Structure- and Property-Based Optimization of Efficient Pan-Bromodomain and Extra Terminal Inhibitors to Identify Oral and Intravenous Candidate I-BET787.

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.

Development of an intracellular quantitative assay to measure compound binding kinetics.

Contemporary drug discovery typically quantifies the effect of a molecule on a biological target using the equilibrium-derived measurements of IC50, EC50, or KD. Kinetic descriptors of drug binding are frequently linked with the effectiveness of a molecule in modulating a disease phenotype; however, these parameters are yet to be fully adopted in early drug discovery. Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) can be used to measure interactions between fluorophore-conjugated probes and luciferase fused target proteins. Here, we describe an intracellular NanoBRET competition assay that can be used to quantify cellular kinetic rates of compound binding to nanoluciferase-fused bromodomain and extra-terminal (BET) proteins. Comparative rates are generated using a cell-free NanoBRET assay and by utilizing orthogonal recombinant protein-based methodologies. A screen of known pan-BET inhibitors is used to demonstrate the value of this approach in the investigation of kinetic selectivity between closely related proteins.

Fragment-based Scaffold Hopping: Identification of Potent, Selective, and Highly Soluble Bromo and Extra Terminal Domain (BET) Second Bromodomain (BD2) Inhibitors.

The profound efficacy of pan-BET inhibitors is well documented, but these epigenetic agents have shown pharmacology-driven toxicity in oncology clinical trials. The opportunity to identify inhibitors with an improved safety profile by selective targeting of a subset of the eight bromodomains of the BET family has triggered extensive medicinal chemistry efforts. In this article, we disclose the identification of potent and selective drug-like pan-BD2 inhibitors such as pyrazole 23 (GSK809) and furan 24 (GSK743) that were derived from the pyrrole fragment 6. We transpose the key learnings from a previous pyridone series (GSK620 2 as a representative example) to this novel class of inhibitors, which are characterized by significantly improved solubility relative to our previous research.

Identification of a Series of N-Methylpyridine-2-carboxamides as Potent and Selective Inhibitors of the Second Bromodomain (BD2) of the Bromo and Extra Terminal Domain (BET) Proteins.

Domain-specific BET bromodomain ligands represent an attractive target for drug discovery with the potential to unlock the therapeutic benefits of antagonizing these proteins without eliciting the toxicological aspects seen with pan-BET inhibitors. While we have reported several distinct classes of BD2 selective compounds, namely, GSK620, GSK549, and GSK046, only GSK046 shows high aqueous solubility. Herein, we describe the lead optimization of a further class of highly soluble compounds based upon a picolinamide chemotype. Focusing on achieving >1000-fold selectivity for BD2 over BD1 ,while retaining favorable physical chemical properties, compound 36 was identified as being 2000-fold selective for BD2 over BD1 (Brd4 data) with >1 mg/mL solubility in FaSSIF media. 36 represents a valuable new in vivo ready molecule for the exploration of the BD2 phenotype.

Discovery of a Highly Selective BET BD2 Inhibitor from a DNA-Encoded Library Technology Screening Hit.

Second-generation bromodomain and extra terminal (BET) inhibitors, which selectively target one of the two bromodomains in the BET proteins, have begun to emerge in the literature. These inhibitors aim to help determine the roles and functions of each domain and assess whether they can demonstrate an improved safety profile in clinical settings compared to pan-BET inhibitors. Herein, we describe the discovery of a novel BET BD2-selective chemotype using a structure-based drug design from a hit identified by DNA-encoded library technologies, showing a structural differentiation from key previously reported greater than 100-fold BD2-selective chemotypes GSK620, GSK046, and ABBV-744. Following a structure-based hypothesis for the selectivity and optimization of the physicochemical properties of the series, we identified 60 (GSK040), an in vitro ready and in vivo capable BET BD2-inhibitor of unprecedented selectivity (5000-fold) against BET BD1, excellent selectivity against other bromodomains, and good physicochemical properties. This novel chemical probe can be added to the toolbox used in the advancement of epigenetics research.

GSK137, a potent small-molecule BCL6 inhibitor with in vivo activity, suppresses antibody responses in mice.

