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

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. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are 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 antiviral 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 50% inhibitory concentrations (IC₅₀s) of 5.99 μg/liter, 1.08 mg/liter, 1.77 μg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Keywords: COVID-19; coronavirus; glycosaminoglycan; heparan sulfate; pseudotyping; spike glycoprotein.

FIG 1

Transduction of HEK293T cells with lentiviral vector (pLV) pseudotyped with (A) VSV-G or (B) CoV-2-SGP. The lentiviral backbone incorporates enhanced green fluorescent protein (eGFP) that is expressed upon integration into target cells. The fluorescence was recorded at 48 h postransduction. Magnification, ×20. Bar, 200 μm.

FIG 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 the UFH-deNS inhibitor assay. (C) Representative fluorescence microscopy of the UFH inhibitor assay.

FIG 3

Structure of anti-SARS-CoV-2 sulfated polysaccharides. Enoxaparin and UFH differ primarily by the average length of the polysaccharide chain (average MW of UFH, ∼15 kDa; average MW of enoxaparin, ∼4.5 kDa). Enoxaparin-de6S and UFH-de6S have H at position R₆. Enoxaparin-deNS and UFH-deNS have H or Ac at R_N. Enoxoparin-fully-deS and UFH-fully-deS have no SO₃⁻ groups. The average MW of marine sulfated glycans is ≥100 kDa.

FIG 4

1D ¹H NMR spectra of (A) UFH, (B) UFH-deNS, (C) UFH-de6S, (D) LMWH, (E) LMWH-deNS, and (F) LMWH-de6S (expansion Δδ_H, 7.0 to 0.4 ppm). The signals which were diagnostic for the desulfation reaction are labeled with letters and numerals according to the monosaccharide types, positions of the protons within the sugar rings, and 6-sulfation (6S) or non-6-sulfation (6nS). The letters A, B, C, D, E, and I denote, respectively, α-GlcN,6diS, α-GlcNAc(6S), α-GlcN6S, α-GlcNAc, α-GlcNS, and α-IdoA2S. The 6-sulfation in the α-GlcNAc(6S) cannot be ensured solely on the basis of the anomeric assignment (signal B1).

FIG 5

Relative IC₅₀ curves for four potent SARS-CoV-2 inhibitors. Curves were modeled using GraphPad Prism 8.4.2. The 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.

FIG 6

SPR measures affinity of pLV-S virion binding to various GAGs. (A) SPR sensorgrams of pLV-S binding to immobilized heparin. Virion concentration is based on an estimated molecular weight of 250 MDa. (B and C) Normalized pLV-S virion binding to surface-immobilized heparin upon competition with different GAGs in solution (B) or different-length heparin-derived oligosaccharides in solution (C). Pseudotype virions were present at 0.35 nM, and GAGs were present at 1,000 nM. All results represent triplicate measurements, with error bars representing one standard deviation.