Large-Scale Plasma Proteome Epitome Profiling is an Efficient Tool for the Discovery of Cancer Biomarkers.

Current proteomic technologies focus on the quantification of protein levels, while little effort is dedicated to the development of system approaches to simultaneously monitor proteome variability and abundance. Protein variants may display different immunogenic epitopes detectable by monoclonal antibodies. Epitope variability results from alternative splicing, posttranslational modifications, processing, degradation, and complex formation and possesses dynamically changing availability of interacting surface structures that frequently serve as reachable epitopes and often carry different functions. Thus, it is highly likely that the presence of some of the accessible epitopes correlates with function under physiological and pathological conditions. To enable the exploration of the impact of protein variation on the immunogenic epitome first, here, we present a robust and analytically validated PEP technology for characterizing immunogenic epitopes of the plasma. To this end, we prepared mAb libraries directed against the normalized human plasma proteome as a complex natural immunogen. Antibody producing hybridomas were selected and cloned. Monoclonal antibodies react with single epitopes, thus profiling with the libraries is expected to profile many epitopes which we define by the mimotopes, as we present here. Screening blood plasma samples from control subjects (n = 558) and cancer patients (n = 598) for merely 69 native epitopes displayed by 20 abundant plasma proteins resulted in distinct cancer-specific epitope panels that showed high accuracy (AUC 0.826-0.966) and specificity for lung, breast, and colon cancer. Deeper profiling (≈290 epitopes of approximately 100 proteins) showed unexpected granularity of the epitope-level expression data and detected neutral and lung cancer-associated epitopes of individual proteins. Biomarker epitope panels selected from a pool of 21 epitopes of 12 proteins were validated in independent clinical cohorts. The results demonstrate the value of PEP as a rich and thus far unexplored source of protein biomarkers with diagnostic potential.

Epitomics: Analysis of Plasma C9 Epitope Heterogeneity in the Plasma of Lung Cancer Patients and Control Subjects.

The human proteome is more complex than the genetic code predicts it to be. Epitomics, or protein epitome profiling, is a tool for understanding sub-protein level variation. With the ultimate goal to explore C9 proteoforms and their relevance to lung cancer, here we report plasma C9 epitope-associated molecular heterogeneity in plasma samples of lung cancer patients and control subjects. We show three C9 epitopes (BSI0449, BSI0581, BSI0639) with markedly different association with lung cancer ("unaltered", "upregulated" and "downregulated"). In order to exclude confounding effects, we show first that the three epitope-defining mAbs recognize C9 in purified form and in the natural context, in the human plasma. Then, we present data demonstrating the lack of major epitope interdependence or overlap. The next experiments represent a quest toward the understanding of the molecular basis of apparent disparate association with lung cancer. Using immunochemistry, SDS PAGE and LC-MS/MS technologies, we demonstrate that epitope-specific immunoprecipitates of plasma C9 seem identical regarding peptide sequence. However, we found epitope-specific posttranslational modification and coprecipitated protein composition differences with respect to control and lung cancer plasma. Epitope profiling enabled the classification of hypothetical C9 proteoforms through differential association with lung cancer.

Comparative proteome analysis identified CD44 as a possible serum marker for docetaxel resistance in castration-resistant prostate cancer.

Baseline or acquired resistance to docetaxel (DOC) represents a significant risk for patients with metastatic prostate cancer (PC). In the last years, novel therapy regimens have been approved providing reasonable alternatives for DOC-resistant patients making prediction of DOC resistance of great clinical importance. We aimed to identify serum biomarkers, which are able to select patients who will not benefit from DOC treatment. DOC-resistant PC3-DR and DU145-DR sublines and their sensitive parental cell lines (DU145, PC3) were comparatively analyzed using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Results were filtered using bioinformatics approaches to identify promising serum biomarkers. Serum levels of five proteins were determined in serum samples of 66 DOC-treated metastatic castration-resistant PC patients (mCRPC) using ELISA. Results were correlated with clinicopathological and survival data. CD44 was subjected to further functional cell culture analyses. We found at least 177 two-fold significantly overexpressed proteins in DOC-resistant cell lines. Our bioinformatics method suggested 11/177 proteins to be secreted into the serum. We determined serum levels of five (CD44, MET, GSN, IL13RA2 and LNPEP) proteins in serum samples of DOC-treated patients and found high CD44 serum levels to be independently associated with poor overall survival (p = 0.001). In accordance, silencing of CD44 in DU145-DR cells resulted in re-sensitization to DOC. In conclusion, high serum CD44 levels may help identify DOC-resistant patients and may thereby help optimize clinical decision-making regarding type and timing of therapy for mCRPC patients. In addition, our in vitro results imply the possible functional involvement of CD44 in DOC resistance.

