The discovery of potent and selective kynurenine 3-monooxygenase inhibitors for the treatment of acute pancreatitis.

A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo.

Increased levels of 3-hydroxykynurenine parallel disease severity in human acute pancreatitis.

Inhibition of kynurenine 3-monooxygenase (KMO) protects against multiple organ dysfunction (MODS) in experimental acute pancreatitis (AP). We aimed to precisely define the kynurenine pathway activation in relation to AP and AP-MODS in humans, by carrying out a prospective observational study of all persons presenting with a potential diagnosis of AP for 90 days. We sampled peripheral venous blood at 0, 3, 6, 12, 24, 48, 72 and 168 hours post-recruitment. We measured tryptophan metabolite concentrations and analysed these in the context of clinical data and disease severity indices, cytokine profiles and C-reactive protein (CRP) concentrations. 79 individuals were recruited (median age: 59.6 years; 47 males, 59.5%). 57 met the revised Atlanta definition of AP: 25 had mild, 23 moderate, and 9 severe AP. Plasma 3-hydroxykynurenine concentrations correlated with contemporaneous APACHE II scores (R2 = 0.273; Spearman rho = 0.581; P < 0.001) and CRP (R2 = 0.132; Spearman rho = 0.455, P < 0.001). Temporal profiling showed early tryptophan depletion and contemporaneous 3-hydroxykynurenine elevation. Furthermore, plasma concentrations of 3-hydroxykynurenine paralleled systemic inflammation and AP severity. These findings support the rationale for investigating early intervention with a KMO inhibitor, with the aim of reducing the incidence and severity of AP-associated organ dysfunction.

Discovery of epigenetic regulator I-BET762: lead optimization to afford a clinical candidate inhibitor of the BET bromodomains.

The bromo and extra C-terminal domain (BET) family of bromodomains are involved in binding epigenetic marks on histone proteins, more specifically acetylated lysine residues. This paper describes the discovery and structure-activity relationships (SAR) of potent benzodiazepine inhibitors that disrupt the function of the BET family of bromodomains (BRD2, BRD3, and BRD4). This work has yielded a potent, selective compound I-BET762 that is now under evaluation in a phase I/II clinical trial for nuclear protein in testis (NUT) midline carcinoma and other cancers.

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.

Discovery of a potent series of non-steroidal non α-trifluoromethyl carbinol glucocorticoid receptor agonists with reduced lipophilicity.

A novel series of indazole non-steroidal glucocorticoid receptor agonist has been discovered. This series features a sulfonamide central core and meta amides which interact with the extended ligand binding domain. This series has produced some of the most potent and least lipophilic agonists of which we are aware such as 20a (NFκB pIC(50) 8.3 (100%), clogP 1.9). Certain analogues in this series also display evidence for modulated pharmacology.

Structure guided design of 5-arylindazole glucocorticoid receptor agonists and antagonists.

Glucocorticoid receptor (GR) agonists have been used for more than half a century as the most effective treatment of acute and chronic inflammatory conditions despite serious side effects that accompany their extended use that include glucose intolerance, muscle wasting, skin thinning, and osteoporosis. As a starting point for the identification of GR ligands with an improved therapeutic index, we wished to discover selective nonsteroidal GR agonists and antagonists with simplified structure compared to known GR ligands to serve as starting points for the optimization of dissociated GR modulators. To do so, we selected multiple chemical series by structure guided docking studies and evaluated GR agonist activity. From these efforts we identified 5-arylindazole compounds that showed moderate binding to the glucocorticoid receptor (GR) with clear opportunities for further development. Structure guided optimization was used to design arrays that led to potent GR agonists and antagonists. Several in vitro and in vivo experiments were utilized to demonstrate that GR agonist 23a (GSK9027) had a profile similar to that of a classical steroidal GR agonist.

Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor.

Crystallography and computer modeling have been used to exploit a previously unexplored channel in the glucocorticoid receptor (GR). Highly potent, nonsteroidal indazole amides showing excellent complementarity to the channel were designed with the assistance of the computational technique AlleGrow. The accuracy of the design process was demonstrated through crystallographic structural determination of the GR ligand-binding domain-agonist complex of the D-prolinamide derivative 11. The utility of the channel was further exemplified through the design of a potent phenylindazole in which structural motifs, seen to interact with the traditional GR ligand pocket, were abandoned and replaced by interactions within the new channel. Occupation of the channel was confirmed with a second GR crystal structure of this truncated D-alaninamide derivative 13. Compound 11 displays properties compatible with development as an intranasal solution formulation, whereas oral bioavailability has been demonstrated with a related truncated exemplar 14. Data with the pyrrolidinone amide 12 demonstrate the potential for further elaboration within the "meta" channel to deliver compounds with selectivity for the desired transrepressive activity of glucocorticoids. The discovery of these interactions with this important receptor offers significant opportunities for the design of novel GR modulators.

Aryl aminopyrazole benzamides as oral non-steroidal selective glucocorticoid receptor agonists.

