Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Development of small molecule inhibitors of natural killer group 2D receptor (NKG2D).

Natural killer group 2D (NKG2D) is a homodimeric activating immunoreceptor whose function is to detect and eliminate compromised cells upon binding to the NKG2D ligands (NKG2DL) major histocompatibility complex (MHC) molecules class I-related chain A (MICA) and B (MICB) and UL16 binding proteins (ULBP1-6). While typically present at low levels in healthy cells and tissue, NKG2DL expression can be induced by viral infection, cellular stress or transformation. Aberrant activity along the NKG2D/NKG2DL axis has been associated with autoimmune diseases due to the increased expression of NKG2D ligands in human disease tissue, making NKG2D inhibitors an attractive target for immunomodulation. Herein we describe the discovery and optimization of small molecule PPI (protein-protein interaction) inhibitors of NKG2D/NKG2DL. Rapid SAR was guided by structure-based drug design and accomplished by iterative singleton and parallel medicinal chemistry synthesis. These efforts resulted in the identification of several potent analogs (14, 21, 30, 45) with functional activity and improved LLE.

Target 2035 - an update on private sector contributions.

Target 2035, an international federation of biomedical scientists from the public and private sectors, is leveraging 'open' principles to develop a pharmacological tool for every human protein. These tools are important reagents for scientists studying human health and disease and will facilitate the development of new medicines. It is therefore not surprising that pharmaceutical companies are joining Target 2035, contributing both knowledge and reagents to study novel proteins. Here, we present a brief progress update on Target 2035 and highlight some of industry's contributions.

Identification of small-molecule protein-protein interaction inhibitors for NKG2D.

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure-activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

Direct-to-Biology Accelerates PROTAC Synthesis and the Evaluation of Linker Effects on Permeability and Degradation.

A platform to accelerate optimization of proteolysis targeting chimeras (PROTACs) has been developed using a direct-to-biology (D2B) approach with a focus on linker effects. A large number of linker analogs-with varying length, polarity, and rigidity-were rapidly prepared and characterized in four cell-based assays by streamlining time-consuming steps in synthesis and purification. The expansive dataset informs on linker structure-activity relationships (SAR) for in-cell E3 ligase target engagement, degradation, permeability, and cell toxicity. Unexpected aspects of linker SAR was discovered, consistent with literature reports on "linkerology", and the method dramatically speeds up empirical optimization. Physicochemical property trends emerged, and the platform has the potential to rapidly expand training sets for more complex prediction models. In-depth validation studies were carried out and confirm the D2B platform is a valuable tool to accelerate PROTAC design-make-test cycles.

Discovery of Highly Potent and Selective IRAK1 Degraders to Probe Scaffolding Functions of IRAK1 in ABC DLBCL.

MyD88 gene mutation has been identified as one of the most prevalent driver mutations in the activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL). The published literature suggests that interleukin-1 receptor-associated kinase 1 (IRAK1) is an essential gene for ABC DLBCL harboring MyD88 mutation. Importantly, the scaffolding function of IRAK1, rather than its kinase activity, is required for tumor cell survival. Herein, we present our design, synthesis, and biological evaluation of a novel series of potent and selective IRAK1 degraders. One of the most potent compounds, Degrader-3 (JNJ-1013), effectively degraded cellular IRAK1 protein with a DC50 of 3 nM in HBL-1 cells. Furthermore, JNJ-1013 potently inhibited IRAK1 downstream signaling pathways and demonstrated strong anti-proliferative effects in ABC DLBCL cells with MyD88 mutation. This work suggests that IRAK1 degraders have the potential for treating cancers that are dependent on the IRAK1 scaffolding function.

Discovery of a Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase with Oral Anti-Inflammatory Activity.

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3H-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype. Compound 27 irreversibly inhibits BTK by targeting a noncatalytic cysteine residue (Cys481) for covalent bond formation. Compound 27 is characterized by selectivity for BTK, potent in vivo BTK occupancy that is sustained after it is cleared from systemic circulation, and dose-dependent efficacy at reducing joint inflammation in a rat collagen-induced arthritis model.

Discovery of a Gut-Restricted JAK Inhibitor for the Treatment of Inflammatory Bowel Disease.

