7-(2-Anilinopyrimidin-4-yl)-1-benzazepin-2-ones Designed by a "Cut and Glue" Strategy Are Dual Aurora A/VEGF-R Kinase Inhibitors.

Although overexpression and hyperactivity of protein kinases are causative for a wide range of human cancers, protein kinase inhibitors currently approved as cancer drugs address only a limited number of these enzymes. To identify new chemotypes addressing alternative protein kinases, the basic structure of a known PLK1/VEGF-R2 inhibitor class was formally dissected and reassembled. The resulting 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones were synthesized and proved to be dual inhibitors of Aurora A kinase and VEGF receptor kinases. Crystal structures of two representatives of the new chemotype in complex with Aurora A showed the ligand orientation in the ATP binding pocket and provided the basis for rational structural modifications. Congeners with attached sulfamide substituents retained Aurora A inhibitory activity. In vitro screening of two members of the new kinase inhibitor family against the cancer cell line panel of the National Cancer Institute (NCI) showed antiproliferative activity in the single-digit micromolar concentration range in the majority of the cell lines.

Novel SAR for quinazoline inhibitors of EHMT1 and EHMT2.

Detailed structure activity relationship of two series of quinazoline EHMT1/EHMT2 inhibitors (UNC0224 and UNC0638) have been elaborated. New and active alternatives are presented for the ubiquitous substitution patterns found in literature for the linker to the lysine mimicking region and the lysine mimic itself. These findings could allow for advancing EHMT1/EHMT2 inhibitors of that type beyond tool compounds by fine-tuning physicochemical properties making these inhibitors more drug-like. .

Bioactive pyrrole alkaloids isolated from the Red Sea: marine sponge Stylissa carteri.

Fifteen pyrrole alkaloids were isolated from the Red Sea marine sponge Stylissa carteri and investigated for their biological activities. Four of them were dibrominated [(+) dibromophakelline, Z-3-bromohymenialdisine, (±) ageliferin and 3,4-dibromo-1H-pyrrole-2-carbamide], nine compounds were monobrominated [(-) clathramide C, agelongine, (+) manzacidin A, (-) 3-bromomanzacidin D, Z-spongiacidin D, Z-hymenialdisine, 2-debromostevensine, 2-bromoaldisine and 4-bromo-1H-pyrrole-2-carbamide)] and finally, two compounds were non-brominated derivatives viz., E-debromohymenialdisine and aldisine. The structure elucidations of isolated compounds were based on 1D & 2D NMR spectroscopic and MS studies, as well as by comparison with literature. In-vitro, Z-spongiacidin D exhibited a moderate activity on (ARK5, CDK2-CycA, CDK4/CycD1, VEGF-R2, SAK and PDGFR-beta) protein kinases. Moreover, Z-3-bromohymenialdisine showed nearly similar pattern. Furthermore, Z-hymenialdisine displayed a moderate effect on (ARK5 & VEGF-R2) and (-) clathramide C showed a moderate activity on AURORA-A protein kinases. While, agelongine, (+) manzacidin A, E-debromohymenialdisine and 3,4-dibromo-1H-pyrrole-2-carbamide demonstrated only marginal inhibitory activities. The cytotoxicity study was evaluated in two different cell lines. The most effective secondary metabolites were (+) dibromophakelline and Z-3-bromohymenialdisine on L5178Y. Finally, Z-hymenialdisine, Z-3-bromohymenialdisine and (±) ageliferin exhibited the highest cytotoxic activity on HCT116. No report about inhibition of AURORA-A and B by hymenialdisine/hymenialdisine analogs existed and no reported toxicity of ageliferin existed in literature.

Cytotoxic effects of the anthraquinone derivatives 1'-deoxyrhodoptilometrin and (S)-(-)-rhodoptilometrin isolated from the marine echinoderm Comanthus sp.

