Selective inhibitors of trypanosomal uridylyl transferase RET1 establish druggability of RNA post-transcriptional modifications

Abstract

Non-coding RNAs are crucial regulators for a vast array of cellular processes and have been implicated in human disease. These biological processes represent a hitherto untapped resource in our fight against disease. In this work we identify small molecule inhibitors of a non-coding RNA uridylylation pathway. The TUTase family of enzymes is important for modulating non-coding RNA pathways in both human cancer and pathogen systems. We demonstrate that this new class of drug target can be accessed with traditional drug discovery techniques. Using the Trypanosoma brucei TUTase, RET1, we identify TUTase inhibitors and lay the groundwork for the use of this new target class as a therapeutic opportunity for the under-served disease area of African Trypanosomiasis. In a broader sense this work demonstrates the therapeutic potential for targeting RNA post-transcriptional modifications with small molecules in human disease.

Keywords: African trypanosomiasis; RET1; RNA modifications; TUTase; drug-discovery; non-coding RNA; post-transcriptional modification; trypanosome; uridylylation.

Figure 1.

Gel based assay for RET1 activity. RET1 catalyzes the addition of terminal uridines resulting in a poly-U RNA product of approximately 150 bases. In the absence of RET1, the 24 base RNA target remains unmodified.

Figure 2.

Schematic for the luciferase coupled HTS assay to detect RET1 activity (PPKD – pyruvate phosphate dikinase).

Figure 3.

Verification of RET1 inhibitors at 50 µM by gel based assay. Lanes from left to right: Positive control, RET1 is active in assay buffer conditions (5% DMSO) and polyuridylates target RNA producing high molecular weight products; Negative control, in the absence of RET1 RNA is not elongated; Compounds 1, 2, and 8 effectively inhibit the ability of RET1 to uridylate RNA.

Figure 4.

Putative binding modes predicted using the RET1 homology model. a) Ataciguat (1) and b) Exifone (2) docked in the RET1 apo binding site showing the most populated cluster. In a) and b), the protein fold is shown in cartoons representation, colored by secondary structure (β-sheet in yellow, α-helix in purple, loops in cyan). The extensive N-terminal domain β-loop containing the Arg358-Glu657 salt bridge (labeled) helps to create a snug binding site for inhibitor. c) Overlay of RET1 homology model (cyan, with sidechains in CPK format) and CID1 crystal structure (all mauve). The residues of CID1 crystal structure are labeled, while those of the RET1 homology model are unlabelled but are shown in the same orientation as in a) and b). The presence of His336 in CID1 increases the electrostatic potential at the top of the binding site, leading to separation of Glu333 and Arg137 (residues corresponding to those that form the Arg358-Glu657 salt bridge in RET1) leading to a more open binding site in CID1.

Figure 5.

Representative curves showing RET1 selectivity of short-listed hit compounds. Compounds exhibited varying degrees of selectivity for inhibiton of RET1 (black curves) over the yeast TUTase CID1 (blue curves).