Lead optimization of novel quinolone chalcone compounds by a structure-activity relationship (SAR) study to increase efficacy and metabolic stability.

Many agents targeting the colchicine binding site in tubulin have been developed as potential anticancer agents. However, none has successfully made it to the clinic, due mainly to dose limiting toxicities and the emergence of multi-drug resistance. Chalcones targeting tubulin have been proposed as a safe and effective alternative. We have shown previously that quinolone chalcones target tubulin and maintain potent anti-proliferative activity vis-à-vis colchicine, while also having high tolerability and low toxicity in mouse models of cancer and refractivity to multi-drug resistance mechanisms. To identify the most effective anticancer chalcone compound, we synthesized 17 quinolone-chalcone derivatives based on our previously published CTR-17 and CTR-20, and then carried out a structure-activity relationship study. We identified two compounds, CTR-21 [((E)-8-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] and CTR-32 [((E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] as potential leads, which contain independent moieties that play a significant role in their enhanced activities. At the nM range, CTR-21 and CTR-32 effectively kill a panel of different cancer cells originated from a variety of different tissues including breast and skin. Both compounds also effectively kill multi-drug resistant cancer cells. Most importantly, CTR-21 and CTR-32 show a high degree of selectivity against cancer cells. In silico, both of them dock near the colchicine-binding site with similar energies. Whereas both CTR-21 and CTR-32 effectively prevents tubulin polymerization, leading to the cell cycle arrest at G2/M, CTR-21 has more favorable metabolic properties. Perhaps not surprisingly, the combination of CTR-21 and ABT-737, a Bcl-2 inhibitor, showed synergistic effect in killing cancer cells, since we previously found the "parental" CTR-20 also exhibited synergism. Taken together, CTR-21 can potentially be a highly effective and relatively safe anticancer drug.

The influence of calculated physicochemical properties of compounds on their ADMET profiles.

We analyzed the influence of calculated physicochemical properties of more than 20,000 compounds on their P-gp and BCRP mediated efflux, microsomal stability, hERG inhibition, and plasma protein binding. Our goal was to provide guidance for designing compounds with desired pharmacokinetic profiles. Our analysis showed that compounds with ClogP less than 3 and molecular weight less than 400 will have high microsomal stability and low plasma protein binding. Compounds with logD less than 2.2 and/or basic pKa larger than 5.3 are likely to be BCRP substrates and compounds with basic pKa less than 5.2 and/or acidic pKa less than 13.4 are less likely to inhibit hERG. Based on these results, compounds with MW < 400, ClogP < 3, basic pKa < 5.2 and acidic pKa < 13.4 are likely to have good bioavailability and low hERG inhibition.

Synthesis and biological evaluation of triterpenoid thiazoles derived from betulonic acid, dihydrobetulonic acid, and ursonic acid.

In this work, 35 new derivatives of betulonic, dihydrobetulonic and ursonic acid were prepared including 30 aminothiazoles and all of them were tested for their in vitro cytotoxic activity in eight cancer cell lines and two non-cancer fibroblasts. Compounds with the IC50 below 5 µM in CCRF-CEM cells and low toxicity in non-cancer fibroblasts (4m, 5c, 5m, 6c, 6m, 7b, and 7c) were further subjected to tests of pharmacological parameters yielding the final set for advanced biological evaluation (4m, 5m, 6m, and 7b). It was proved by several methods, that all of them trigger apoptosis via the intrinsic pathway and derivatives 5m and 7b are the most effective (IC50 2.4 µM and 3.6 µM). They are the best candidates to become potentially new anticancer drugs and will be subjected to in vivo tests in mice. In addition, compounds 6b and 6c deserve more attention because their activity is not limited only to chemosensitive CCRF-CEM cell line. Specifically, compound 6b is highly active against K562 leukemic cell line (0.7 µM) and its IC50 activity in colon cancer HCT116 cell line is 1.0 µM. Compound 6c is active in both normal K562 and resistant K562-TAX cell lines (IC50 3.4 µM and 5.4 µM) and both colon cancer cell lines (HCT116 and HCT116p53-/-, IC50 3.5 µM and 3.4 µM).

