ABSTRACT
P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAF-deficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multidomain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.
Subject(s)
Benzoates/pharmacology , Piperidines/pharmacology , Pyridazines/pharmacology , p300-CBP Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing , Animals , Benzoates/chemical synthesis , Benzoates/chemistry , Cell Differentiation/drug effects , Cytokines/metabolism , Dendritic Cells/metabolism , Humans , Inflammation/chemically induced , Inflammation/metabolism , Lipopolysaccharides , Macrophages/metabolism , Mice , Monocytes/metabolism , Peptide Hydrolases/metabolism , Piperidines/chemical synthesis , Piperidines/chemistry , Protein Domains , Proteolysis , Pyridazines/chemical synthesis , Pyridazines/chemistry , Stereoisomerism , Ubiquitin-Protein Ligases , p300-CBP Transcription Factors/chemistryABSTRACT
High-throughput screening (HTS) hits include compounds with undesirable properties. Many filters have been described to identify such hits. Notably, pan-assay interference compounds (PAINS) has been adopted by the community as the standard term to refer to such filters, and very useful guidelines have been adopted by the American Chemical Society (ACS) and subsequently triggered a healthy scientific debate about the pitfalls of draconian use of filters. Using an inhibitory frequency index, we have analyzed in detail the promiscuity profile of the whole GlaxoSmithKline (GSK) HTS collection comprising more than 2 million unique compounds that have been tested in hundreds of screening assays. We provide a comprehensive analysis of many previously published filters and newly described classes of nuisance structures that may serve as a useful source of empirical information to guide the design or growth of HTS collections and hit triaging strategies.
Subject(s)
Drug Discovery/methods , High-Throughput Screening Assays/methods , Small Molecule Libraries/chemistry , Biological Assay/methodsABSTRACT
The correct folding of proteins is a fundamental process in the normal physiological functioning of cells, and is mediated by cellular chaperones including members of the Hsp70 family. Many diseases are caused by a failure of cellular chaperones to adequately maintain correct protein folding, and has led to the development of a therapeutic strategy to upregulate the activity of cellular chaperones in order to ameliorate intrinsic folding deficits. A large range of pharmacological agents that can induce cellular chaperones and correct deficits associated with misfolded proteins are known. This review surveys the mechanisms and compounds that have been used to modulate cellular chaperones, and discusses the continuing challenges in translating this approach into clinical improvements in the treatment of protein misfolding disorders.
