Dataset Information

Development of a small molecule that corrects misfolding and increases secretion of Z ?₁ -antitrypsin.

ABSTRACT: Severe ?₁ -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant ?₁ -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z ?₁ -antitrypsin. The lead compound blocks Z ?₁ -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z ?₁ -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z ?₁ -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z ?₁ -antitrypsin deficiency.

SUBMITTER: Lomas DA

PROVIDER: S-EPMC7933930 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Development of a small molecule that corrects misfolding and increases secretion of Z α1 -antitrypsin.

Lomas David A DA Irving James A JA Arico-Muendel Christopher C Belyanskaya Svetlana S Brewster Andrew A Brown Murray M Chung Chun-Wa CW Dave Hitesh H Denis Alexis A Dodic Nerina N Dossang Anthony A Eddershaw Peter P Klimaszewska Diana D Haq Imran I Holmes Duncan S DS Hutchinson Jonathan P JP Jagger Alistair M AM Jakhria Toral T Jigorel Emilie E Liddle John J Lind Ken K Marciniak Stefan J SJ Messer Jeff J Neu Margaret M Olszewski Allison A Ordonez Adriana A Ronzoni Riccardo R Rowedder James J Rüdiger Martin M Skinner Steve S Smith Kathrine J KJ Terry Rebecca R Trottet Lionel L Uings Iain I Wilson Steve S Zhu Zhengrong Z Pearce Andrew C AC

EMBO molecular medicine 20210129 3

Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduc ...[more]

PMID: 33512066

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Project description:Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP) and interleukin-6 (IL-6) via effects on Smad4 or Stat3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone genistein from 28−52 hours post-fertilization in zebrafish embryos enhanced Hepcidin transcript levels as assessed by whole mount in situ hybridization and quantitative realtime RT-PCR. Genistein’s stimulatory effect was conserved in human hepatocytes: genistein treatment of HepG2 cells increased both Hepcidin transcript levels and Hepcidin promoter activity. We found that genistein’s effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the BMP response elements or the Stat3 binding site in the Hepcidin promoter. RNA-sequencing of transcripts from genistein-treated hepatocytes indicated that genistein upregulated 68% of the transcripts that were upregulated by BMP6, however genistein raised the levels of several transcripts involved in Stat3 signaling that were not upregulated by BMP6. Chromatin-immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. CONCLUSION: Genistein is the first small molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. RNA-seq of HepG2 cells treated with DMSO 1%, BMP6 50 ng/ml, or genistein 10 micromolar. The numbers of biological replicates were 3, 2, and 3.

Project description:UnlabelledHepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells.ConclusionGenistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.

Dataset Information

Development of a small molecule that corrects misfolding and increases secretion of Z ?₁ -antitrypsin.

Publications

Development of a small molecule that corrects misfolding and increases secretion of Z α<sub>1</sub> -antitrypsin.

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Dataset Information

Development of a small molecule that corrects misfolding and increases secretion of Z ?1 -antitrypsin.

Publications

Development of a small molecule that corrects misfolding and increases secretion of Z α<sub>1</sub> -antitrypsin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Development of a small molecule that corrects misfolding and increases secretion of Z ?₁ -antitrypsin.