Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

Summary

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

Abstract

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN‘s endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

Introduction

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all approved for the treatment of multiple myeloma, bind to the E3 ubiquitin ligase cereblon (CRBN), a substrate adaptor for cullin4A-RING E3 ubiquitin ligase (CRL4^CRBN)^1,2,3. Binding of IMiDs enhances the affinity of CRL4^CRBN to the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), leading to their ubiquitination and degradation (Figure 1)^4,5,....

Protocol

1. Preparation of PROTAC molecules

CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in these syntheses are toxic and carcinogenic. Please use all appropriate safety practices and personal protective equipment.

1. Preparation of tert-butyl N-(2,6-dioxo-3-piperidyl)carbamate (compound 1)
   1. Add 1,1'-carbonyldiimidazole (1.95 g, 12 mmol) and a catalytic amount of 4-(dimethylam.......

Discussion

The design of such homo-PROTACs as described here for CRBN relies on the specific affinity of pomalidomide to CRBN, which has been successfully utilized in numerous heterobifunctional PROTACs and resulted in the development of PROTAC 8 as a highly selective CRBN degrader. The specificity of our molecule has already been confirmed by proteomic analyses²⁴. For genetically mediated knockout, exclusion and validation of side effects is challenging and time consuming. In addition, a ch.......

Materials

Name	Company	Catalog Number	Comments
1,1'-Carbonyldiimidazole	TCI chemicals	C0119
2,2′-(Ethylenedioxy)-bis(ethylamine)	Sigma-Aldrich	385506	Compound 6
2-Mercaptoethanol	Sigma-Aldrich	M6250
3-Fluorophthalic anhydride, 98 %	Alfa Aesar	A12275
4-Dimethylaminopyridine, 99 %	Acros	148270250	Toxic
Acrylamidstammlösung/ Bisacrylamid (30%/0,8%)	Carl Roth	3029.1
Aiolos (D1C1E) mAB	Cell signaling	15103S
Anti-CRBN antibody produced in rabbit	Sigma	HPA045910
Anti-rabbit IgG HRP-linked antibody	Sigma	7074S
Ammonium Persulfate	Roth	9592.2
Boc-Gln-OH	TCI chemicals	B1649
Bovine Serum Albumin	Sigma-Aldrich	A7906-100G
CellTiter-Glo Luminescent Cell Viability Assay	Promega	G7571
ChemiDoc XRS+	Bio-Rad	1708265
DMF, anhydrous, 99.8 %	Acros	348435000	Extra Dry over Molecular Sieve
DMSO, anhydrous, 99.7 %	Acros	348445000	Extra Dry over Molecular Sieve
Glycine	Sigma-Aldrich	15523-1L-R
Goat anti-mouse (HRP conjugated)	Santa Cruz biotechnology	sc-2005
Halt Protease & Phosphatase Inhibitor Single-use Cocktail (100X)	Thermo Scientific	1861280
Ikaros (D6N9Y) Mab	Cell signaling	14859S
ImmobilonP Transfer Membrane (0,45µm)	Merck	IPVH000010
Iodomethane, 99 %	Sigma-Aldrich	I8507	Highly toxic
Methanol	Sigma-Aldrich	32213-2.5L
Mg132	Selleckchem	S2619
Mini Trans-Blot electrophoretic transfer cell	Bio-Rad	1703930
Mini-PROTEAN Tetra Vertical Electrophoresis Cell	Bio-Rad	1658004
MLN4942	biomol (cayman)	Cay15217-1
Monoclonal Anti-α-Tubulin antibody produced in mouse (B512)	Sigma	T5168
N-Ethyldiisopropylamine, 99 %	Alfa Aesar	A11801
Nonfat dried milk powder	PanReac AppliChem	A0830,0500
Nunc F96 MicroWell White Polystyrene Plate	Thermo Scientific	136101
NuPAGE LDS Sample Buffer (4X)	Thermo Scientific	NP0008
Pierce BCA Protein Assay kit	Thermo Scientific	23225
Pomalidomide	Selleckchem	S1567
RestoreTM Western Blot Stripping Buffer	Thermo Scientific	46430
sodium dodecyl sulfate	Carl Roth	183.1
Sodium Chloride	Sigma-Aldrich	A9539-500g
TEMED	Carl Roth	2367.3
tert-Butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate	Sigma-Aldrich	89761	Compound 5
Tricin	Carl Roth	6977.4
Trizma base	Sigma-Aldrich	T1503-1kg
Tween-20	Sigma-Aldrich	P7949-500ml
WesternBright ECL spray	Advansta	K-12049-D50