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB-POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB-POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB-POZ domain. The compound has good solubility (128 µg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

Reducing False Positives through the Application of Fluorescence Lifetime Technology: A Comparative Study Using TYK2 Kinase as a Model System.

The predominant assay detection methodologies used for enzyme inhibitor identification during early-stage drug discovery are fluorescence-based. Each fluorophore has a characteristic fluorescence decay, known as the fluorescence lifetime, that occurs throughout a nanosecond-to-millisecond timescale. The measurement of fluorescence lifetime as a reporter for biological activity is less common than fluorescence intensity, even though the latter has numerous issues that can lead to false-positive readouts. The confirmation of hit compounds as true inhibitors requires additional assays, cost, and time to progress from hit identification to lead drug-candidate optimization. To explore whether the use of fluorescence lifetime technology (FLT) can offer comparable benefits to label-free-based approaches such as RapidFire mass spectroscopy (RF-MS) and a superior readout compared to time-resolved fluorescence resonance energy transfer (TR-FRET), three equivalent assays were developed against the clinically validated tyrosine kinase 2 (TYK2) and screened against annotated compound sets. FLT provided a marked decrease in the number of false-positive hits when compared to TR-FRET. Further cellular screening confirmed that a number of potential inhibitors directly interacted with TYK2 and inhibited the downstream phosphorylation of the signal transducer and activator of transcription 4 protein (STAT4).

Template-Hopping Approach Leads to Potent, Selective, and Highly Soluble Bromo and Extraterminal Domain (BET) Second Bromodomain (BD2) Inhibitors.

A number of reports have recently been published describing the discovery and optimization of bromo and extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 (3) and GSK620 (5). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds 28 (GSK452), 39 (GSK737), and 36 (GSK217).

GSK973 Is an Inhibitor of the Second Bromodomains (BD2s) of the Bromodomain and Extra-Terminal (BET) Family.

Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.

The Optimization of a Novel, Weak Bromo and Extra Terminal Domain (BET) Bromodomain Fragment Ligand to a Potent and Selective Second Bromodomain (BD2) Inhibitor.

The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (15, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, 20 (GSK620) and 56 (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.

Cellular Target Engagement Approaches to Monitor Epigenetic Reader Domain Interactions.

Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling are implicated in a number of cancers and immunological and neurodegenerative conditions. Within GlaxoSmithKline (GSK) we have utilized a number of variations of the NanoBRET technology for the direct measurement of compound-target engagement within native cellular environments to drive high-throughput, routine structure-activity relationship (SAR) profiling across differing epigenetic targets. NanoBRET is a variation of the bioluminescence resonance energy transfer (BRET) methodology utilizing proteins of interest fused to either NanoLuc, a small, high-emission-intensity luciferase, or HaloTag, a modified dehalogenase enzyme that can be selectively labeled with a fluorophore. The combination of these two technologies has enabled the application of NanoBRET to biological systems such as epigenetic protein-protein interactions, which have previously been challenging. By synergizing target engagement assays with more complex primary cell phenotypic assays, we have been able to demonstrate compound-target selectivity profiles to enhance cellular potency and offset potential liability risks. Additionally, we have shown that in the absence of a robust, cell phenotypic assay, it is possible to utilize NanoBRET target engagement assays to aid chemistry in progressing at a higher scale than would have otherwise been achievable. The NanoBRET target engagement assays utilized have further shown an excellent correlation with more reductionist biochemical and biophysical assay systems, clearly demonstrating the possibility of using such assay systems at scale, in tandem with, or in preference to, lower-throughput cell phenotypic approaches.

A Photoaffinity Displacement Assay and Probes to Study the Cyclin-Dependent Kinase Family.

The CDK family plays a crucial role in the control of the cell cycle. Dysregulation and mutation of the CDKs has been implicated in cancer and the CDKs have been investigated extensively as potential therapeutic targets. Selective inhibition of specific isoforms of the CDKs is crucial to achieve therapeutic effect while minimising toxicity. We present a group of photoaffinity probes designed to bind to the family of CDKs. The site of crosslinking of the optimised probe, as well as its ability to enrich members of the CDK family from cell lysates, was investigated. In a proof of concept study, we subsequently developed a photoaffinity probe-based competition assay to profile CDK inhibitors. We anticipate that this approach will be widely applicable to the study of small molecule binding to protein families of interest.