Investigation of Janus Kinase (JAK) Inhibitors for Lung Delivery and the Importance of Aldehyde Oxidase Metabolism.

The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.

Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase.

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.

Identification of KasA as the cellular target of an anti-tubercular scaffold.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis ß-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related ß-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.

Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin.

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.

Crystallization and initial crystallographic analysis of covalent DNA-cleavage complexes of Staphyloccocus aureus DNA gyrase with QPT-1, moxifloxacin and etoposide.

Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Šresolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.

The Discovery of in Vivo Active Mitochondrial Branched-Chain Aminotransferase (BCATm) Inhibitors by Hybridizing Fragment and HTS Hits.

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.

Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire mass spectrometry.

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

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.

Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance.

Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.

A multi-centre evaluation of the intra-assay and inter-assay variation of commercial and in-house anti-cardiolipin antibody assays.

AIMS: To assess the intra-assay (intra-run) and inter-assay (inter-run) variation of commercial and in-house IgG and IgM anti-cardiolipin antibody (aCL) assays/kits, and to determine an appropriate maximum value for inclusion in consensus guidelines. METHODS: Frozen aliquots of two patient specimens and one commercial control were sent to nine laboratories for the evaluation of eight commercial kits and one in-house assay. Intra-assay and inter-assay evaluations were performed with all three samples for IgG aCL, and one patient specimen for IgM aCL. RESULTS: The IgG and IgM aCL values varied considerably between the nine assays/kits. The majority of assays/kits demonstrated less than 20% intra-assay and inter-assay variation, with lower intra-assay and inter-assay variation observed with the commercial control. Single calibrator assays were not consistently associated with higher inter-assay variation than multi-point calibrator assays. CONCLUSIONS: An inter-assay coefficient of variation of 20% was determined to be an appropriate maximum value for inclusion in the Australasian aCL Working Party consensus guidelines. Improved standardisation between different assay/kits is still required.

Consensus guidelines on anti-cardiolipin antibody testing and reporting.

UNLABELLED: Consensus guidelines on anti-cardiolipin antibody (aCL) testing have been developed to help minimise laboratory variation in the performance and reporting of aCL assays. These guidelines include minimum, optimum and optional recommendations for the following aspects of aCL testing and reporting: (1) isotype of aCL tested; (2) specimen type; (3) controls and assay precision; (4) calibrators; (5) patient samples; (6) rheumatoid factors and IgM aCL testing; (7) reporting of results; (8) cut-off values; and (9) interpretative comments. ABBREVIATIONS: aCL, anti-cardiolipin antibodies; APS, anti-phospholipid antibody syndrome; ASCIA, Australasian Society of Clinical Immunology and Allergy; ASTH, Australasian Society of Thrombosis and Haemostasis; beta2-GPI=beta2-glycoprotein I; ELISA, enzyme-linked immunosorbent assay; NCCLS, National Committee for Clinical Laboratory Standards; HSANZ, Haematology Society of Australia and New Zealand; QAP, Quality Assurance Program; RCPA, Royal College of Pathologists of Australasia; %CV, inter-assay inter-run coefficient of variation.