A Novel Pyrazolopyrimidine Ligand of Human PGK1 and Stress Sensor DJ1 Modulates the Shelterin Complex and Telomere Length Regulation

Alan E Bilsland,Yu Liu,Andrew Turnbull,David Sumpton,Katrina Stevenson,Claire J Cairney,Susan M Boyd,Jon Roffey,David Jenkinson,W Nicol Keith

doi:10.1016/j.neo.2019.07.008

Alan E Bilsland, Yu Liu + Show 8 more

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https://doi.org/10.1016/j.neo.2019.07.008

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Abstract

Telomere signaling and metabolic dysfunction are hallmarks of cell aging. New agents targeting these processes might provide therapeutic opportunities, including chemoprevention strategies against cancer predisposition. We report identification and characterization of a pyrazolopyrimidine compound series identified from screens focused on cell immortality and whose targets are glycolytic kinase PGK1 and oxidative stress sensor DJ1. We performed structure–activity studies on the series to develop a photoaffinity probe to deconvolute the cellular targets. In vitro binding and structural analyses confirmed these targets, suggesting that PGK1/DJ1 interact, which we confirmed by immunoprecipitation. Glucose homeostasis and oxidative stress are linked to telomere signaling and exemplar compound CRT0063465 blocked hypoglycemic telomere shortening. Intriguingly, PGK1 and DJ1 bind to TRF2 and telomeric DNA. Compound treatment modulates these interactions and also affects Shelterin complex composition, while conferring cellular protection from cytotoxicity due to bleomycin and desferroxamine. These results demonstrate therapeutic potential of the compound series.

Highlights

Telomere attrition, genomic instability and cellular senescence are hallmarks of aging [1]
The telomere length regulation effects observed with hypoglycemia and CRT0063465 treatment may involve regulation of telomere binding factors. We investigated this hypothesis by co-immunoprecipitation and western blotting of the core shelterin factor TRF2 in HCT116 cells treated in the presence or absence of hypoglycemia and 10 nM CRT0063465
We investigated whether phosphoglycerate kinase 1 (PGK1) and DJ1 proteins are localized to telomeres by chromatin immunoprecipitation and telomere QPCR in cells treated in the presence or absence of hypoglycemia and CRT0063465

Summary

Introduction

Genomic instability and cellular senescence are hallmarks of aging [1]. In normal somatic cells the end replication problem, oxidative stress, and processing by nucleases cause progressive telomere shortening during each round of cell division, compromising protection at critically shortened telomeres causing DNA damage signaling and senescence [3]. The shelterin complex, comprising TRF1, TRF2, POT1, TIN2, TPP1, and RAP1, is the fundamental controller of telomere protection and its composition and activities have been extensively reviewed [4]. In aged normal cells with critically shortened telomeres these protective functions of shelterin are abrogated. Telomere dysfunction results from polymorphism or mutation of telomerase or shelterin components. These variants associate with cancer susceptibility, idiopathic pulmonary fibrosis, and several accelerated aging syndromes, most notably dyskeratosis congenita [5,6,7,8,9,10]. Restoring telomere protection is an important goal in aging research and cancer prevention

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