Aryl aminopyrazole amides capped with N-alkylbenzamides 13-16 are selective glucocorticoid receptor agonists. 2,6-Disubstituted benzamides have prednisolone-like potency or better in vitro. Good oral exposure was demonstrated in the rat, with compounds with lower lipophilicity, for example N-hydroxyethyl benzamides (e.g., 16e).

The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist.

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).

Pharmacological properties of the enhanced-affinity glucocorticoid fluticasone furoate in vitro and in an in vivo model of respiratory inflammatory disease.

Fluticasone furoate (FF) is a novel enhanced-affinity glucocorticoid that has been developed as topical therapy for allergic rhinitis. The pharmacological properties of FF have been investigated using a number of in vitro experimental systems. FF demonstrated very potent glucocorticoid activity in several key pathways downstream of the glucocorticoid receptor (GR) as follows: the transrepression nuclear factor-kappaB (NF-kappaB) pathway, the transactivation glucocorticoid response element pathway, and inhibition of the proinflammatory cytokine tumor necrosis factor-alpha. Furthermore, FF showed the greatest potency compared with other glucocorticoids for preserving epithelial integrity and reducing epithelial permeability in response to protease- and mechanical-induced cell damage. FF showed a 30- to >330,000-fold selectivity for GR-mediated inhibition of NF-kappaB vs. the other steroid hormone receptors, substantially better than a number of other clinically used glucocorticoids. In studies examining the respiratory tissue binding properties of glucocorticoids, FF had the largest cellular accumulation and slowest rate of efflux compared with other clinically used glucocorticoids, consistent with greater tissue retention. The in vivo anti-inflammatory activity of FF was assessed in the Brown Norway rat ovalbumin-induced lung eosinophilial model of allergic lung inflammation. At a dose of only 30 microg, FF achieved almost total inhibition of eosinophil influx in the lung, an inhibition that was greater than that seen with the same dose of fluticasone propionate. In conclusion, the potent and selective pharmacological profile of FF described here could deliver an effective, safe, and sustained topical treatment of respiratory inflammatory diseases such as allergic rhinitis and asthma.

A pharmacological approach to enhancing the therapeutic index of corticosteroids in airway inflammatory disease.

Inhaled corticosteroids are highly effective agents for the treatment of asthma and chronic obstructive pulmonary disease. Although local delivery to the lung has led to significant progress towards minimising side effects associated with systemic exposure, additional opportunities exist for further enhancement of the therapeutic index. Recent developments in the molecular and structural understanding of corticosteroid biology have made it increasingly clear that novel corticosteroids with intrinsically different pharmacology can be designed.

Functional analysis of the interleukin-5 receptor antagonist peptide, AF18748.

AF18748 is a dimeric peptide of 2 x 19 amino acids that specifically binds to the alpha-chain of the human IL-5 receptor (IL-5R), preventing binding of IL-5 and acting as a receptor antagonist. However, the peptide acts by inducing alpha-chain dimerization, and we therefore set out to investigate whether this peptide would have any residual agonist activity. AF18748 was unable to induce activation of a number of signal transduction pathways, but was able to specifically block IL-5-dependent signal transduction. The peptide was unable to support the survival and proliferation of the cytokine dependent cell line, Ba/F3-IL-5R, and was unable to prime the respiratory burst, or induce adhesion, migration or survival of primary human eosinophils. In each case the AF18748 functioned as an antagonist. These data suggest that AF18748 may be useful to specifically modulate eosinophil function in vivo.

Inhibition of nuclear factor-kappaB activation un-masks the ability of TNF-alpha to induce human eosinophil apoptosis.

Apoptosis renders eosinophils functionally effete and marks them for "silent" removal from inflamed sites by macrophages. We show, for the first time, that eosinophils exposed to TNF-alpha rapidly lose their cytoplasmic levels of IkappaBalpha, the inhibitory subunit of NF-kappaB. Consequently, TNF-alpha triggers NF-kappaB mobilization from the cytoplasm to the nucleus, as determined by tracking the NF-kappaB subunit p65 by immunofluorescence and Western blot analysis. Inhibition of TNF-alpha-mediated IkappaBalpha degradation and NF-kappaB activation by gliotoxin or the proteasome inhibitor MG-132 un-masks the caspase-dependent pro-apoptotic properties of TNF-alpha. In addition, cycloheximide similarly renders TNF-alpha pro-apoptotic, suggesting that NF-kappaB activation controls the production of a protein(s) that protects eosinophils from the cytotoxic effects of TNF-alpha. Evidence is presented suggesting that TNF-alpha triggered apoptosis is more susceptible to NF-kappaB inhibition than constitutive apoptosis, leading to the possibility of the specific targeting of apoptosis in eosinophil sub-populations. Prior to morphological signs of apoptosis, TNF-alpha-induced IL-8 synthesis is abrogated by inhibition of NF-kappaB. We propose that NF-kappaB activation plays a critical role in controlling eosinophil responsiveness and apoptosis, and speculate that selective inhibitors of eosinophil NF-kappaB activation may ultimately provide alternative therapeutic agents for the treatment of eosinophilic diseases, including asthma and allergic rhinitis.