Preclinical Evaluation of Gastrin-Releasing Peptide Receptor Antagonists Labeled with 161Tb and 177Lu: A Comparative Study.

To elucidate potential benefits of the Auger-electron-emitting radionuclide 161Tb, we compared the preclinical performance of the gastrin-releasing peptide receptor antagonists RM2 (DOTA-Pip5-d-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) and AMTG (α-Me-Trp8-RM2), each labeled with both 177Lu and 161Tb. Methods: 161Tb/177Lu labeling (90°C, 5 min) and cell-based experiments (PC-3 cells) were performed. In vivo stability (30 min after injection) and biodistribution studies (1-72 h after injection) were performed on PC-3 tumor-bearing CB17-SCID mice. Results: Gastrin-releasing peptide receptor affinity was high for all compounds (half-maximal inhibitory concentration [nM]: [161Tb]Tb-RM2, 2.46 ± 0.16; [161Tb]Tb-AMTG, 2.16 ± 0.09; [177Lu]Lu-RM2, 3.45 ± 0.18; [177Lu]Lu-AMTG, 3.04 ± 0.08), and 75%-84% of cell-associated activity was receptor-bound. In vivo, both AMTG analogs displayed distinctly higher stability (30 min after injection) and noticeably higher tumor retention than their RM2 counterparts. Conclusion: On the basis of preclinical results, [161Tb]Tb-/[177Lu]Lu-AMTG might reveal a higher therapeutic efficacy than [161Tb]Tb-/[177Lu]Lu-RM2, particularly [161Tb]Tb-AMTG because of additional Auger-electron emissions at the cell membrane level.

Pharmacological inhibition of TRPV2 attenuates phagocytosis and lipopolysaccharide-induced migration of primary macrophages.

BACKGROUND AND PURPOSE: In macrophages, transient receptor potential vanilloid 2 (TRPV2) channel contributes to various cellular processes such as cytokine production, differentiation, phagocytosis and migration. Due to a lack of selective pharmacological tools, its function in immunological processes is not well understood and the identification of novel and selective TRPV2 modulators is highly desirable. EXPERIMENTAL APPROACH: Novel and selective TRPV2 modulators were identified by screening a compound library using Ca2+ influx assays with human embryonic kidney 293 (HEK293) cells heterologously expressing rat TRPV2. Hits were further characterized and validated with Ca2+ influx and electrophysiological assays. Phagocytosis and migration of macrophages were analysed and the contribution of TRPV2 to the generation of Ca2+ microdomains was studied by total internal reflection fluorescence microscopy (TIRFM). KEY RESULTS: The compound IV2-1, a dithiolane derivative (1,3-dithiolan-2-ylidene)-4-methyl-5-phenylpentan-2-one), is a potent inhibitor of heterologously expressed TRPV2 channels (IC50 = 6.3 ± 0.7 µM) but does not modify TRPV1, TRPV3 or TRPV4 channels. IV2-1 also inhibits TRPV2-mediated Ca2+ influx in macrophages. IV2-1 inhibits macrophage phagocytosis along with valdecoxib and after siRNA-mediated knockdown. Moreover, TRPV2 inhibition inhibits lipopolysaccharide-induced migration of macrophages whereas TRPV2 activation promotes migration. After activation, TRPV2 shapes Ca2+ microdomains predominantly at the margin of macrophages, which are important cellular regions to promote phagocytosis and migration. CONCLUSIONS AND IMPLICATIONS: IV2-1 is a novel TRPV2-selective blocker and underline the role of TRPV2 in macrophage-mediated phagocytosis and migration. Furthermore, we provide evidence that TRPV2 activation generates Ca2+ microdomains, which may be involved in phagocytosis and migration of macrophages.

Development of the First 18F-Labeled Radiohybrid-Based Minigastrin Derivative with High Target Affinity and Tumor Accumulation by Substitution of the Chelating Moiety.