Proteome profiling of enzalutamide-resistant cell lines and serum analysis identified ALCAM as marker of resistance in castration-resistant prostate cancer.

Enzalutamide (ENZA) is a frequently used therapy in metastatic castration-resistant prostate cancer (mCRPC). Baseline or acquired resistance to ENZA have been observed, but the molecular mechanisms of resistance are poorly understood. We aimed to identify proteins involved in ENZA resistance and to find therapy-predictive serum markers. We performed comparative proteome analyses on ENZA-sensitive parental (LAPC4, DuCaP) and -resistant prostate cancer cell lines (LAPC4-ENZA, DuCaP-ENZA) using liquid chromatography tandem mass spectrometry (LC-MS/MS). The top four most promising candidate markers were selected using bioinformatic approaches. Serum concentrations of selected markers (ALCAM, AGR2, NDRG1, IDH1) were measured in pretreatment samples of 72 ENZA-treated mCRPC patients using ELISA. In addition, ALCAM serum levels were measured in 101 Abiraterone (ABI) and 100 Docetaxel (DOC)-treated mCRPC patients' baseline samples. Results were correlated with clinical and follow-up data. The functional role of ALCAM in ENZA resistance was assessed in vitro using siRNA. Our proteome analyses revealed 731 significantly differentially abundant proteins between ENZA-sensitive and -resistant cells and our filtering methods identified four biomarker candidates. Serum analyses of these proteins revealed only ALCAM to be associated with poor patient survival. Furthermore, higher baseline ALCAM levels were associated with poor survival in ABI- but not in DOC-treated patients. In LAPC4-ENZA resistant cells, ALCAM silencing by siRNA knockdown resulted in significantly enhanced ENZA sensitivity. Our analyses revealed that ALCAM serum levels may help to identify ENZA- and ABI-resistant patients and may thereby help to optimize future clinical decision-making. Our functional analyses suggest the possible involvement of ALCAM in ENZA resistance.

Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries.

Serum human epididymis protein 4 (HE4) as a tumor marker in men with lung cancer.

BACKGROUND: Human epididymis protein 4 (HE4) is a reliable tumor marker for ovarian cancer, but only limited data are available on HE4 levels in lung malignancies. METHODS: HE4 levels were measured at diagnosis in 98 men with lung cancer at different stages of the disease, and these results were compared to an age-matched healthy male cohort (n=98). The concentrations of classical tumor markers were also determined, and their efficacy was compared to that of HE4. RESULTS: Compared to healthy controls, patients with lung neoplasm showed significantly higher HE4 levels [118.2 (80.6-150.1) pmol/L vs. 62.2 (47.2-76.1) pmol/L; p<0.001]. Although age and smoking modulated HE4 levels in the healthy cohort, no such effect was observed in the patient population. The area under the receiver operating characteristic curve (ROC-AUC) for HE4 was 0.848 (95% CI 0.792-0.904) for differentiating lung cancer patients from healthy controls, with a cut-off value of 97.6 pmol/L (sensitivity: 64.3%, specificity: 95.9%). HE4 levels were significantly elevated in all stages of lung cancer, and even in patients without clinical symptoms (p<0.05), but no difference was found between the different histological subgroups. A significant correlation was found between HE4 values and the tumor size determined by CT/MRI (Spearman's ρ=0.227, p=0.030). The combination of HE4 with CEA and CA 125 considerably enhanced the diagnostic efficacy [ROC-AUC: 0.963 (95% CI 0.937-0.990), sensitivity: 91.8%, specificity: 92.8%]. CONCLUSIONS: Our data suggest that serum HE4, especially in combination with CEA and CA 125, qualifies as a surrogate diagnostic marker in men with lung cancer.