To identify Janus kinase (JAK) inhibitors that selectively target gastrointestinal tissues with limited systemic exposures, a class of imidazopyrrolopyridines with a range of physical properties was prepared and evaluated. We identified compounds with low intrinsic permeability and determined a correlation between permeability and physicochemical properties, clogP and tPSA, for a subset of compounds. This low intrinsic permeability translated into compounds displaying high colonic exposure and low systemic exposure after oral dosing at 25 mg/kg in mouse. In a mouse PK/PD model, oral dosing of lead compound 2 demonstrated dose-dependent inhibition of pSTAT phosphorylation in colonic explants post-oral dose but low systemic exposure and no measurable systemic pharmacodynamic activity. We thus demonstrate the utility of JAK inhibitors with low intrinsic permeability as a feasible approach to develop gut-restricted, pharmacologically active molecules with a potential advantage over systemically available compounds that are limited by systemic on-target adverse events.

Immunological characterization of HM5023507, an orally active PI3Kδ/γ inhibitor.

Phosphoinositide 3-kinases, delta (PI3Kδ) and gamma (PI3Kγ) are enriched in immune cells and regulate the development and function of innate and adaptive immunity. Dual PI3Kδγ inhibitors are considered high value targets for their potential to treat a variety of immune-mediated diseases, but their discovery has been challenging. Here we describe the preclinical pharmacology of HM5023507, an orally active dual inhibitor of δγ isoforms in immune signaling. HM5023507 inhibited PI3Kδ and PI3Kγ isoforms with greater than 100-fold selectivity against PI3Kα and PI3Kß in recombinant enzymatic assays and in primary human immune cells with an exquisite selectivity against other targets. HM5023507 attenuated the PI3Kδ/γ signaling in human basophils (IC50: 42/340 nmol/L; selectivity ratio ~1:8). HM5023507 attenuated the activation and function of human B and T cells, Th17 differentiation of CD4 T cells in the blood of healthy donors and rheumatoid arthritis patients, and cytokine and IgG production in human T and B cell cocultures, in vitro. Orally dosed HM5023507 attenuated PI3K δ/γ-mediated immune signaling in the rat in a dose-related manner. In addition, HM5023507 inhibited semiestablished collagen-induced arthritic inflammation in the rats (ED50 of 0.25mg/kg, p.o. BID or 0.5 mg/kg, QD, AUC: 1422 ng/mL*h), improved histopathology- and micro-computed tomography (µCT)-based indices of joint damage, bone destruction, and attenuated the levels of anti-collagen antibody, with an overall anti-inflammatory profile matching that of a TNFα neutralizing antibody. The PI3K δγ inhibitory profile of HM5023507 and its selectivity make it a useful tool to further delineate immunobiology of dual PI3K δγ targeting.

Discovery, Optimization, and Evaluation of Potent and Highly Selective PI3Kγ-PI3Kδ Dual Inhibitors.

An electronic density model was developed and used to identify a novel pyrrolotriazinone replacement for a quinazolinone, a commonly used moiety to impart selectivity in inhibitors for PI3Kγ and PI3Kδ. Guided by molecular docking, this new specificity piece was then linked to the hinge-binding region of the inhibitor using a novel cyclic moiety. Further structure-activity relationship optimization around the hinge region led to the discovery of candidate 26, a highly potent and selective PI3Kγ-PI3Kδ dual inhibitor with favorable drug metabolism and pharmacokinetic properties in preclinical species.

Clinical Development of Histamine H4 Receptor Antagonists.

The discovery of the histamine H4 receptor (H4R) provided a new avenue for the exploration of the physiological role of histamine, as well as providing a new drug target for the development of novel antihistamines. The first step in this process was the identification of selective antagonists to help unravel the pharmacology of the H4R relative to other histamine receptors. The discovery of the selective H4R antagonist JNJ 7777120 was vital for showing a role for the H4R in inflammation and pruritus. While this compound has been very successful as a tool for understanding the function of the receptor, it has drawbacks, including a short in vivo half-life and hypoadrenocorticism toxicity in rats and dogs, that prevented advancing it into clinical studies. Further research let to the discovery of JNJ 39758979, which, similar to JNJ 7777120, was a potent and selective H4R antagonist and showed anti-inflammatory and anti-pruritic activity preclinically. JNJ 39758979 advanced into human clinical studies and showed efficacy in reducing experimental pruritus and in patients with atopic dermatitis. However, development of this compound was terminated due to the occurrence of drug-induced agranulocytosis. This was overcome by developing another H4R antagonist with a different chemical structure, toreforant, that does not appear to have this side effect. Toreforant has been tested in clinical studies in patients with rheumatoid arthritis, asthma, or psoriasis. In conclusions there have been many H4R antagonists reported in the literature, but only a few have been studied in humans underscoring the difficulty in finding ligands with all of the properties necessary for testing in the clinic. Nevertheless, the clinical data to date suggests that H4R antagonists can be beneficial in treating atopic dermatitis and pruritus.

