Heparin's potential role in blocking SARS-CoV-2 cellular entry was studied in real time using portable SPR — demonstrating the platform's value in rapid pandemic response.
Introduction
In search of therapeutic treatments that can inhibit the infection of SARS-CoV-2 viruses, heparin, as well as heparan sulfate (HS) and its derivatives, may be potential drug candidates due to their known binding interactions with surface viral proteins. Furthermore, they are inexpensive and widely available. Heparin is a glucosaminoglycan (GAG), which is a complex carbohydrate found on animal cells. It is known that these GAGs bind to surface viral proteins which result in viral adhesion and cell entry.
In the study by Mycroft-West et al., SPR — specifically Affinité's quad inlet P4SPR™ — was used to confirm the binding between SARS-CoV-2 S1 receptor-binding domain (RBD) and heparin. A competitive assay also demonstrated the extent to which each heparin derivative could inhibit the binding between the RBD and immobilized heparin on the SPR sensor chip.
SPR Experimental Details
A quad inlet P4SPR™ (4 channels) was used to measure the binding interactions between heparin and RBD. This was done by immobilizing heparin onto SPR sensor chips via a biotin-streptavidin linkage and measuring the SPR shift upon introduction of the RBD. The P4SPR gold sensor chips were functionalized with a mixture of polyethylene glycol methyl ether (mPEG) thiol and biotin-mPEG thiol. Streptavidin was injected into the channels followed by biotin-heparin.
Confirmation of Binding with FGF2 and SARS-CoV-2 S1 RBD Protein
FGF2 was first introduced to the heparin-modified SPR sensor chips to validate the SPR assay as it is known to bind to heparin or HS. Injection of 100 nM FGF2 led to a SPR shift of 1.60 nm (or 567 RU). The control channel, which had no heparin, did not elicit a signal. In a separate experiment, the injection of 800 nM SARS-CoV-2 S1 RBD led to a signal shift of 0.22 nm (or 78 RU). These signals could be observed in real-time and measured within 3 min.
An SPR Competition Assay with Heparin and other Heparin Derivatives
Mixtures of 800 nM SARS-CoV-2 S1 RBD and heparin of various concentrations were prepared and injected into the P4SPR. Starting at 1.7 µg/mL of heparin, a reduction in the binding of SARS-CoV-2 S1 RBD to the heparin was observed. The signal further decreased in a dose-dependent manner, and the binding was completely inhibited at 1.7 mg/mL of heparin.
A panel of heparin derivatives of different levels and patterns of sulfation was used to compete with surface-immobilized heparin to bind to SARS-CoV-2 S1 RBD. Some derivatives such as enoxaparin and Hep9 prevented more than 50% of RBD from binding to immobilized heparin, while others did not have such a significant effect. Native heparin inhibited binding of RBD most effectively. This demonstrates that the degree and location of sulfation dictate the binding between SARS-CoV-2 S1 RBD and heparin derivatives.
Conclusions
By immobilizing heparin onto SPR sensor chips via a biotin-streptavidin linkage, the binding between the SARS-CoV-2 S1 RBD and heparin was quickly validated. Furthermore, the extent of binding inhibition was measured for heparin, HS, and its derivatives using a user-friendly SPR setup. The quad inlet P4SPR provides a simple yet informative tool that can pave the way to discover therapeutic drugs to prevent infection by SARS-CoV-2 and other viruses.
Reference
[1] Mycroft-West CJ, et al. Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin. Thromb. Haemost., 2020, 120, 1700.