SHAPE-enabled fragment-based ligand discovery for RNA

Abstract

The transcriptome represents an attractive but underused set of targets for small-molecule ligands. Here, we devise a technology that leverages fragment-based screening and SHAPE-MaP RNA structure probing to discover small-molecule fragments that bind an RNA structure of interest. We identified fragments and cooperatively binding fragment pairs that bind to the thiamine pyrophosphate (TPP) riboswitch with millimolar to micromolar affinities. We then used structure-activity relationship information to efficiently design a linked-fragment ligand, with no resemblance to the native ligand, with high ligand efficiency and druglikeness, that binds to the TPP thiM riboswitch with high nanomolar affinity and that modulates RNA conformation during cotranscriptional folding. Principles from this work are broadly applicable, leveraging cooperativity and multisite binding, for developing high-quality ligands for diverse RNA targets.

Keywords: RNA-targeted ligand discovery; SHAPE-MaP; cooperativity; fragment linking.

Fig. 1.

Schemes for RNA construct and fragment screening workflow. (A) RNA motifs 1 and 2 and the barcode helix are shown in colors; structure cassette helices are gray. (B) RNA is probed using SHAPE in the presence or absence of a small-molecule fragment. (C) Chemical modifications corresponding to ligand-dependent structural information are read out by multiplexed MaP sequencing. (D) Fragment hits are identified as multiple, statistically significant differences in SHAPE reactivities.

Fig. 2.

Representative SHAPE mutation rate comparisons for fragment hits and nonhits. Normalized mutation rates for fragment-exposed samples (orange) are compared to no-ligand traces (blue). Statistically significant changes in mutation rate are denoted with green triangles (see SI Appendix, Fig. S2 for SHAPE confirmation data). (Top) Mutation rate comparison for a representative fragment that does not bind the test construct. (Middle) Fragment hit to the TPP riboswitch region of the RNA. (Bottom) Nonspecific hit that induces reactivity changes across the entire test construct. RNA motif 1 and 2 landmarks are shown below SHAPE profiles.

Fig. 3.

Ligands and affinities for fragments that bind the TPP riboswitch. (A) Fragments that bind as detected in the initial round of SHAPE-enabled screening. Dissociation constants were determined by ITC; ^‡ SE is derived from ≥3 replicates; other error estimates are calculated based on 95% confidence intervals for the least-squares regression of the binding curve. The native TPP ligand [9] is included for comparison. (B and C) Structure-activity relationships for analogs of fragment 2. Modifications to the (B) quinoxaline core and (C) pendant groups. (D) Fragments that bind in the presence of a prebound primary partner, as detected by SHAPE in round two screening. Hits were validated by replicate SHAPE analysis. (E) Structure-activity relationships for analogs of fragment 28 binding to the TPP riboswitch RNA, in the presence and absence of prebound fragment 2. und, undetermined due to inability to fit ITC binding curve; insoluble, compound insoluble at concentrations required for ITC.

Fig. 4.

Thermodynamic cycle and stepwise ligand binding affinities for fragments 2 and 31. (A) Summary of binding by 2 (blue, K₁) and 31 (orange, K₂) fragments and the linked compound Z1. K_d values determined by ITC. (B) Representative ITC data showing single-compound and cooperative binding by 2 and 31. Linking the two fragments shows an additive effect in binding energies, resulting in a submicromolar ligand, Z1 (K_L). ITC traces are shown with experimental traces in dark blue and background traces (ligand titrated into buffer) as light blue. Confidence intervals (95%) are in purple shading.

Fig. 5.

Covalent linking of 17 and 31 as a function of linker type and length, terminal group chemotype, and terminal group orientation. Modifications that increase RNA binding affinity are colored green; negative and neutral modifications are colored red and yellow, respectively. Dissociation constants determined by ITC.

Fig. 6.

Structures of the TPP riboswitch bound by compounds identified in this study and by the native TPP ligand. Nucleotides in direct contact with the organic ligands are beige. Mg²⁺ and Mn²⁺ cations and water molecules are depicted as green, lavender, and red spheres, respectively; spheres do not represent physical sizes of ions and water molecules. Intermolecular hydrogen and metal ion coordination bonds are shown as gray and green dashed lines, respectively. Crystal structures of riboswitch bound by (A) compound 17, (B) TPP, and (C) compound 38. Arrow in B shows rotation of G72 in the 17-bound structure. (D) Relative positions of 38 and TPP ligands. (E) Model of Z1 bound to riboswitch. Arrow shows postulated displacement of Mn²⁺ atom (Mn2). (F) Surface view for model of Z1 bound to RNA. TPP (light green color and thin sticks) is shown as a reference; for clarity, a few nucleotides at the front of the structure were removed in this image. Py, pyrimidine; Tz, thiazole; PP, pyrophosphate moieties of TPP; Ox, quinoxaline; Pi, pyridine; Pip, piperazine of 38. TPP-bound structure is from 2GDI (28).

Fig. 7.

Cotranscriptional riboswitch ligand-binding assays. (A) DNA template for cotranscriptional assay with the thiM riboswitch. Ligand binding is detected as the presence, or not, of an RNA conformation that protects the final transcript from RNase H cleavage by a DNA oligonucleotide that binds the P1 helix. (B) Representative cleavage assay, performed in the presence of Z1. (C) K_switch determination for TPP, Z1, and 38. Compounds Z1 and 38 differ by a single –CH₂– group (Fig. 5). Data were fit to a single-site binding equation. Data shown are representative of experiments performed in triplicate.

Fig. 8.

Comparison of fragment-linker-fragment ligands, ordered by their linking coefficient (E). Cooperative (more efficient) linking corresponds to smaller E values (top of vertical axis). Values are shown on a logarithmic axis. Z1 is highlighted in blue. Dissociation constants for individual fragments and linked ligand are denoted below component fragments; E-value and LE are shown (see Key). Covalent linkage introduced between fragments is highlighted in red. Structures for component fragments are detailed in SI Appendix, Table S3, and values for Z1 are reported relative to initial fragment hits.