Aptamer BC 007 - Efficient binder of spreading-crucial SARS-CoV-2 proteins

Abstract

Corona virus disease 2019 (COVID-19) is a respiratory disease caused by a new coronavirus (SARS-CoV-2) which causes significant morbidity and mortality. The emergence of this novel and highly pathogenic SARS-CoV-2 and its rapid international spread poses a serious global public health emergency. To date 32,174,627 cases, of which 962,613 (2.99%) have died, have been reported (https://www.who.int/westernpacific/health-topics/coronavirus, accessed 23 Sep 2020). The outbreak was declared a Public Health Emergency of International Concern on 30 January 2020. There are still not many SARS-CoV-2-specific and effective treatments or vaccines available. A second round of infection is obviously unavoidable. Aptamers had already been at the centre of interest in the fight against viruses before now. The selection and development of a new aptamer is, however, a time-consuming process. We therefore checked whether a clinically developed aptamer, BC 007, which is currently in phase 2 of clinical testing for a different indication, would also be able to efficiently bind DNA-susceptible peptide structures from SARS-CoV-2-spreading crucial proteins, such as the receptor binding domain (RBD) of the spike protein and the RNA dependent RNA polymerase of SARS-CoV-2 (re-purposing). Indeed, several such sequence-sections have been identified. In particular for two of these sequences, BC 007 showed specific binding in a therapy-relevant concentration range, as shown in Nuclear magnetic resonance (NMR)- and Circular dicroism (CD)-spectroscopy and isothermal titration calorimetry (ITC). The excellent clinical toxicity and tolerability profile of this substance opens up an opportunity for rapid clinical testing of its COVID-19 effectiveness.

Keywords: Antiviral; Aptamer; BC 007; COVID-19; Clinical research; Health sciences; Infectious disease; Pharmaceutical science; Pharmacology; Re-purposing; SARS-CoV-2.

Figure 1

NMR spectroscopic evaluation of the molecular interaction of BC 007 with sequence sections of the spike protein and the RNA dependent RNA polymerase of SARS-CoV-2. A: The upper NMR spectrum of BC 007 (1 mM) in combination with 0.9 mM YRLFRK (in blue colour) shows formation of the quadruplex structure of BC 007 induced by molecular interaction with the peptide which is clearly recognisable by eight imino signals between 11.5 and 12.5 ppm which are comparable to the well-known potassium-induced fold of BC 007 [32] (lower green spectrum). Substances were dissolved in 0.5 ml pure H₂O/D₂O mixture without any additives. The peptide signals in the spectrum are strongly shifted and broadened in comparison to the spectrum taken from the peptide alone without BC 007 (red colour). B: Same spectra zoomed into the range between 8.5 and 5.5 ppm. It shows that the peptide signals (red spectrum) are then broadened and shifted in their position in the upper spectrum (blue) due to the interaction between peptide and BC 007. C: BC 007 in presence of KIKRMK and NRKRISN showing an almost negligible intermolecular interaction. D: Ranking of the NMR spectroscopic evaluation of the molecular interaction of BC 007 with sequence sections of the ACE2-receptor binding domain (RBD) of SARS-CoV-2 (YRLFRK), of the S1/S2 (TNSPRRARSV) and S2′ (SKPSKRSF) cleavage sites of the spike protein, of the RNA dependent RNA polymerase (LYRNRDV and HRFYRLAN) and of the SARS-CoV spike protein (KYRYLRHGK).

Figure 2

2D NMR spectroscopic evaluation of the YRLFRK-BC 007 binding. Comparison of the NOESY-spectra between A: BC 007-YRLFRK and B: BC 007-K⁺, both recorded in in H₂O/D₂O 90/10 at 298K. Dotted lines enclose the binding relevant residues T3/T4 and T12/T13 of the TT-loops. C: ¹H-TOCSY spectrum (600 MHZ) of the 1mM mixture BC 007-YRLFRK at 298K in H2O/D2O 90/10. Pulse program mlevgpphw5, size of fid 4096 × 512, number of scans 64, spectral width 24 ppm, TOCSY mixing time 70 ms.

Figure 3

Characterization of BC 007 binding to peptidesequence-sections of SARS-CoV-2 spike protein (YRLFRK, NRKRISN) and RNA dependent RNA polymerase (HRFYRLAN) with ITC. A: Binding isotherms of BC 007–peptides interaction, derived by the integration of raw data, heat of injection, Q, vs peptide–BC 007 M ratio; B: Exemplarily ITC raw data for the representative titration of HRFYRLAN (3.6 mM) into BC 007 (200 μM), differential heating power, DP vs time C: Thermodynamic signature plot of the HRFYRLAN-BC 007 binding, representing the balance entalpic (red) and entropic (green) contributions to the Gibbs free energy (blue).

Figure 4

CD spectroscopic evaluation of the molecular interaction of BC 007 with sequence sections of the spike protein (YRLFRK, NRKRISN) and the RNA dependent RNA polymerase (HRFYRLAN) of SARS-CoV-2. Resulting mixtures of ITC-experiments were diluted in water down to a concentration of 21.5 μM BC 007 and the corresponding peptide in the molar excess of 1:4.

Figure 5

CD spectroscopic evaluation of the molecular interaction of BC 007 with sequence sections of the spike protein (YRLFRK, NRKRISN) and the RNA dependent RNA polymerase (HRFYRLAN) of SARS-CoV-2. A: spectra of the resulting mixtures of ITC-experiments were diluted in water down to a concentration of 21.5 μM BC 007 and the corresponding peptide in the molar excess of 1:4 in the presence of the final concentration of 5 mM KCl. B: The RBD-binding protein occurring peptide (YRLFRK) with and without 5 mM KCl. The grey curve shows 21.5 μM BC 007 in presence of the same amount of diluted ITC-buffer corresponding to 19.3 mM Na⁺.

Figure 6

Visualization of the accessibility of the BC 007-binding peptide sequences of the RNA dependent RNA polymerase of SARS-CoV-2 and thrombin in comparison for BC 007 binding. A: Localization of “HRFYRLAN” on the surface of the RNA dependent RNA polymerase was checked using the PBD entry ID: 6M71 [36] and inserted BC 007 from PDB ID entry 1HAO [29]. B: visualization of the thrombin-BC 007 binding, taken from PDB ID entry 1HAO [29] using the PyMOL Molecular Graphics System, Version 2.4.1. Schrödinger, LLC.