Cesar Ramirez-Molina

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Discovery of GSK143, a highly potent, selective and orally efficacious spleen tyrosine kinase inhibitor.

The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.

The use of partially porous particle columns for the routine, generic analysis of biological samples for pharmacokinetic studies in drug discovery by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry.

Recent years have seen the introduction of new high performance liquid chromatography (HPLC) instruments and columns that are capable of achieving high resolution, high speed liquid chromatographic separations at back pressures up to 1000 bar, so-called ultra-high performance liquid chromatography (UHPLC). Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is gaining widespread use for this purpose, and for this approach to be successful a generically applicable, robust column is required. Here, data are presented showing the robustness of a partially porous 2.7 microm diameter particle material in this application and the accuracy and precision of an assay for a typical pharmaceutical in plasma. This stationary phase material is evaluated for performance and compared with other materials frequently used for similar analyses using a test mix currently used routinely in our laboratories to assess the performance of UHPLC-MS/MS systems. The partially porous material demonstrates similar resolving power to sub-2 microm materials under the ballistic gradient chromatography conditions employed and exhibits excellent resilience over the analysis of thousands of protein precipitated plasma extracts. It is suggested that this stationary phase material can be an invaluable tool in generic, high throughput assays for pharmaceutical bioanalysts.

Inhibition of spleen tyrosine kinase as treatment of postoperative ileus

Objective Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. Design In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. Results In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4±0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2±0.7; GSK143 (1 mg/kg): 7.6±0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0–90 μM) and trinitrophenyl (0–4 μg/ml) induced a concentration-dependent release of β-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03–10 μM) concentration dependently blocked substance P and trinitrophenyl induced β-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. Conclusions The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI.

4-Phenyl-7-Azaindoles as Potent, Selective and Bioavailable Ikk2 Inhibitors Demonstrating Good in Vivo Efficacy.

The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.

Screening strategy for the rapid detection of in vitro generated glutathione conjugates using high‐performance liquid chromatography and low‐resolution mass spectrometry in combination with LightSight® software for data processing

The knowledge of drug metabolism in the early phases of the drug discovery process is vital for minimising compound failure at later stages. As chemically reactive metabolites may cause adverse drug reactions, it is generally accepted that avoiding formation of reactive metabolites increases the chances of success of a molecule. In order to generate this important information, a screening strategy for the rapid detection of in vitro generated reactive metabolites trapped by glutathione has been developed. The bioassay incorporated the use of native glutathione and its close analogue the glutathione ethyl ester. The generic conditions for detecting glutathione conjugates that undergo constant neutral loss of 129 Da were optimised using a glutathione-based test mix of four compounds. The final liquid chromatography/tandem mass spectrometry constant neutral loss method used low-resolution settings and a scanning window of 200 amu. Data mining was rapidly and efficiently performed using LightSight software. Unambiguous identification of the glutathione conjugates was significantly facilitated by the analytical characteristics of the conjugate pairs formed with glutathione and glutathione ethyl ester, i.e. by chromatographic retention time and mass differences. The reliability and robustness of the screening strategy was tested using a number of compounds known to form reactive metabolites. Overall, the developed screening strategy provided comprehensive and reliable identification of glutathione conjugates and is well suited for rapid routine detection of trapped reactive metabolites. This new approach allowed the identification of a previously unreported diclofenac glutathione conjugate.

Optimisation of a novel series of potent and orally bioavailable azanaphthyridine SYK inhibitors

The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.

Discovery of potent and selective Spleen Tyrosine Kinase inhibitors for the topical treatment of inflammatory skin disease

The discovery and lead optimisation of a novel series of SYK inhibitors is described. These were optimised for SYK potency and selectivity against Aurora B. Compounds were profiled in a human skin penetration study to identify a suitable candidate molecule for pre-clinical development. Compound 44 (GSK2646264) was selected for progression and is currently in Phase I clinical trials.

Tu1629 Spleen Tyrosine Kinase Inhibition Reduces Intestinal Inflammation and Prevents Postoperative Ileus

Background: Recent advances underline intestinal inflammation, induced by handling of the gut during surgery, as crucial mechanism in the pathophysiology of postoperative ileus (POI). Macrophages, in the muscularis layer, and mast cells are the key players in the induction of this inflammatory process. Spleen tyrosine kinase (Syk) is an important kinase involved in macrophage activation as well as in mast cell degranulation and therefore inhibition of Syk pathway may represent an interesting therapeutic approach for POI. In the current study, we have evaluated the effect of the Syk-inhibitor GSK143 as potential treatment to shorten POI. Methods: The effect of an oral single dose (3mg/kg, 1.5 hours before surgery) was evaluated in a mouse model of POI, by analyzing gastrointestinal transit and intestinal muscularis inflammation. The in vitro effect of GSK143 (1 and 3 μM) was evaluated on cultured peritoneal mast cells (pMCs) and bone marrow derived macrophages (BMDMs) stimulated with immune complexes with IgE-anti-TNP (40 ng/mL) and LPS (100 ng/ml) respectively. Results: Administration of GSK143 (3 mg/kg) resulted in a serum level of 0.38±0.24 μM 1.5 hours after oral delivery. Treatment with a single dose of GSK143 (3 mg/kg) significantly improved gastrointestinal transit after surgical intestinal manipulated mice compared to placebo (Geometric Center (GC): Placebo, 4.2 ±0.4 vs GSK143, GC; 6.9±0.6). In addition, GSK143 was able to reduce the number of recruited myeloperoxidase (MPO) positive cells (placebo, 187±30 vs GSK143, 44±8, per 0.5mm2), neutrophils (placebo, 4.6x10-5±0.2 x10-5 vs GSK143, 1.7x10-5 ±0.7 x10-5) and monocytes (placebo, 1.6x106±0.4 x10-6 vs GSK143, 0.8x10-6 ±0.25 x10-6) in the muscularis externa compared to placebo. To define the possible target cells affected, GSK143 was tested in vitro on pMCs and BMDMs. Thirty minutes after GSK143 (1 and 3 μM) or vehicle treatment, pMCs degranulation, quantified by assessment of β-hexosaminidase, was induced by immune complexes with IgE-anti-TNP. Interestingly, GSK143 significantly reduced pMCs degranulation compared to vehicle in a dose dependent manner (GSK143 1μM, 62±7.5% and GSK143 3μM, 76±3.3% inhibition vs vehicle). In line, pre-treatment of BMDMs with GSK143 (1 and 3 μM) prior to LPS challenge significantly lowered the expression of IL-6 (21±4% to 31± 3 % inhibition vs vehicle ), TNFα (39±0.8% to 54±0.7% inhibition vs vehicle ), IL1 β (31±1.3% to 39±1.06% inhibition vs vehicle) and CCL2 (33±5.3% to 43±7% inhibition vs vehicle). Conclusion: Pre-treatment with the Syk-inhibitor GSK143 attenuates intestinal inflammation and consequently restores gastrointestinal transit in a model of POI. In vitro data suggests that both macrophages and mast cells are targeted by GSK143. These findings strongly suggest that Syk inhibition may be a new therapeutical tool to shorten POI.