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.     

PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?

Dysregulated signal transduction in innate and adaptive immune cells is known to be associated with the development of various autoimmune and inflammatory diseases. Consequently, targeting intracellular signalling of the pro-inflammatory cytokine network heralds hope for the next generation of anti-inflammatory drugs. Phosphoinositide 3-kinases (PI3Ks) generate lipid-based second messengers that control an array of intracellular signalling pathways that are known to have important roles in leukocytes. In light of the recent progress in the development of selective PI3K inhibitors, and the beneficial effects of these inhibitors in models of acute and chronic inflammatory disorders, we discuss the therapeutic potential of blocking PI3K isoforms for the treatment of rheumatoid arthritis and other immune-mediated diseases.     

Insulin-Driven PI3K-AKT Signaling in the Hepatocyte Is Mediated by Redundant PI3Kα and PI3Kβ Activities and Is Promoted by RAS

1. Download : Download high-res image (248KB)
2. Download : Download full-size image

     

CoA Synthase is in complex with p85αPI3K and affects PI3K signaling pathway

The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA Synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85αPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85αPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110α and regulatory p85α subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85αPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction.     

Influence of liposome composition and membrane binding on protein kinase activity of PI3Kγ

Phosphoinositide 3-kinase γ (PI3Kγ) has been implicated in a variety of cellular signaling processes. It is a multifunctional enzyme with lipid and protein kinase activity that also acts as a scaffold protein. Although it is well known that membrane recruitment is essential for the phosphorylation of phosphoinositides, the cellular localization of PI3Kγ as a protein kinase remains unclear. It has merely been described that PI3Kγ protein kinase activity leading to MAPK activation seems to be restricted to a cytosolic localization. Here, we demonstrate that a hybrid-PI3Kγ having protein kinase, but not lipid kinase activity shows a similar cellular distribution with a high membrane association and comparable liposome binding behavior to wild-type PI3Kγ. Binding of PI3Kγ to liposomes mimicking the natural plasma membrane slightly stimulates autophosphorylation of PI3Kγ. However, liposomes containing an unphysiologically high amount of PI inhibit autophosphorylation of PI3Kγ. Finally, PI3Kγ bound to membrane fragments does not show autophosphorylation which is possibly due to protein–protein-interactions at the plasma membrane. This indicates that not only MAPK activation, but PI3Kγ protein kinase activity in general is localized in the cytosol.     

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.     

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.     

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.     

Calmodulin and IQGAP1 activation of PI3Kα and Akt in KRAS,HRAS and NRAS-driven cancers

Calmodulin (CaM) binds only oncogenic KRas, but not HRas or NRas, and thus contributes only to KRAS-driven cancers. How CaM interacts with KRas and how it boosts KRAS cancers are among the most coveted aims in cancer biology. Here we address this question, and further ask: Are there proteins that can substitute for CaM in HRAS- and NRAS-driven cancers? Can scaffolding protein IQGAP1 be one? Data suggest that formation of a CaM–KRas–PI3Kα ternary complex promotes full PI3Kα activation, and thereby potent PI3Kα/Akt/mTOR proliferative signaling. CaM binds PI3Kα at the cSH2 and nSH2 domains of its regulatory p85 subunit; the WW domain of IQGAP1 binds cSH2. This raises the question whether IQGAP1, together with an oncogenic Ras isoform, can partially activate PI3Kα. Activated, membrane-bound PI3Kα generates PIP₃. CaM shuttles Akt to the plasma membrane; CaM's release and concomitant phosphoinositide binding stimulates Akt activation. Notably, IQGAP1 directly interacts with, and helps juxtapose, PI3Kα and Akt as well as mTOR. Our mechanistic review aims to illuminate CaM's actions, and help decipher how oncogenic Ras isoforms – not only KRas4B – can activate the PI3Kα/Akt/mTOR pathway at the membrane and innovate drug discovery, including blocking the PI3Kα–IQGAP1 interaction in HRAS- and NRAS-driven cancers.     

Discovery and biological evaluation of novel pyrazolopyridine derivatives as potent and orally available PI3Kδ inhibitors

Phosphatidylinositol-3-kinase (PI3K)δ inhibition is one of the most attractive approaches to the treatment of autoimmune diseases and leukocyte malignancies. Through the exploration of pyrazolopyridine derivatives as potential PI3Kδ inhibitors, compound 12a was identified as a potent PI3Kδ inhibitor but suffered from poor oral exposure in mice. With a modified amide linkage group, compound 15a was developed as an orally available PI3Kδ inhibitor with reduced selectivity against other PI3Ks. To improve the trade-off between selectivity and PK profile, structure–activity relationship (SAR) studies of terminal substituents on the pyrolidine ring were conducted. As a result, we developed potent PI3Kδ inhibitors with good oral availability. In particular, the representative compound 15j showed excellent selectivity for PI3Kδ over other PI3Ks with good oral exposure in mice.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Syk-Mediated Translocation of PI3Kδ to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in β₂ integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during β₂ integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr³²³) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I_A. Syk-Tyr³²³ was found to be critical for the enrichment of the catalytic subunit p110δ of PI3K class I_A as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110δ to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of β₂ integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110δ signaling for β₂ integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Design and synthesis of a novel pyrrolidinyl pyrido pyrimidinone derivative as a potent inhibitor of PI3Kα and mTOR

Lead optimization efforts that employed structure base drug design and physicochemical property based optimization leading to the discovery of a novel series of 4-methylpyrido pyrimidinone (MPP) are discussed. Synthesis and profile of 1, a PI3Kα/mTOR dual inhibitor, is highlighted.     

