Structure-activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal.

The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR+ CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (KM) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar KM of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2200-fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity.

A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells.

The Kinetic Intra-Cellular Assay (KICA) is a recombinant cell-based technique that utilizes NanoBRET technology. KICA enables the measurement of intracellular binding kinetics. This protocol describes steps for cellular transfection and expression, followed by addition of a target specific fluorophore conjugated probe and a range of concentrations of competitor compounds, followed by the measurement of BRET in a 384 well format. Fitting the BRET data allows measurement of forward and reverse binding rates and the determination of KD. For complete details on the use and execution of this profile, please refer to Lay et al. (2021).

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.

Design and Synthesis of a Highly Selective and In Vivo-Capable Inhibitor of the Second Bromodomain of the Bromodomain and Extra Terminal Domain Family of Proteins.

Pan-bromodomain and extra terminal domain (BET) inhibitors interact equipotently with the eight bromodomains of the BET family of proteins and have shown profound efficacy in a number of in vitro phenotypic assays and in vivo pre-clinical models in inflammation or oncology. A number of these inhibitors have progressed to the clinic where pharmacology-driven adverse events have been reported. To better understand the contribution of each domain to their efficacy and improve their safety profile, selective inhibitors are required. This article discloses the profile of GSK046, also known as iBET-BD2, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive pre-clinical in vitro and in vivo characterization.

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.

1-Aryl-3,4-dihydroisoquinoline inhibitors of JNK3.

A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38alpha and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.

A Qualified Success: Discovery of a New Series of ATAD2 Bromodomain Inhibitors with a Novel Binding Mode Using High-Throughput Screening and Hit Qualification.

The bromodomain of ATAD2 has proved to be one of the least-tractable proteins within this target class. Here, we describe the discovery of a new class of inhibitors by high-throughput screening and show how the difficulties encountered in establishing a screening triage capable of finding progressible hits were overcome by data-driven optimization. Despite the prevalence of nonspecific hits and an exceptionally low progressible hit rate (0.001%), our optimized hit qualification strategy employing orthogonal biophysical methods enabled us to identify a single active series. The compounds have a novel ATAD2 binding mode with noncanonical features including the displacement of all conserved water molecules within the active site and a halogen-bonding interaction. In addition to reporting this new series and preliminary structure-activity relationship, we demonstrate the value of diversity screening to complement the knowledge-based approach used in our previous ATAD2 work. We also exemplify tactics that can increase the chance of success when seeking new chemical starting points for novel and less-tractable targets.

GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain.

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.

Structure-Based Optimization of Naphthyridones into Potent ATAD2 Bromodomain Inhibitors.

ATAD2 is a bromodomain-containing protein whose overexpression is linked to poor outcomes in a number of different cancer types. To date, no potent and selective inhibitors of the bromodomain have been reported. This article describes the structure-based optimization of a series of naphthyridones from micromolar leads with no selectivity over the BET bromodomains to inhibitors with sub-100 nM ATAD2 potency and 100-fold BET selectivity.

Fragment-Based Discovery of Low-Micromolar ATAD2 Bromodomain Inhibitors.

Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.

1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain.

The BRPF (bromodomain and PHD finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. Here, we report the discovery, binding mode, and structure-activity relationship (SAR) of the first potent, selective series of inhibitors of the BRPF1 bromodomain.

Discovery and characterization of small molecule inhibitors of the BET family bromodomains.

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.