ABSTRACT
The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.
Subject(s)
Administration, Intravenous , Animals , Administration, Oral , Mice , Structure-Activity Relationship , Humans , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Molecular StructureABSTRACT
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
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
Subject(s)
Benzimidazoles/pharmacology , Enzyme Inhibitors/pharmacology , Hydrolases/antagonists & inhibitors , Neutrophils/drug effects , Animals , Benzimidazoles/chemical synthesis , Binding, Competitive , Calcium/metabolism , Citrulline/metabolism , Enzyme Inhibitors/chemical synthesis , HEK293 Cells , Histones/metabolism , Humans , In Vitro Techniques , Mice , Models, Molecular , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Small Molecule Libraries , Substrate SpecificityABSTRACT
Protein sulfenic acids (SOHs) are the principal oxidation products formed when redox active proteins interact with peroxide molecules. We have developed a new antibody reagent that detects protein SOHs derivatized with dimedone. Using this new antibody, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is the predominant protein sulfenate present in isolated rat ventricular myocytes under basal conditions. During oxidative stress with hydrogen peroxide (H(2)O(2)), GAPDH SOH labeling is lost, but a number of secondary dimedone-reactive protein sulfenates then appear. As the sulfenate labeling is lost, the Cys-149 sulfinic/sulfonic acid oxidation states of GAPDH appear. This hyperoxidized GAPDH is associated with both the inhibition of glycolysis and its ability to reduce H(2)O(2). We examined whether inactivation of GAPDH was causative in the generation of secondary protein sulfenates that coincide with its hyperoxidation. The selective GAPDH inhibitor koningic acid (which functions by forming a covalent adduct at Cys-149) fully prevented basal SOH labeling, as well as subsequent peroxide-induced hyperoxidation. However, koningic acid-mediated inhibition of GAPDH alone did not induce the formation of intracellular H(2)O(2) or secondary protein sulfenates and also failed to potentiate their peroxide-induced formation. Overall, GAPDH appears to have peroxidase-like properties, but its inhibition failed to impact on downstream oxidant signaling involving secondary protein sulfenation.