Comparative proteome and serum analysis identified FSCN1 as a marker of abiraterone resistance in castration-resistant prostate cancer.

BACKGROUND: Abiraterone (Abi) is an androgen receptor signaling inhibitor that significantly improves patients' life expectancy in metastatic prostate cancer (PCa). Despite its beneficial effects, many patients have baseline or acquired resistance against Abi. The aim of this study was to identify predictive serum biomarkers for Abi treatment. METHODS: We performed a comparative proteome analysis on three Abi sensitive (LNCaPabl, LAPC4, DuCaP) and resistant (LNCaPabl-Abi, LAPC4-Abi, DuCaP-Abi) PCa cell lines using liquid chromatography tandem mass spectrometry (LC-MS/MS) technique. Two bioinformatic selection workflows were applied to select the most promising candidate serum markers. Serum levels of selected proteins were assessed in samples of 100 Abi-treated patients with metastatic castration-resistant disease (mCRPC) using ELISA. Moreover, FSCN1 serum concentrations were measured in samples of 69 Docetaxel (Doc) treated mCRPC patients. RESULTS: Our proteome analysis identified 68 significantly, at least two-fold upregulated proteins in Abi resistant cells. Using two filtering workflows four proteins (AMACR, KLK2, FSCN1 and CTAG1A) were selected for ELISA analyses. We found high baseline FSCN1 serum levels to be significantly associated with poor survival in Abi-treated mCRPC patients. Moreover, the multivariable analysis revealed that higher ECOG status (>1) and high baseline FSCN1 serum levels (>10.22 ng/ml by ROC cut-off) were independently associated with worse survival in Abi-treated patients (p < 0.001 and p = 0.021, respectively). In contrast, no association was found between serum FSCN1 concentrations and overall survival in Doc-treated patients. CONCLUSIONS: Our analysis identified baseline FSCN1 serum levels to be independently associated with poor survival of Abi-treated, but not Doc-treated mCRPC patients, suggesting a therapy specific prognostic value for FSCN1.

N-4-t-Butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamide TRPV1 antagonists: Structure-activity relationships in the A-region.

Structure-activity relationships for the A-region in a series of N-4-t-butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamides as TRPV1 antagonists have been investigated. Among them, the 3-fluoro analogue 54 showed high binding affinity and potent antagonism for both rTRPV1 and hTRPV1 in CHO cells. Its stereospecific activity was demonstrated with marked selectivity for the (S)-configuration (54S versus 54R). A docking study of 54S with our hTRPV1 homology model highlighted crucial hydrogen bonds between the ligand and the receptor contributing to its potency.

2-(4-Methylsulfonylaminophenyl) propanamide TRPV1 antagonists: Structure-activity relationships in the B and C-regions.

On the basis of the previous lead N-4-t-butylbenzyl 2-(3-fluoro-4-methylsulfonylaminophenyl) propanamide (3) as a potent TRPV1 antagonist, structure-activity relationships for the B (propanamide part) and C-region (4-t-butylbenzyl part) have been investigated for rTRPV1 in CHO cells. The B-region was modified with dimethyl, cyclopropyl and reverse amides and then the C-region was replaced with 4-substituted phenyl, aryl alkyl and diaryl alkyl derivatives. Among them, compound 50 showed high binding affinity with K(i)=21.5nM, which was twofold more potent than 3 and compound 54 exhibited potent antagonism with K(i(ant))=8.0nM comparable to 3.

