Discovery of MK-1454: A Potent Cyclic Dinucleotide Stimulator of Interferon Genes Agonist for the Treatment of Cancer.

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

STimulator of INterferon Genes Agonism Accelerates Antitumor Activity in Poorly Immunogenic Tumors.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.

Establishing and Validating Cellular Functional Target Engagement Assay for Selective IRAK4 Inhibitor Discovery.

One of the main reasons for the lack of drug efficacy in late-stage clinical trials is the lack of specific and selective target engagement. To increase the likelihood of success of new therapeutics, one approach is to conduct proximal target engagement testing during the early phases of preclinical drug discovery. To identify and optimize selective IRAK4 inhibitors, a kinase that has been implicated in multiple inflammatory and autoimmune diseases, we established an electrochemiluminescence (ECL)-based cellular endogenous IRAK1 activation assay as the most proximal functional evaluation of IRAK4 engagement to support structure-activity relationship (SAR) studies. Since IRAK1 activation is dependent on both the IRAK4 scaffolding function in Myddosome formation and IRAK4 kinase activity for signal transduction, this assay potentially captures inhibitors with different mechanisms of action. Data from this IRAK1 assay with compounds representing different structural classes showed statistically significant correlations when compared with results from both IRAK4 biochemical kinase activity and functional peripheral blood mononuclear cell (PBMC)-derived tumor necrosis factor α (TNFα) secretion assays, validating the biological relevancy of the IRAK1 target engagement as a biomarker of the IRAK4 activity. Plate uniformity and potency reproducibility evaluations demonstrated that this assay is amenable to high throughput. Using Bland-Altman assay agreement analysis, we demonstrated that incorporating such proximal pharmacological assessment of cellular target engagement to an in vitro screening funnel for SAR studies can prevent compound optimization toward off-target activity.

Structural insights on ligand recognition at the human leukotriene B4 receptor 1.

The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Potent, non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.

Determination of EPAC2 function using EPAC2 null Min6 sublines generated through CRISPR-Cas9 technology.

Min6 cells, a mouse ß cell line derived from transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells, are commonly utilized as an in vitro cellular model for investigating targets involved in insulin secretion. Epac2, an exchange protein that can be directly activated by cyclic AMP (cAMP), is critical for pharmacologic stimuli-induced insulin secretion and has been hypothesized to be a direct target of sulfonylurea. Previous loss of function studies only specifically knocked out EPAC2 isoform A, leaving the other two isoforms intact. In this study, we investigated the function of EPAC2 in Min6 cells by generating EPAC2 knock-out sublines using CRISPR-Cas9 technology, by removing all three isoforms of EPAC2. Our results indicate that Min6 cells can be successfully cloned from a single cell after electroporation with plasmids expressing EPAC2 specific guide RNA, Cas9 and GFP, followed by sorting for GFP expressing single cells. Two clones were found to have a single nucleotide deletion in targeted site of EPAC2 gene by sequencing, therefore creating a frame shift in exon 13. The EPAC2 null clones have an unexpectedly increased secretion of insulin at basal level and an elevated total intracellular insulin content. However, EPAC2 deficiency impaires glucose and sulfonylurea induced insulin secretion without affecting sulfonylurea binding to cells. Potassium chloride induced insulin secretion remains intact. Interestingly, cAMP levels remained unchanged in EPAC2 null cells during these processes. To understand the global function of EPAC2, RNA Seq study was performed, which reveals that EPAC2 deficiency affects expression of multiple previously unrecognized genes, suggesting that EPAC2 can function through multiple pathways in addition to being a cAMP sensor.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Discovery of 3-morpholino-imidazole[1,5-a]pyrazine BTK inhibitors for rheumatoid arthritis.

8-Amino-imidazo[1,5-a]pyrazine-based Bruton's tyrosine kinase (BTK) inhibitors, such as 6, exhibited potent inhibition of BTK but required improvements in both kinase and hERG selectivity (Liu et al., 2016; Gao et al., 2017). In an effort to maintain the inhibitory activity of these analogs and improve their selectivity profiles, we carried out SAR exploration of groups at the 3-position of pyrazine compound 6. This effort led to the discovery of the morpholine group as an optimized pharmacophore. Compounds 13, 23 and 38 displayed excellent BTK potencies, kinase and hERG selectivities, and pharmacokinetic profiles.

