Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS‐CoV‐2 Genome

Figure 1. NMR‐based fragment screening and hit identification: A, Schematic representation of the experiments and criteria used to identify binders/ non‐binders. B, Chemical structures of Binder 3 and Binder 8 along with an overlay of their corresponding NMR spectra [1D 1H (i), waterLOGSY (ii) and T 2‐CPMG (iii); (5 ms and 100 ms)] of the mixture in absence (blank) and presence of RNA. The top 1D 1H‐spectrum corresponds to the single fragment used for chemical shift deconvolution in the mixture. The inset highlights the signal corresponding to the hit used for analysis. Visual inspection of the overlay indicates that Binder 3 displays CSP (≥6 Hz) and T 2 reduction of ca. 50 %. Binder 8 shows no CSP, but a clear sign change in waterLOGSY and T 2‐reduction of ca. 80 %. C, 1D 1H‐NMR spectral regions of fragments with strong (top row), weak (middle row) or no binding (lower row) to 3_SL2. Binder 1 shows a CSP of 6.75 Hz, a positive waterLOGSY signal and a T 2‐reduction of 64 % suggesting that it binds to 3_SL2 (top row). 708236–68‐4 shows a minor CSP of 3.43 Hz, but neither a positive waterLOGSY signal nor a T 2‐reduction, indicating either very weak or no binding to 3_SL2 (middle row).…

Figure 2. NMR‐based fragment screening identifies 69 fragment hits across the SCoV‐2 RNAs. Tabular column summarizing screened SCoV‐2 RNA elements and their corresponding hits. Dark green indicates hits for individual RNA elements; light green indicates hits for larger RNAs. The last column (C.) lists the number of binders to the investigated RNA target. Chemical structures of respective fragment hits are shown.

Scheme 1. Molecular descriptors and chemical diversity of RNA binders in the DSI‐poised library. About 75 % of the fragments showing binding to SCoV‐2 RNA contain two functional units, connected by four different linking units. In case of LU3, the urea functional group likely contributes to binding affinity.

Scheme 2. Classification into five classes of the 40 ligands that bind to the 15 regulatory RNAs. LU=linking unit.

Scheme 3. Comparison of binding and non‐binding fragments with closely related chemical structure.

Figure 3. Affinities of binders and mapping of binding sites in SCoV‐2 RNAs. A) NMR‐based (ligand‐observed; protons chosen to follow CSP are indicated) titration curves for the interaction of fragment 4 with PK and 3_s2m. B) Fluorescence‐based titration curve and dissociation constant of D01 with PK. C) Interaction of D01 with PK monitored by RNA‐observed NMR. 1H, 15 N‐HSQC overlay of PK alone and in presence of four equivalents of D01 (green). Addition of D01 to PK results in CSPs or line broadening beyond detection. Affected nucleotides are mapped onto the PK secondary and 3D structures (PDB:7O7Z[61]).