Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction

Abstract

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

Figure 1

Molecular dynamics modeling defines direct interactions of pixatimod with S1 RBD: (A) Structure of pixatimod. (B) Model (pose-a) showing interactions of pixatimod on the Coulombic surface of the RBD domain of spike protein. The sulfated tetrasaccharide partially occupies the HS/heparin binding site I. (C) Model (pose-b) showing interactions of pixatimod with the RBD domain of spike protein wherein the sulfated tetrasaccharide partially occupies the HS/heparin binding site III. The RBD is rendered using the Coulombic surface whereas the ligand is shown as a stick. (D) The lipophilic tail of pixatimod in both models wraps around the hydrophobic residues, thereby creating a steric clash with the helix of ACE2 protein. (E) The RBD is shown as a ribbon colored from the N- to C-terminal (blue to red). The residues of RBD (shown as a ball and stick) responsible for binding to ACE2 (shown in a light blue ribbon) are labeled. Pixatimod uses similar residues from the receptor binding motif on the RBD and thereby inhibits binding of ACE2. The Coulombic surface was rendered using UCSF Chimera coloring defaults: ε = 4r, thresholds ±10 kcal/mol. Red corresponds to negative charges, white to neutral, and blue to positive charges. Hydrogens are not shown for clarity.

Figure 2

Pixatimod interacts with SARS-CoV-2 S1-RBD and inhibits binding to cells and the ACE2 receptor. (A) Circular dichroism spectra (190–260 nm) of SARS-CoV-2 m(His)S1-RBD alone (black), or with pixatimod (blue) or heparin (red). The vertical dotted line indicates 193 nm. (B) The same spectra expanded between 200 and 240 nm. Vertical dotted lines indicate 222 and 208 nm. (C) Secondary structure content analyzed using BeStSel for SARS-CoV-2 m(His)S1-RBD (analyses using BeStSel were performed on smoothed data between 200 and 260 nm). (D) Differential scanning fluorimetry of binding of pixatimod (blue, 10 μg) or heparin (red, 10 μg) to mS1-RBD (1 μg, black line, protein-only control). *T_m values for RBD alone (48.4 °C, SD = 0.3) and in the presence of pixatimod (39.3 °C, SD = 1) were statistically different, t(4) = 15.25, p = 0.0001. (E) Effects of pixatimod and unfractionated porcine mucosal heparin on binding of mammalian expressed mS1-RBD-monomeric Fc to A549 human lung epithelial cells. Data were normalized to the control with no addition of mammalian expressed mS1-RBD-mFc (n = 3 ± CV). (F) Competitive ELISA assay using biotinylated human ACE2 protein immobilized on streptavidin coated plates to measure the inhibition of binding of mS1-RBD in the presence of various concentrations of inhibitor compounds. Pixatimod (IC₅₀, 10.1 μg/mL) and porcine mucosal heparin (IC₅₀, 24.6 μg/mL). n = 3, ±SD; representative example shown.

