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.

A Phenotypic Approach for the Identification of New Molecules for Targeted Protein Degradation Applications.

Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin-proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel-Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.

Targeting IRAK4 for Degradation with PROTACs.

Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) is a key mediator of innate immunity. IRAK4 overactivation is linked with several autoimmune diseases. To date, many IRAK4 inhibitors have been developed to block the protein's kinase activity with the most advanced reaching Phase II clinical trials. Nevertheless, several reports suggest kinase activity is not disease-relevant in certain cell types, so removing scaffolding signaling in addition to IRAK4 kinase activity may offer a better therapeutic outcome. Herein, we describe the design and synthesis of an IRAK4 Proteolysis Targeted Chimera (PROTAC). We show that IRAK4 degradation induced by compound 9 leads to the inhibition of multiple cytokines in PBMCs. However, in IL-1ß stimulated human dermal fibroblasts, inhibition of IL-6 and TNF-α release was not observed despite IRAK4 degradation. Nonetheless, the possibility of targeting both IRAK4 kinase and scaffolding function could potentially lead to new therapeutic opportunities to treat autoimmune, inflammatory, and oncological diseases.

Evaluation of a crystallographic surrogate for kallikrein 5 in the discovery of novel inhibitors for Netherton syndrome.

The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.

Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10-100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.

Discovery of 6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one (GSK2245035), a Highly Potent and Selective Intranasal Toll-Like Receptor 7 Agonist for the Treatment of Asthma.

Induction of IFNα in the upper airways via activation of TLR7 represents a novel immunomodulatory approach to the treatment of allergic asthma. Exploration of 8-oxoadenine derivatives bearing saturated oxygen or nitrogen heterocycles in the N-9 substituent has revealed a remarkable selective enhancement in IFNα inducing potency in the nitrogen series. Further potency enhancement was achieved with the novel (S)-pentyloxy substitution at C-2 leading to the selection of GSK2245035 (32) as an intranasal development candidate. In human cell cultures, compound 32 resulted in suppression of Th2 cytokine responses to allergens, while in vivo intranasal administration at very low doses led to local upregulation of TLR7-mediated cytokines (IP-10). Target engagement was confirmed in humans following single intranasal doses of 32 of ≥20 ng, and reproducible pharmacological response was demonstrated following repeat intranasal dosing at weekly intervals.

Novel methylene-linked heterocyclic EP1 receptor antagonists.

We describe the SAR, in terms of heterocyclic replacements, for a series of pyrazole EP(1) receptor antagonists. This study led to the identification of several aromatic heterocyclic replacements for the pyrazole in the original compound. Investigation of replacements for the methylene linker uncovered disparate SAR in the thiazole and pyridine series.

Identification of novel glycine sulfonamide antagonists for the EP1 receptor.

A high-throughput screen targeting the EP(1) receptor identified non-acidic glycine sulfonamide derivative 2a with a pK(i) of 6.2. Analogue synthesis allowed a thorough investigation of the structure-activity relationship (SAR) and led to a 100-fold increase in recombinant potency.

Evaluation of machine-learning methods for ligand-based virtual screening.

Machine-learning methods can be used for virtual screening by analysing the structural characteristics of molecules of known (in)activity, and we here discuss the use of kernel discrimination and naive Bayesian classifier (NBC) methods for this purpose. We report a kernel method that allows the processing of molecules represented by binary, integer and real-valued descriptors, and show that it is little different in screening performance from a previously described kernel that had been developed specifically for the analysis of binary fingerprint representations of molecular structure. We then evaluate the performance of an NBC when the training-set contains only a very few active molecules. In such cases, a simpler approach based on group fusion would appear to provide superior screening performance, especially when structurally heterogeneous datasets are to be processed.

Identification of novel pyrazole acid antagonists for the EP1 receptor.

The discovery, synthesis and structure-activity relationship (SAR) of a novel series of EP1 receptor antagonists is described. Pyrazole acid 4, identified from a chemical array, had desirable physicochemical properties, an excellent in vitro microsomal inhibition and cytochrome P450 (CYP450) profile and good exposure levels in blood. This compound had an ED50 of 1.3 mg/kg in a rat pain model. A range of more potent analogues in the in vitro assay was identified using efficient array chemistry. These EP1 antagonists have potential as agents in the treatment of PGE2 mediated pain.

Virtual screening using binary kernel discrimination: effect of noisy training data and the optimization of performance.

Binary kernel discrimination (BKD) uses a training set of compounds, for which structural and qualitative activity data are available, to produce a model that can then be applied to the structures of other compounds in order to predict their likely activity. Experiments with the MDL Drug Data Report database show that the optimal value of the smoothing parameter, and hence the predictive power of BKD, is crucially dependent on the number of false positives in the training set. It is also shown that the best results for BKD are achieved using one particular optimization method for the determination of the smoothing parameter that lies at the heart of the method and using the Jaccard/Tanimoto coefficient in the kernel function that is used to compute the similarity between a test set molecule and the members of the training set.

Discovery of novel biaryl heterocyclic EP1 receptor antagonists.

We describe the generation of novel EP(1) receptor antagonists by investigation of thiophene isosteres. In addition, we disclose preliminary in vitro and in vivo DMPK for selected compounds.

Drug rings database with web interface. A tool for identifying alternative chemical rings in lead discovery programs.

This paper describes the development of a drug rings database and Web-based search tools. The database contains ring structures from both corporate and commercial databases, along with characteristic descriptors including frequency of occurrence as an indicator of synthetic accessibility and calculated property and geometric parameters. Analysis of the rings in several major databases is described, with illustrations of applications of the database in lead discovery programs where bioisosteres and geometric isosteres are sought.