Kynurenine monooxygenase (KMO) blockade protects against multiple organ failure caused by acute pancreatitis (AP), but the link between KMO and systemic inflammation has eluded discovery until now. Here, we show that the KMO product 3-hydroxykynurenine primes innate immune signaling to exacerbate systemic inflammation during experimental AP. We find a tissue-specific role for KMO, where mice lacking Kmo solely in hepatocytes have elevated plasma 3-hydroxykynurenine levels that prime inflammatory gene transcription. 3-Hydroxykynurenine synergizes with interleukin-1β to cause cellular apoptosis. Critically, mice with elevated 3-hydroxykynurenine succumb fatally earlier and more readily to experimental AP. Therapeutically, blockade with the highly selective KMO inhibitor GSK898 rescues the phenotype, reducing 3-hydroxykynurenine and protecting against critical illness and death. Together, our findings establish KMO and 3-hydroxykynurenine as regulators of inflammation and the innate immune response to sterile inflammation. During critical illness, excess morbidity and death from multiple organ failure can be rescued by systemic KMO blockade.

GSK3335065 is an inhibitor of kynurenine monooxygenase (KMO) being developed for the treatment of acute pancreatitis. Healthy male volunteers were administered ascending doses of GSK3335065 or matched placebo as a single intravenous bolus injection to assess safety, tolerability, pharmacokinetics and pharmacodynamics. GSK3335065 displayed an apparent volume of distribution between 20.6 L and 44.6 L, a clearance between 0.462 L/h and 0.805 L/hr and a terminal half-life between 31.3 and 34.5 hr. In the single subject who received 1.3 mg GSK3335065, changes in tryptophan pathway metabolites were observed consistent with the changes seen in preclinical species suggesting that KMO enzyme activity was partially inhibited. However, a broad complex ventricular tachycardia was observed in this subject, which was judged to be a Serious Adverse Event (SAE) and resulted in early termination of the study. While development of GSK3335065 was subsequently discontinued, significant confounding factors hinder a clear interpretation that the tachycardia was directly related to administration of the compound.

Objective: Acute Pancreatitis (AP) is sudden onset pancreas inflammation that causes systemic injury with a wide and markedly heterogeneous range of clinical consequences. Here, we hypothesized that this observed clinical diversity corresponds to diversity in molecular subtypes that can be identified in clinical and multiomics data. Summary Background Data: Observational cohort study. n = 57 for the discovery cohort (clinical, transcriptomics, proteomics, and metabolomics data) and n = 312 for the validation cohort (clinical and metabolomics data). Methods: We integrated coincident transcriptomics, proteomics, and metabolomics data at serial time points between admission to hospital and up to 48 hours after recruitment from a cohort of patients presenting with acute pancreatitis. We systematically evaluated 4 different metrics for patient similarity using unbiased mathematical, biological, and clinical measures of internal and external validity. We next compared the AP molecular endotypes with previous descriptions of endotypes in a critically ill population with acute respiratory distress syndrome (ARDS). Results: Our results identify 4 distinct and stable AP molecular endotypes. We validated our findings in a second independent cohort of patients with AP. We observed that 2 endotypes in AP recapitulate disease endotypes previously reported in ARDS. Conclusions: Our results show that molecular endotypes exist in AP and reflect biological patterns that are also present in ARDS, suggesting that generalizable patterns exist in diverse presentations of critical illness.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Severe α 1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α 1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high throughput screen to identify small molecules that bind to, and stabilise Z α 1 -antitrypsin. The lead compound blocks Z α 1 -antitrypsin polymerisation in vitro , reduces intracellular polymerisation and increases the secretion of Z α 1 -antitrypsin three-fold in mammalian cells including an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerization pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α 1 -antitrypsin into the plasma by 7-fold. There was no observable clearance of hepatic inclusions with respect to controls. This study provides proof-of-principle that ‘mutation ameliorating’ small molecules are a viable approach to treat protein conformational diseases.