Discovery of a New Isoxazole-3-hydroxamate-Based Histone Deacetylase 6 Inhibitor SS-208 with Antitumor Activity in Syngeneic Melanoma Mouse Models.

Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.

Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies.

Subcellular fractionation of tissue homogenate provides enriched in vitro models (e.g., microsomes, cytosol, or membranes), which are routinely used in the drug metabolism or transporter activity and protein abundance studies. However, batch-to-batch or interlaboratory variability in the recovery, enrichment, and purity of the subcellular fractions can affect performance of in vitro models leading to inaccurate in vitro to in vivo extrapolation (IVIVE) of drug clearance. To evaluate the quality of subcellular fractions, we developed a simple, targeted, and sensitive LC-MS/MS proteomics-based strategy, which relies on determination of protein markers of various cellular organelles, i.e., plasma membrane, cytosol, nuclei, mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, cytoskeleton, and exosomes. Application of the quantitative proteomics method confirmed a significant effect of processing variables (i.e., homogenization method and centrifugation speed) on the recovery, enrichment, and purity of isolated proteins in microsomes and cytosol. Particularly, markers of endoplasmic reticulum lumen and mitochondrial lumen were enriched in the cytosolic fractions as a result of their release during homogenization. Similarly, the relative recovery and composition of the total membrane fraction isolated from cell vs tissue samples was quantitatively different and should be considered in IVIVE. Further, analysis of exosomes isolated from sandwich-cultured hepatocyte media showed the effect of culture duration on compositions of purified exosomes. Therefore, the quantitative proteomics-based strategy developed here can be applied for efficient and simultaneous determination of multiple protein markers of various cellular organelles when compared to antibody- or activity-based assays and can be used for quality control of subcellular fractionation procedures including in vitro model development for drug metabolism and transport studies.

Discovery and structure-activity-relationship study of novel conformationally restricted indane analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors.

The discovery and optimization of various of indane amides as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, favorable PK properties and great selectivity against IDH1wt and IDH2R140Q.

Expression of Plant Receptor Kinases in Tobacco BY-2 Cells.

Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

Purification of Plant Receptor Kinases from Plant Plasma Membranes.

Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein. Here we describe two different optimized protein purification protocols, batch and on-chip immunoprecipitation, which efficiently isolate plant membrane receptor kinases for functional analysis.

Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor.

Ca2+ regulates ryanodine receptor's (RyR) activity through an activating and an inhibiting Ca2+-binding site located on the cytoplasmic side of the RyR channel. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. We used lanthanide ions (Ln3+) as probes to investigate the Ca2+ sensors of RyR, because they specifically bind to Ca2+-binding proteins and they are impermeable to the channel. Eu3+'s and Sm3+'s action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 µM Ca2+, Ln3+ potently inhibited RyR's open probability (Kd Eu3+ = 167 ± 5 nM and Kd Sm3+ = 63 ± 3 nM), but in nominally 0 [Ca2+], low [Eu3+] activated the channel. These results suggest that Ln3+ acts as an agonist of both Ca2+-binding sites. More importantly, the voltage-dependent characteristics of Ln3+'s action led to the conclusion that the activating Ca2+ binding site is located within the electrical field of the channel (in the vestibule). This idea was tested by applying the pore blocker toxin maurocalcine on the cytoplasmic side of RyR. These experiments showed that RyR lost reactivity to changing cytosolic [Ca2+] from 50 µM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca2+ from dissociating from its binding site and support our vestibular Ca2+ sensor-model further.

Inhibition and conformational change of SERCA3b induced by Bcl-2.

