Abstract
SARS-CoV-2 is a virus that causes Severe Acute Respiratory Syndrome Coronavirus, Type 2. After its emergence in 2019, it quickly spread to global pandemic levels. Understanding how the virus interacts with and enters human cells has been critical for developing effective therapeutic strategies. The virus enters human cells by binding to HSPG (Heparan Sulfate Proteoglycan) and ACE2 (Angiotensin Converting Enzyme 2) receptors through the surface spike protein. HSPGs are protein-carbohydrate complexes found on the cell surfaces responsible for cell adhesion and migration. The current treatment options don’t prevent the infection, but instead treat patients already infected. Thus, a wider range of anti-viral agents needs to be explored, in particular topical broad spectrum anti-viral drugs.
One of the promising classes of broad spectrum anti-viral agents are sulfoglycodendrimers (SGDs). SGDs are highly branched polyanionic synthetic macromolecules with a polymer core and terminal sulfated carbohydrates. SGDs have previously shown anti-viral activity against the HIV virus. Here, the positively charged polybasic amino acids in the spike protein of HIV interact with the negatively charged sulfate groups on HSPGs via electrostatic interactions. These interactions can be blocked by SGDs. Since SARS-CoV-2 shares similarities with the HIV virus in initial binding to the target cells through HSPGs, SGDs are also being explored for their anti-viral activity against SARS-CoV-2.
Given the role of electrostatic interactions between the SARS-Cov-2 virus and HSPGs in the early stages of viral binding and infection, a competitive ELISA (Enzyme Linked Immunosorbent Assay) was developed to evaluate the interaction between the spike protein and the SGDs. ELISA is a common biochemistry technique used to quantify proteins and antigens. The SGDs were tested against SARS-CoV-2 using ELISA. In this assay, biotinylated heparin sulfate (HS) was immobilized onto a streptavidin-coated plate, followed by the addition of the spike protein and SGDs, creating a competitive design. The signal was detected using a 6x His-tag monoclonal antibody and/or HisProbe-HRP.
The HisProbe-HRP is a nickel (Ni2+)-activated derivative of horseradish peroxidase (HRP) used for the detection of His-tagged proteins whose epitope specific antibody isn’t known. The following SGDs were tested: cellobiose, maltotriose, melibiose, and maltose. All SGDs showed inhibition between HS and the spike protein. The average IC50 values obtained were: 14.4 µM for cellobiose, 26.7 µM for maltotriose, 4.4 µM for melibiose, 18.3 µM for maltose. Here, IC50 values represent the concentration of SGDs needed to inhibit the viral protein binding by 50%.
A major challenge encountered was protein aggregation. While testing a lactose-SGD via a HIV ELISA assay, protein aggregation was observed. The protein aggregation was mitigated by the addition of detergent in the assay buffer. Testing unsulfated vs sulfated SGDs revealed that sulfated SGDs causes aggregation through interactions with negatively charged heparin sulfate (HS) and positively charged spike protein in the SARS ELISA assay. The efficacy of SGDs depends on: % sulfation, molecular weight, polar group orientation and the electrostatic interactions. Further testing of SGDs against SARS-CoV-2 via ELISA will explore their potential as inhibitors as they show promising anti-viral properties and need to develop new therapeutics treating COVID-19.