We investigated cytotoxic effects of the anthraquinone derivatives 1'-deoxyrhodoptilometrin (SE11) and (S)-(-)-rhodoptilometrin (SE16) isolated from the marine echinoderm Comanthus sp. in two tumor cell lines (C6 glioma, Hct116 colon carcinoma). Both compounds showed cytotoxic effects, with SE11 [IC50-value (MTT assay): 13.1 µM in Hct116 cells] showing a higher potency to induce apoptotic and necrotic cell death. No generation of oxidative stress was detectable (DCF assay), and also no modulation of Nrf2/ARE and NFκB signaling could be shown. Investigation of 23 protein kinases associated with cell proliferation, survival, metastasis, and angiogenesis showed that both compounds were potent inhibitors of distinct kinases, e.g., IGF1-receptor kinase, focal adhesion kinase, and EGF receptor kinase with SE11 being a more potent compound (IC50 values: 5, 18.4 and 4 µM, respectively). SE11 caused a decrease in ERK phosphorylation which may be a consequence of the inhibition of EGF receptor kinase by this compound. Since an inhibition of the EGF receptor/MAPK pathway is an important target for diverse cytostatic drugs, we suggest that the anthraquinone derivative 1'-deoxyrhodoptilometrin (SE11) may be an interesting lead structure for the development of new anticancer drugs.

Cytotoxic and protein kinase inhibiting nakijiquinones and nakijiquinols from the sponge Dactylospongia metachromia.

Chemical investigation of the sponge Dactylospongia metachromia afforded five new sesquiterpene aminoquinones (1-5), two new sesquiterpene benzoxazoles (6 and 7), the known analogue 18-hydroxy-5-epi-hyrtiophenol (8), and a known glycerolipid. The structures of all compounds were unambiguously elucidated by one- and two-dimensional NMR and by MS analyses, as well as by comparison with the literature. Compounds 1-5 showed potent cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 1.1 to 3.7 µM. When tested in vitro for their inhibitory potential against 16 different protein kinases, compounds 5, 6, and 8 exhibited the strongest inhibitory activity against ALK, FAK, IGF1-R, SRC, VEGF-R2, Aurora-B, MET wt, and NEK6 kinases (IC50 0.97-8.62 µM).

Trimeric hemibastadin congener from the marine sponge Ianthella basta.

The first naturally occurring trimeric hemibastadin congener, sesquibastadin 1 (1), and the previously reported bastadins 3, 6, 7, 11, and 16 (2-6) were isolated from the marine sponge Ianthella basta, collected in Indonesia. The structure of 1 was elucidated on the basis of 1D and 2D NMR measurements and by HRMS. Among all the isolated compounds, the linear sesquibastadin 1 (1) and bastadin 3 (2) showed the strongest inhibition rates for at least 22 protein kinases (IC(50) = 0.1-6.5 µM), while the macrocyclic bastadins (3-6) demonstrated a strong cytotoxic potential against the murine lymphoma cell line L5178Y (IC(50) = 1.5-5.3 µM).

Regulation of HDM2 activity by the ribosomal protein L11.

The HDM2 protein plays an important role in regulating the stability and function of the p53 tumor suppressor protein. In this report, we show that the ribosomal protein L11 can interact with HDM2 and inhibit HDM2 function, thus leading to the stabilization and activation of p53. The inhibition of HDM2 activity by L11 shows some similarity to the previously described activity of ARF, and expression of either ARF or L11 can induce a p53 response. Enhancement of the interaction between endogenous L11 and HDM2 following treatment of cells with low levels of actinomycin-D suggests that the HDM2/L11 interaction represents a novel pathway for p53 stabilization in response to perturbations in ribosome biogenesis.

Arthrinins A-D: novel diterpenoids and further constituents from the sponge derived fungus Arthrinium sp.