An interaction of Bcl-2 with SERCA had been documented in vitro using the SERCA1a isoform isolated from rat skeletal muscle [Dremina, E. S., Sharov, V. S., Kumar, K., Azidi, A., Michaelis, E. K., Schöneich, C. (2004) Biochem. J. 383 (361-370)]. Here, we demonstrate the interaction of Bcl-2 with the SERCA3b isoform both in vitro and in cell culture. In vitro, the interaction of Bcl-2 with SERCA3b was studied using Bcl-2∆21, a truncated form of human Bcl-2, and microsomes isolated from SERCA3b-overexpressing HEK-293 cells. For these experiments, SERCA3b was quantified by a combination of amino acid analysis and Western blotting. We observed that Bcl-2∆21 both inactivates SERCA3b and co-immunoprecipitates with SERCA3b. The incubation with Bcl-2∆21 changes the distribution of SERCA3b during sucrose density gradient centrifugation, likely as the result of Bcl-2∆21-induced conformational change of SERCA3b. When SERCA3b-overexpressing HEK-293 cells were co-transfected with Bcl-2, Bcl-2-dependent SERCA3b inactivation was observed. In these cells, Bcl-2 interaction with SERCA3b was demonstrated by co-immunoprecipitation. Furthermore, overexpression of Bcl-2 reduced fluorescein isothiocyanate (FITC) labeling of SERCA3b. Together, our data provide evidence for the interaction of Bcl-2 with SERCA3b in vitro and in cell culture, and for Bcl-2-dependent conformational and functional changes of SERCA3b.

Isolation of Endoplasmic Reticulum and Its Membrane.

The association of ribosomes with the rough endoplasmic reticulum (ER) is dependent on Mg2+. The ribosomes can be stripped from the ER by removal of Mg2+ from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when ER vesicles are analyzed by equilibrium density gradient centrifugation. Here, I describe the isolation of microsomes from Arabidopsis, followed by the use of the density shift approach in conjunction with equilibrium density gradient centrifugation as a means to diagnose whether a protein is associated with the ER. The same approach can also be used as a means to enrich for ER membranes.

The C-terminal Domains of Apoptotic BH3-only Proteins Mediate Their Insertion into Distinct Biological Membranes.

Changes in the equilibrium of pro- and anti-apoptotic members of the B-cell lymphoma-2 (Bcl-2) protein family in the mitochondrial outer membrane (MOM) induce structural changes that commit cells to apoptosis. Bcl-2 homology-3 (BH3)-only proteins participate in this process by either activating pro-apoptotic effectors or inhibiting anti-apoptotic components and by promoting MOM permeabilization. The association of BH3-only proteins with MOMs is necessary for the activation and amplification of death signals; however, the nature of this association remains controversial, as these proteins lack a canonical transmembrane sequence. Here we used an in vitro expression system to study the insertion capacity of hydrophobic C-terminal regions of the BH3-only proteins Bik, Bim, Noxa, Bmf, and Puma into microsomal membranes. An Escherichia coli complementation assay was used to validate the results in a cellular context, and peptide insertions were modeled using molecular dynamics simulations. We also found that some of the C-terminal domains were sufficient to direct green fluorescent protein fusion proteins to specific membranes in human cells, but the domains did not activate apoptosis. Thus, the hydrophobic regions in the C termini of BH3-only members associated in distinct ways with various biological membranes, suggesting that a detailed investigation of the entire process of apoptosis should include studying the membranes as a setting for protein-protein and protein-membrane interactions.

Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers.

Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients.

A microsomal based method to detect aromatase activity in different brain regions of the rat using ultra performance liquid chromatography-mass spectrometry.

Aromatase (ARO) is a cytochrome P450 enzyme that accounts for local estrogen production in the brain. The goal of this study was to develop a microsomal based assay to sensitively and reliably detect the low levels of ARO activity in different brain regions. Enzyme activity was detected based on the conversion of testosterone to estradiol. Quantity of estradiol was measured using ultra performance liquid chromatography-mass spectrometry. Detection was linear over a range of 2.5-200pg/ml estradiol, and was reproducible with intra- and inter-assay coefficients of variation (CV) <15%. Estradiol production using isolated microsomes was linear with time up to 30min as well as linearly related to amount of microsome. Substrate concentration curves revealed enzymatic kinetics (hippocampus: Vmax and Km: 0.57pmol estradiol/h per mg microsome and 48.58nM; amygdala: Vmax and Km: 1.69pmol estradiol/h per mg microsome and 48.4nM; preoptic area: Vmax and Km: 0.96pmol estradiol/h per mg microsome and 44.31nM) with testosterone used at a saturating concentration of 400nM. Anastrozole treatment blocked ARO activity in hippocampal and ovarian microsomes, indicating that the assay is specific for ARO. Also, we showed that the distribution of the long form ARO mRNA (CYP19A1) in different regions of the brain is correlated with ARO activity, with highest levels in the amygdala, followed by preoptic area and hippocampus. In the frontal cortex, very little long form ARO mRNA, and little to no ARO activity, were detected. These findings demonstrate that the microsomal incubation (MIB) assay is a sensitive and reliable method for quantifying ARO activity in discrete brain regions.

