Supplementary Table 1. Emergence of BRAF mutations in cfDNA; Supplementary Table 2. Patients with BRAF V600K mutations in FFPE tumor and plasma cfDNA samples; Supplementary Table 3. Experimental therapies in 34 patients with longitudinal collection of plasma cfDNA; Supplementary Figure 1. A. Among the 160 patients whose cfDNA samples were tested for BRAF mutations, the median overall survival duration of the 113 patients with a BRAF-mutant cfDNA percentage of 0% (10.0 months, 95% confidence interval [CI], 7.2-12.8 months; blue) was longer than that of the 47 patients with a BRAF-mutant cfDNA percentage of >0% (7.0 months, 95% CI, 2.5-11.5 months; red), but this difference was not significant (P=0.09). B. Among these same 160 patients, the median overall survival duration of the 126 patients with a BRAF-mutant cfDNA percentage of <1% (10.5 months, 95% CI, 8.4-12.6 months; blue) was significantly longer than that of the 34 patients with a BRAF-mutant cfDNA percentage of greater than or equal to 1%(5.5 months, 95% CI, 3.4-7.6 months; red; P=0.006). C. Among the 47 patients with cfDNA BRAF mutations, the median overall survival duration of the 21 patients with a BRAF-mutant cfDNA percentage of less than or equal to 2% (11.2 months, 95% CI, 10.4-12.0 months; blue) was longer than that of the 26 patients with a BRAF-mutant cfDNA percentage of >2% (4.4 months, 95% CI, 3.2-5.6 months; red), but this difference was not statistically significant (P=0.05). D. Among the 62 patients whose formalin-fixed paraffin-embedded tumor samples had BRAF mutations, the median overall survival duration of the 37 patients with a BRAF-mutant cfDNA percentage of less than or equal to 2% (14.6 months, 95% CI, 9.4-19.8 months; blue) was significantly longer than that of 25 patients with a BRAF-mutant cfDNA percentage of >2% (4.5 months, 95% CI, 3.0-6.0 months; P=0.001). Supplementary Figure 2. A. The median amount of cfDNA in plasma of the 160 patients whose cfDNA was tested for BRAF mutations was 60 ng/ml. The median overall survival duration of the 81 patients with a cfDNA amount in plasma less than or equal to 60 ng/ml (8.9 months, 95% confidence interval [CI], 4.8-13.0 months; blue)was similar to that of the 79 patients with a cfDNA amount in plasma >60 ng/ml (8.7 months, 95% CI, 5.2-12.2 months; red; P=0.91). B. The median cfDNA concentration of these same 160 patients was 4.6 ng/μl. The median overall survival duration of the 82 patients with a cfDNA concentration <5 ng/IJI (9.5 months, 95% Cl, 5.8-13.2 months; blue) was similar to that of the 78 patients with cfDNA concentrations 5 ng/ml (8.7 months, 95% Cl, 5.4-12.0 months; red), but this difference was not significant (P=0.89).

e15112 Background: To validate the Idylla MSI Test to detect microsatellite instability (MSI) in colorectal cancer (CRC) samples in comparison with Promega MSI Analysis System v1.2 (Promega MSI) and in conditions similar to normal use. Methods: The study was performed on residual formalin-fixed and paraffin-embedded (FFPE) samples obtained from routine diagnostics by two centers, University Hospital Aarhus and University Hospital Antwerp. Samples originated from CRC patients (all stages). Both centers performed the Idylla MSI Test (with 7 novel homopolymer deletion markers) at their premises on 150 and 180 samples, respectively. The comparator method, Promega MSI, was performed for all 330 samples by University Hospital Antwerp. Results: Seven (n=7) of 330 samples were excluded from the concordance analysis due to invalid or error (failed) results for Idylla MSI Test and/or Promega MSI. For the 323 valid results, overall, positive and negative percentage agreement were 99.7% (98.3%-100%;), 98.7% (92.9%-99.8%) and 100% (98.5%-100%), respectively. A higher number of invalids was observed for Promega MSI (2.1%) compared to the Idylla MSI Test (0.6%) taking into account per protocol retesting (18 retests for Promega MSI (6%) and 3 for Idylla MSI Test (1%)). Conclusions: This study validated the Idylla MSI Test with high performance and low invalid rate to discriminate MSI-H from MSS status on a clinical routine set of CRC samples.

Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 μM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.

Abstract Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAFV600 status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAFV600 mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63–0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60–0.83) and specificity of 98% (95% CI, 0.93–1.00). A higher percentage, but not concentration, of BRAFV600 cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAFV600 cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAFV600 mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAFV600 in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397–404. ©2016 AACR.

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTMBRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction-based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTMBRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation-detecting tests.

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.

11086 Background: Mutations in the BRAF gene provide actionable targets for cancer therapy in melanoma and other tumor types. Novel, fast, and accurate diagnostic systems are needed for further implementation of personalized therapy. Methods: The molecular diagnostics (MDx) prototype platform (Biocartis, Mechelen, Belgium) is a fully integrated real-time PCR-based system with high sensitivity (1% mutant in wild-type [wt] background) and fast turnaround time (< 90 minutes), which requires no sample preparation and <2 min hands-on time. Archival formalin-fixed paraffin-embedded tumor samples (1 to 5 shavings of 10 µm) from patients (pts) with advanced cancers previously tested for V600 BRAF mutations in a CLIA-certified Molecular Diagnostic Laboratory (PCR-based sequencing or Sequenom MassARRAY) were tested for BRAF V600 mutations using the MDx prototype platform. Concordance between methods and treatment outcomes with BRAF/MEK inhibitors were analyzed. Results: Forty-seven pts (melanoma, n=26; colorectal, n=8; papillary thyroid, n=3; other cancers, n=10) with available tissue and CLIA laboratory BRAF results were selected (BRAF V600 mutant, n=37; BRAF V600 wt, n=10). Of the 40 pts for whom the same tissue block was used for MDx and CLIA, BRAF status was concordant in 38 (95%; kappa 0.87; 95% CI 0.69-1.05) of them. BRAF status by MDx was discordant with CLIA in 3 of 47 cases (mutant by CLIA, but not MDx); one discrepant case contained a different mutation subtype (resp. V600E vs. V600K/R), and in another case different tissue blocks were used for MDx vs. CLIA testing. Of 34 pts with BRAF mutations detected by MDx, 28 were treated on protocols (on the basis of the CLIA results) with BRAF/MEK inhibitors and 8 (29%) had a partial (n=7) or complete response (n=1). Of interest, 1 pt with prostate cancer (V600E by CLIA, wt by MDx) received a BRAF/MEK inhibitor and did not respond. Detailed patient characteristics, mutation types and discrepancy analysis will be presented. Conclusions: The BRAF V600 mutation MDx prototype assay is a fast (turn-around time about 1.5 hours) and simple (<2 minutes hands-on time) test to determine BRAF mutation status with 95% concordance with CLIA laboratory if identical tissue blocks are used.