Bioassay-guided fractionation of a methanolic extract of the fungus Arthrinium sp., isolated from the Mediterranean sponge Geodia cydonium, afforded 10 natural products including five new diterpenoids, arthrinins A-D (1-4) and myrocin D (5). In addition, five known compounds were obtained, which included myrocin A (6), norlichexanthone (7), anomalin A (8), decarboxycitrinone (9) and 2,5-dimethyl-7-hydroxychromone (10). The structures of all isolated compounds were unambiguously elucidated based on extensive 1D and 2D NMR and HR-MS analyzes. The absolute configuration of arthrinins A-D (1-4) was established by the convenient Mosher method performed in NMR tubes and by interpretation of the ROESY spectra. Antiproliferative activity of the isolated compounds was assessed in vitro against four different tumor cell lines, including mouse lymphoma (L5178Y), human chronic myelogenous leukemia (K562), human ovarian cancer (A2780) and cisplatin-resistant ovarian cancer cells (A2780CisR), using the MTT assay. Norlichexanthone (7) and anomalin A (8) exhibited the strongest activities with IC50 values ranging from 0.40 to 74.0 µM depending on the cell line investigated. This was paralleled by the inhibitory activity of both compounds against 16 cancer related protein kinases including aurora-B, PIM1, and VEGF-R2. In vitro IC50 values of 7 and 8 against these three protein kinases ranged from 0.3 to 11.7 µM. Further investigation of the potential antitumoral activity of compounds 5-8 was performed in an in vitro angiogenesis assay against human umbilical vascular endothelial cells (HUVEC) sprouting induced by vascular endothelial growth factor A (VEGF-A). Anomalin A (8), myrocin D (5) and myrocin A (6) inhibited VEGF-A dependent endothelial cell sprouting with IC50 values of 1.8, 2.6 and 3.7 µM, respectively, whereas norlichexanthone (7) was inactive.

Workflow comparison for label-free, quantitative secretome proteomics for cancer biomarker discovery: method evaluation, differential analysis, and verification in serum.

The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed.

A novel roscovitine derivative potently induces G1-phase arrest in platelet-derived growth factor-BB-activated vascular smooth muscle cells.

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to the pathogenesis of restenosis. Thus, drugs interfering with cell cycle progression in VSMC are promising candidates for an antirestenotic therapy. In this study, we pharmacologically characterize N-5-(2-aminocyclohexyl)-N-7-benzyl-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine-5,7-di-amine (LGR1406), a novel derivative of the cyclin-dependent kinase (CDK) inhibitor roscovitine (ROSC), in PDGF-BB-activated VSMC. Cell proliferation was quantified measuring DNA synthesis via 5-bromo-2'-deoxyuridine incorporation. Analysis of cell cycle distribution was done by flow cytometry using propidium iodide-stained nuclei. Key regulators of the cell cycle and relevant signaling pathways were dissected by Western blot analyses. In addition, in vitro kinase assays and in silico studies regarding the pharmacokinetic profile of both compounds were performed. LGR1406 shows a stronger (IC(50) = 3.0 muM) antiproliferative activity than ROSC (IC(50) = 16.9 muM), halting VSMCs in G(0)/G(1) phase of the cell cycle, whereas ROSC does not arrest but rather delays cell cycle progression. Neither of the compounds interferes with early PDGF-BB-induced signaling pathways (p38, extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, Akt, signal transducer and activator of transcription 3), and both inhibit CDKs, with LGR1406 exerting a slightly higher potency against CDK1/2 and 4 than ROSC. Expression of cyclins A and E as well as hyperphosphorylation of the pocket proteins retinoblastoma protein and p107 are negatively affected by both compounds, although to a different extent. In silico calculations predicted a much higher metabolic stability for LGR1406 compared with ROSC. Altogether, ROSC derivatives, such as LGR1406 seem to be promising compounds for further development in antirestenotic therapy.

N-substituted 2'-(aminoaryl)benzothiazoles as kinase inhibitors: hit identification and scaffold hopping.

Starting with a hit from vHTS attained by a docking procedure of virtual compounds into ATP pockets of different kinases applying the 4SCan technology, variations of the adenine mimic resulted in the identification of promising scaffolds, giving rise to in vitro IC(50) values in the nanomolar range on different kinases down to 63nM.

Substituted 2-arylbenzothiazoles as kinase inhibitors: hit-to-lead optimization.

Based on an (aminoaryl)benzothiazole quinazoline hit structure for kinase inhibition, a systematic optimization program resulted in a lead structure allowing for inhibitory activities in cellular phosphorylation assays in the low nanomolar range.

Bioactive metabolites from the endophytic fungus Stemphylium globuliferum isolated from Mentha pulegium.