In order to optimize elevated kidney retention of previously reported minigastrin derivatives, we substituted (R)-DOTAGA by DOTA in (R)-DOTAGA-rhCCK-16/-18. CCK-2R-mediated internalization and affinity of the new compounds were determined using AR42J cells. Biodistribution and µSPECT/CT imaging studies at 1 and 24 h p.i. were carried out in AR42J tumor-bearing CB17-SCID mice. Both DOTA-containing minigastrin analogs exhibited 3- to 5-fold better IC50 values than their (R)-DOTAGA-counterparts. natLu-labeled peptides revealed higher CCK-2R affinity than their natGa-labeled analogs. In vivo, tumor uptake at 24 h p.i. of the most affine compound, [19F]F-[177Lu]Lu-DOTA-rhCCK-18, was 1.5- and 13-fold higher compared to its (R)-DOTAGA derivative and the reference compound, [177Lu]Lu-DOTA-PP-F11N, respectively. However, activity levels in the kidneys were elevated as well. At 1 h p.i., tumor and kidney accumulation of [19F]F-[177Lu]Lu-DOTA-rhCCK-18 and [18F]F-[natLu]Lu-DOTA-rhCCK-18 was high. We could demonstrate that the choice of chelators and radiometals has a significant impact on CCK-2R affinity and thus tumor uptake of minigastrin analogs. While elevated kidney retention of [19F]F-[177Lu]Lu-DOTA-rhCCK-18 has to be further addressed with regard to radioligand therapy, its radiohybrid analog, [18F]F-[natLu]Lu-DOTA-rhCCK-18, might be ideal for positron emission tomography (PET) imaging due to its high tumor accumulation at 1 h p.i. and the attractive physical properties of fluorine-18.

Synthesis and preclinical evaluation of novel 99mTc-labeled PSMA ligands for radioguided surgery of prostate cancer.

BACKGROUND: Radioguided surgery (RGS) has recently emerged as a valuable new tool in the management of recurrent prostate cancer (PCa). After preoperative injection of a 99mTc-labeled prostate-specific membrane antigen (PSMA) inhibitor, radioguided intraoperative identification and resection of lesions is facilitated by means of suitable γ-probes. First clinical experiences show the feasibility of RGS and suggest superiority over conventional lymph node dissection in recurrent PCa. However, commonly used [99mTc]Tc-PSMA-I&S exhibits slow whole-body clearance, thus hampering optimal tumor-to-background ratios (TBR) during surgery. We therefore aimed to develop novel 99mTc-labeled, PSMA-targeted radioligands with optimized pharmacokinetic profile to increase TBR at the time of surgery. METHODS: Three 99mTc-labeled N4-PSMA ligands were preclinically evaluated and compared to [99mTc]Tc-PSMA-I&S. PSMA affinity (IC50) and internalization were determined on LNCaP cells. Lipophilicity was assessed by means of the distribution coefficient logD7.4 and an ultrafiltration method was used to determine binding to human plasma proteins. Biodistribution studies and static µSPECT/CT-imaging were performed at 6 h p.i. on LNCaP tumor-bearing CB17-SCID mice. RESULTS: The novel N4-PSMA tracers were readily labeled with [99mTc]TcO4- with RCP > 95%. Comparable and high PSMA affinity was observed for all [99mTc]Tc-N4-PSMA-ligands. The ligands showed variable binding to human plasma and medium to low lipophilicity (logD7.4 - 2.6 to - 3.4), both consistently decreased compared to [99mTc]Tc-PSMA-I&S. Biodistribution studies revealed comparable tumor uptake among all [99mTc]Tc-N4-PSMA-ligands and [99mTc]Tc-PSMA-I&S, while clearance from most organs was superior for the novel tracers. Accordingly, increased TBR were achieved. [99mTc]Tc-N4-PSMA-12 showed higher TBR than [99mTc]Tc-PSMA-I&S for blood and all evaluated tissue. In addition, a procedure suitable for routine clinical production of [99mTc]Tc-N4-PSMA-12 was established. Labeling with 553 ± 187 MBq was achieved with RCP of 98.5 ± 0.6% (n = 10). CONCLUSION: High tumor accumulation and favorable clearance from blood and non-target tissue make [99mTc]Tc-N4-PSMA-12 an attractive tracer for RGS, possibly superior to currently established [99mTc]Tc-PSMA-I&S. Its GMP-production according to a method presented here and first clinical investigations with this novel radioligand is highly recommended.