Abstract Introduction Activating mutations in the BRAF gene are common in skin and colorectal tumors. In more than 90% of the cases, these mutations are located in the BRAF V600 codon (V600E and V600K). Targeted therapies like vemurafenib and dabrafenib have shown objective response rates in up to half of metastatic melanomas harboring these BRAF V600 mutations. Tumor mutation status is usually assessed starting from formalin-fixed, paraffin-embedded (FFPE) tumor tissue shavings. Typically, the test involves shipment of the paraffin block from the local pathology lab to a specialized molecular lab, where several steps need to be performed. Methods The Biocartis molecular diagnostics (MDx) prototype platform is a novel, random access, sample-in result-out automated qPCR system. It uses a disposable cartridge which can simultaneously detect and quantify up to 30 molecular markers from a wide range of solid and liquid sample types, including blood, feces, and sputum. For FFPE-based assays, a single curl or shaving is directly placed into the cartridge. The complete process time for sample preparation, PCR and reporting is less than 90 min, with <2 minutes hands-on time. We hereby present a concordance study of the Biocartis BRAF V600 Mutation prototype assay (which detects BRAF V600E, V600K, V600R, V600E2, V600D and V600M) with the Roche cobas® 4800 BRAF V600 Mutation Test (Cobas). Sixty-four FFPE samples from melanoma and other tumors, with varying amounts of melanin, % tumor, and % BRAF V600 mutated tumor cells were randomly selected and tested. Both the Biocartis MDx prototype platform and the Biocartis BRAF V600 Mutation prototype assay were used for research use only purposes. Deep sequencing was performed using MiSeq (Illumina) with over 5,000-fold coverage and sensitivity of 1% of minority species for each sample. Results One sample was not eligible for Cobas testing because of low tumor content. Three other samples failed on both platforms. For the remaining 60 samples, results on the Biocartis MDx platform and Cobas were concordant in 58 (96.7%) samples (44 BRAF V600 WT and 14 BRAF V600 mutant samples, of which 36 samples were randomly selected and confirmed by deep sequencing). In 2 (3.3%) samples, no BRAF V600 mutation was detected by Cobas, while a BRAF V600 mutation was detected by both the Biocartis MDx platform and by deep sequencing. Both were melanoma samples (one of which was highly pigmented), excised in 1993 and 2005, and contained 4.6% V600E and 5.6% V600K (as determined by deep sequencing), respectively. Conclusions The new MDx platform is a fast and reliable method for BRAF V600 mutation testing directly on FFPE tumor shavings with superior analytical sensitivity, ease of use, and turnaround time compared to existing diagnostic tests. The BRAF V600 Mutation prototype assay provided excellent concordance with both Cobas (96.7%) and deep sequencing (100%). Citation Format: Benoit Devogelaere, Koen Van Acker, Inky De Baere, Pascale Holemans, Tania Ivens, Bart Claes, Evelien Rondelez, Geneviève Vandercruyssen, Marijke Van der Auwera, Mark Kockx, Isabelle Vanden Bempt, Ina Vandenbroucke, Geert Maertens, Erwin Sablon. BRAF V600 mutation testing on FFPE samples using a novel fully integrated molecular diagnostics platform: A concordance study with reference methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4213. doi:10.1158/1538-7445.AM2013-4213

