ABSTRACT
Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that co-opt the cell's natural proteasomal degradation mechanisms to degrade undesired proteins. A challenge associated with PROTACs is the time and resource-intensive optimization; thus, the development of high-throughput platforms for their synthesis and biological evaluation is required. In this study, we establish an ultra-high-throughput experimentation (ultraHTE) platform for PROTAC synthesis, followed by direct addition of the crude reaction mixtures to cellular degradation assays without any purification. This 'direct-to-biology' (D2B) approach was validated and then exemplified in a medicinal chemistry campaign to identify novel BRD4 PROTACs. Using the D2B platform, the synthesis of 650 PROTACs was carried out in a 1536-well plate, and subsequent biological evaluation was performed by a single scientist in less than 1 month. Due to its ability to hugely accelerate the optimization of new degraders, we anticipate our platform will transform the synthesis and testing of PROTACs.
Subject(s)
Nuclear Proteins , Proteolysis Targeting Chimera , Transcription Factors , Biological Assay , Biology , Proteolysis , Ubiquitin-Protein LigasesABSTRACT
Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96â h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology inâ vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Focal Adhesion Kinase 1/antagonists & inhibitors , Proteolysis/drug effects , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Benzamides/chemistry , Benzamides/pharmacokinetics , Benzamides/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Dipeptides/chemistry , Dipeptides/pharmacokinetics , Dipeptides/pharmacology , Focal Adhesion Kinase 1/metabolism , Humans , Mice , Molecular Structure , Ubiquitin-Protein Ligases/metabolismABSTRACT
The bromodomain and extra-terminal domain (BET) family of proteins bind acetylated lysine residues on histone proteins. The four BET bromodomains-BRD2, BRD3, BRD4, and BRDT-each contain two bromodomain modules. BET bromodomain inhibition is a potential therapy for various cancers and immunoinflammatory diseases, but few reported inhibitors show selectivity within the BET family. Inhibitors with selectivity for the first or second bromodomain are desired to aid investigation of the biological function of these domains. Focused library screening identified a series of tetrahydroquinoxalines with selectivity for the second bromodomains of the BET family (BD2). Structure-guided optimization of the template improved potency, selectivity, and physicochemical properties, culminating in potent BET inhibitors with BD2 selectivity.
Subject(s)
Drug Discovery , Nuclear Proteins/antagonists & inhibitors , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinoxalines/chemistry , Quinoxalines/pharmacology , Transcription Factors/antagonists & inhibitors , Amino Acid Sequence , Binding Sites , Cell Cycle Proteins , Humans , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , Protein Conformation , Quantitative Structure-Activity Relationship , Sequence HomologyABSTRACT
The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.
ABSTRACT
The Chan-Evans-Lam reaction is a valuable C-N bond forming process. However, aryl boronic acid pinacol (BPin) ester reagents can be difficult coupling partners that often deliver low yields, in particular in reactions with aryl amines. Herein, we report effective reaction conditions for the Chan-Evans-Lam amination of aryl BPin with alkyl and aryl amines. A mixed MeCN/EtOH solvent system was found to enable effective C-N bond formation using aryl amines while EtOH is not required for the coupling of alkyl amines.
ABSTRACT
A modular synthesis of functionalised biaryl phenols from two boronic acid derivatives has been developed via one-pot Suzuki-Miyaura cross-coupling, chemoselective control of boron solution speciation to generate a reactive boronic ester in situ, and oxidation. The utility of this method has been further demonstrated by application in the synthesis of drug molecules and components of organic electronics, as well as within iterative cross-coupling.
