C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules (i.e. PROTACs). E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the >500 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-NRF2 activator bardoxolone to a BRD4 inhibitor JQ1. Notably, this work reports the first covalent, reversible E3 ligase recruiter for TPD applications.

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Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

10.26434/chemrxiv.12055935.v1 ◽

2020 ◽

Author(s):

Bingqi Tong ◽

Mai Luo ◽

Yi Xie ◽

Jessica Spradlin ◽

John A. Tallarico ◽

...

Keyword(s):

Drug Discovery ◽

Protein Binding ◽

Protein Degradation ◽

Small Molecules ◽

Small Molecule ◽

E3 Ligase ◽

E3 Ligases ◽

Protein Levels ◽

Nrf2 Activator

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules (i.e. PROTACs). E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the >500 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-NRF2 activator bardoxolone to a BRD4 inhibitor JQ1. Notably, this work reports the first covalent, reversible E3 ligase recruiter for TPD applications.

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Bardoxolone conjugation enables targeted protein degradation of BRD4

Scientific Reports ◽

10.1038/s41598-020-72491-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Bingqi Tong ◽

Mai Luo ◽

Yi Xie ◽

Jessica N. Spradlin ◽

John A. Tallarico ◽

...

Keyword(s):

Drug Discovery ◽

Protein Binding ◽

Protein Degradation ◽

Small Molecules ◽

Small Molecule ◽

E3 Ligase ◽

E3 Ligases ◽

Protein Levels ◽

Nrf2 Activator

Abstract Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

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PHOTACs Enable Optical Control of Protein Degradation

10.26434/chemrxiv.8206688 ◽

2019 ◽

Cited By ~ 2

Author(s):

Martin Reynders ◽

Bryan Matsuura ◽

Marleen Bérouti ◽

Daniele Simoneschi ◽

Antonio Marzio ◽

...

Keyword(s):

Protein Degradation ◽

E3 Ligase ◽

Optical Control ◽

Cellular Proteins ◽

Bifunctional Molecules ◽

Protein Levels ◽

Lead Compounds ◽

Subsequent Degradation ◽

Temporal And Spatial ◽

Precision Therapeutics

PROTACs (proteolysis targeting chimeras) are bifunctional molecules that tag proteins for ubiquitylation by an E3 ligase complex and subsequent degradation by the proteasome. They have emerged as powerful tools to control the levels of specific cellular proteins and are on the verge of being clinically used. We now introduce photoswitchable PROTACs that can be activated with the temporal and spatial precision that light provides. These trifunctional molecules, which we named PHOTACs, consist of a ligand for an E3 ligase, a photoswitch, and a ligand for a protein of interest. We demonstrate this concept by using PHOTACs that target either BET family proteins (BRD2,3,4) or FKBP12. Our lead compounds display little or no activity in the dark but can be reversibly activated to varying degrees with different wavelengths of light. Our modular and generalizable approach provides a method for the optical control of protein levels with photopharmacology and could lead to new types of precision therapeutics that avoid undesired systemic toxicity.

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Screening antiproliferative drug for breast cancer from bisbenzylisoquinoline alkaloid tetrandrine and fangchinoline derivatives by targeting BLM helicase

BMC Cancer ◽

10.1186/s12885-019-6146-7 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Wangming Zhang ◽

Shuang Yang ◽

Jinhe Liu ◽

Linchun Bao ◽

He Lu ◽

...

Keyword(s):

