Discovery and Lead-Optimization of 4,5-Dihydropyrazoles as Mono-Kinase Selective, Orally Bioavailable and Efficacious Inhibitors of Receptor Interacting Protein 1 (RIP1) Kinase.

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.

RIP1 Kinase Drives Macrophage-Mediated Adaptive Immune Tolerance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.

Structural and Functional Basis of C-Methylation of Coumarin Scaffolds by NovO.

C-methylation of aromatic small molecules by C-methyltransferases (C-MTs) is an important biological transformation that involves C-C bond formation using S-adenosyl-l-methionine (SAM) as the methyl donor. Here, two advances in the mechanistic understanding of C-methylation of the 8-position of coumarin substrates catalyzed by the C-MT NovO from Streptomyces spheroides are described. First, a crystal structure of NovO reveals the Arg116-Asn117 and His120-Arg121 motifs are essential for coumarin substrate binding. Second, the active-site His120 is responsible for deprotonation of the phenolic 7-hydroxyl group on the coumarin substrate, activating the rate-determining methyl transfer step from SAM. This work expands our mechanistic knowledge of C-MTs, which could be used in the downstream development of engineered biocatalysts for small molecule C-alkylations.

Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone Lysine Demethylases. 1. 3-Amino-4-pyridine Carboxylate Derivatives.

Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 µM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response.

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Integration of lead optimization with crystallography for a membrane-bound ion channel target: discovery of a new class of AMPA receptor positive allosteric modulators.

A novel series of AMPAR positive modulators is described that were identified by high throughput screening. The molecules of the series have been optimized from a high quality starting point hit to afford excellent developability, tolerability, and efficacy profiles, leading to identification of a clinical candidate. Unusually for an ion channel target, this optimization was integrated with regular generation of ligand-bound crystal structures and uncovered a novel chemotype with a unique and highly conserved mode of interaction via a trifluoromethyl group.

Discovery of N-[(2S)-5-(6-fluoro-3-pyridinyl)-2,3-dihydro-1H-inden-2-yl]-2-propanesulfonamide, a novel clinical AMPA receptor positive modulator.

A series of AMPA receptor positive allosteric modulators has been optimized from poorly penetrant leads to identify molecules with excellent preclinical pharmacokinetics and CNS penetration. These discoveries led to 17i, a potent, efficacious CNS penetrant molecule with an excellent pharmacokinetic profile across preclinical species, which is well tolerated and is also orally bioavailable in humans.

BACE-1 hydroxyethylamine inhibitors using novel edge-to-face interaction with Arg-296.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described a series of potent inhibitors based on an hydroxyethylamine (HEA) transition state mimetic template. These inhibitors interact with the non prime side of the enzyme using a novel edge-to-face interaction with Arg-296.

Second generation of BACE-1 inhibitors. Part 1: The need for improved pharmacokinetics.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.

Second generation of BACE-1 inhibitors part 2: Optimisation of the non-prime side substituent.

Our first generation of hydroxyethylamine transition-state mimetic BACE-1 inhibitors allowed us to validate BACE-1 as a key target for Alzheimer's disease by demonstrating amyloid lowering in an animal model, albeit at rather high doses. Finding a molecule from this series which was active at lower oral doses proved elusive and demonstrated the need to find a novel series of inhibitors with improved pharmacokinetics. This Letter describes the discovery of such inhibitors.

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics.

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.

1-Aryl-3,4-dihydroisoquinoline inhibitors of JNK3.

A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38alpha and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.

Second generation of hydroxyethylamine BACE-1 inhibitors: optimizing potency and oral bioavailability.

BACE-1 inhibition has the potential to provide a disease-modifying therapy for the treatment of Alzheimer's disease. Optimization of a first generation of BACE-1 inhibitors led to the discovery of novel hydroxyethylamines (HEAs) bearing a tricyclic nonprime side. These derivatives have nanomolar cell potency and are orally bioavailable.

BACE-1 inhibitors part 1: identification of novel hydroxy ethylamines (HEAs).

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described the lead generation effort which resulted, with the support of X-ray crystallography, in the discovery of potent inhibitors based on a hydroxy ethylamine (HEA) transition-state mimetic. These inhibitors were capable of lowering amyloid production in a cell-based assay.

BACE-1 inhibitors part 3: identification of hydroxy ethylamines (HEAs) with nanomolar potency in cells.

This article is focusing on further optimization of previously described hydroxy ethylamine (HEA) BACE-1 inhibitors obtained from a focused library with the support of X-ray crystallography. Optimization of the non-prime side of our inhibitors and introduction of a 6-membered sultam substituent binding to Asn-294 as well as a fluorine in the C-2 position led to derivatives with nanomolar potency in cell-based assays.

BACE-1 inhibitors part 2: identification of hydroxy ethylamines (HEAs) with reduced peptidic character.

This paper describes the discovery of non-peptidic, potent, and selective hydroxy ethylamine (HEA) inhibitors of BACE-1 by replacement of the prime side of a lead di-amide 2. Inhibitors with nanosmolar potency and high selectivity were identified. Depending on the nature of the P(1)(') and P(2)(') substituents, two different binding modes were observed in X-ray co-crystal structures.