Identification of compounds inhibiting a Leishmania RNA editing reaction.

Abstract

Several species of Leishmania are human pathogens that afflict more than 12 million people worldwide, and the current treatment options are limited. An RNA editing reaction that is both essential and specific to the parasites is an attractive target for new drug development. The editing reaction involves the post-transcriptional modification of specific mitochondrial mRNAs through the precise deletion or insertion of uridylates. Many aspects of the editing mechanism are still unclear, and the lack of specific inhibitors to probe the reaction has hindered the field. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify inhibitors, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A novel editing assay was developed in this thesis that overcame previous limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter for the assay consists of an RNA editing substrate linked to a streptavidin-binding domain that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding domain is only activated by an editing-induced conformational change. The reporter RNA is labeled with a ruthenium complex, and an electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis and binding affinity measurements were used to characterize the reporter. This highly sensitive assay was optimized and validated for use in high-throughput screening, and a pilot screen of a 1280 compound library identified compounds that are the first specific inhibitors of the editing reaction. Some of the identified inhibitors will have value as probes of the editing reaction and have already provided insights into possible regulatory mechanisms. The identification of novel drugs through screens of large chemical libraries is now possible with the new assay.