1. Introduction
Due to the emergence of pandemic diffusion of SARS-CoV-2, health care systems and emergency medical services have become overwhelmed [
1]. Some measures, such as lock-down of communities, social distancing, and quarantine-type for those suspected to be infected, can, at least in part, slow the COVID-19 (
COronaVIrus Disease 19) spread [
2] and therefore enable the health systems to cope. In the worldwide search for a response to the COVID-19 pandemic, different natural remedies against COVID-19 have been reported [
3,
4,
5]. Among these,
Pelargonium sidoides and lactoferrin showed important anti-inflammatory, anti-oxidative, and antiviral properties, administered alone or in combination [
3,
4,
5,
6,
7,
8].
Pelargonium sidoides (
PEL; Geraniaceae) is an African medicinal plant, traditionally used for curing different diseases, including diarrhea, colic, gastritis, tuberculosis, cough, hepatic disorders, menstrual complaints, and gonorrhea [
9]. The common name,
umckaloabo, represents the Zulu (“
Umkuhlune”—coughing and fever; and “
Uhlabo” = pain in the chest) [
10] word describing ‘severe cough’. Indeed, its extracts are successfully employed in modern phytotherapy in Europe to cure infectious diseases of the respiratory tract [
5,
8,
9].
Pelargonium sidoides is indicated for the common cold [
11], cough, and bronchitis [
12].
Pelargonium sidoides root extracts preparations are available in some European Countries with a full marketing authorization (e.g., Bulgaria, Czech Republic, Germany) or registered as a traditional herbal medicinal product (e.g., Austria, Hungary, Italy, The Netherlands, Poland, Spain, Sweden), and are widely used for acute bronchitis and other respiratory infections [
11]. Relevant key metabolites assumed to be active are hydrolysable tannins, catechins, gallic acid, and methyl gallate, including some unusual O-galloyl-C-glucosyl flavones, scopoletin, 6,8-dihydroxy-5,7-dimethoxycoumarin, 5,6,7-trimethoxycoumarin. Other coumarins, as well as, quercetin 3-O-b-D-glucoside, myricetin, and other flavonoids, have been isolated. This herbal medicine has been experimentally proven for anti-viral activity as reported for
Pelargonium sidoides that interfere in vitro with the replication of different respiratory viruses, including human coronaviruses [
13], influenza virus (in vitro and in vivo) [
14], and Rhinovirus isolated from patients with severe asthma [
15], by stimulating IFN-b in vitro, while gallic acid enhanced the expression of iNOS and TNF-α [
16].
Lactoferrin (Lf) is a glycoprotein of the transferrin family [
17,
18], synthetized by exocrine glands and neutrophils and is present in human milk and in all secretions [
17,
18]. This protein is one of the most important factors of innate immunity, constituting a barrier against pathogens colonizing both mother and fetal habitats [
19], and it was demonstrated that it could also act as a potential nutraceutical capable of contrasting SARS-CoV-2 infection [
6,
7].
Lactoferrin has four essential activities: chelation of two ferric ions per molecule, interaction with anionic compounds, translocation into the nucleus, and modulation of inflammation and iron homeostasis [
3,
17,
18]. Lf’s capability to chelate two ferric ions per molecule influences bacterial and viral replication and hinders reactive oxygen species formation (ROS) [
17,
20,
21]. The binding of Lf to anionic surface components, thanks to its cationic features, is associated with the host protection against bacterial and viral adhesion and entry [
17]. Moreover, the entrance of Lf into host cells, and its translocation into the nucleus [
22,
23], is related to its anti-inflammatory function [
24]. In a recent study, we demonstrated that bovine Lf (bLf) exerts its antiviral activity either by direct binding to the SARS-CoV-2 particles or by obscuring their host cell receptors [
6]. Moreover, the results obtained through the computational approaches strongly supported the hypothesis of a direct recognition between the bLf and the spike glycoprotein [
6].
As reported in literature [
5], the combination
Pelargonium sidoides + Lf (
PELIRGOSTIM), can reduce in vitro the release of proinflammatory cytokines, oxidants, and bacteria growth, most likely preventing leukocyte chemiotaxis with a reduced inflammatory pattern.
PEL and Lf used alone were able to reduce LPS-induced proinflammatory IL-1β, as well as reduce ROS, nitrite, and bacteria growth. It can be hypothesized that this synergistic effect may counteract SARS-CoV-2 infection.
In view of this context, it is important to understand if the compounds contained in PEL extracts can interfere with the lactoferrin structure and function and what types of interactions can be established with the key components of SARS-CoV-2.
To this aim, in this study we applied computational methods to verify the hypothesis of a direct interaction between
PEL compounds and the Lf protein and with SARS-CoV-2 Spike, 3CLPro, RdRp proteins, and membrane. Selected high-score complexes obtained with molecular docking were structurally investigated through classical molecular dynamics (MD) simulations, rescoring their interaction energies using the molecular mechanics energies combined with generalized Born and surface area continuum solvation (MM/GBSA) method [
25]. The results obtained from the computational analyses suggest that
PEL compounds and Lf could synergistically interfere with the mechanism of infection of SARS-CoV-2, especially in the early stages.
4. Discussion
In this study, we applied computational methods to check for the occurrence of interactions of the PEL compounds with Lf and SARS-CoV-2 components.
Pelargonium sidoides preparations have been trialed clinically for cough, even if the clinical evidence is high only for bronchitis and the common cold. [
61,
62,
63].