Multiplex G Protein-Coupled Receptor Screen Reveals Reliably Acting Agonists and a Gq-Phospholipase C Coupling Mode of GPR30/GPER1.

G protein-coupled receptors (GPCRs) constitute the most versatile family of pharmacological target proteins. For some "orphan" GPCRs, no ligand or drug-like modulator is known. In this study, we have established and applied a parallelized assay to coscreen 29 different human GPCRs. Three compounds, chlorhexidine, Lys-05, and 9-aminoacridine, triggered transient Ca2+ signals linked to the expression of GPR30. GPR30, also named G protein-coupled estrogen receptor 1 (GPER1), was reported to elicit increases in cAMP in response to 17ß-estradiol, 4-hydroxytamoxifen, or G-1. These findings could, however, not be reproduced by other groups, and the deorphanization of GPR30 is, therefore, intensely disputed. The unbiased screen and following experiments in transiently or stably GPR30-overexpressing HEK293 cells did not show responses to 17ß-estradiol, 4-hydroxytamoxifen, or G-1. A thorough analysis of the activated signaling cascade revealed a canonical Gq-coupled pathway, including phospholipase C, protein kinase C and ERK activation, receptor internalization, and sensitivity to the Gq inhibitor YM-254890. When expressed in different cell lines, the localization of a fluorescent GPR30 fusion protein appeared variable. An efficient integration into the plasma membrane and stronger functional responses were found in HEK293 and in MCF-7 cells, whereas GPR30 appeared mostly retained in endomembrane compartments in Cos-7 or HeLa cells. Thus, conflicting findings may result from the use of different cell lines. The newly identified agonists and the finding that GPR30 couples to Gq are expected to serve as a starting point for identifying physiologic responses that are controlled by this GPCR. SIGNIFICANCE STATEMENT: This study has identified and thoroughly characterized novel and reliably acting agonists of the G protein-coupled receptor GPER1/GPR30. Applying these agonists, this study demonstrates that GPR30 couples to the canonical Gq-phospholipase C pathway and is rapidly internalized upon continuous exposure to the agonists.

BTDAzo: A Photoswitchable TRPC5 Channel Activator.

Photoswitchable reagents can be powerful tools for high-precision biological control. TRPC5 is a Ca2+ -permeable cation channel with distinct tissue-specific roles, from synaptic function to hormone regulation. Reagents giving spatiotemporally-resolved control over TRPC5 activity may be key to understanding and harnessing its biology. Here we develop the first photoswitchable TRPC5-modulator, BTDAzo, to address this goal. BTDAzo can photocontrol TRPC5 currents in cell culture, as well as controlling endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. BTDAzos are also the first reported azo-benzothiadiazines, an accessible and conveniently derivatised azoheteroarene with strong two-colour photoswitching. BTDAzo's ability to control TRPC5 across relevant channel biology settings makes it suitable for a range of dynamically reversible photoswitching studies in TRP channel biology, with the aim to decipher the various biological roles of this centrally important ion channel.

De novo variants in ATP2B1 lead to neurodevelopmental delay.

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.

Valdecoxib blocks rat TRPV2 channels.