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.     

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.     

PI3K 抑制剂抗肿瘤临床研究进展

磷脂酰肌醇-3-激酶 (PI3K) 是一种胞内磷脂酰肌醇激酶,在介导细胞生长、发育、分裂、分化和凋亡等过程中发挥重要作用,因此 PI3K 抑制剂的开发已成为当前抗癌新药研究的热点之一。目前已有多个 PI3K 抑制剂进入临床研究阶段或已上市,其单用或与其他药物联 用的疗效和安全性有待进一步临床验证。综述 PI3K 抑制剂作为抗肿瘤药物的临床研究进展,为其进一步研究与应用提供参考。     

Inhibition of Wnt and PI3K Signaling Modulates GSK-3β Activity and Induces Morphological Changes in Cortical Neurons: Role of Tau Phosphorylation

Glycogen synthase kinase GSK-3β has been identified as one of the major candidates mediating tau hyperphosphorylation at the same sites as those present in tau protein in brain from Alzheimer′s disease (AD) patients. However, the signal transduction pathways involved in the abnormal activation of GSK-3β, have not been completely elucidated. GSK-3β activity is repressed by the canonical Wnt signaling pathway, but it is also modulated through the PI3K/Akt route. Recent studies have suggested that Wnt signaling might be involved in the pathophysiology of AD. On the other hand, modulators of the PI3K pathway might be reduced during aging leading to a sustained activation of GSK-3β, which in turn would increase the risk of tau hyperphosphorylation. The role of Wnt and PI3K signaling inhibition on the extent of tau phosphorylation and neuronal morphology has not been completely elucidated. Thus, in the present investigation we analyzed the effects of different negative modulators of the Wnt and the PI3K pathways on GSK-3β activation and phosphorylation of tau at the PHF-1 epitope in cortical cultured neurons and hippocampal slices from adult rat brain. Changes in the microtubule network were also studied. We found that a variety of Wnt and PI3K inhibitors, significantly increased tau phosphorylation at the PHF-1 site, induced the disarrangement of the microtubule network and the accumulation of tau within cell bodies. These changes correlated with alterations in neuronal morphology. Special issue article in honor of Dr. Ricardo Tapia.     

CpG-ODN Attenuates Pathological Cardiac Hypertrophy and Heart Failure by Activation of PI3Kα-Akt Signaling

Phosphoinositide-3-kinase α (PI3Kα) represents a potential novel drug target for pathological cardiac hypertrophy (PCH) and heart failure. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are classic agonists of Toll-like receptor 9 (TLR9), which typically activates PI3K-Akt signaling in immune cells; however, the role of the nucleotide TLR9 agonists in cardiac myocytes is largely unknown. Here we report that CpG-ODN C274 could both attenuate PCH and improve cardiac dysfunction by activating PI3Kα-Akt signaling cascade. In vitro studies indicated that C274 could blunt reactivation of fetal cardiac genes and cell enlargement induced by a hypertrophic agent, isoproterenol. The anti-hypertrophic effect of C274 was suppressed by a pan-PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or a small interfering RNA targeting PI3Kα. In vivo studies demonstrated that PCH, as marked by increased heart weight (HW) and cardiac ANF mRNA, was normalized by pre-administration with C274. In addition, Doppler echocardiography detected cardiac ventricular dilation, and contractile dysfunction in isoproterenol-treated animals, consistent with massive replacement fibrosis, reflecting cardiac cell death. As expected, pre-treatment of mice with C274 could prevent cardiac dysfunction associated with diminished cardiac cell death and fibrosis. In conclusion, CpG-ODNs are novel cardioprotective agents possessing antihypertrophic and anti-cell death activity afforded by engagement of the PI3Kα-Akt signaling. CpG-ODNs may have clinical use curbing the progression of PCH and preventing heart failure.     

PI3K/Akt-dependent phosphorylation of GSK3β and activation of RhoA regulate Wnt5a-induced gastric cancer cell migration

Wnt5a, a non-transforming Wnt family member, plays complicated roles in oncogenesis and cancer metastasis. However, Wnt5a signaling in gastric cancer progression remains poorly defined. In this study, we found that Wnt5a dose-dependently stimulated the migration of human gastric cancer cells (SGC-7901), with the maximal effect at 100 ng/mL, via enhancing phosphorylation of PI3K/Akt and GSK3β and activating RhoA. Pharmaceutical inhibition of PI3K with LY294002 or Akt siRNA significantly decreased Wnt5a-induced GSK3β phosphorylation and consequently cell migration. Additionally, GSK3β siRNA remarkably inhibited Wnt5a-induced RhoA activation, stress fiber formation and cell migration. Analogously, pre-treatment with LiCl, which induced phosphorylation of GSK3β at Ser9, increased Wnt5a-induced cell migration. Finally, ectopic expression of dominant negative RhoA (N19) suppressed Wnt5a-induced cell migration. Taken together, we demonstrated for the first time that Wnt5a promoted gastric cancer cell migration via the PI3K/Akt/GSK3β/RhoA signaling pathway. These findings could provide a rationale for designing new therapy targeting gastric cancer metastasis.     

Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKT(Ser473) phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.