Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140.

BACKGROUND: SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn's disease (CD), suggesting a role in inflammation. RESULTS: We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa. CONCLUSIONS: This study identifies SP140 as a druggable epigenetic therapeutic target for CD.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening condition that has become a global issue. Current treatment is limited to two medicines that require prolonged dosing and are associated with multiple side effects, which often lead to treatment discontinuation and failure. One way to address these shortcomings is through target-based drug discovery on validated T. cruzi protein targets. One such target is the proteasome, which plays a crucial role in protein degradation and turnover through chymotrypsin-, trypsin-, and caspase-like catalytic activities. In order to initiate a proteasome drug discovery program, we isolated proteasomes from T. cruzi epimastigotes and characterized their activity using a commercially available glow-like luminescence-based assay. We developed a high-throughput biochemical assay for the chymotrypsin-like activity of the T. cruzi proteasome, which was found to be sensitive, specific, and robust but prone to luminescence technology interference. To mitigate this, we also developed a counterscreen assay that identifies potential interferers at the levels of both the luciferase enzyme reporter and the mechanism responsible for a glow-like response. Interestingly, we also found that the peptide substrate for chymotrypsin-like proteasome activity was not specific and was likely partially turned over by other catalytic sites of the protein. Finally, we utilized these biochemical tools to screen 18,098 compounds, exploring diverse drug-like chemical space, which allowed us to identify 39 hits that were active in the primary screening assay and inactive in the counterscreen assay.

The Mechanism of Acetyl Transfer Catalyzed by Mycobacterium tuberculosis GlmU.

The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridylyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP- N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine 1-phosphate, acetyl-coenzyme A, and uridine 5'-triphosphate. GlmU utilizes ternary complex formation to transfer an acetyl from acetyl-coenzyme A to glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl-coenzyme A binding first, which triggers a conformational change in GlmU, followed by glucosamine 1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.

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.

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.

Use of NanoBiT and NanoBRET to characterise interleukin-23 receptor dimer formation in living cells.

BACKGROUND AND PURPOSE: Interleukin-23 (IL-23) and its receptor are important drug targets for the treatment of auto-inflammatory diseases. IL-23 binds to a receptor complex composed of two single transmembrane spanning proteins IL23R and IL12Rß1. In this study, we aimed to gain further understanding of how ligand binding induces signalling of IL-23 receptor complexes using the proximity-based techniques of NanoLuc Binary Technology (NanoBiT) and Bioluminescence Resonance Energy Transfer (BRET). EXPERIMENTAL APPROACH: To monitor the formation of IL-23 receptor complexes, we developed a split luciferase (NanoBiT) assay whereby heteromerisation of receptor subunits can be measured through luminescence. The affinity of NanoBiT complemented complexes for IL-23 was measured using NanoBRET, and cytokine-induced signal transduction was measured using a phospho-STAT3 AlphaLISA assay. KEY RESULTS: NanoBiT measurements demonstrated that IL-23 receptor complexes formed to an equal degree in the presence and absence of ligand. NanoBRET measurements confirmed that these complexes bound IL-23 with a picomolar binding affinity. Measurement of STAT3 phosphorylation demonstrated that pre-formed IL-23 receptor complexes induced signalling following ligand binding. It was also demonstrated that synthetic ligand-independent signalling could be induced by high affinity (HiBit) but not low affinity (SmBit) NanoBiT crosslinking of the receptor N-terminal domains. CONCLUSIONS AND IMPLICATIONS: These results indicate that receptor complexes form prior to ligand binding and are not sufficient to induce signalling alone. Our findings indicate that IL-23 induces a conformational change in heteromeric receptor complexes, to enable signal transduction. These observations have direct implications for drug discovery efforts to target the IL-23 receptor.

Characterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes.

Association of single nucleotide polymorphisms in the IL-23 receptor with several auto-inflammatory diseases, led to the heterodimeric receptor and its cytokine-ligand IL-23, becoming important drug targets. Successful antibody-based therapies directed against the cytokine have been licenced and a class of small peptide antagonists of the receptor have entered clinical trials. These peptide antagonists may offer therapeutic advantages over existing anti-IL-23 therapies, but little is known about their molecular pharmacology. In this study, we use a fluorescent version of IL-23 to characterise antagonists of the full-length receptor expressed by living cells using a NanoBRET competition assay. We then develop a cyclic peptide fluorescent probe, specific to the IL23p19:IL23R interface and use this molecule to characterise further receptor antagonists. Finally, we use the assays to study the immunocompromising C115Y IL23R mutation, demonstrating that the mechanism of action is a disruption of the binding epitope for IL23p19.

Structure-Guided Design of a Domain-Selective Bromodomain and Extra Terminal N-Terminal Bromodomain Chemical Probe.