A Prospective Virtual Screening Study: Enriching Hit Rates and Designing Focus Libraries To Find Inhibitors of PI3Kδ and PI3Kγ.

Here, we report a high-throughput virtual screening (HTVS) study using phosphoinositide 3-kinase (both PI3Kγ and PI3Kδ). Our initial HTVS results of the Janssen corporate database identified small focused libraries with hit rates at 50% inhibition showing a 50-fold increase over those from a HTS (high-throughput screen). Further, applying constraints based on "chemically intuitive" hydrogen bonds and/or positional requirements resulted in a substantial improvement in the hit rates (versus no constraints) and reduced docking time. While we find that docking scoring functions are not capable of providing a reliable relative ranking of a set of compounds, a prioritization of groups of compounds (e.g., low, medium, and high) does emerge, which allows for the chemistry efforts to be quickly focused on the most viable candidates. Thus, this illustrates that it is not always necessary to have a high correlation between a computational score and the experimental data to impact the drug discovery process.

Functional Profiling of 2-Aminopyrimidine Histamine H4 Receptor Modulators.

Histamine is an important endogenous signaling molecule that is involved in a number of physiological processes including allergic reactions, gastric acid secretion, neurotransmitter release, and inflammation. The biological effects of histamine are mediated by four histamine receptors with distinct functions and distribution profiles (H1-H4). The most recently discovered histamine receptor (H4) has emerged as a promising drug target for treating inflammatory diseases. A detailed understanding of the role of the H4 receptor in human disease remains elusive, in part because low sequence similarity between the human and rodent H4 receptors complicates the translation of preclinical pharmacology to humans. This review provides an overview of H4 drug discovery programs that have studied cross-species structure-activity relationships, with a focus on the functional profiling of the 2-aminopyrimidine chemotype that has advanced to the clinic for allergy, atopic dermatitis, asthma, and rheumatoid arthritis.

The effect of pK(a) on pyrimidine/pyridine-derived histamine H4 ligands.

During the course of our efforts toward the discovery of human histamine H4 antagonists from a series of 2-aminiopyrimidines, it was noted that a 6-trifluoromethyl group dramatically reduced affinity of the series toward the histamine H4 receptor. This observation was further investigated by synthesizing a series of ligands that varied in pKa of the pyrimidine derived H4 ligands by over five orders of magnitude and the effect on histamine H4 affinity. This trend was then extended to the discovery of C-linked piperidinyl-2-amino pyridines as histamine H4 receptor antagonists.

Agonist/antagonist modulation in a series of 2-aryl benzimidazole H4 receptor ligands.

The present work details the transformation of a series of human histamine H(4) agonists into potent functional antagonists. Replacement of the aminopyrrolidine diamine functionality with a 5,6-fused pyrrolopiperidine ring system led to an antagonist. The dissection of this fused diamine led to the eventual replacement with heterocycles. The incorporation of histamine as the terminal amine led to a very potent and selective histamine H(4) agonist; whereas incorporation of the constrained histamine analog, spinacamine, modulated the functional activity to give a partial agonist. In two separate series, we demonstrate that constraining the terminal amino portion modulated the spectrum of functional activity of histamine H(4) ligands.

Phosphoinositide 3-kinase gamma (PI3Kgamma) inhibitors for the treatment of inflammation and autoimmune disease.