Detection of leucine-rich alpha-2-glycoprotein 1 containing immunocomplexes in the plasma of lung cancer patients with epitope-specific mAbs.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. With the expectation of improved survival, tremendous efforts and resources have been invested in the discovery of specific biomarkers for early detection of the disease. Several investigators have reported the presence of cancer-associated autoantibodies in the plasma or serum of lung cancer patients. Previously, we used a monoclonal antibody (mAb) proteomics technology platform for the discovery of novel lung cancer-associated proteins. OBJECTIVE: The identification of specific protein epitopes associated with various cancers is a promising method in biomarker discovery. Here, in a preliminary study, we aimed to detect autoantibody-leucine-rich alpha-2-glycoprotein 1 (LRG1) immunocomplexes using epitope-specific monoclonal antibodies (mAbs). METHODS: We performed sandwich ELISA assays using the LRG1 epitope-specific capture mAbs, Bsi0352 and Bsi0392, and an IgG-specific polyclonal antibody coupled to a reporter system as the detection reagent. We tested the plasma of lung cancer patients and apparently healthy controls. RESULTS: Depending on the epitope specificity of the capture mAb, we were either unable to distinguish the control from LC-groups or showed a higher level of LRG1 and IgG autoantibody containing immunocomplexes in the plasma of non-small cell lung cancer and small cell lung cancer subgroups of lung cancer patients than in the plasma of control subjects. CONCLUSIONS: Our findings underline the importance of protein epitope-specific antibody targeted approaches in biomarker research, as this may increase the accuracy of previously described tests, which will need further validation in large clinical cohorts.

Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery.

mAb proteomics, a reversed biomarker discovery approach, is a novel methodology to recognize the proteins of biomarker potential, but requires subsequent antigen identification steps. While in case of high-abundant proteins, it generally does not represent a problem, for medium or lower abundant proteins, the identification step requires a large amount of sample to assure the proper amount of antigen for the ID process. In this article, we report on the use of combined chromatographic and precipitation techniques to generate a large set of fractions representing the human plasma proteome, referred to as the Analyte Library, with the goal to use the relevant library fractions for antigen identification in conjunction with mAb proteomics. Starting from 500 mL normal pooled human plasma, this process resulted in 783 fractions with the average protein concentration of 1 mg/mL. First, the serum albumin and immunoglobulins were depleted followed by prefractionation by ammonium sulfate precipitation steps. Each precipitate was then separated by size exclusion chromatography, followed by cation and anion exchange chromatography. The 20 most concentrated ion exchange chromatography fractions were further separated by hydrophobic interaction chromatography. All chromatography and precipitation steps were carefully designed aiming to maintain the native forms of the intact proteins throughout the fractionation process. The separation route of vitamin D-binding protein (an antibody proteomics lead) was followed in all major fractionation levels by dot blot assay in order to identify the library fraction it accumulated in and the identity of the antigen was verified by Western blot.

Structural insights into transient receptor potential vanilloid type 1 (TRPV1) from homology modeling, flexible docking, and mutational studies.

The transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel composed of four monomers with six transmembrane helices (TM1-TM6). TRPV1 is found in the central and peripheral nervous system, and it is an important therapeutic target for pain relief. We describe here the construction of a tetrameric homology model of rat TRPV1 (rTRPV1). We experimentally evaluated by mutational analysis the contribution of residues of rTRPV1 contributing to ligand binding by the prototypical TRPV1 agonists, capsaicin and resiniferatoxin (RTX). We then performed docking analysis using our homology model. The docking results with capsaicin and RTX showed that our homology model was reliable, affording good agreement with our mutation data. Additionally, the binding mode of a simplified RTX (sRTX) ligand as predicted by the modeling agreed well with those of capsaicin and RTX, accounting for the high binding affinity of the sRTX ligand for TRPV1. Through the homology modeling, docking and mutational studies, we obtained important insights into the ligand-receptor interactions at the molecular level which should prove of value in the design of novel TRPV1 ligands.

Apico-basal inhomogeneity in distribution of ion channels in canine and human ventricular myocardium.

