Heparin: A simplistic repurposing to prevent SARS-CoV-2 transmission in light of its in-vitro nanomolar efficacy

Abstract

The world is currently facing a novel coronavirus (SARS-CoV-2) pandemic. The greatest threat that is disrupting the normal functioning of society is the exceptionally high species independent transmission. Drug repurposing is understood to be the best strategy to immediately deploy well-characterized agents against new pathogens. Several repurposable drugs are already in evaluation for determining suitability to treat COVID-19. One such promising compound includes heparin, which is widely used in reducing thrombotic events associated with COVID-19 induced pathology. As part of identifying target-specific antiviral compounds among FDA and world-approved libraries using high-throughput virtual screening (HTVS), we previously evaluated top hits for anti-SARS-CoV-2 activity. Here, we report results of highly efficacious viral entry blocking properties of heparin (IC₅₀ = 12.3 nM) in the complete virus assay, and further, propose ways to use it as a potential transmission blocker. Exploring further, our in-silico analysis indicated that the heparin interacts with post-translational glycoconjugates present on spike proteins. The patterns of accessible spike-glycoconjugates in open and closed states are completely contrasted by one another. Heparin-binding to the open conformation of spike structurally supports the state and may aid ACE2 binding as reported with cell surface-bound heparan sulfate. We also studied spike protein mutant variants' heparin interactions for possible resistance. Based on available data and optimal absorption properties by the skin, heparin could potentially be used to block SARS-CoV-2 transmission. Studies should be designed to exploit its nanomolar antiviral activity to formulate heparin as topical or inhalation-based formulations, particularly on exposed areas and sites of primary viremia e.g. ACE2 rich epithelia of the eye (conjunctiva/lids), nasal cavity, and mouth.

Keywords: Anticoagulant; COVID-19; Heparin; Lipid-based formulations; SARS-CoV2; Spike protein; Sulfated polysaccharide; Topical delivery; Transmission-blocking.

Fig. 1

ACE2 rich Mucocutaneous tissue and upper respiratory mucosa are the entry points for the SARS-CoV-2 virus. The proposed intervention with heparin formulation should be the key to block transmission.

Fig. 2

Evaluation of antiviral activities of heparin. (A) against virus attachment and entry/fusion. The experimental procedure, virus concentration (PFU/well or MOI), and the time of addition and treatment with the test compounds are presented in the method sections. (B) Nonobservance of inactivation of viral infections by the test compounds heparin. A virus surface spike protein mediates SARS-CoV-2 entry into cells. Our results are in agreement with previous reports and show the contrast in antiviral activity (Ic50 = 12.3 nM in entry assay vs. nil activity in spread assay in given concentration range) in different formats confirming the mechanism of action of heparin.

Fig. 3

Schematic representation of spike trimer structure (PDB_ID:7CAI) [20]. Poses represented in Panel A and B are 180-degree rotation of the model on the vertical plane. Green and Turquoise surface representations are chains A and C with open state spike with RBD regions extended away from the main spike structure. The purple surface model of Chain B represents a closed state in which RBD is facing inwards and seems to be inactive for ACE2 binding. The processing sites are described according to earlier reports [74]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Molecular docking analysis of heparin against spike protein of SARS-CoV-2 envelope. The trimeric protein complex had two high scoring attachment sites (A) Heparin-binding to an open state i.e. RBD (brown highlights on green) is protruding exposing the ACE2 binding region. This binding is more supportive of spike-ACE2 interaction and possibly stabilizes the open state. This interaction could explain the role of heparan sulfate in facilitating ACE2 receptor binding as reported earlier [15] (B) heparin-binding to the closed domain: This binding has a similarity to open state binding as heparin molecule traces the sugar moieties (glycoconjugates) but due to different topology heparin seems to masks the RBD domain (yellow circle) and probably this is the reason for heparin (Unbound heparan sulfate) blocking spike-Ace2 interactions as also reported earlier [13]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Cartoon representation of the proposed mechanism of heparan sulfate and heparin neutralization of SARS-CoV-2 virion. (A) This is a topological map of the domain architecture of spike protein showing different functional regions. (B) Based on the essential role of Heparan sulfate [17] and the docking results suggest an open state of spike protein upon interacting with HSPGs. (C) Interactions of open state spike protein with ACE2 and/or NRP1 receptors. (D) Cleavage of a spike by TMPRSS2 exposing viral fusion domains. (E) Fusion: Spike protein-mediated fusion of viral envelope with host plasma membrane resulting in delivery of nucleocapsid to host cell cytoplasm, Internalization: Formation of endosomes like inclusions mediated by Spike-Heparan sulfate-ACE2/NRP1 interactions leading to nucleocapsid delivery to host cytoplasm through further processing of spike by Furin and/or Cathepsin L and Viral envelope and endosomal membrane fusion similar to the plasma membrane.

Fig. 6

Relative mutation site analysis with respect to heparin binding to the closed state. Inset table lists the mutations reported as of February 2021 (GISAID-hcov-19 with entropy greater than 0.1). The three peptides constituting the spike trimer are colored red, blue and gray. Heparin mostly interacts with blue peptide in this pose, therefore, all the mutant positions are labelled on the same to avoid confusion. Interestingly most of the mutant locations are away from the heparin binding site which suggests important and highly conserved role of sulfated polysaccharides in the viral invasion as well as usability of heparin as a universal blocker of all the mutant spike variants. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Molecular docking analysis of heparin against spike protein (Closed state RBD) of SARS-CoV-2 envelope with different mutations. Position/location of the heparin molecule is pointed out with a circular purple ligand symbol. The trimeric protein complex had three mutant structures from PDB (7AD1, 7NDB, and 7KDL) and supercomputer predicted structures for mutants from UK, Brazil, South Africa, and Danish minks with differences in vaccine efficacies reported recently (Table 3). All the structure showed strong binding with closed state RBD. Lower docking score observed with Danish mink might not be necessarily be due to mutation but could be due to flexible state of the relatively large trimeric complex and also predicted structures might lack proper glycosylation's that seem to play an important role in spike stabilizations as seen in wild type structure 7CAI. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)