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. 2015;12(8):792-800.
doi: 10.1080/15476286.2015.1058478.

Identification of small molecule inhibitors of Zcchc11 TUTase activity

Shuibin Lin  1 , Richard I Gregory
Affiliations

Identification of small molecule inhibitors of Zcchc11 TUTase activity

Shuibin Lin et al. RNA Biol. 2015.

Abstract

The RNA-binding protein Lin28 regulates the expression of the let-7 family of microRNAs (miRNAs) during early embryonic development. Lin28 recruits the 3' terminal uridylyl transferase (TUTase) Zcchc11 (TUT4) and/or Zcchc6 (TUT7) to precursor let-7 RNA (pre-let-7) to selectively block let-7 biogenesis. Uridylated pre-let-7 is targeted for decay by the downstream exonuclease Dis3l2 thereby preventing processing to mature let-7. Activation of this oncogenic pathway via up-regulation of Lin28 expression promotes cellular transformation, drives tumorigenesis in mouse models, and is frequently observed in a wide variety of cancer. Recent proof-of-principle experiments showed that Zcchc11 knockdown inhibits the tumorigenicity of Lin28-expressing human cancer cells and established this enzyme as a possible new therapeutic target for human malignancies. However, there are currently no known pharmacological agents capable of targeting this novel enzyme. In this study we developed and applied a sensitive biochemical assay that monitors Zcchc11 activity. Using this assay we performed an automated high-throughput screen of ∼ 15,000 chemicals to identify putative TUTase inhibitors. Several of these small molecules were validated as specific inhibitors of Zcchc11 activity. Our results demonstrate the feasibility of screening for TUTase inhibitors and present a relatively simple platform that can be exploited for future drug discovery efforts aimed at restoring let-7 expression in cancer.

Keywords: Lin28; TUT4; TUTase; Zcchc11; cancer; let-7; miRNA; microRNA; uridylation.

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Figures

Figure 1.
Figure 1.
Purification of rZcchc11. (A) Schematic representation of Zcchc11 and the minimal region of Zcchc11 (rZcchc11) that is required for Lin28 mediated uridylation of pre-let-7. (B) SDS-PAGE and coomassie blue staining analysis of purified rZcchc11. (C and D) Uridylation of pre-let-7a1 by Lin28 and rZcchc11. (E and F) Uridylation of mature let-7i guide RNA by rZcchc11.
Figure 2.
Figure 2.
Optimization of PPi light assay to measure Zcchc11 activity. (A) Schematic demonstration of the PPi light assay. The PPi generated by rZcchc11-mediated uridylation is converted into luciferase signal by the PPiLight™ Inorganic Pyrophosphate Assay kit. (B) Optimization of PPi light assay for high throughput screening. Included (as indicated) is a titration of UTP, let-7i guide RNA, and rZcchc11 for the optimization of PPi light assay.
Figure 3.
Figure 3.
High-throughput screening of Zcchc11 inhibitors. (A) Flow chart of the high throughput screening stratgey. 91 of 14,822 screened compounds were cherry picked for secondary assay. (B) A representative screening plate showing the correlation of luciferase reading between 2 repeats. (C) A representative screening plate showing the luciferase reading of each well. B and C were generated with visualization software Vortex. Red: positive control; Dark blue: Negative control; Light blue: screening samples; Gray: empty well.
Figure 4.
Figure 4.
Secondary assay for TUTase inhibitors. (A) 91 cherry-picked compounds were analyzed with the Lin28 dependent secondary assay to determine their functions in regulation of Zcchc11 activity in uridylation of pre-let-7-a1. Concentrations used for the assay: known bioactive compounds: 33 μM; natural product extracts: 50 μg/ml. B and C: Test of diluted natural product extracts (1.5 μg/ml) (B) and known bioactive compounds (C) in uridylation assay. (D) Test of re-purchased compounds in inhibiting Lin28 and Zcchc11-mediated uridylation of pre-let-7a1.
Figure 5.
Figure 5.
Mechanisms and specificity of Zcchc11 inhibition. (A) Structure of the identified compounds. (B) Test of compound inhibition in reducing conditions with 100 mM β-ME. (C) IC50 calculation from dose curve experiments with compounds N6, L6 and F7 in regulating Zcchc11 mediated uridylation. Signal intensitiy of each band was calculated by Image J and the inhibitory curves generated by Excel. (D) Specificity of compounds N6, L6 and F7 in regulating Zcchc11 activity in uridylation of mature let-7i guide RNA. Upper panel: roles of compounds in inhibiting rZcchc11 activity; Lower panel: roles of compounds in inhibiting PUP activity.
Figure 6.
Figure 6.
Test of Zcchc11 inhibitors in cells. (A) Dis3l2 knockdown (left panel) in V6.5 mESCs results in accumulations of uridylated pre-let7 (U-tailed pre-let-7, right panel). (B) Zcchc11 knockdown (left) in Dis3l2 depleted cells decreases uridylated pre-let7 level (U-tailed pre-let-7, right panel). (C) Treatment of Dis3l2 depleted mESCs with compound L6 (500 μM) results in decreased level of U-tailed pre-let-7, while have little effect on pri-let-7 expression. **, P < 0.01.
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