Biochemical characterization of tirabrutinib and other irreversible inhibitors of Bruton's tyrosine kinase reveals differences in on - and off - target inhibition.

BACKGROUND: Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies. METHODS: Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC50 studies) or following a time course where inactivation kinetics were measured. RESULTS: Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M-1 s-1 for BTK with selectivity against important off-targets. CONCLUSIONS: For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. GENERAL SIGNIFICANCE: This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.

Discovery of Orally Efficacious Phosphoinositide 3-Kinase δ Inhibitors with Improved Metabolic Stability.

Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.

Dose-Dependent Suppression of Cytokine production from T cells by a Novel Phosphoinositide 3-Kinase Delta Inhibitor.

There remains a significant need for development of effective small molecules that can inhibit cytokine-mediated inflammation. Phosphoinositide 3 kinase (PI3K) is a direct upstream activator of AKT, and plays a critical role in multiple cell signaling pathways, cell cycle progression, and cell growth, and PI3K inhibitors have been approved or are in clinical development. We examined novel PI3Kdelta inhibitors, which are highly selective for the p110delta isoform of in CD3/CD28 stimulated T-cell cytokine production. In vitro generated CD4+ T effector cells stimulated in the presence of a PI3Kdelta inhibitor demonstrated a dose-dependent suppression of cytokines produced by Th1, Th2, and Th17 cells. This effect was T-cell intrinsic, and we observed similar effects on human PBMCs. Th17 cells expressing a constitutively activated form of AKT were resistant to PI3Kdelta inhibition, suggesting that the inhibitor is acting through AKT signaling pathways. Additionally, PI3Kdelta inhibition decreased IL-17 production in vivo and decreased neutrophil recruitment to the lung in a murine model of acute pulmonary inflammation. These experiments show that targeting PI3Kdelta activity can modulate T-cell cytokine production and reduce inflammation in vivo, suggesting that PI3Kdelta inhibition could have therapeutic potential in treating inflammatory diseases.

2,4,6-Triaminopyrimidine as a Novel Hinge Binder in a Series of PI3Kδ Selective Inhibitors.

Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) is an appealing target for several hematological malignancies and inflammatory diseases. Herein, we describe the discovery and optimization of a series of propeller shaped PI3Kδ inhibitors comprising a novel triaminopyrimidine hinge binder. Combinations of electronic and structural strategies were employed to mitigate aldehyde oxidase mediated metabolism. This medicinal chemistry effort culminated in the identification of 52, a potent and highly selective inhibitor of PI3Kδ that demonstrates efficacy in a rat model of arthritis.

Randomized phase 1 study of the phosphatidylinositol 3-kinase δ inhibitor idelalisib in patients with allergic rhinitis.

BACKGROUND: Phosphatidylinositol 3-kinase p110δ isoform (PI3K p110δ) activity is essential for mast cell activation, suggesting that inhibition of PI3K p110δ might be useful in treating allergic diseases. OBJECTIVE: We sought to determine the effect of the PI3K p110δ-selective inhibitor idelalisib on allergic responses. METHODS: This phase 1 randomized, double-blind, placebo-controlled, 2-period crossover study was conducted with the Vienna Challenge Chamber. Grass pollen-induced allergic symptoms were documented during screening. Eligible subjects received idelalisib (100 mg twice daily) or placebo for 7 days, with allergen challenge on day 7. After a 2-week washout period, subjects received the alternate treatment and repeated allergen challenge. Study measures included safety, nasal and nonnasal symptoms, nasal airflow, nasal secretions, basophil activation, and plasma cytokine levels. RESULTS: Forty-one patients with allergic rhinitis received idelalisib/placebo (n = 21) or placebo/idelalisib (n = 20). Idelalisib treatment was well tolerated. Mean total nasal symptom scores were lower during the combined idelalisib treatment periods compared with placebo (treatment difference [idelalisib - placebo], -1.78; 95% CI, -2.53 to -1.03; P < .001). Statistically significant differences were also observed for the combined treatment periods for total symptom scores, nasal airflow, nasal secretion weight, and nasal congestion scores. The percentage of ex vivo-activated basophils (CD63(+)/CCR3(+) cells; after stimulation with grass pollen) was substantially lower for idelalisib-treated compared with placebo-treated subjects. Plasma CCL17 and CCL22 levels were reduced after idelalisib treatment. CONCLUSION: Idelalisib treatment was well tolerated in patients with allergic rhinitis and appears to reduce allergic responses clinically and immunologically after an environmental allergen challenge.

Inhibition of PI3Kδ reduces kidney infiltration by macrophages and ameliorates systemic lupus in the mouse.

Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease generated and maintained throughout life by autoreactive T and B cells. Class I phosphoinositide 3-kinases (PI3K) are heterodimers composed of a regulatory and a catalytic subunit that catalyze phosphoinositide-3,4,5-P3 formation and regulate cell survival, migration, and division. Activity of the PI3Kδ isoform is enhanced in human SLE patient PBLs. In this study, we analyzed the effect of inhibiting PI3Kδ in MRL/lpr mice, a model of human SLE. We found that PI3Kδ inhibition ameliorated lupus progression. Treatment of these mice with a PI3Kδ inhibitor reduced the excessive numbers of CD4(+) effector/memory cells and B cells. In addition, this treatment reduced serum TNF-α levels and the number of macrophages infiltrating the kidney. Expression of inactive PI3Kδ, but not deletion of the other hematopoietic isoform PI3Kγ, reduced the ability of macrophages to cross the basement membrane, a process required to infiltrate the kidney, explaining MRL/lpr mice improvement by pharmacologic inhibition of PI3Kδ. The observations that p110δ inhibitor prolonged mouse life span, reduced disease symptoms, and showed no obvious secondary effects indicates that PI3Kδ is a promising target for SLE.

Effects of isoform-selective phosphatidylinositol 3-kinase inhibitors on osteoclasts: actions on cytoskeletal organization, survival, and resorption.

Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (ß), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15-20 min to 65-75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kß and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.

CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability.

Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ as a treatment approach for patients with B-cell malignancies. We thus identified 5-fluoro-3-phenyl-2-[(S)-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (CAL-101), a highly selective and potent p110δ small molecule inhibitor (half-maximal effective concentration [EC(50)] = 8nM). Using tumor cell lines and primary patient samples representing multiple B-cell malignancies, we have demonstrated that constitutive phosphatidylinositol-3-kinase pathway activation is p110δ-dependent. CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decreased phosphorylation of Akt and other downstream effectors, an increase in poly(ADP-ribose) polymerase and caspase cleavage and an induction of apoptosis. These effects have been observed across a broad range of immature and mature B-cell malignancies, thereby providing a rationale for the ongoing clinical evaluation of CAL-101.