OBJECTIVES: The aim of the present study was to compare the apico-basal distribution of ion currents and the underlying ion channel proteins in canine and human ventricular myocardium. METHODS: Ion currents and action potentials were recorded in canine cardiomyocytes, isolated from both apical and basal regions of the heart, using whole-cell voltage clamp techniques. Density of channel proteins in canine and human ventricular myocardium was determined by Western blotting. RESULTS: Action potential duration was shorter and the magnitude of phase-1 repolarization was significantly higher in apical than basal canine myocytes. No differences were observed in other parameters of the action potential or cell capacitance. Amplitude of the transient outward K(+) current (29.6+/-5.7 versus 16.5+/-4.4 pA/pF at +65 mV) and the slow component of the delayed rectifier K(+) current (5.61+/-0.43 versus 2.14+/-0.18 pA/pF at +50 mV) were significantly larger in apical than in basal myocytes. Densities of the inward rectifier K(+) current, rapid delayed rectifier K(+) current, and L-type Ca(2+) current were similar in myocytes of apical and basal origin. Apico-basal differences were found in the expression of only those channel proteins which are involved in mediation of the transient outward K(+) current and the slow delayed rectifier K(+) current: expression of Kv1.4, KChIP2, KvLQT1 and MinK was significantly higher in apical than in basal myocardium in both canine and human hearts. CONCLUSIONS: The results suggest that marked apico-basal electrical inhomogeneity exists in the canine-and probably in the human-ventricular myocardium, which may result in increased dispersion, and therefore, cannot be ignored when interpreting ECG recordings, pathological alterations, or drug effects.

Specific detection and quantitation of bovine IgG in bioreactor derived mouse mAb preparations.

Monoclonal antibody and recombinant protein production benefits greatly from bovine serum as an additive. The caveat is that bovine serum IgG, co-purifies with mAbs and IgG Fc-containing fusion proteins and it presents a contaminant in the end products. In order to analytically validate the products, species specific reagents are needed that react with bovine IgG exclusively. Our attempts to find such commercially available reagents failed. Here, we report the production of species specific mAbs which recognize bovine IgG even in the presence of excess amount of mouse IgG. We present five mAbs: Bsi4028, Bsi4032, Bsi4033, Bsi4034 and Bsi4035 suitable to determine the presence of bovine IgG contamination via ELISA or immunoblotting in bioreactor derived mouse mAb preparations. To quantitate bovine IgG content we developed sensitive sandwich ELISAs capable to detect bovine IgG contaminant in the ng/ml (~10-11M/l) range. Finally, we show that bovine IgG is efficiently removed from bioreactor produced mouse mAb preparation via affinity depletion columns prepared with Bsi4028, Bsi4032, Bsi4033, Bsi4034, Bsi4035 mAbs.

Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release.

We investigated the contribution of dexamethasone treatment on the recovery of postischemic cardiac function and the development of reperfusion-induced arrhythmias in ischemic/reperfused isolated rat hearts. Rats were treated with 2 mg/kg of intraperitoneal injection of dexamethasone, and 24 hours later, hearts were isolated according to the 'working' mode, perfused, and subjected to 30 min global ischemia followed by 120 min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 60 min and 120 min reperfusion, 2 mg/kg of dexamethasone significantly improved the postischemic recovery of aortic flow and left ventricular developed pressure from their control values of 10.7 +/- 0.3 ml/min and 10.5 +/- 0.3 kPa to 22.2 +/- 0.3 ml/min (p < 0.05) and 14.3 +/- 0.5 kPa (p < 0.05), 19.3 +/- 0.3 ml/min (p < 0.05) and 12.3 +/- 0.5 kPa (p < 0.05), respectively. Heart rate and coronary flow did not show a significant change in postischemic recovery after 60 or 120 min reperfusion. In rats treated with 0.5 mg/kg of actinomycin D injected i.v., one hour before the dexamethasone injection, suppressed the dexamethasone-induced cardiac protection. Electrocardiograms were monitored to determine the incidence of reperfusion-induced ventricular fibrillation. Dexamethasone pretreatment significantly reduces the occurrence of ventricular fibrillation. Cytochrome c release was also observed in the cytoplasm. The results suggest that the inhibition of cytochrome c release is involved in the dexamethasone-induced cardiac protection.

Sensitization of recombinant vanilloid receptor-1 by various neurotrophic factors.