Gungabissoon, Usha MSc; Delgado, Megan MPH; Cooper, Selin PhD; Ma, Liyuan MS; Uings, Iain BSc Author Information

ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

The wild type protein, transthyretin (TTR), and over 120 genetic TTR variants are amyloidogenic and cause, respectively, sporadic and hereditary systemic TTR amyloidosis. The homotetrameric TTR contains two identical thyroxine binding pockets, occupation of which by specific ligands can inhibit TTR amyloidogenesis in vitro. Ligand binding stabilizes the tetramer, inhibiting its proteolytic cleavage and its dissociation. Here, we show with solution-state NMR that ligand binding induces long-distance conformational changes in the TTR that have not previously been detected by X-ray crystallography, consistently with the inhibition of the cleavage of the DE loop. The NMR findings, coupled with surface plasmon resonance measurements, have identified dynamic exchange processes underlying the negative cooperativity of binding of "monovalent" ligand tafamidis. In contrast, mds84, our prototypic "bivalent" ligand, which is a more potent stabilizer of TTR in vitro that occupies both thyroxine pockets and the intramolecular channel between them, has greater structural effects.

Acute pancreatitis (AP) is sudden onset pancreas inflammation that causes multiple organ dysfunction syndrome (MODS) and death in certain individuals who develop AP yet minimal systemic inflammation in others. Here, we show that this observed diversity in systemic response and outcome is accompagnied by diversity in molecular subtypes that can be identified using computational analysis of clinical and multiomic data. We integrated co-incident whole blood transcriptomic, plasma proteomic, and serum metabolomic data at serial time points from a cohort of patients presenting with AP and systematically evaluated four different metrics for patient similarity, using unbiased mathematical, biological and clinical measures of internal and external validity. Our results identify four distinct and stable AP endotypes that are characterized by pathway and biomarker combination stereotypes into hypermetabolic, hepatopancreaticobiliary, catabolic and innate immune endotypes. The catabolic endotype in AP strikingly recapitulates a disease endotype previously reported in acute respiratory distress syndrome, a recognized complication of AP. Our findings demonstrate that clinically-relevant and generalizable endotypes exist in AP.

Expression of mutant Huntingtin (HTT) protein is central to the pathophysiology of Huntington's Disease (HD). The E3 ubiquitin ligase MID1 appears to have a key role in facilitating translation of the mutant HTT mRNA suggesting that interference with the function of this complex could be an attractive therapeutic approach. Here we describe a peptide that is able to disrupt the interaction between MID1 and the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A). By fusing this peptide to a sequence from the HIV-TAT protein we demonstrate that the peptide can disrupt the interaction within cells and show that this results in a decrease in levels of ribosomal S6 phosphorylation and HTT expression in cultures of cerebellar granule neurones derived from HdhQ111/Q7 mice. This data serves to validate this pathway and paves the way for the discovery of small molecule inhibitors of this interaction as potential therapies for HD.

Recently, we reported a novel role for KMO in the pathogenesis of acute pancreatitis (AP). A number of inhibitors of kynurenine 3-monooxygenase (KMO) have previously been described as potential treatments for neurodegenerative conditions and particularly for Huntington's disease. However, the inhibitors reported to date have insufficient aqueous solubility relative to their cellular potency to be compatible with the intravenous (iv) dosing route required in AP. We have identified and optimized a novel series of high affinity KMO inhibitors with favorable physicochemical properties. The leading example is exquisitely selective, has low clearance in two species, prevents lung and kidney damage in a rat model of acute pancreatitis, and is progressing into preclinical development.

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.

