SAR studies around a series of triazolopyridines as potent and selective PI3Kγ inhibitors

Herein we describe the SAR of a novel series of 6-aryl-2-amino-triazolopyridines as potent and selective PI3Kγ inhibitors. The 6-aryl-triazolopyridine core was identified by chemoproteomic screening of a kinase focused library. Rapid chemical expansion around a bi-functional core identified the key features required for PI3Kγ activity and selectivity. The series was optimized to afford 43 (CZC19945), a potent PI3Kγ inhibitor with high oral bioavailability and selectivity over PI3Kα and PI3Kδ. Modification to the core afforded 53 (CZC24832) which showed increased selectivity over the entire kinome in particular over PI3Kβ.     

Optimization and in vivo evaluation of pyrazolopyridines as a potent and selective PI3Kδ inhibitor

Chemical optimization of pyrazolopyridine 1, focused on cellular potency, isoform selectivity and microsomal stability, led to the discovery of the potent, selective and orally available PI3Kδ inhibitor 5d. On the basis of its desirable potency, selectivity and pharmacokinetic profiles, 5d was tested in the trinitrophenylated aminoethylcarboxymethyl-Ficoll (TNP-Ficoll)-induced antibody production model, and showed higher antibody inhibition than a 4-fold oral dose of the starting compound 1. These excellent results suggest that 5d is a potential candidate for further studies in the treatment of autoimmune diseases and leukocyte malignancies.     

Identification of highly potent and selective PI3Kδ inhibitors

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.     

Research advances on selective phosphatidylinositol 3 kinase δ (PI3Kδ) inhibitors

PI3Kδ in B cells mediates antigen receptor signaling and promote neutrophil chemotaxis. The activation of PI3Kδ can cause mast cell maturation and degranulation, myeloid cell dysfunction, and cytokine release. As a key signal molecule, PI3Kδ interacts with the lipid binding domain of a variety of cellular proteins as a secondary messenger, ultimately affecting a series of significant cellular pathways in disease pathology. Therefore, many research organizations and pharmaceutical companies have studied it to develop effectively selective PI3Kδ inhibitors as therapeutics. This review summarizes research advances in varying chemical classes of selective PI3Kδ inhibitors and the structure-activity relationship, and it mainly focuses on the propeller- versus flat-type class of inhibitors.     

PI3Kγ activation by CXCL12 regulates tumor cell adhesion and invasion

Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3Kγ regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.     

Optimization of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines to generate a highly selective PI3Kδ inhibitor

Chemical optimization of the 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP) scaffold was conducted with a focus on cellular potency while maintaining high selectivity against PI3K isoforms. Compound 11f was identified as a potent, highly selective and orally available PI3Kδ inhibitor. In addition, 11f exhibited efficacy in an in vivo antibody production model. The desirable drug-like properties and in vivo efficacy of 11f suggest its potential as a drug candidate for the treatment of autoimmune diseases and leukocyte malignancies.     

Genetic enhancement of behavioral itch responses in mice lacking phosphoinositide 3-kinase-γ (PI3Kγ)

Protein interacting with C Kinase 1 (PICK1), a PDZ domain-containing scaffolding protein, interacts with multiple different proteins in the mammalian nervous system and is believed to play important roles in diverse physiological and pathological conditions. In this study, we report that PICK1 is expressed in neurons of the dorsal root ganglion (DRG) and spinal cord dorsal horn, two major pain-related regions. PICK1 was present in approximately 29.7% of DRG neurons, most of which were small-less than 750 μm² in cross-sectional area. Some of these PICK1-positive cells co-labeled with isolectin B4 or calcitonin-gene-related peptide. In the dorsal horn, PICK1 immunoreactivity was concentrated in the superficial dorsal horn, where it was prominent in the postsynaptic density, axons, and dendrites. Targeted disruption of PICK1 gene did not affect basal paw withdrawal responses to acute noxious thermal and mechanical stimuli or locomotor reflex activity, but it completely blocked the induction of peripheral nerve injury-induced mechanical and thermal pain hypersensitivities. PICK1 appears to be required for peripheral nerve injury-induced neuropathic pain development and to be a potential biochemical target for treating this disorder.     

Biophysical and Structural Characterization of Novel RAS-Binding Domains (RBDs) of PI3Kα and PI3Kγ

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 Å X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.     

Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow

PI3Kδ mediates key immune cell signaling pathways and is a target of interest for multiple indications in immunology and oncology. Here we report a structure-based scaffold-hopping strategy for the design of chemically diverse PI3Kδ inhibitors. Using this strategy, we identified several scaffolds that can be combined to generate new PI3Kδ inhibitors with high potency and isoform selectivity. In particular, an oxindole-based scaffold was found to impart exquisite selectivity when combined with several hinge binding motifs.     

Discovery of a novel aminopyrazine series as selective PI3Kα inhibitors

We report the discovery of a novel aminopyrazine series of PI3Kα inhibitors, designed by hybridizing two known scaffolds of PI3K inhibitors. We describe the progress achieved from the first compounds plagued with poor general kinase selectivity to compounds showing high selectivity for PI3Kα over PI3Kβ and excellent general kinase selectivity. This effort culminated with the identification of compound 5 displaying high potency and selectivity, and suitable physiochemical and pharmacokinetic properties for oral administration. In vivo, compound 5 showed good inhibition of tumour growth (86% tumour growth inhibition at 50 mg/kg twice daily orally) in the MCF7 xenograft model in mice.     