The vanilloid receptor (VR1) is a central integrator molecule of nociceptive stimuli. In this study, we have measured the effects of various neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and -4) on recombinant rat VR1-mediated intracellular calcium rise in response to capsaicin in VR1/C6 cells. Our results clearly show that all neurotrophins sensitize the VR1 to capsaicin. Furthermore, using K252a, an inhibitor of tyrosine kinases, we present that actions of neurotrophins are mediated by the trk (A, B, C) receptors expressed in these cells. These data argue for the putative roles of neurotrophins in inducing inflammatory (thermal) hyperalgesia via VR1.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.

Screening TRPV1 antagonists for the treatment of pain: lessons learned over a decade.

BACKGROUND: The capsaicin receptor TRPV1, a polymodal nociceptor whose expression is up-regulated in a number of painful inflammatory disorders, represents a promising therapeutic target for pain relief. Potent small molecule TRPV1 antagonists are now undergoing clinical trials in patients with inflammatory or neuropathic pain. This review focuses on the multiplicity of factors regulating this channel and on their contributions to the emerging complexity of responses to TRPV1 and partial antagonists. For example, it is now clear that antagonists of capsaicin response can also antagonize, have no effect, or stimulate response to heat or protons. The complexity of TRPV1 regulation affords the potential to optimize agents for a specific therapeutic indication. An encouraging advance is the dissection of therapeutic efficacy of antagonists from induction of hyperthermia, a side effect that initially had raised concerns about the suitability of systemically administered TRPV1 antagonists for therapy. OBJECTIVES AND METHODS: To discuss the challenges facing the development of clinically useful TRPV1 antagonists based on our experience and a comprehensive review of the literature. RESULTS/CONCLUSIONS: TRPV1 is a polymodal receptor. Some antagonists block all modalities of TRPV1 stimulation whereas others are more selective in their pharmacological profile. A number of antagonists can, conversely, potentiate certain modes of TRPV1 activation (e.g., protons and heat). The selectivity of TRPV1 antagonists is species-dependent, posing a problem for extrapolation from animal models to patients. At present, this rich pharmacology of TRPV1 antagonists complicates drug development but for the future it promises great opportunities for drug design.

Characterization of the interaction of phorbol esters with the C1 domain of MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase) alpha/beta.

C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP. MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), a member of the dystrophia myotonica protein kinase family that functions downstream of Cdc42, contains a C1 domain with substantial homology to that of the diacylglycerol/phorbol ester-responsive C1 domains and has been reported to bind phorbol ester. We have characterized here the interaction of the C1 domains of the two MRCK isoforms alpha and beta with phorbol ester. The MRCK C1 domains bind [20-(3)H]phorbol 12,13-dibutyrate with K(d) values of 10 and 17 nm, respectively, reflecting 60-90-fold weaker affinity compared with the protein kinase C delta C1b domain. In contrast to binding by the C1b domain of PKCdelta, the binding by the C1 domains of MRCK alpha and beta was fully dependent on the presence of phosphatidylserine. Comparison of ligand binding selectivity showed resemblance to that by the C1b domain of PKCalpha and marked contrast to that of the C1b domain of PKCdelta. In intact cells, as in the binding assays, the MRCK C1 domains required 50-100-fold higher concentrations of phorbol ester for induction of membrane translocation. We conclude that additional structural elements within the MRCK structure are necessary if the C1 domains of MRCK are to respond to phorbol ester at concentrations comparable with those that modulate PKC.

Halogenation of 4-hydroxy-3-methoxybenzyl thiourea TRPV1 agonists showed enhanced antagonism to capsaicin.

Selected potent TRPV1 agonists (1-6) have been modified by 5- or 6-halogenation on the aromatic A-region to analyze their effects on potency and efficacy (agonism versus antagonism). The halogenation caused enhanced functional antagonism at TRPV1 compared to the corresponding prototype agonists. The analysis of SAR indicated that the antagonism was enhanced as the size of the halogen increased (I>Br>Cl) and when the 6-position was halogenated. Compounds 23c and 31b were found to be potent full antagonists with K(i) (as functional antagonist)=23.1 and 30.3 nM in rTRPV1/CHO system, respectively.