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.

CSAR 2014: A Benchmark Exercise Using Unpublished Data from Pharma.

The 2014 CSAR Benchmark Exercise was the last community-wide exercise that was conducted by the group at the University of Michigan, Ann Arbor. For this event, GlaxoSmithKline (GSK) donated unpublished crystal structures and affinity data from in-house projects. Three targets were used: tRNA (m1G37) methyltransferase (TrmD), Spleen Tyrosine Kinase (SYK), and Factor Xa (FXa). A particularly strong feature of the GSK data is its large size, which lends greater statistical significance to comparisons between different methods. In Phase 1 of the CSAR 2014 Exercise, participants were given several protein-ligand complexes and asked to identify the one near-native pose from among 200 decoys provided by CSAR. Though decoys were requested by the community, we found that they complicated our analysis. We could not discern whether poor predictions were failures of the chosen method or an incompatibility between the participant's method and the setup protocol we used. This problem is inherent to decoys, and we strongly advise against their use. In Phase 2, participants had to dock and rank/score a set of small molecules given only the SMILES strings of the ligands and a protein structure with a different ligand bound. Overall, docking was a success for most participants, much better in Phase 2 than in Phase 1. However, scoring was a greater challenge. No particular approach to docking and scoring had an edge, and successful methods included empirical, knowledge-based, machine-learning, shape-fitting, and even those with solvation and entropy terms. Several groups were successful in ranking TrmD and/or SYK, but ranking FXa ligands was intractable for all participants. Methods that were able to dock well across all submitted systems include MDock,1 Glide-XP,2 PLANTS,3 Wilma,4 Gold,5 SMINA,6 Glide-XP2/PELE,7 FlexX,8 and MedusaDock.9 In fact, the submission based on Glide-XP2/PELE7 cross-docked all ligands to many crystal structures, and it was particularly impressive to see success across an ensemble of protein structures for multiple targets. For scoring/ranking, submissions that showed statistically significant achievement include MDock1 using ITScore1,10 with a flexible-ligand term,11 SMINA6 using Autodock-Vina,12,13 FlexX8 using HYDE,14 and Glide-XP2 using XP DockScore2 with and without ROCS15 shape similarity.16 Of course, these results are for only three protein targets, and many more systems need to be investigated to truly identify which approaches are more successful than others. Furthermore, our exercise is not a competition.

Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Discovered through X-ray Fragment Screening.

Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

Discovery and Optimization of Potent, Selective, and in Vivo Efficacious 2-Aryl Benzimidazole BCATm Inhibitors.

To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-(5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched chain amino acids as a consequence of BCATm inhibition.

Structurally Diverse Mitochondrial Branched Chain Aminotransferase (BCATm) Leads with Varying Binding Modes Identified by Fragment Screening.

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.

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.

Identification of a Novel and Selective Series of Itk Inhibitors via a Template-Hopping Strategy.

Inhibition of Itk potentially constitutes a novel, nonsteroidal treatment for asthma and other T-cell mediated diseases. In-house kinase cross-screening resulted in the identification of an aminopyrazole-based series of Itk inhibitors. Initial work on this series highlighted selectivity issues with several other kinases, particularly AurA and AurB. A template-hopping strategy was used to identify a series of aminobenzothiazole Itk inhibitors, which utilized an inherently more selective hinge binding motif. Crystallography and modeling were used to rationalize the observed selectivity. Initial exploration of the SAR around this series identified potent Itk inhibitors in both enzyme and cellular assays.

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.

p38alpha mitogen-activated protein kinase inhibitors: optimization of a series of biphenylamides to give a molecule suitable for clinical progression.

p38alpha MAP kinase is a key anti-inflammatory target for rheumatoid arthritis, influencing biosynthesis of pro-inflammatory cytokines TNFalpha and IL-1beta at a translational and transcriptional level. In this paper, we describe how we have optimized a series of novel p38alpha/beta inhibitors using crystal structures of our inhibitors bound to p38alpha, classical medicinal chemistry, and modeling of virtual libraries to derive a molecule suitable for progression into clinical development.

Biphenyl amide p38 kinase inhibitors 1: Discovery and binding mode.

The biphenyl amides (BPAs) are a novel series of p38 MAP kinase inhibitors. The discovery of the series through structure-based focused screening is described, and the binding mode of the compounds is explained with reference to X-ray crystal structures.

N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)amides as potent, selective, inhibitors of JNK2 and JNK3.

The identification and exploration of a novel, potent and selective series of N-(3-cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)amide inhibitors of JNK2 and JNK3 kinases is described. Compounds 5a and 11a were identified as potent inhibitors of JNK3 (pIC50 6.7 and 6.6, respectively), with essentially equal potency against JNK2 (pIC50 6.5). Selectivity within the mitogen-activated protein kinase (MAPK) family, against JNK1, p38alpha and ERK2, was observed for the series. X-ray crystallography of 5e and 8a in JNK3 revealed a unique binding mode, with the 3-cyano substituent forming an H-bond acceptor interaction with the hinge region of the ATP-binding site.

Pyrrolidine-5,5-trans-lactams. 4. Incorporation of a P3/P4 urea leads to potent intracellular inhibitors of hepatitis C virus NS3/4A protease.

[reaction: see text] In this, the first of two Letters, we describe how a P3/P4 urea linking unit was used to greatly enhance the biochemical and replicon potency of inhibitors based upon the pyrrolidine-5,5-trans-lactam template. Compound 7b demonstrated a 100 nM IC(50) in the replicon cell-based surrogate HCV assay.

Pyrrolidine-5,5-trans-lactams. 2. The use of X-ray crystal structure data in the optimization of P3 and P4 substituents.

[reaction: see text] In this, the second of two letters, we describe the elaboration of the pyrrolidine-5,5-trans-lactam template to delineate the requirements for optimal substitution of the pyrrolidine and lactam nitrogen atoms. Central to the strategy is the use of rapid iterative synthesis in conjunction with X-ray crystal structure determination of ligand-protein complexes.