Abstract Background: Mutations in the BRAF gene provide actionable targets for cancer therapy, and can be found in diverse cancers including melanoma, papillary thyroid and colorectal cancers. Novel, fast, and accurate diagnostic systems are needed for further implementation of personalized cancer therapy. Methods: The molecular diagnostics (MDx) prototype platform (Biocartis, Mechelen, Belgium) is a fully integrated real-time PCR-based system with high sensitivity (1%) and quick turnaround time (< 90 minutes), which requires no sample preparation and <2 min hands-on time. Archival formalin-fixed paraffin-embedded tumor samples (1x to 5x of 10 μm shavings) from patients with advanced cancers previously tested for V600 BRAF mutations in the CLIA-certified Molecular Diagnostic Laboratory (PCR-based sequencing or Sequenom MassARRAY) were tested with a BRAF V600 mutation prototype assay using the MDx prototype platform for research only purposes. Concordance between methods and treatment outcomes with BRAF/MEK inhibitors were analyzed. Results: Thirty-eight patients (melanoma, n=21; colorectal, n=7; other cancers, n=10) with available tissue and CLIA laboratory BRAF results were identified (BRAF V600 mutation, n=28; wild-type BRAF, n=10). Of the 36 patients for whom the same tissue block was used for MDx and CLIA, BRAF results were concordant in 35 (97%; kappa 0.93; 95% CI 0.80-1.06) of them. BRAF results by MDx were discordant with CLIA in a total of 3 of 38 patients (mutations found by CLIA, but not MDx), but in two of these individuals, different tissue blocks were used for MDx versus CLIA interrogation. Of 25 patients with BRAF mutations detected by MDx, 21 had previously been treated (on the basis of the CLIA lab results) with BRAF/MEK targeted therapies and 7 (33%) had a partial response (PR). Both of the patients with discrepant BRAF status (mutations found by CLIA, but not MDx) who were treated with a BRAF/MEK inhibitor did not respond. Detailed patient characteristics, mutation types and additional discrepancy analysis will be presented. Conclusions: The BRAF V600 mutation MDx prototype platform is a fast (turn-around time about 1.5 hours) and simple (<2 minutes hands-on time) test to determine BRAF mutation status. When identical tissue blocks are used, results from the MDx prototype platform and from CLIA laboratory PCR-based sequencing or Sequenom MassARRAY demonstrate 97% concordance. Citation Format: Filip Janku, Laura S. Angelo, Benoit Devogelaere, Gerald S. Fachook, Siqing Fu, Helen J. Huang, Apostolia M. Tsimberidou, David S. Hong, Vanda M. Stepanek, Veronica R. Holley, Mark J. Routbort, Erwin Sablon, Geert Maertens, Razelle Kurzrock. BRAF mutation testing with a novel, rapid, fully-automated molecular diagnostics prototype platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4126. doi:10.1158/1538-7445.AM2013-4126

The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.

Surface plasmon resonance (SPR)-based optical biosensors have been widely used to study biomolecular interactions, and applied to many areas of drug discovery including target identification, fragment screening, lead compound selection, early ADME (absorption, distribution, metabolism and excretion), and quality control. These biosensors allow the following of a biomolecular interaction in real time to monitor kinetics and determine affinity. In this chapter, we describe an SPR-based assay to measure the interaction between hepatitis C virus NS5B polymerase (wild type and/or mutants) and a small-molecule inhibitor. Viral polymerase proteins are captured on a Ni(2+)-nitrilotriacetic acid sensor surface while the small--molecule inhibitors are passed over the surface. In this way kinetics and affinity of the enzyme-inhibitor interactions can be measured, making it possible to select potent and promising lead candidates.

Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 μM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.

Das Konzept der Wirkstoffähnlichkeit fasst die physikochemischen Eigenchaften niedermolekularer Wirkstoffe zu einem Satz von Regeln zusammen. Von makrocyclischen Wirkstoffen ist bekannt, dass sie diese Regeln brechen. Eine strukturbasierte Makrocyclisierungsstrategie wurde angewendet, um HCV-NS5B-Inhibitoren (HCV=Hepatitis-C-Virus) mit verbesserten pharmakokinetischen Eigenschaften zu entwerfen, die ein Beispiel einer rationalen Strategie darstellt, um die Grenzen des standardmäßigen „wirkstoffähnlichen chemischen Raums“ zu überwinden. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Autosomal dominant polycystic kidney disease (ADPKD) arises as a consequence of mutations of the genes PKD1 and PKD2, encoding respectively the integral membrane proteins polycystin-1 and polycystin-2 (TRPP2), resulting in a disturbance in intracellular Ca(2+) signaling. Previously we investigated the interaction between TRPP2 and the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum (ER). We identified the molecular determinants of this interaction and observed an enhanced IP(3)-induced Ca(2+) release (IICR). Since we found that TRPP2 strongly bound to a cluster of positively charged amino acids in the N-terminal ligand-binding domain (LBD) of the IP(3)R, we now investigated whether TRPP2 would interfere with the binding of IP(3) to the IP(3)R. In in vitro experiments we observed that TRPP2 partially inhibited the binding of IP(3) to the LBD of the IP(3)R with an IC(50) of ∼350 nM. The suppressor domain, i.e., the N-terminal 225 amino acids of the LBD of the IP(3)R, mediated this inhibitory effect of TRPP2 on IP(3) binding. The observation that the interaction between the IP(3)R and TRPP2 decreased IP(3) binding is in apparent contrast to the increased IICR. The data can be explained however by a subsequent activation of Ca(2+)-induced Ca(2+) release (CICR) via TRPP2. Implications of this mechanism for cellular Ca(2+) signaling are discussed in this addendum.