The transient receptor potential vanilloid 2 (TRPV2) channel is broadly expressed in a multitude of different tissues and is implicated in the pathology of several diseases, such as the progression of different cancer types. However, a lack of specific, potent and non-toxic TRPV2 activators and inhibitors complicate further studies to clarify the role of TRPV2. We here present valdecoxib as a novel inhibitor of heterologously expressed rat TRPV2 channels in HEK293 cells and native TRPV2 channels, endogenously expressed in the rat basophilic leukemia (RBL-2H3) cell line. Fluorometric assays reveal an IC50 of 9 µM and 11 µM for TRPV2 in HEK293 and RBL-2H3 cells, respectively. Closely related TRPV1, TRPV3 or TRPV4 channels are not blocked by valdecoxib. The inhibition is reversible and direct as confirmed by whole-cell and excised inside-out electrophysiological recordings. Other cyclooxygenase-2 inhibitors do not affect TRPV2 activity. Furthermore, we demonstrate that the combined application of 2-aminoethoxydiphenyl borate (2-APB) and probenecid at concentrations, which, on their own, elicit only small TRPV2 currents, act in a highly synergistic manner when applied simultaneously. Taken together, we here provide novel tools and chemical lead structures for further studying TRPV2 channel function in native tissues.

The ethoxycarbonyl group as both activating and protective group in N-acyl-Pictet-Spengler reactions using methoxystyrenes. A short approach to racemic 1-benzyltetrahydroisoquinoline alkaloids.

We present a systematic investigation on an improved variant of the N-acyl-Pictet-Spengler condensation for the synthesis of 1-benzyltetrahydroisoquinolines, based on our recently published synthesis of N-methylcoclaurine, exemplified by the total syntheses of 10 alkaloids in racemic form. Major advantages are a) using ω-methoxystyrenes as convenient alternatives to arylacetaldehydes, and b) using the ethoxycarbonyl residue for both activating the arylethylamine precursors for the cyclization reaction, and, as a significant extension, also as protective group for phenolic residues. After ring closure, the ethoxycarbonyl-protected phenols are deprotected simultaneously with the further processing of the carbamate group, either following route A (lithium alanate reduction) to give N-methylated phenolic products, or following route B (treatment with excess methyllithium) to give the corresponding alkaloids with free N-H function. This dual use of the ethoxycarbonyl group shortens the synthetic routes to hydroxylated 1-benzyltetrahydroisoquinolines significantly. Not surprisingly, these ten alkaloids did not show noteworthy effects on TPC2 cation channels and the tumor cell line VCR-R CEM, and did not exhibit P-glycoprotein blocking activity. But due to their free phenolic groups they can serve as valuable intermediates for novel derivatives addressing all of these targets, based on previous evidence for structure-activity relationships in this chemotype.

Quantitative global lipidomics analysis of patients with ovarian cancer versus benign adnexal mass.

Altered lipid metabolism has emerged as an important feature of ovarian cancer (OC), yet the translational potential of lipid metabolites to aid in diagnosis and triage remains unproven. We conducted a multi-level interrogation of lipid metabolic phenotypes in patients with adnexal masses, integrating quantitative lipidomics profiling of plasma and ascites with publicly-available tumor transcriptome data. Using Sciex Lipidyzer, we assessed concentrations of > 500 plasma lipids in two patient cohorts-(i) a pilot set of 100 women with OC (50) or benign tumor (50), and (ii) an independent set of 118 women with malignant (60) or benign (58) adnexal mass. 249 lipid species and several lipid classes were significantly reduced in cases versus controls in both cohorts (FDR < 0.05). 23 metabolites-triacylglycerols, phosphatidylcholines, cholesterol esters-were validated at Bonferroni significance (P < 9.16 × 10-5). Certain lipids exhibited greater alterations in early- (diacylglycerols) or late-stage (lysophospholipids) cases, and multiple lipids in plasma and ascites were positively correlated. Lipoprotein receptor gene expression differed markedly in OC versus benign tumors. Importantly, several plasma lipid species, such as DAG(16:1/18:1), improved the accuracy of CA125 in differentiating early-stage OC cases from benign controls, and conferred a 15-20% increase in specificity at 90% sensitivity in multivariate models adjusted for age and BMI. This study provides novel insight into systemic and local lipid metabolic differences between OC and benign disease, further implicating altered lipid uptake in OC biology, and advancing plasma lipid metabolites as a complementary class of circulating biomarkers for OC diagnosis and triage.