Inhibition of protein phosphatase 2A activity by PI3Kγ regulates β-adrenergic receptor function

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Highlights? PI3Kγ inhibits βAR resensitization by regulating PP2A activity ? PI3Kγ inhibits PP2A activity by phosphorylating I2PP2A on Serine 9 and 93 ? Phosphorylation of I2PP2A leads to its agonist-mediated interaction with PP2A ? siRNA-targeted depletion of I2PP2A results in βAR resensitization     

Discovery of new aminopyrimidine-based phosphoinositide 3-kinase beta (PI3Kβ) inhibitors with selectivity over PI3Kα

Phosphatidylinositol-3-kinase beta (PI3Kβ) is an important therapeutic target in arterial thrombosis and special types of cancer. In this study, a new series of aminopyridine-based PI3Kβ selective inhibitors have been developed by the structure-based design strategy. When incorporated with the phenyl ring on sulfonamide moiety, aminopyrimidine analogs showed good potency on PI3Kβ and selectivity over PI3Kα. Intriguingly, replacement of phenyl group on sulfonamide with naphthyl group enhanced selectivity over PI3Kα while retaining submicromolar PI3Kβ potency. Molecular modeling suggests that increased PI3Kβ specificity is caused by the interaction with salt bridge (Lys782-Asp923) and Asp862 that creat a unique pocket in PI3Kβ. These results clearly provide useful insight in the design of new PI3Kβ inhibitors with high potency and selectivity.     

PI3Kγ Drives Priming and Survival of Autoreactive CD4+ T Cells during Experimental Autoimmune Encephalomyelitis

The class IB phosphoinositide 3-kinase gamma enzyme complex (PI3Kγ) functions in multiple signaling pathways involved in leukocyte activation and migration, making it an attractive target in complex human inflammatory diseases including MS. Here, using pik3cg ^−/− mice and a selective PI3Kγ inhibitor, we show that PI3Kγ promotes development of experimental autoimmune encephalomyelitis (EAE). In pik3cg^−/− mice, EAE is markedly suppressed and fewer leukocytes including CD4⁺ and CD8⁺ T cells, granulocytes and mononuclear phagocytes infiltrate the CNS. CD4⁺ T cell priming in secondary lymphoid organs is reduced in pik3cg^−/− mice following immunisation. This is attributable to defects in DC migration concomitant with a failure of full T cell activation following TCR ligation in the absence of p110γ. Together, this results in suppressed autoreactive T cell responses in pik3cg^−/− mice, with more CD4⁺ T cells undergoing apoptosis and fewer cytokine-producing Th1 and Th17 cells in lymphoid organs and the CNS. When administered from onset of EAE, the orally active PI3Kγ inhibitor AS605240 caused inhibition and reversal of clinical disease, and demyelination and cellular pathology in the CNS was reduced. These results strongly suggest that inhibitors of PI3Kγ may be useful therapeutics for MS.     

Hsp90 chaperones PPARγ and regulates differentiation and survival of 3T3-L1 adipocytes

Adipose tissue dysregulation has a major role in various human diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) is a key regulator of adipocyte differentiation and function, as well as a target of insulin-sensitizing drugs. The Hsp90 chaperone stabilizes a diverse set of signaling ‘client'' proteins, thereby regulates various biological processes. Here we report a novel role for Hsp90 in controlling PPARγ stability and cellular differentiation. Specifically, we show that the Hsp90 inhibitors geldanamycin and novobiocin efficiently impede the differentiation of murine 3T3-L1 preadipocytes. Geldanamycin at higher concentrations also inhibits the survival of both developing and mature adipocytes, respectively. Further, Hsp90 inhibition disrupts an Hsp90-PPARγ complex, leads to the destabilization and proteasomal degradation of PPARγ, and inhibits the expression of PPARγ target genes, identifying PPARγ as an Hsp90 client. A similar destabilization of PPARγ and a halt of adipogenesis also occur in response to protein denaturing stresses caused by a single transient heat-shock or proteasome inhibition. Recovery from stress restores PPARγ stability and adipocyte differentiation. Thus, our findings reveal Hsp90 as a critical stress-responsive regulator of adipocyte biology and offer a potential therapeutic target in obesity and the metabolic syndrome.     

Discovery and optimization of heteroaryl piperazines as potent and selective PI3Kδ inhibitors

A high-throughput screening (HTS) campaign identified a class of heteroaryl piperazines with excellent baseline affinity and selectivity for phosphoinositide 3-kinase δ (PI3Kδ) over closely related isoforms. Rapid evaluation and optimization of structure-activity relationships (SAR) for this class, leveraging the modular nature of this scaffold, facilitated development of this hit class into a series of potent and selective inhibitors of PI3Kδ. This effort culminated in the identification of 29, which displayed excellent potency in enzyme and cell-based assays, as well as favorable pharmacokinetic and off-target profiles.     

Identification of GNE-293, a potent and selective PI3Kδ inhibitor: Navigating in vitro genotoxicity while improving potency and selectivity

In an effort to identify potent and isoform selective inhibitors of PI3Kδ, GNE-293 (34) was identified. Inhibitor 2 was found to induce micronuclei formation in both the MNT and HCA in vitro assays. Compounds testing negative for genotoxicity were successfully identified through modifications of the 2-benzimidazole substituent and the methylene moiety to disrupt planarity. A variety of heteroatom linkers were explored to examine their effect on potency and isoform selectivity by restricting torsional angles to favor ligand interactions with PI3Kδ’s Trp760. These modifications also resulted in an improved in vivo pharmacokinetic profile.