Small-molecule-mediated disruption of the protein-protein interactions between acetylated histone tails and the tandem bromodomains of the bromodomain and extra-terminal (BET) family of proteins is an important mechanism of action for the potential modulation of immuno-inflammatory and oncology disease. High-quality chemical probes have proven invaluable in elucidating profound BET bromodomain biology, with seminal publications of both pan- and domain-selective BET family bromodomain inhibitors enabling academic and industrial research. To enrich the toolbox of structurally differentiated N-terminal bromodomain (BD1) BET family chemical probes, this work describes an analysis of the GSK BRD4 bromodomain data set through a lipophilic efficiency lens, which enabled identification of a BD1 domain-biased benzimidazole series. Structure-guided growth targeting a key Asp/His BD1/BD2 switch enabled delivery of GSK023, a high-quality chemical probe with 300-1000-fold BET BD1 domain selectivity and a phenotypic cellular fingerprint consistent with BET bromodomain inhibition.

Structure-Guided Design and Synthesis of a Pyridazinone Series of Trypanosoma cruzi Proteasome Inhibitors.

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.

Bottlenecks and opportunities in antibiotic discovery against Mycobacterium tuberculosis.

Tuberculosis (TB) persists as a major global health issue and a leading cause of death by a single infectious agent. The global burden of TB is further exacerbated by the continuing emergence and dissemination of strains of Mycobacterium tuberculosis resistant to multiple antibiotics. The need for novel drugs that can be used to shorten the course for current TB drug regimens as well as combat the persistent threat of antibiotic resistance has never been greater. There have been significant advances in the discovery of de novo TB treatments, with the first TB-specific drugs in 45 years approved for use. However, there are still issues that restrict the pipeline of new antitubercular chemotherapies. The rate of failure of TB drug candidates in clinical trials remains high, while the validation of new TB drug targets and subsequent identification of novel inhibitors remains modest.

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).

Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET.

Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rß1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rß1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure.

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.

Identification and Optimization of a Ligand-Efficient Benzoazepinone Bromodomain and Extra Terminal (BET) Family Acetyl-Lysine Mimetic into the Oral Candidate Quality Molecule I-BET432.

The bromodomain and extra terminal (BET) family of proteins are an integral part of human epigenome regulation, the dysregulation of which is implicated in multiple oncology and inflammatory diseases. Disrupting the BET family bromodomain acetyl-lysine (KAc) histone protein-protein interaction with small-molecule KAc mimetics has proven to be a disease-relevant mechanism of action, and multiple molecules are currently undergoing oncology clinical trials. This work describes an efficiency analysis of published GSK pan-BET bromodomain inhibitors, which drove a strategic choice to focus on the identification of a ligand-efficient KAc mimetic with the hypothesis that lipophilic efficiency could be drastically improved during optimization. This focus drove the discovery of the highly ligand-efficient and structurally distinct benzoazepinone KAc mimetic. Following crystallography to identify suitable growth vectors, the benzoazepinone core was optimized through an explore-exploit structure-activity relationship (SAR) approach while carefully monitoring lipophilic efficiency to deliver I-BET432 (41) as an oral candidate quality molecule.

Design, Synthesis, and Characterization of I-BET567, a Pan-Bromodomain and Extra Terminal (BET) Bromodomain Oral Candidate.

Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.

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).

Optimization of Naphthyridones into Selective TATA-Binding Protein Associated Factor 1 (TAF1) Bromodomain Inhibitors.

Bromodomain containing proteins and the acetyl-lysine binding bromodomains contained therein are increasingly attractive targets for the development of novel epigenetic therapeutics. To help validate this target class and unravel the complex associated biology, there has been a concerted effort to develop selective small molecule bromodomain inhibitors. Herein we describe the structure-based efforts and multiple challenges encountered in optimizing a naphthyridone template into selective TAF1(2) bromodomain inhibitors which, while unsuitable as chemical probes themselves, show promise for the future development of small molecules to interrogate TAF1(2) biology. Key to this work was the introduction and modulation of the basicity of a pendant amine which had a substantial impact on not only bromodomain selectivity but also cellular target engagement.

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.

Application of Atypical Acetyl-lysine Methyl Mimetics in the Development of Selective Inhibitors of the Bromodomain-Containing Protein 7 (BRD7)/Bromodomain-Containing Protein 9 (BRD9) Bromodomains.

Non-BET bromodomain-containing proteins have become attractive targets for the development of novel therapeutics targeting epigenetic pathways. To help facilitate the target validation of this class of proteins, structurally diverse small-molecule ligands and methodologies to produce selective inhibitors in a predictable fashion are in high demand. Herein, we report the development and application of atypical acetyl-lysine (KAc) methyl mimetics to take advantage of the differential stability of conserved water molecules in the bromodomain binding site. Discovery of the n-butyl group as an atypical KAc methyl mimetic allowed generation of 31 (GSK6776) as a soluble, permeable, and selective BRD7/9 inhibitor from a pyridazinone template. The n-butyl group was then used to enhance the bromodomain selectivity of an existing BRD9 inhibitor and to transform pan-bromodomain inhibitors into BRD7/9 selective compounds. Finally, a solvent-exposed vector was defined from the pyridazinone template to enable bifunctional molecule synthesis, and affinity enrichment chemoproteomic experiments were used to confirm several of the endogenous protein partners of BRD7 and BRD9, which form part of the chromatin remodeling PBAF and BAF complexes, respectively.