Identification of quinazoline based inhibitors of IRAK4 for the treatment of inflammation.

Interleukin-1 receptor associated kinase 4 (IRAK4) has been implicated in IL-1R and TLR based signaling. Therefore selective inhibition of the kinase activity of this protein represents an attractive target for the treatment of inflammatory diseases. Medicinal chemistry optimization of high throughput screening (HTS) hits with the help of structure based drug design led to the identification of orally-bioavailable quinazoline based IRAK4 inhibitors with excellent pharmacokinetic profile and kinase selectivity. These highly selective IRAK4 compounds show activity in vivo via oral dosing in a TLR7 driven model of inflammation.

Discovery of novel BTK inhibitors with carboxylic acids.

We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.

Discovery of 8-Amino-imidazo[1,5-a]pyrazines as Reversible BTK Inhibitors for the Treatment of Rheumatoid Arthritis.

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition and for the treatment of B cell related diseases. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-ray crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.

Identification of N-(1H-pyrazol-4-yl)carboxamide inhibitors of interleukin-1 receptor associated kinase 4: Bicyclic core modifications.

IRAK4 plays a critical role in the IL-1R and TLR signalling, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of permeable N-(1H-pyrazol-4-yl)carboxamides was developed by introducing lipophilic bicyclic cores in place of the polar pyrazolopyrimidine core of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides. Replacement of the pyrazolo[1,5-a]pyrimidine core with the pyrrolo[2,1-f][1,2,4]triazine, the pyrrolo[1,2-b]pyridazine, and thieno[2,3-b]pyrazine cores guided by cLogD led to the identification of highly permeable IRAK4 inhibitors with excellent potency and kinase selectivity.

Discovery of 5-Amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Inhibitors of IRAK4.

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing.

Optimisation of pharmacokinetic properties to afford an orally bioavailable and selective V1A receptor antagonist.

The previously described lead compound 5 is a potent and selective V(1A) antagonist with affinity at both the rat and human receptor, but displays poor oral bioavailability and moderate clearance. We report herein the successful optimisation of the pharmacokinetic (PK) properties to afford the potent, selective, orally bioavailable and CNS penetrant compound 15f. A custom optimisation approach was required which demonstrated the value of using early, rapid in vivo PK studies to show improvements in oral exposure. Such assays may be of particular value where low oral bioavailability is anticipated to be multifactorial (e.g., permeability, gut wall metabolism and/or transport) where satisfactory modelling of in vitro data is likely to be difficult within a drug discovery context.

Identification and optimisation of novel sulfonamide, selective vasopressin V1B receptor antagonists.

The synthesis and preliminary structure-activity relationships (SAR) of a novel class of vasopressin V(1B) receptor antagonists are described. Hit compound 5, identified via high throughput screening of the corporate collection, showed good activity in a V(1B) binding assay (K(i) 63 nM) but did not possess the lead-like physicochemical properties typically required in a hit compound. A 'deletion approach' on the HTS hit 5 was performed, with the focus on improvement of physicochemical properties, yielding the selective V(1B) antagonist 9f (K(i) 190 nM), with improved druglike characteristics.

Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

The discovery of novel 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamides as vasopressin V1A receptor antagonists.

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V(1A) receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V(1A) antagonists.

Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) (V(3)) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and oxytocin (OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

The discovery and SAR of indoline-3-carboxamides--a new series of 5-HT6 antagonists.

Antagonists of the 5-HT(6) receptor have been shown to improve cognitive function in a wide range of animal models and as such may prove to be attractive agents for the symptomatic treatment of cognitive disorders such as Alzheimer's disease (AD) and schizophrenia. We report herein the identification and SAR around N-(2-aminoalkyl)-1-(arylsulfonyl)indoline-3-carboxamides-a novel chemotype of 5-HT(6) antagonists.