Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

Ritesh Tandon, Joshua S Sharp, Fuming Zhang, Vitor H Pomin, Nicole M Ashpole, Dipanwita Mitra, Weihua Jin, Hao Liu, Poonam Sharma, Robert J Linhardt, Ritesh Tandon, Joshua S Sharp, Fuming Zhang, Vitor H Pomin, Nicole M Ashpole, Dipanwita Mitra, Weihua Jin, Hao Liu, Poonam Sharma, Robert J Linhardt

Abstract

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 μg/L, 1.08 mg/L, 1.77 μg/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

Keywords: COVID-19; Coronavirus; Glycosaminoglycans; Pseudotyping; Spike glycoprotein.

Figures

Figure 1:. Model of SARS-CoV-2 attachment and…
Figure 1:. Model of SARS-CoV-2 attachment and entry.
Binding of virus to HS in the glycocalyx increases the local concentration of virus, improving binding to hACE2.
Figure 2.. SARS-CoV-2 SGP pseudotyped lentiviral screen…
Figure 2.. SARS-CoV-2 SGP pseudotyped lentiviral screen for inhibition of attachment and entry.
A. Quantitation of GFP-transduced cells in the presence of each inhibitor at three concentrations. Average GFP transduction of control was 200.2 cells per well. B. Representative fluorescence microscopy of UFH-deNS inhibitor assay. C. Representative fluorescence microscopy of UFH inhibitor assay.
Figure 3.. Structure of anti-SARS-CoV-2 sulfated polysaccharides.
Figure 3.. Structure of anti-SARS-CoV-2 sulfated polysaccharides.
Enoxaparin and UFH differ primarily by average length of the polysaccharide (Avg. MW UFH ~15 kDa; Avg. MW enoxaparin ~4.5 kDa). Enoxaparin/UFH −6S have H at position R4. Enoxaparin/UFH –NS have H or Ac at R3. Enoxaparin/UFH desulf have no SO3− groups. Avg. MW of marine sulfated glycans is ≥ 100 kDa.
Figure 4.. Relative IC 50 curves for…
Figure 4.. Relative IC50 curves for four potent SARS-CoV-2 inhibitors.
Curves were modeled using GraphPad Prism 8.4.2. Top limit was set at the average vehicle-only control level for this assay batch (200.2), with the bottom limit allowed to float independently for each inhibitor. Details are shown in Table 1.
Figure 5.. SPR sensorgrams of pLV-S virions…
Figure 5.. SPR sensorgrams of pLV-S virions bound to immobilized heparin.
Virion concentration is based on an estimated molecular weight of 250 MDa.

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