Identification of specific inhibitors for a trypanosomatid RNA editing reaction

Abstract

Several mitochondrial mRNAs of the trypanosomatid protozoa are edited through the post-transcriptional insertion and deletion of uridylates. The reaction has provided insights into basic cellular biology and is also important as a potential therapeutic target for the diseases caused by trypanosomatid pathogens. Despite this importance, the field has been hindered by the lack of specific inhibitors that could be used as probes of the reaction mechanism or developed into novel therapeutics. In this study, an electrochemiluminescent aptamer-switch was utilized in a high-throughput screen for inhibitors of a trypanosomatid RNA editing reaction. The screen identified GW5074, mitoxantrone, NF 023, protoporphyrin IX, and D-sphingosine as inhibitors of insertion editing, with IC(50) values ranging from 1 to 3 μM. GW5074 and protoporphyrin IX are demonstrated to inhibit at or before the endonuclease cleavage that initiates editing and will be valuable biochemical probes for the early events of the in vitro reaction. Since protoporphyrin IX and sphingosine are both naturally present within the trypanosomatids, their effectiveness as in vitro inhibitors is also suggestive of the potential for in vivo modulatory roles.

FIGURE 1.

Inhibitors of a trypanosomatid editing reaction. (A) The RNA reporter for the HTS contains a streptavidin aptamer (yellow) that is only active after the insertion of three U's (red) by the in vitro editing reaction. Upon immobilization of the RNA at the bottom of a streptavidin-coated microtiter plate and electrical stimulation, an ECL signal is generated from a ruthenium complex attached to the reporter. (B) Confirmation of the inhibitory activity using a radiolabeled reporter that is based on the cytochrome b mRNA sequence. The gRNA used for the assay has two guiding nucleotides. Compounds were dissolved in DMSO and used at a final concentration of 10 μM. Reactions contained 0.01% TX-100, and products were analyzed after electrophoresis on a denaturing gel. No editing activity was detected in the presence of any of the inhibitors (n = 3). (C) Confirmation of the inhibitory activity of compounds obtained from the HTS using a radiolabeled SELEX-optimized reporter with two guiding nucleotides. Reactions contained 0.01% TX-100, and products were analyzed after electrophoresis on a denaturing gel. The percent inhibition for correct editing (+2) in the presence of each compound is relative to the DMSO-only control (n = 3).

FIGURE 2.

Inhibition of in vitro editing by several derivatives and precursors of protoporphyrin IX using the radiolabeled SELEX-optimized reporter. The percent inhibition for the formation of correctly edited product (+2) is relative to the DMSO-only control (n = 4). Minus signs denote reaction stimulation rather than inhibition. (DME) Dimethyl ester; (TME) trimethyl ester.

FIGURE 3.

The inhibitors affect different aspects of the editing reaction. The radiolabeled RNA reporter for these reactions corresponds to the intermediate formed by endonuclease cleavage at an editing site. Since the reporter for the full-reaction is normally circular in order to prevent U-additions to the 3′ end, the intermediate resulting from endonuclease cleavage at the editing site is the indicated full-length linear molecule (input). In the absence of inhibitor, two guided U's are added to the linear molecule, and ligated circular product containing two U-insertions is formed. The percent inhibition for the formation of the edited circular product is relative to the DMSO-only control (n = 3). GW5074 and protoporphyrin IX only weakly inhibit the editing of the post-cleavage substrate as compared to the corresponding full reaction indicated in Figure 1C.