ABSTRACT The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca2+ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca2+ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP3R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca2+ release in intact cells and IP3-induced Ca2+ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca2+ signal by a local Ca2+-induced Ca2+-release mechanism, which only occurred in the presence of the TRPP2-IP3R interaction, and not via altered IP3R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca2+ microdomains necessary for initiating Ca2+-induced Ca2+ release. The data strongly suggest that defects in this mechanism may account for the altered Ca2+ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) arises as a consequence of mutations of the genes PKD1 and PKD2, encoding respectively the integral membrane proteins polycystin-1 and polycystin-2 (TRPP2), resulting in a disturbance in intracellular Ca(2+) signaling. Previously we investigated the interaction between TRPP2 and the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum (ER). We identified the molecular determinants of this interaction and observed an enhanced IP(3)-induced Ca(2+) release (IICR). Since we found that TRPP2 strongly bound to a cluster of positively charged amino acids in the N-terminal ligand-binding domain (LBD) of the IP(3)R, we now investigated whether TRPP2 would interfere with the binding of IP(3) to the IP(3)R. In in vitro experiments we observed that TRPP2 partially inhibited the binding of IP(3) to the LBD of the IP(3)R with an IC(50) of ∼350 nM. The suppressor domain, i.e., the N-terminal 225 amino acids of the LBD of the IP(3)R, mediated this inhibitory effect of TRPP2 on IP(3) binding. The observation that the interaction between the IP(3)R and TRPP2 decreased IP(3) binding is in apparent contrast to the increased IICR. The data can be explained however by a subsequent activation of Ca(2+)-induced Ca(2+) release (CICR) via TRPP2. Implications of this mechanism for cellular Ca(2+) signaling are discussed in this addendum.

BACKGROUND:Serum samples from patients with autoimmune connective tissue diseases that show a finely speckled antinuclear antibody (ANA) on indirect immune-fluorescence often have antibodies against unknown nuclear target antigens. To search for such autoantigens we applied a proteomic approach using sera from patients with a high ANA titer (>or=640) and finely speckled fluorescence but in whom no antibodies to extractable nuclear antigens (ENA) could be identified.METHODS:Using an immunoproteomics approach we identified heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) as a novel nuclear target of autoantibody response.RESULTS:Recombinant rat hnRNP H1 reacted in Western blot analyses with 48% of 93 sera from patients with primary Sjögren syndrome and with 5.2% of 153 sera from patients with other connective tissue diseases (diseased controls). For comparison, the diagnostic sensitivity and specificity of anti-Sjögren syndrome A (SSA) antibodies for primary Sjögren syndrome in the same patient cohort were 88.2% and 76.3%, respectively. Interestingly, 5 of 11 primary Sjögren syndrome patients with no anti-SSA or anti-SSB antibodies had anti-hnRNP H1 antibodies. Anti-hnRNP H1 antibodies were preabsorbed by hnRNP H1, as demonstrated by indirect immunofluorescence. In an evaluation of the presence of anti-hnRNP H1 antibodies in 188 consecutive samples submitted to the clinical laboratory with positive ANA (titer >or=160), anti-hnRNP H1 antibodies were found in 3 of 7 (2 primary and 5 secondary) Sjögren syndrome patients and in 8.3% of the diseased controls.CONCLUSIONS:HnRNP H1 is a newly discovered autoantigen that could become an additional diagnostic marker.