Umckaloabo preparations are generally considered to be safe, although gastrointestinal discomfort (stomach pain, heartburn, nausea, or diarrhea) might occur [
8].
PEL extracts are commonly employed in modern phytotherapy in Europe to cure infectious diseases of the respiratory tract [
4,
5].
On the other hand, lactoferrin has been proven to act as a scavenger against iron overload and inflammation in lung epithelium of mice infected by
Pseudomonas aeruginosa [
64,
65] and was found to rebalance lung iron-handling proteins and to decrease broncho-alveolar iron overload, one of the main actors in infection progression and exacerbation [
3]. Moreover, several studies described Lf’s antiviral activity towards enveloped and naked viruses, related to different virus families, such as
Retroviridae (human immunodeficiency viruses),
Papillomaviridae (human papillomavirus),
Herpersviridae (Cytomegalovirus, Herpes simplex virus), Caliciviridae (feline calicivirus),
Flaviviridae (hepatitis C virus, Japanese encephalitis virus),
Reoviridae (rotavirus),
Adenoviridae (adenovirus),
Pneumoviridae (respiratory syncytial virus),
Paramixoviridae (parainfluenza virus),
Orthomixoviridae (influenza A virus), and other viruses [
3]. bLf has been found to hinder viral entry into host cells through its competitive binding to the cell surface receptors, mainly negatively charged compounds such as glycosaminoglycans (GAGs) [
66,
67,
68,
69,
70,
71]. In addition, Lf was found to prevent viral infections by binding to dendritic cell-specific intercellular adhesion. Overall, the antiviral effect of Lf occurs in the early phase of infection, preventing the entry of viral particles into the host cells, either by blocking cellular receptors and/or by directly binding to the viral particles. Further, Lf is also able to exert an antiviral activity when it is added in the post-infection phase, as demonstrated in Rotavirus infection by Superti et al., [
72] and in HIV infection by Puddu et al. [
73].
In a previous study, Terlizzi et al. demonstrated that the combination of
PEL and Lf could exert additive/synergistic pharmacological activities as anti-inflammatory, antioxidant, and antimicrobial agents compared with the single components [
5]. They found that
PEL and Lf used alone were able to reduce LPS-induced proinflammatory IL-1β, as well as reduce ROS, nitrite, and bacteria growth. More importantly, the combination of
PEL with Lf showed an additive pharmacological activity in terms of antioxidant and antimicrobial activities. Data demonstrated that the combination of
PEL + Lf significantly reduced the levels of IL-1β after LPS stimulation. This effect was an innovative and hitherto unknown combination, able to attenuate inflammation-related pathways [
3,
5,
6,
7].
Molecular docking and molecular dynamics simulation approaches strongly supported the hypothesis of a direct recognition between the bLf and the SARS-CoV-2 spike glycoprotein [
6,
28]. The affinity between their molecular surfaces, the large number of atomistic interactions detected, and their persistence during the simulation suggested that this recognition is very likely to occur and that bLf may hinder the spike binding to the ACE2 receptor, thus blocking virus entry into host cells [
6].
In this scenario, we have carried out a series of molecular docking and molecular dynamics simulations to identify possible interactions between PEL and Lf and between PEL and some of the SARS-CoV-2 components.
First, we analyzed by molecular docking if the interaction between
PEL compounds and Lf could alter its functional properties, hampering the interaction with other macromolecules as Spike. Our results are fully in agreement with literature since it has been demonstrated that the structure and activity of lactoferrin is not altered by the presence of organic molecules or metal ions different from iron [
17,
18]. Based on these results, the combined use of
PEL and Lf in a dietary supplement has been acknowledged.
Subsequently, computational studies have been carried out to evaluate a possible interaction between
PEL compounds and the SARS-CoV-2 3CLpro protein. In a recent work it has been shown that compounds capable of interacting with both residues of the 3CLpro catalytic dyad can inhibit the activity of this enzyme between 50 and 88% [
74]. Moreover, other flavonoids have shown inhibitory activity against this protein [
75]. In general, the results confirms that several
PEL compounds can stably interact with the active site of the protease in proximity of the catalytic dyad, suggesting an inhibitory activity against the virus protease.
The interaction of
PEL compounds with the SARS-CoV-2 Spike glycoprotein has been also investigated. The molecular docking experiments were carried out on the trimeric structure of the Spike, selecting as research area the HR1 domains, which are responsible for the conformational change that allows the entry of the virus inside cells [
28]. Interfering with these domains should block the glycoprotein in its prefusion state, before it can enter the cells by recognizing the ACE2 receptor. Based on these results, we could assume that several analyzed compounds could interfere with the conformational changes of the Spike glycoprotein.
The interaction of PEL compounds with the SARS-CoV-2 RdRp polymerase has also been checked, but from the results no molecules of the PEL extracts may have the ability to interfere with the viral polymerase.
Finally, we verified whether the PEL compounds could interact with the membrane of the SARS-CoV-2 virus. In fact, being rich in phenolic acids, PEL extracts could interfere with the lipid membrane dynamical properties, consequently affecting the motions of the proteins inserted in the double layer. The MD simulation analyses suggest that the interaction of PEL compounds with the membrane can alter its external electrostatic potential, inducing a curvature of the bilayer. However, this effect extends to the entire membrane, suggesting that PEL compounds can penetrate the viral lipid bilayer, alter its physical properties, and also interact with viral proteins in infected cells.