High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRASG12C by Intact Protein MS and Targeted MRM.

With recent advances and success in several drugs designed to treat acute and chronic diseases, targeted covalent inhibitors show a resurgence in drug discovery. As covalent inhibition is time-dependent, the preferred quantitative potency metric of irreversible inhibitors is the second-order rate constant kinact/Ki, rather than IC50. Here, we present the development of a mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors. Using a simple liquid handling robot for automated sample preparation and a solid-phase extraction-based RapidFire-MS system for rapid MS analysis, kinetic characterization of covalent inhibitors was performed in high throughput both by intact protein analysis and targeted multiple reaction monitoring (MRM). In addition, a bimolecular reaction model was applied to extract kinact/Ki in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRASG12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of kinact/Ki values from single digit to 3 × 105 M-1 s-1.

Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

Design, synthesis and activity of a potent, selective series of N-aryl pyridinone inhibitors of p38 kinase.

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.

Discovery of PH-797804, a highly selective and potent inhibitor of p38 MAP kinase.

The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.

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.

Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain.

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation.

Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.

Selective inhibition of the p38α MAPK-MK2 axis inhibits inflammatory cues including inflammasome priming signals.

p38α activation of multiple effectors may underlie the failure of global p38α inhibitors in clinical trials. A unique inhibitor (CDD-450) was developed that selectively blocked p38α activation of the proinflammatory kinase MK2 while sparing p38α activation of PRAK and ATF2. Next, the hypothesis that the p38α-MK2 complex mediates inflammasome priming cues was tested. CDD-450 had no effect on NLRP3 expression, but it decreased IL-1ß expression by promoting IL-1ß mRNA degradation. Thus, IL-1ß is regulated not only transcriptionally by NF-κB and posttranslationally by the inflammasomes but also posttranscriptionally by p38α-MK2. CDD-450 also accelerated TNF-α and IL-6 mRNA decay, inhibited inflammation in mice with cryopyrinopathy, and was as efficacious as global p38α inhibitors in attenuating arthritis in rats and cytokine expression by cells from patients with cryopyrinopathy and rheumatoid arthritis. These findings have clinical translation implications as CDD-450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non-MK2 substrates involved in antiinflammatory and housekeeping responses.

Discovery of a Potent and Selective Sphingosine Kinase 1 Inhibitor through the Molecular Combination of Chemotype-Distinct Screening Hits.

Sphingosine kinase (SphK) is the major source of the lipid mediator and G protein-coupled receptor agonist sphingosine-1-phosphate (S1P). S1P promotes cell growth, survival, and migration and is a key regulator of lymphocyte trafficking. Inhibition of S1P signaling has been proposed as a strategy for treatment of inflammatory diseases and cancer. Two different formats of an enzyme-based high-throughput screen yielded two attractive chemotypes capable of inhibiting S1P formation in cells. The molecular combination of these screening hits led to compound 22a (PF-543) with 2 orders of magnitude improved potency. Compound 22a inhibited SphK1 with an IC50 of 2 nM and was more than 100-fold selective for SphK1 over the SphK2 isoform. Through the modification of tail-region substituents, the specificity of inhibition for SphK1 and SphK2 could be modulated, yielding SphK1-selective, potent SphK1/2 dual, or SphK2-preferential inhibitors.

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.

Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis.

BACKGROUND: The Janus kinase (JAK) family of tyrosine kinases includes JAK1, JAK2, JAK3 and TYK2, and is required for signaling through Type I and Type II cytokine receptors. CP-690,550 is a potent and selective JAK inhibitor currently in clinical trials for rheumatoid arthritis (RA) and other autoimmune disease indications. In RA trials, dose-dependent decreases in neutrophil counts (PBNC) were observed with CP-690,550 treatment. These studies were undertaken to better understand the relationship between JAK selectivity and PBNC decreases observed with CP-690,550 treatment. METHODS: Potency and selectivity of CP-690,550 for mouse, rat and human JAKs was evaluated in a panel of in vitro assays. The effect of CP-690,550 on granulopoiesis from progenitor cells was also assessed in vitro using colony forming assays. In vivo the potency of orally administered CP-690,550 on arthritis (paw edema), plasma cytokines, PBNC and bone marrow differentials were evaluated in the rat adjuvant-induced arthritis (AIA) model. RESULTS: CP-690,550 potently inhibited signaling through JAK1 and JAK3 with 5-100 fold selectivity over JAK2 in cellular assays, despite inhibiting all four JAK isoforms with nM potency in in vitro enzyme assays. Dose-dependent inhibition of paw edema was observed in vivo with CP-690,550 treatment. Plasma cytokines (IL-6 and IL-17), PBNC, and bone marrow myeloid progenitor cells were elevated in the context of AIA disease. At efficacious exposures, CP-690,550 returned all of these parameters to pre-disease levels. The plasma concentration of CP-690,550 at efficacious doses was above the in vitro whole blood IC50 of JAK1 and JAK3 inhibition, but not that of JAK2. CONCLUSION: Results from this investigation suggest that CP-690,550 is a potent inhibitor of JAK1 and JAK3 with potentially reduced cellular potency for JAK2. In rat AIA, as in the case of human RA, PBNC were decreased at efficacious exposures of CP-690,550. Inflammatory end points were similarly reduced, as judged by attenuation of paw edema and cytokines IL-6 and IL-17. Plasma concentration at these exposures was consistent with inhibition of JAK1 and JAK3 but not JAK2. Decreases in PBNC following CP-690,550 treatment may thus be related to attenuation of inflammation and are likely not due to suppression of granulopoiesis through JAK2 inhibition.