The endophytic fungus Stemphylium globuliferum was isolated from stem tissues of the Moroccan medicinal plant Mentha pulegium. Extracts of the fungus, which was grown on solid rice medium, exhibited considerable cytotoxicity when tested in vitro against L5178Y cells. Chemical investigation yielded five new secondary metabolites, alterporriol G (4) and its atropisomer alterporriol H (5), altersolanol K (11), altersolanol L (12), stemphypyrone (13), and the known compounds 6-O-methylalaternin (1), macrosporin (2), altersolanol A (3), alterporriol E (6), alterporriol D (7), alterporriol A (8), alterporriol B (9), and altersolanol J (10). The structures were determined on the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry. Among the alterporriol-type anthranoid dimers, the mixture of alterporriols G and H (4/5) exhibited considerable cytotoxicity against L5178Y cells with an EC(50) value of 2.7 microg/mL, whereas the other congeners showed only modest activity. The compounds were also tested for kinase inhibitory activity in an assay involving 24 different kinases. Compounds 1, 2, 3, and the mixture of 4 and 5 were the most potent inhibitors, displaying EC(50) values between 0.64 and 1.4 microg/mL toward individual kinases.

Hexa-, hepta- and nonaprenylhydroquinones isolated from marine sponges Sarcotragus muscarum and Ircinia fasciculata inhibit NF-kappaB signalling in H4IIE cells.

OBJECTIVES: Marine organisms have proven to be a rich source of potent pharmacologically active compounds. Three polyprenyl-1,4-hydroquinone derivates (hexaprenyl-1,4-hydroquinone, heptaprenyl-1,4-hydroquinone and nonaprenyl-1,4-hydroquinone) were isolated from the Zoobenthos-inhabiting sponges Sarcotragus muscarum and Ircinia fasciculata from the Eastern Mediterranean Sea (phylum: Porifera; class: Demospongiae). METHODS: Hexa-, hepta- and nonaprenylhydroquinone were identified by (1)H-NMR, H,H-COSY, heteronuclear multiple bond correlation, FAB-MS and UV spectroscopy. The effects of the compounds on cell viability was determined using the MTT assay; anti-oxidative potential was measured using the Trolox equivalent antioxidative capacity assay. Inhibition of nuclear factor-kappaB activity was detected by secreted alkaline phosphatase assay. Activity against an array of protein kinases was determined in 96-well FlashPlates. KEY FINDINGS: All compounds had prominent antioxidative activity, comparable to that of the synthetic vitamin E derivate Trolox. Hexaprenylhydroquinone showed the greatest cytotoxicity in H4IIE hepatoma cells (EC50 2.5 muM). All three compounds inhibited NF-kappaB signalling in this cell line, with heptaprenylhydroquinone being the most active. Screening of 23 kinases involved in signal transduction pathways (cell proliferation, survival, angiogenesis and metastasis) showed that hexaprenylhydroquinone and heptaprenylhydroquinone inhibited the activity of the epidermal growth factor receptor (IC50 1.6 and 1.4 mug/ml, respectively), and heptaprenylhydroquinone also inhibited the activity of other kinases (Src tyrosine kinase, vascular endothelial growth factor receptor 3 and insulin-like growth factor 1 receptor). CONCLUSIONS: The prenylated hydroquinones isolated from the marine sponges S. muscarum and I. fasciculata showed cytotoxic and antioxidative activities and inhibited NF-kappaB signalling in H4IIE hepatoma cells and protein kinases. These findings may result in the generation of new lead substances in cancer therapy.

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

Targeting hormone refractory prostate cancer by in vivo selected DNA libraries in an orthotopic xenograft mouse model.