Phosphoinositide 3-kinase gamma (PI3Kgamma) is a lipid kinase in leukocytes that generates phosphatidylinositol 3,4,5-trisphosphate to recruit and activate downstream signaling molecules. Distinct from other members in the PI3K family, PI3Kgamma is activated by G-protein coupled-receptors responding to chemotactic ligands. PI3Kgamma plays an important role in migration of both myeloid and lymphoid cells. It is also required for other leukocyte functions such as neutrophil oxidative burst, T cell proliferation and mast degranulation. Mice with PI3Kgamma inactivated by genetic or pharmacological approaches are protected from disease development in a number of inflammation and autoimmune disease models. The function of PI3Kgamma depends on its kinase activity and therefore it has been suggested by many reports that small molecules inhibiting its kinase activity could be promising for the treatment of inflammation and autoimmune diseases. Over the last five years, a number of pharmaceutical companies have reported a wide variety of PI3Kgamma inhibitors, of which several x-ray crystal structures with PI3Kgamma have been elucidated. The structural characteristics and selectivity profiles of these inhibitors, in particular thiazolidinones and 2-aminoheterocycles, and those disclosed in related patent applications are summarized in this review.

Small molecule inhibitors of phosphoinositide 3-kinase (PI3K) delta and gamma.

In recent years, pharmaceutical companies have increasingly focused on phosphoinositide 3-kinases delta (PI3Kdelta) and gamma (PI3Kgamma) as therapeutic targets for the treatment of inflammatory and autoimmune diseases. All class 1 PI3-kinases (alpha/beta/gamma/delta) generate phospholipid second messengers that help govern cellular processes such as migration, proliferation, and apoptosis. PI3K delta/ gamma lipid kinases are mainly restricted to the hematopoetic system whereas PI3K alpha/beta are ubiquitously expressed, thus raising potential toxicity concerns for chronic indications such as asthma and rheumatoid arthritis. Therefore, the challenge in developing a small molecule inhibitor of PI3K is to define and attain the appropriate isoform selectivity profile. Significant advances in the design of such compounds have been achieved by utilizing x-ray crystal structures of various inhibitors bound to PI3Kgamma in conjunction with pharmacophore modeling and high-throughput screening. Herein, we review the history and challenges involved with the discovery of small molecule isoform-specific PI3K inhibitors. Recent progress in the design of selective PI3Kdelta, PI3Kgamma, and PI3Kdelta/gamma dual inhibitors will be presented.

TLR7/9 antagonists as therapeutics for immune-mediated inflammatory disorders.

There is an increasing interest in ligands of nucleic acid-sensing Toll-like receptors (TLR), especially TLR7 and TLR9, for pharmacological intervention in various diseases. The TLR7 agonist imiquimod is currently used as a topical treatment for genital warts caused by human papillomavirus (HPV), actinic keratosis (AK) and superficial basal cell carcinoma. Oligodeoxynucleotides (ODN) TLR9 agonists are currently in clinical trials for use in lung cancer, as anti-viral therapy, as adjuvants and as immune modulators in asthma and allergies. TLR7/9 antagonists, such as the anti-malaria drugs chloroquine, hydroxychloroquine and quinacrine, have been used since the 1950s to treat immune-mediated inflammatory disorders (IMID) such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sjögren's syndrome. However, the use of these anti-malarials in IMID is limited due to the side effects or suboptimal efficacy. Pre-clinical animal models as well as genetic linkage studies have indicated that TLR7/9 play a pivotal role in the aforementioned as well as other IMID such as multiple sclerosis (MS), inflammatory bowl disease (IBD)/colitis and psoriasis. Recent evidence has suggested that selective, specific antagonists for TLR7 and/or 9 might be more beneficial in certain diseases, such as SLE. Thus, the use of suppressive ODN or novel small molecule TLR7/9 inhibitors with a larger safety window and differentiated selectivity may potentially have significant clinical utility in these IMID. Herein, we review efforts to develop novel TLR7/9 antagonists and the rationale for the use of such therapeutics in a variety of IMID.

The histamine H4 receptor in autoimmune disease.

Histamine exerts its actions through four known receptors. The recently cloned histamine receptor, H4R, has been shown to have a role in chemotaxis and mediator release in various types of immune cells including mast cells, eosinophils, dendritic cells and T cells. H4R antagonists have been shown to have anti-inflammatory properties and efficacy in a number of disease models, such as those for asthma and colitis in vivo. Recently, H4R antagonists have been developed with high receptor affinity and specificity, which make them good tools for further characterisation of the receptor in animal models and, eventually, in humans. Histamine and the cells that produce it, such as mast cells and basophils, have long been thought to be involved in allergic conditions but there has recently been recognition that they may also play a role in various autoimmune diseases. Given this and the fact that the H4R has function in mast cells, dendritic cells and T cells, antagonists for the receptor may be useful in treating autoimmune diseases in addition to allergy.