Anticancer Activity of Pyrimethamine via Ubiquitin Mediated Degradation of AIMP2-DX2.

Dae Gyu Kim,Chul Min Park,Srigouri Huddar,Sunkyung Lee,Semi Lim,Sunghoon Kim

doi:10.3390/molecules25122763

Abstract

While aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is a tumor suppressor, its exon 2-depleted splice variant (AIMP2-DX2 or shortly DX2) is highly expressed in human lung cancer, and the ratio of DX2 to AIMP2 increases according to the progression of lung cancer. In this study, pyrimethamine inhibited the level of DX2 (IC50 = 0.73 µM) in A549 cells expressing nanoluciferase-tagged DX2. In a panel of 5 lung cancer cell lines with various DX2 levels, pyrimethamine most potently suppressed the growth of H460 cells, which express high levels of DX2 (GI50 = 0.01 µM). An immunoblot assay in H460 cells showed that pyrimethamine decreased the DX2 level dose-dependently but did not affect the AIMP2 level. Further experiments confirmed that pyrimethamine resulted in ubiquitination-mediated DX2 degradation. In an in vivo mouse xenograft assay using H460 cells, intraperitoneal administration of pyrimethamine significantly reduced the tumor size and weight, comparable with the effects of taxol, without affecting body weight. Analysis of tumor tissue showed a considerably high concentration of pyrimethamine with a decreased levels of DX2. These results suggest that pyrimethamine, currently used as anti-parasite drug, could be repurposed to treat lung cancer patients expressing high level of DX2.

Highlights

Aminoacyl-tRNA synthetases (ARSs) are the enzymes that catalyze the ligation of specific amino acids to their cognate tRNAs in the first step of protein translation [1]
We describe the effects of pyrimethamine in a nanoluciferase assay, a viability assay in 5 lung cancer cell lines with different expression levels of DX2, a mechanism of action study, and an in vivo xenograft assay
For establishment of more sensitive high-throughput screening, we changed the luciferase tag to a nanoluciferase tag because the nanoluciferase is three times smaller and 2,500,000 times brighter than the previously used luciferase, and 763 compounds were screened in the drug repositioning library from Korea Chemical Bank (KCB) at KRICT by our screening system

Summary

Introduction

Aminoacyl-tRNA synthetases (ARSs) are the enzymes that catalyze the ligation of specific amino acids to their cognate tRNAs in the first step of protein translation [1]. Nine ARSs and three aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are assembled into a multi-tRNA synthetase complex (MSC) that serves as a hub for various regulatory pathways in addition to playing a role in protein synthesis. While three AIMPs, AIMP1/p43, AIMP2/p38 and AIMP3/p18, function as scaffold proteins for the assembly and integrity of MSCs [2,3,4], they participate in diverse regulatory roles that are not directly linked to protein synthesis. Among the AIMPs, AIMP2 works as a potent tumor suppressor and induces growth arrest by transforming growth factor-β (TGF β) signaling via ubiquitin-mediated degradation of FUSE-binding protein (FBP) and downregulation of c-myc [5]. AIMP2 functions as a proapoptotic factor by protecting the tumor suppressor p53 under DNA damage stress [7]. AIMP2 heterozygous mice showed increased susceptibility to tumorigenesis driven by lung, colon, and skin carcinogens, showing that the antiproliferative and proapoptotic activities of AIMP2 are evident in vivo [8]

Methods

Results

Conclusion