Mass Spectrometry-Based Protein Footprinting Defines the Binding Pocket of Crotonylated H3K14 in the PHD1 Domain of BAF45D within the BAF Chromatin Remodeling Complex.

The BRG-/BRM-associated factor (BAF) chromatin remodeling complex is a central actor in transcription. One mechanism by which BAF affects gene expression is via its various histone mark readers, including double plant homeodomains (DPF), located in the BAF45D subunit. DPF domains recognize lysine acetyl and acylations, including crotonylation, localized at promoters and enhancers.

GNE-064: A Potent, Selective, and Orally Bioavailable Chemical Probe for the Bromodomains of SMARCA2 and SMARCA4 and the Fifth Bromodomain of PBRM1.

Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins.

GNE-371, a Potent and Selective Chemical Probe for the Second Bromodomains of Human Transcription-Initiation-Factor TFIID Subunit 1 and Transcription-Initiation-Factor TFIID Subunit 1-like.

The biological functions of the dual bromodomains of human transcription-initiation-factor TFIID subunit 1 (TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure-activity-relationship studies that ultimately led to our in vitro tool compound, 27 (GNE-371).

Quantitative Translational Analysis of Brain Kynurenic Acid Modulation via Irreversible Kynurenine Aminotransferase II Inhibition.

Kynurenic acid (KYNA) plays a significant role in maintaining normal brain function, and abnormalities in KYNA levels have been associated with various central nervous system disorders. Confirmation of its causality in human diseases requires safe and effective modulation of central KYNA levels in the clinic. The kynurenine aminotransferases (KAT) II enzyme represents an attractive target for pharmacologic modulation of central KYNA levels; however, KAT II and KYNA turnover kinetics, which could contribute to the duration of pharmacologic effect, have not been reported.

GNE-886: A Potent and Selective Inhibitor of the Cat Eye Syndrome Chromosome Region Candidate 2 Bromodomain (CECR2).

The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.

Inhibition of bromodomain-containing protein 9 for the prevention of epigenetically-defined drug resistance.

Bromodomain-containing protein 9 (BRD9), an epigenetic "reader" of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375).

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue.

Diving into the Water: Inducible Binding Conformations for BRD4, TAF1(2), BRD9, and CECR2 Bromodomains.

The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed.

Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637).

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

Regulatory T Cell Modulation by CBP/EP300 Bromodomain Inhibition.

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors.

Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma.

Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC.

PF-04859989 as a template for structure-based drug design: identification of new pyrazole series of irreversible KAT II inhibitors with improved lipophilic efficiency.

The structure-based design, synthesis, and biological evaluation of a new pyrazole series of irreversible KAT II inhibitors are described herein. The modification of the inhibitor scaffold of 1 and 2 from a dihydroquinolinone core to a tetrahydropyrazolopyridinone core led to discovery of a new series of potent KAT II inhibitors with excellent physicochemical properties. Compound 20 is the most potent and lipophilically efficient of these new pyrazole analogs, with a k(inact)/K(i) value of 112,000 M(-1)s(-1) and lipophilic efficiency (LipE) of 8.

Structure-Based Design of Irreversible Human KAT II Inhibitors: Discovery of New Potency-Enhancing Interactions.

A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The utilization of structure-activity relationships (SAR) in conjunction with X-ray crystallography led to the discovery of hydroxamate 4, a disubstituted analogue that has a significant potency enhancement due to a novel interaction with KAT II. The use of k inact/K i to assess potency was critical for understanding the SAR in this series and for identifying compounds with improved pharmacodynamic profiles.

Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer.

Discovery of Brain-Penetrant, Irreversible Kynurenine Aminotransferase II Inhibitors for Schizophrenia.

Kynurenine aminotransferase (KAT) II has been identified as a potential new target for the treatment of cognitive impairment associated with schizophrenia and other psychiatric disorders. Following a high-throughput screen, cyclic hydroxamic acid PF-04859989 was identified as a potent and selective inhibitor of human and rat KAT II. An X-ray crystal structure and (13)C NMR studies of PF-04859989 bound to KAT II have demonstrated that this compound forms a covalent adduct with the enzyme cofactor, pyridoxal phosphate (PLP), in the active site.

Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 (CK1) enzymes.

Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) epsilon or delta can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1epsilon and CK1delta using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking.

High-throughput screening in embryonic stem cell-derived neurons identifies potentiators of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type glutamate receptors.

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells.

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes.

Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology.

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively.

Targeting the MraY and MurG bacterial enzymes for antimicrobial therapeutic intervention.

Assays for two enzymes from Escherichia coli were developed and validated as antibacterial inhibitor screens. The MraY and MurG enzymes were overexpressed and purified as the membrane fraction or to homogeneity, respectively. The MurG enzyme was expressed with a six-histidine tag using an optimized minimal-medium protocol for subsequent purification.