Figure 3

Pixatimod inhibits infection of Vero E6 and human bronchial epithelial cells with different SARS-CoV-2 virus isolates. Live virus infectivity assays were performed as described in the Materials and Methods section for 3 different SARS-CoV-2 isolates (representative data shown). (A) Plaque reduction neutralization assay of Victoria isolate (VIC01) (EC₅₀ 8.1 μg/mL; n = 3, ±SD). (B) Plaque reduction assay of DE isolate, EC₅₀ 2.7 μg/mL; n = 3, ±SD. (C) Cytopathic assay of Queensland isolates, EC₅₀ 13.2 (QLD02) and 0.9 (QLD935 with D614G mutation) μg/mL n = 6, ±SEM. Representative examples are shown in each case. Results of pixatimod inhibition of SARS-CoV-2 infectivity are expressed as percent plaque reduction (A), plaque number as a percent of control (B), or percent inhibition from the cytopathic effect (C). Panel B also shows cytotoxicity data for Vero cells for a calculation of the CC₅₀ value (>236 μg/mL). In panel C, data is also shown for octyl β-maltotetraoside tridecasulfate (Octyl-βMTTS; Figure S2), an analogue of pixatimod which lacks the steroid side-chain. (D) Plaque assay to measure the inhibitory effect of pixatimod on viral shedding in BCi-NS1.1 human bronchial epithelial cells grown in an air liquid interface (ALI). ALI cultures were infected for 2 h with SARS-CoV-2 (VIC01, MOI = 0.2) previously preincubated for 1 h at 37 °C with 0.4, 4, and 40 μg/mL pixatimod or HBSS for untreated. After 72 h, an apical rinse was performed with 200 μL of HBSS, and 100 μL of the wash was used in a plaque assay in Vero E6 cells. Values are expressed as plaque forming unit (PFU)/mL, n = 2, ±SD. (E) Cytotoxicity data for BCi-NS1.1 human bronchial epithelial cells. The CC₅₀ value for pixatimod is >300 μg/mL. Values correspond to the mean of n = 3 independent experiments with technical duplicates, ±SEM.

Figure 4

Pixatimod inhibits SARS-CoV-2 infection in K18-hACE2 transgenic mice. (A) Mean percentage weight change relative to day 1 postinfection (QLD02 isolate). Statistics by repeat measures ANOVA for days 4 and 5. n = 4 mice per group, ±SE. Mice were euthanized on day 5. (B) Mean tissue titers on day 5 postinfection. Statistics by Kolmogorov–Smirnov test (nasal turbinates) and t test (brain). n = 4 mice per group, ±SE.

Figure 5

Pixatimod retains potency against multiple SARS-CoV-2 virus variants of concern. Live virus infectivity assays were performed as described in the Materials and Methods section for multiple different SARS-CoV-2 isolates (representative data shown). (A) Microinhibitory assay to assess the % viral foci reduction caused by increasing concentrations of pixatimod when incubated with live Wuhan-like SARS-CoV-2 (VIC01), B.1.1.7 Alpha variant, B.1.351 Beta variant, or B.1.617.2 Delta variant, in vitro with Vero E6 cells. Data represent 3 independent experiments per variant with n = 2 technical replicates, ±SD, except for the B.1.617.2 Delta variant where only 2 independent experiments were performed with n = 2 technical replicates. The EC₅₀ values for VIC01 (Victoria isolate), B.1.1.7 variant, B.1.351 variant, and B.1.617.2 variant were 3.6 ± 1.8, 6.5 ± 0.8, 2.1 ± 1.7, and 1.0 ± 0.2 μg/mL, respectively. (B) Inhibitory effect of pixatimod (4 μg/mL) on the B.1.1.7 (Alpha) SARS-CoV-2 variant used to infect BCi-NS1.1 bronchial epithelial cells (ALI). Viral load in the cells was determined by an RT-PCR analysis and compared to a 2 h infected (untreated) control (n = 2 with technical duplicates, ±SD). (C) Data for SARS-CoV2 microneutralization assays for Omicron (B.1.1.529) and Vic01 variants treated with pixatimod (Oxford assay). IC₅₀ values obtained were 0.74 ± 0.28 and 2.63 ± 0.93 μg/mL for Omicron and Vic01, respectively (n = 3, ±SD).

Figure 6

Proposed multimodal mechanisms of pixatimod activity against SARS-CoV-2 and other viruses. The principal mode of action demonstrated here is that pixatimod acts as a decoy receptor [1], blocking S1-RBD binding to HS coreceptors and inhibiting viral attachment to host cells, thus blocking viral infection. Additional potential modes of action include: [2] virucidal activity of pixatimod, dependent upon the cholestanol moiety, which may lead to degradation and permanent inactivation of SARS-CoV-2 virus particles; [3] suppression of IL-6 secretion by antigen presenting cells, primarily macrophages; and [4] blocking viral escape from host cells by inhibiting heparanase which otherwise promotes viral escape by cleaving HS receptors.^,