The targeting of specific tissue is a major challenge for the effective use of therapeutics and agents mediating this targeting are strongly demanded. We report here on an in vivo selection technology that enables the de novo identification of pegylated DNA aptamers pursuing tissue sites harbouring a hormone refractory prostate tumour. To this end, two libraries, one of which bearing an 11 kDa polyethylene glycol (PEG) modification, were used in an orthotopic xenograft prostate tumour mouse model for the selection process. Next-generation sequencing revealed an in vivo enriched pegylated but not a naïve DNA aptamer recognising prostate cancer tissue implanted either subcutaneous or orthotopically in mice. This aptamer represents a valuable and cost-effective tool for the development of targeted therapies for prostate cancer. The described selection strategy and its analysis is not limited to prostate cancer but will be adaptable to various tissues, tumours, and metastases. This opens the path towards DNA aptamers being experimentally and clinically engaged as molecules for developing targeted therapy strategies.

A novel reverse transduction adenoviral array for the functional analysis of shRNA libraries.

BACKGROUND: The identification of novel drug targets by assessing gene functions is most conveniently achieved by high-throughput loss-of-function RNA interference screening. There is a growing need to employ primary cells in such screenings, since they reflect the physiological situation more closely than transformed cell lines do. Highly miniaturized and parallelized approaches as exemplified by reverse transfection or transduction arrays meet these requirements, hence we verified the applicability of an adenoviral microarray for the elucidation of gene functions in primary cells. RESULTS: Here, we present microarrays of infectious adenoviruses encoding short hairpin RNA (shRNA) as a new tool for gene function analysis. As an example to demonstrate its application, we chose shRNAs directed against seven selected human protein kinases, and we have performed quantitative analysis of phenotypical responses in primary human umbilical vein cells (HUVEC). These microarrays enabled us to infect the target cells in a parallelized and miniaturized procedure without significant cross-contamination: Viruses were reversibly immobilized in spots in such a way that the seeded cells were confined to the area of the viral spots, thus simplifying the subsequent addressing of genetically modified cells for analysis. Computer-assisted image analysis of fluorescence images was applied to analyze the cellular response after shRNA expression. Both the expression level of knock-down target proteins as well as the functional output as measured by caspase 3 activity and DNA fractionation (TUNEL) were quantified. CONCLUSION: We have developed an adenoviral microarray technique suitable for miniaturized and parallelized analysis of gene function. The practicability of this technique was demonstrated by the analysis of several kinases involved in the activation of programmed cell death, both in tumor cells and in primary cells.

Characterization of compound 584, an Abl kinase inhibitor with lasting effects.

BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

Inverse in silico screening for identification of kinase inhibitor targets.

Protein kinases are clinically relevant, attractive drug targets for cancer. One major problem with kinase inhibitors is broad promiscuity, causing off-target actions and side effects. In silico prediction of targets of a compound would immensely facilitate and accelerate drug development. Using a virtual "inverse" screening approach, where single compounds are docked into protein structures from a database, we identify among known targets of indirubin derivatives phosphoinositide-dependent kinase 1 (PDK1) as a target of one derivative (6BIO) in particular. This prediction is functionally supported by an in vitro kinase assay, inhibition of intracellular phosphorylation of PDK1-substrates, and inhibition of endothelial cell migration, which highly depends on PDK1. Virtual inverse screening combined with biological tests, thus, is proposed as a valuable tool for the drug discovery process and re-examination of already established kinase inhibitors.

Cytotoxic metabolites from the fungal endophyte Alternaria sp. and their subsequent detection in its host plant Polygonum senegalense.

From the Egyptian medicinal plant Polygonum senegalense the fungal endophyte Alternaria sp. was isolated. Extracts of the fungus grown either in liquid culture or on solid rice media exhibited cytotoxic activity when tested in vitro against L5178Y cells. Chromatographic separation of the extracts yielded 15 natural products, out of which seven were new compounds, with both fungal extracts differing considerably with regard to their secondary metabolites. Compounds 1, 2, 3, 6, and 7 showed cytotoxic activity with EC 50 values ranging from 1.7 to 7.8 microg/mL. When analyzed in vitro for their inhibitory potential against 24 different protein kinases, compounds 1- 3, 5- 8, and 15 inhibited several of these enzymes (IC 50 values 0.22-9.8 microg/mL). Interestingly, compounds 1, 3, and 6 were also identified as constituents of an extract derived from healthy leaves of the host plant P. senegalense, thereby indicating that the production of natural products by the endophyte proceeds also under in situ conditions within the plant host.