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells.

BACKGROUND: Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. METHODOLOGY: Immature Leydig cells isolated from 35 day-old rats were cultured with 30 µM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 µM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. RESULTS AND CONCLUSIONS: In intact Leydig cells, 30 µM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 µM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 µM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 µM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.

[Phosphoinositides: lipidic essential actors in the intracellular traffic]. / Les phosphoinositides, des lipides acteurs essentiels du trafic intracellulaire.

Phosphoinositides (PPIn) are lipids involved in the vesicular transport of proteins between the different intracellular compartments. They act by recruiting and/or activating effector proteins and are thus involved in crucial cellular functions including vesicle budding, fusion and dynamics of membranes and regulation of the cytoskeleton. Although they are present in low concentrations in membranes, their activity is essential for cell survival and needs to be tightly controlled. Therefore, phosphatases and kinases specific of the various cellular membranes can phosphorylate/dephosphorylate their inositol ring on the positions D3, D4 and/or D5. The differential phosphorylation determines the intracellular localisation and the activity of the PPIn. Indeed, non-phosphorylated phosphatidylinositol (PtdIns) is the basic component of the PPIn and can be found in all eukaryotic cells at the cytoplasmic face of the ER, the Golgi, mitochondria and microsomes. It can get phosphorylated on position D4 to obtain PtdIns4P, a PPIn enriched in the Golgi compartment and involved in the maintenance of this organelle as well as anterograde and retrograde transport to and from the Golgi. PtdIns phosphorylation on position D3 results in PtdIns3P that is required for endosomal transport and multivesicular body (MVB) formation and sorting. These monophosphorylated PtdIns can be further phosphorylated to produce bisphophorylated PtdIns. Thus, PtdIns(4,5)P2, mainly produced by PtdIns4P phosphorylation, is enriched in the plasma membrane and involved in the regulation of actin cytoskeleton and endocytosis. PtdIns(3,5)P2, mainly produced by PtdIns3P phosphorylation, is enriched in late endosomes, MVBs and the lysosome/vacuole and plays a role in endosome to vacuole transport. PtdIns(3,4)P2 is absent in yeast, cells and mainly produced by PtdIns4P phosphorylation in human cells; PtdIns(3,4)P2 is localised in the plasma membrane and plays an important role as a second messenger by recruiting specific protein kinases (Akt and PDK1). Finally the triple phosphorylated PPIn, PtdIns(3,4,5)P3 also absent in yeast, is produced by the phosphorylation of PtdIns(3,4)P2 and localized at the plasma membrane of human cells where it binds proteins via their PH domain. Interaction partners include members of the Arf (ADP-ribosylation factors) family, PDK1 (Phosphoinositide Dependent Kinase 1) and Akt. Therefore this last PPIn is essential for the control of cell proliferation and its deregulation leads to the development of numerous cancers. In conclusion, the regulation of PPIn phosphorylation/dephosphorylation is complex and needs to be very precisely regulated. Indeed phosphatases and kinases allow the maintenance of the equilibrium between the different forms. PPIn play a crucial role in numerous cellular functions and a loss in their synthesis or regulation results in severe genetic diseases.

Proton myo-inositol cotransporter is a novel γ-secretase associated protein that regulates Aß production without affecting Notch cleavage.

γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid ß-peptide (Aß), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aß production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aß and these proteins were selected for further studies on Aß production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aß secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aß40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aß production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aß lowering therapy aimed at treating Alzheimer's disease.

Functional and pharmacological induced structural changes of the cystic fibrosis transmembrane conductance regulator in the membrane solved using SAXS.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to the ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. To investigate the conformation of the CFTR in the membrane, we applied the small-angle x-ray scattering (SAXS) technique on microsomal membranes extracted from NIH/3T3 cells permanentely transfected with wild-type (WT) CFTR and with CFTR carrying the ΔF508 mutation. The electronic density profile of the membranes was calculated from the SAXS data, assuming the lipid bilayer electronic density to be composed by a series of Gaussian shells. The data indicate that membranes in the microsome vesicles, that contain mostly endoplasmic reticulum membranes, are oriented in the outside-out conformation. Phosphorylation does not change significantly the electronic density profile, while dephosphorylation produces a significant modification in the inner side of the profile. Thus, we conclude that the CFTR and its associated protein complex in microsomes are mostly phosphorylated. The electronic density profile of the ΔF508-CFTR microsomes is completely different from WT, suggesting a different assemblage of the proteins in the membranes. Low-temperature treatment of cells rescues the ΔF508-CFTR protein, resulting in a conformation that resembles the WT. Differently, treatment with the corrector VX-809 modifies the electronic profile of ΔF508-CFTR membrane, but does not recover completely the WT conformation. To our knowledge, this is the first report of a direct physical measurement of the structure of membranes containing CFTR in its native environment and in different functional and pharmacological conditions.

Pyrazolopyridine inhibitors of B-Raf(V600E). Part 4: rational design and kinase selectivity profile of cell potent type II inhibitors.

Cell potent inhibitors of B-Raf(V600E) that bind to the kinase in the DFG-out conformation are reported. These compounds utilize the hinge-binding group and lipophilic linker from a previously disclosed series of B-Raf(V600E) inhibitors that bind to the kinase in an atypical DFG-in, αC-helix-out conformation. This new series demonstrates that DFG-out kinase inhibitors can be rationally designed from related inhibitors which utilize an unconventional binding mode. Kinase selectivity profiles are compared. The pattern of kinase selectivity was found to be determined by the feature of the inhibitor which extends into the back pocket of the kinase and leads to the kinase conformation, rather than by the hinge-binding group or other minor modifications.

Physiologically based kinetic models for the alkenylbenzene elemicin in rat and human and possible implications for risk assessment.

The present study describes physiologically based kinetic (PBK) models for the alkenylbenzene elemicin (3,4,5-trimethoxyallylbenzene) in rat and human, based on the PBK models previously developed for the structurally related alkenylbenzenes estragole, methyleugenol, and safrole. Using the newly developed models, the level of metabolic activation of elemicin in rat and human was predicted to obtain insight in species differences in the bioactivation of elemicin and read across to the other methoxy allylbenzenes, estragole and methyleugenol. Results reveal that the differences between rat and human in the formation of the proximate carcinogenic metabolite 1'-hydroxyelemicin and the ultimate carcinogenic metabolite 1'-sulfoxyelemicin are limited (<3.8-fold). In addition, a comparison was made between the relative importance of bioactivation for elemicin and that of estragole and methyleugenol. Model predictions indicate that compound differences in the formation of the 1'-sulfoxymetabolites are limited (<11-fold) in rat and human liver. The insights thus obtained were used to perform a risk assessment for elemicin using the margin of exposure (MOE) approach and read across to the other methoxy allylbenzene derivatives for which in vivo animal tumor data are available. This reveals that elemicin poses a lower priority for risk management as compared to its structurally related analogues estragole and methyleugenol. Altogether, the results obtained indicate that PBK modeling provides an important insight in the occurrence of species differences in the metabolic activation of elemicin. Moreover, they provide an example of how PBK modeling can facilitate a read across in risk assessment from compounds for which in vivo toxicity studies are available to a compound for which only limited toxicity data have been described, thus contributing to the development of alternatives for animal testing.