval for a clinical study of intratumoral injection of LL-37 for the condition of melanoma (ClinicalTrials.gov Identifier:

NCT02225366). At high concentrations in vitro, LL-37 has been found cytotoxic to different eukaryotic cells, 60 but when human serum is present, the cytotoxic activity of LL-37 is inhibited, and this could be the reason why LL-37 is not cytotoxic in the body. Indeed, the cytotoxicity of LL-37 secreted into the blood is negated by the binding of LL-37 to plasma proteins such as apolipoprotein A-I. 61 It is well elucidated how the cytotoxic effects of LL-37 are counteracted by its binding to apolipoprotein A-I. 62 The K D of the binding is such that shields mammalian plasma membranes from lysis. 63 Now, it has been reported that excision of the N-terminal hydrophobic residues from LL-37 reduces its cytotoxicity. 61 From Figure 2(Panel I,B) , it can be seen that the first three N-terminal amino acids (LLG) of LL-37 do not align with the NTH residues. Also, from the docking data, these three amino acids do not form interactions with RBD. This means that, for the development of LL-37 as a therapeutic, the option of removing two-three amino acids from the N-terminus is available.

The predicted non-allergenicity of LL-37 can mean that LL-37 could be administered to patients through the respiratory tract route, which would have more immediate efficacy in treating the disease.

Also, this would greatly improve the deliverability to large populations in a shorter time, due to ease of administration compared to an injectable. If administered intravenously, being a peptide, LL-37 could migrate from the circulation to the infected regions in the lungs. Peptide therapeutics enter the tissues from the vasculature by diffusion or convective extravasation, and transfer from the circulation to the tissues also depends on the properties of specific peptides. 64 In relation to the lungs, as respiratory epithelial cells express hCap-18, LL-37 has been found to be present in the bronchial alveolar lavage fluid, 65 and increased levels have been detected in tracheal aspirates during infection. In the lungs, the normal role of LL-37 is to provide innate immunity against bacterial infections. In the case of Covid-19, SARS-CoV-2 infects the respiratory epithelial cells, 66 

Immunology of COVID-19: current state of the science

Towards effective COVID-19 vaccines: updates, perspectives and challenges (review)

Epidemiology, genome, and clinical features of the pandemic SARS-CoV-2: a recent view

Structure of the SARS-CoV-2 spike receptorbinding domain bound to the ACE2 receptor

Computational biophysical characterization of the SARS-CoV-2 spike protein binding with the ACE2 receptor and implications for infectivity

Cathelicidin impact on inflammatory cells

Host defense peptides: frontline immunomodulators

Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3

P2X7 receptor regulates internalization of antimicrobial peptide LL-37 by human macrophages that promotes intracellular pathogen clearance

The host defense peptide LL-37 triggers release of nucleic acids from human mast cells

Dual functions of the human antimicrobial peptide LL-37-target membrane perturbation and host cell cargo delivery

Antiviral activity of the human cathelicidin, LL-37, and derived peptides on seasonal and pandemic influenza a viruses

Human antimicrobial peptides as therapeutics for viral infections

Vitamin D deficiency and air pollution exacerbate COVID-19 through suppression of antiviral peptide LL37. Front Public Health

In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2

Cathelicidins have direct antiviral activity against respiratory syncytial virus in vitro and protective function in vivo in mice and humans

Cathelicidin antimicrobial peptides suppress EV71 infection via regulating antiviral response and inhibiting viral binding

Modulation of growth factor/cytokine synthesis and signaling by 1alpha,25-dihydroxyvitamin D(3): implications in cell growth and differentiation

Vitamin D3 modulates the innate immune response through regulation of the hCAP-18/LL-37 gene expression and cytokine production

Low plasma 25(OH) vitamin D level is associated with increased risk of COVID-19 infection: an Israeli population-based study

Association of vitamin D status and other clinical characteristics with COVID-19 test results