Kynurenine-3-monooxygenase (KMO) is a key FAD-dependent enzyme of tryptophan metabolism. In animal models, KMO inhibition has shown benefit in neurodegenerative diseases such as Huntington’s and Alzheimer’s. Most recently it has been identified as a target for acute pancreatitis multiple organ dysfunction syndrome (AP-MODS); a devastating inflammatory condition with a mortality rate in excess of 20%. Here we report and dissect the molecular mechanism of action of three classes of KMO inhibitors with differentiated binding modes and kinetics. Two novel inhibitor classes trap the catalytic flavin in a previously unobserved tilting conformation. This correlates with picomolar affinities, increased residence times and an absence of the peroxide production seen with previous substrate site inhibitors. These structural and mechanistic insights culminated in GSK065(C1) and GSK366(C2), molecules suitable for preclinical evaluation. Moreover, revising the repertoire of flavin dynamics in this enzyme class offers exciting new opportunities for inhibitor design.

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 (R-2 = 0.273; Spearman rho = 0.581; P < 0.001) and CRP (R-2 = 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.

Blocking the enzyme KMO with a small molecule reduces the levels of toxic tryptophan metabolites and reduces multiple extrapancreatic organ failure in a rat model of acute pancreatitis. Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death1,2. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population4. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism5, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.

Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z' value 0.8) and provided several tractable hit series for further investigation.

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.

Inhaled glucocorticoids are highly effective therapies for respiratory diseases. Progress in characterising the complex molecular mechanisms that control glucocorticoid action raises the possibility that anti-inflammatory molecules with reduced side-effect liabilities can be identified. Here we describe the discovery of a novel glucocorticoid, GW 870086, with unique biological properties, and demonstrate the potential of gene expression profiling in the characterisation of its pharmacology. This molecule is under clinical evaluation as a novel respiratory agent. The pharmacological properties of GW870086 were assessed against fluticasone propionate (FP) using cellular and in vivo model systems, including extensive gene expression profiling. GW870086 repressed inflammatory cytokine release from primary lung epithelial cells with similar efficacy to FP, but in marked contrast to FP antagonised the effect of dexamethasone on MMTV driven reporter gene transactivation. GW870086 had a very significant effect on the expression of specific glucocorticoid regulated genes while having minimal impact on the expression of other known target genes, demonstrating unique regulation of gene transcription. In experimental in vivo models of irritant-induced contact dermatitis and ovalbumin-induced allergic inflammation models, GW870086 showed comparable anti-inflammatory efficacy to FP. GW870086 is a potent anti-inflammatory compound that has a unique ability to regulate a specific subset of genes that are normally effected by classical glucocorticoids. It has clear potential to be developed as a new topical steroid with a distinct safety profile to existing therapies.

Background and PurposeGlucocorticoids are highly effective therapies for a range of inflammatory diseases. Advances in the understanding of the diverse molecular mechanisms underpinning glucocorticoid action suggest that anti‐inflammatory molecules with reduced side effect liabilities can be discovered. Here we set out to explore whether modification of the 17α position of the steroid nucleus could generate molecules with a unique pharmacological profile and to determine whether such molecules would retain anti‐inflammatory activity.Experimental ApproachThe pharmacological properties of GW870086 were compared with fluticasone propionate (FP) using a range of cellular and in vivo model systems, including extensive gene expression profiling.Key ResultsGW870086 repressed inflammatory cytokine release from lung epithelial cells in a similar manner to FP but antagonized the effect of dexamethasone on MMTV‐driven reporter gene transactivation. GW870086 had a strong effect on the expression of some glucocorticoid‐regulated genes (such as PTGS2), while having minimal impact on the expression of other known target genes (such as SGK). GW870086 retained the ability to strengthen tight junctions in epithelial cell culture but, unlike FP, was unable to protect the culture from elastase‐mediated damage. In murine models of irritant‐induced contact dermatitis and ovalbumin‐induced allergic inflammation, GW870086 showed comparable anti‐inflammatory efficacy to FP.Conclusion and ImplicationsGW870086 is a potent anti‐inflammatory compound with a unique ability to regulate only a subset of those genes that are normally affected by classical glucocorticoids. It has the potential to become a new topical steroid with a different safety profile to existing therapies.