Breast Cancer ◽

Dna Binding ◽

Small Molecules ◽

Cancer Cells ◽

Small Molecule ◽

Breast Cancer Cells ◽

Dna Unwinding ◽

Protein Levels ◽

Blm Helicase ◽

Bisbenzylisoquinoline Alkaloids

Abstract Background The high expression of BLM (Bloom syndrome) helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to screen potential antiproliferative small molecules from 12 small molecules (the derivatives of bisbenzylisoquinoline alkaloids tetrandrine and fangchinoline) by targeting BLM642–1290 helicase. Then we explore the inhibitory mechanism of those small molecules on proliferation of MDA-MB-435 breast cancer cells. Methods Fluorescence polarization technique was used to screen small molecules which inhibited the DNA binding and unwinding of BLM642–1290 helicase. The effects of positive small molecules on the ATPase and conformation of BLM642–1290 helicase were studied by the malachite green-phosphate ammonium molybdate colorimetry and ultraviolet spectral scanning, respectively. The effects of positive small molecules on growth of MDA-MB-435 cells were studied by MTT method, colony formation and cell counting method. The mRNA and protein levels of BLM helicase in the MDA-MB-435 cells after positive small molecule treatments were examined by RT-PCR and ELISA, respectively. Results The compound HJNO (a tetrandrine derivative) was screened out which inhibited the DNA binding, unwinding and ATPase of BLM642–1290 helicase. That HJNO could bind BLM642–1290helicase to change its conformationcontribute to inhibiting the DNA binding, ATPase and DNA unwinding of BLM642–1290 helicase. In addition, HJNO showed its inhibiting the growth of MDA-MB-435 cells. The values of IC50 after drug treatments for 24 h, 48 h and 72 h were 19.9 μmol/L, 4.1 μmol/L and 10.9 μmol/L, respectively. The mRNA and protein levels of BLM helicase in MDA-MB-435 cells increased after HJNO treatment. Those showed a significant difference (P < 0.05) compared with negative control when the concentrations of HJNO were 5 μmol/L and 10 μmol/L, which might contribute to HJNO inhibiting the DNA binding, ATPase and DNA unwinding of BLM helicase. Conclusion The small molecule HJNO was screened out by targeting BLM642–1290 helicase. And it showed an inhibition on MDA-MB-435 breast cancer cells expansion.

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Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20246238 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6238 ◽

Cited By ~ 1

Author(s):

Anna L. Gill ◽

Monica Z. Wang ◽

Beth Levine ◽

Alan Premasiri ◽

Fernando G. Vieira

Keyword(s):

Motor Neurons ◽

Laser Scanning ◽

Neuronal Cell ◽

Fluorescein Isothiocyanate ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Primary Neurons ◽

Repeat Protein ◽

Neuronal Cell Lines ◽

Dipeptide Repeat

A repeat expansion mutation in the C9orf72 gene is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this study, using multiple cell-based assay systems, we reveal both increased dipeptide repeat protein (DRP) toxicity in primary neurons and in differentiated neuronal cell lines. Using flow cytometry and confocal laser scanning microscopy of cells treated with fluorescein isothiocyanate (FITC)-labeled DRPs, we confirm that poly-glycine-arginine (GR) and poly-proline-arginine (PR) DRPs entered cells more readily than poly-glycine-proline (GP) and poly-proline-alanine (PA) DRPs. Our findings suggest that the toxicity of C9-DRPs may be influenced by properties associated with differentiated and aging motor neurons. Further, our findings provide sensitive cell-based assay systems to test phenotypic rescue ability of potential interventions.

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MAP1B Interaction with the FW Domain of the Autophagic Receptor Nbr1 Facilitates Its Association to the Microtubule Network

International Journal of Cell Biology ◽

10.1155/2012/208014 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 12

Author(s):

Katie Marchbank ◽

Sarah Waters ◽

Roland G. Roberts ◽

Ellen Solomon ◽

Caroline A. Whitehouse

Keyword(s):

Protein Degradation ◽

Microtubule Network ◽

Protein Levels ◽

Ubiquitinated Proteins ◽

Autophagy Induction ◽

Evolutionarily Conserved ◽

Cargo Proteins ◽

Autophagic Degradation ◽

Membrane Bound ◽

Lysosomal Acidification

Selective autophagy is a process whereby specific targeted cargo proteins, aggregates, or organelles are sequestered into double-membrane-bound phagophores before fusion with the lysosome for protein degradation. It has been demonstrated that the microtubule network is important for the formation and movement of autophagosomes. Nbr1 is a selective cargo receptor that through its interaction with LC3 recruits ubiquitinated proteins for autophagic degradation. This study demonstrates an interaction between the evolutionarily conserved FW domain of Nbr1 with the microtubule-associated protein MAP1B. Upon autophagy induction, MAP1B localisation is focused into discrete vesicles with Nbr1. This colocalisation is dependent upon an intact microtubule network as depolymerisation by nocodazole treatment abolishes starvation-induced MAP1B recruitment to these vesicles. MAP1B is not recruited to autophagosomes for protein degradation as blockage of lysosomal acidification does not result in significant increased MAP1B protein levels. However, the protein levels of phosphorylated MAP1B are significantly increased upon blockage of autophagic degradation. This is the first evidence that links the ubiquitin receptor Nbr1, which shuttles ubiquitinated proteins to be degraded by autophagy, to the microtubule network.

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