Modulation of the immune response to respiratory viruses by vitamin D

Molecular dynamics simulations of human antimicrobial peptide LL-37 in model POPC and POPG lipid bilayers

Deducing the N-and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2

Distinct conformational states of SARS-CoV-2 spike protein

Protein structure analysis of the interactions between SARS-CoV-2 spike protein and the human ACE2 receptor: from conformational changes to novel neutralizing antibodies

Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles

Structural and functional basis of SARS-CoV-2 entry by using human ACE2

The solution structure of micelle-bound peptide

Dynamic alpha-helix structure of micelle-bound human amylin

Conformational and molecular interaction studies of glucagon-like peptide-2 with its N-terminal extracellular receptor domain

Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments

TM-align: a protein structure alignment algorithm based on the TM-score

How significant is a protein structure similarity with TM-score = 0.5?

Systematic comparison of SCOP and CATH: a new gold standard for protein structure analysis

The HDOCK server for integrated protein-protein docking

Neutralization of SARS-CoV-2 by destruction of the Prefusion spike

Scalable algorithms for molecular dynamics simulations on commodity clusters

How accurate is the description of ligand-protein interactions by a hybrid QM/MM approach?

A hierarchical approach to all-atom protein loop prediction

Structural basis of receptor recognition by SARS-CoV-2

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Nextstrain: real-time tracking of pathogen evolution

SkipCPP-Pred: an improved and promising sequence-based predictor for predicting cell-penetrating peptides

Machinelearning-based prediction of cell-penetrating peptides and their uptake efficiency with improved accuracy

CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides

CPPsite: a curated database of cell penetrating peptides. Database (Oxford)

Development and challenges of antimicrobial peptides for therapeutic applications

Pulmonary and cardiovascular safety of inhaled insulin in routine practice: the Exubera large simple trial (VOLUME)

Inhaled insulin: overview of a novel route of insulin administration. Vasc Health Risk Manag

AllergenFP: allergenicity prediction by descriptor fingerprints

AllerCatPro-prediction of protein allergenicity potential from the protein sequence

In silico approach for predicting toxicity of peptides and proteins

HAPPENN is a novel tool for hemolytic activity prediction for therapeutic peptides which employs neural networks

An in silico scientific basis for LL-37 as a therapeutic and vitamin D as preventive for Covid-19. (Version 1)

LL-37 fights SARS-CoV-2: the vitamin D-inducible peptide LL-37 inhibits binding of SARS-CoV-2 spike protein to its cellular receptor angiotensin converting enzyme 2 in vitro

Vitamin D deficiency 2.0: an update on the current status worldwide

Safety of high-dose vitamin D supplementation: secondary analysis of a randomized controlled trial

Treatment with LL-37 is safe and effective in enhancing healing of hard-to-heal venous leg ulcers: a randomized, placebo-controlled clinical trial

Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37

Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37

The human cathelicidin LL-37-a pore-forming antibacterial peptide and host-cell modulator

Cooperative function of LL-37 and HNP1 protects mammalian cell membranes from lysis

Pharmacokinetics and pharmacokineticpharmacodynamic correlations of therapeutic peptides

The human cathelicidin LL-37: a multifunctional peptide involved in infection and inflammation in the lung

Pathogenesis of COVID-19 from a cell biology perspective

Evidence that vitamin D supplementation could reduce risk of influenza and COVID-19 infections and deaths

Preventing vitamin D deficiency during the COVID-19 pandemic: UK definitions of vitamin D sufficiency and recommended supplement dose are set too low

An in silico scientific basis for LL-37

as a therapeutic for Covid-19

The authors declare no potential conflict of interest.

Manisha Deshpande conceived, designed and led the project and carried out the Structural Alignment study, the Safety studies, CPP prediction, and data analyses. She has also written most of the 

Data are available on request from the authors.

https://orcid.org/0000-0001-6945-8288Manisha Deshpande https://orcid.org/0000-0002-5786-0066