Selectivity switch between FAK and Pyk2: Macrocyclization of FAK inhibitors improves Pyk2 potency.

Herein, we describe the synthesis of Pyk2 inhibitors via macrocyclization of FAK and dual Pyk2-FAK inhibitors. We identified macrocycle 25a as a highly potent Pyk2 inhibitor (IC50=0.7nM), with ∼175-fold improvement in Pyk2 potency as compared to its acyclic counterpart. In many cases, macrocyclization improved Pyk2 potency while weakening FAK potency, thereby improving the Pyk2/FAK selectivity ratio for this structural class of inhibitors. Various macrocyclic linkers were studied in an attempt to optimize Pyk2 selectivity. We observed macrocyclic atropisomerism during the synthesis of 19-membered macrocycles 10a-d, and successfully obtained crystallographic evidence of one atropisomer (10a-AtropB) preferentially bound to Pyk2.

Inhibition of hepatobiliary transporters by a novel kinase inhibitor contributes to hepatotoxicity in beagle dogs.

PF-022 (1) is a novel polycyclic benzothiophene kinase inhibitor selective for mitogen-activated protein kinase-activated protein kinase 2 (MK2). Compound 1 emerged as an inhibitor bearing submicromolar potency against MK2 (IC50 5 nM) and demonstrated projected human pharmacokinetics sufficient for oral dosing. However, following a single, oral administration of 1 to beagle dogs, animals experienced an acute liver injury characterized by increases in biomarkers associated with hepatotoxicity; particularly noteworthy was the reversible elevation in bile salts and total bilirubin. Accompanying this observation was an ADME appraisal which included hepatic bioactivation of 1 in multiple species and the in vitro inhibition of P-glycoprotein (P-gp; IC50 21 µM). Simply attenuating the bioactivation via structural modification proved ineffective in improving the in vivo tolerability of this polycyclic scaffold. Hence, disruption of hepatobiliary transporters by the compound series was hypothesized as the likely mechanism contributing to the acute hepatotoxicity. Indeed, closer in vitro examination employing transporter gene overexpressing MDCK cell lines and membrane vesicles revealed potent compound-dependent inhibition of human multi-drug resistance-associated protein 2 (MRP2/ABCC2; IC50 38 µM) and bile salt export pump (BSEP/ABCB11; IC50 10 µM), two crucial hepatobiliary transport proteins accountable for bilirubin and bile salt homeostasis, respectively. Subsequent introduction of pKa-altering modifications to a second generation compound PF029 proved successful in reducing its affinity for these key efflux transporters (MRP2 IC50 >>80 µM; BSEP IC50 > 70 µM; P-gp > 90 µM), consequently mitigating this overt organ toxicity in dogs.

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury.

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

A benzothiophene inhibitor of mitogen-activated protein kinase-activated protein kinase 2 inhibits tumor necrosis factor alpha production and has oral anti-inflammatory efficacy in acute and chronic models of inflammation.

Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cytokines. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), a direct downstream substrate of p38 kinase, regulates lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) production through modulating the stability and translation of these mRNAs. Developing small-molecule inhibitors of MK2 may yield anti-inflammatory efficacy with a different safety profile relative to p38 kinase inhibitors. This article describes the pharmacologic properties of a benzothiophene MK2 inhibitor, PF-3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8-one]. PF-3644022 is a potent freely reversible ATP-competitive compound that inhibits MK2 activity (K(i) = 3 nM) with good selectivity when profiled against 200 human kinases. In the human U937 monocytic cell line or peripheral blood mononuclear cells, PF-3644022 potently inhibits TNFalpha production with similar activity (IC(50) = 160 nM). PF-3644022 blocks TNFalpha and IL-6 production in LPS-stimulated human whole blood with IC(50) values of 1.6 and 10.3 microM, respectively. Inhibition of TNFalpha in U937 cells and blood correlates closely with inhibition of phospho-heat shock protein 27, a target biomarker of MK2 activity. PF-3644022 displays good pharmacokinetic parameters in rats and is orally efficacious in both the rat acute LPS-induced TNFalpha model and the chronic streptococcal cell wall-induced arthritis model. Dose-dependent inhibition of TNFalpha production in the acute model and inhibition of paw swelling in the chronic model is observed with ED(50) values of 6.9 and 20 mg/kg, respectively. PF-3644022 efficacy in the chronic inflammation model is strongly correlated with maintaining a C(min) higher than the EC(50) measured in the rat LPS-induced TNFalpha model.

Biochemical, cellular, and anti-inflammatory properties of a potent, selective, orally bioavailable benzamide inhibitor of Rho kinase activity.

Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.

Benzothiophene inhibitors of MK2. Part 2: improvements in kinase selectivity and cell potency.

Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

Benzothiophene inhibitors of MK2. Part 1: structure-activity relationships, assessments of selectivity and cellular potency.

Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.

Aqueous versus non-aqueous salt delivery strategies to enhance oral bioavailability of a mitogen-activated protein kinase-activated protein kinase (MK-2) inhibitor in rats.

A potent pyridine-containing MK2 inhibitor has recently been internally discovered. In pre-clinical dosing, the low solubility of the neutral form limited oral bioavailability and dose escalation in toxicity studies. A mesylate salt was developed as part of a formulation strategy to enhance both oral bioavailability and dose escalation orally in pre-clinical rat studies. Several non-aqueous systems were used to deliver the mesylate salt, which resulted in varied oral bioavailability. It was found that administration of an aqueous chaser immediately after dosing drastically increased the oral bioavailability of the salt. This finding implies that the quantity of water present in vivo is an important consideration when evaluating salts of free bases with low aqueous solubility in pre-clinical in vivo rat models where limited aqueous vehicle may be presented.

Synthesis, crystal structure, and activity of pyrazole-based inhibitors of p38 kinase.

A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in vivo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule induces an interaction with Asp112 of p38 alpha. A compound identified from this series was efficacious in an animal model of rheumatic disease.

Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2.

The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.