Estradiol analogs attenuate autophagy, cell migration and invasion by direct and selective inhibition of TRPML1, independent of estrogen receptors.

The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17ß-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion.

Gene editing and synthetically accessible inhibitors reveal role for TPC2 in HCC cell proliferation and tumor growth.

The role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of cancer cells in vitro, affects their energy metabolism, and successfully abrogates tumor growth in vivo. Concurrently, we have developed simplified analogs of the alkaloid tetrandrine as potent TPC2 inhibitors by screening a library of synthesized benzyltetrahydroisoquinoline derivatives. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against cancer cells and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing a TPC2 inhibitor with antitumor efficacy in mice. Hence, our study unveils TPC2 as valid target for cancer therapy and provides easily accessible tetrandrine analogs as a promising option for effective pharmacological interference.

Chemical and pharmacological characterization of the TRPML calcium channel blockers ML-SI1 and ML-SI3.

The members of the TRPML subfamily of non-selective cation channels (TRPML1-3) are involved in the regulation of important lysosomal and endosomal functions, and mutations in TRPML1 are associated with the neurodegenerative lysosomal storage disorder mucolipidosis type IV. For in-depth investigation of functions and (patho)physiological roles of TRPMLs, membrane-permeable chemical tools are urgently needed. But hitherto only two TRPML inhibitors, ML-SI1 and ML-SI3, have been published, albeit without clear information about stereochemical details. In this investigation we developed total syntheses of both inhibitors. ML-SI1 was only obtained as a racemic mixture of inseparable diastereomers and showed activator-dependent inhibitory activity. The more promising tool is ML-SI3, hence ML-SI1 was not further investigated. For ML-SI3 we confirmed by stereoselective synthesis that the trans-isomer is significantly more active than the cis-isomer. Separation of the enantiomers of trans-ML-SI3 further revealed that the (-)-isomer is a potent inhibitor of TRPML1 and TRPML2 (IC50 values 1.6 and 2.3 µM) and a weak inhibitor (IC50 12.5 µM) of TRPML3, whereas the (+)-enantiomer is an inhibitor on TRPML1 (IC50 5.9 µM), but an activator on TRPML 2 and 3. This renders the pure (-)-trans-ML-SI3 more suitable as a chemical tool for the investigation of TRPML1 and 2 than the racemate. The analysis of 12 analogues of ML-SI3 gave first insights into structure-activity relationships in this chemotype, and showed that a broad variety of modifications in both the N-arylpiperazine and the sulfonamide moiety is tolerated. An aromatic analogue of ML-SI3 showed an interesting alternative selectivity profile (strong inhibitor of TRPML1 and strong activator of TRPML2).

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

Correction to: Restoration of MARCKS enhances chemosensitivity in cancer.

In the original article, the title of the article is "Restoration of MARCK enhances chemosensitivity in cancer". The authors would like to change the article title to "Restoration of MARCKS enhances chemosensitivity in cancer" by adding a letter "S" to the word MARCK.

Proteome Profiling Uncovers an Autoimmune Response Signature That Reflects Ovarian Cancer Pathogenesis.

Harnessing the immune response to tumor antigens in the form of autoantibodies, which occurs early during tumor development, has relevance to the detection of cancer at early stages. We conducted an initial screen of antigens associated with an autoantibody response in serous ovarian cancer using recombinant protein arrays. The top 25 recombinants that exhibited increased reactivity with cases compared to controls revealed TP53 and MYC, which are ovarian cancer driver genes, as major network nodes. A mass spectrometry based independent analysis of circulating immunoglobulin (Ig)-bound proteins in ovarian cancer and of ovarian cancer cell surface MHC-II bound peptides also revealed a TP53-MYC related network of antigens. Our findings support the occurrence of a humoral immune response to antigens linked to ovarian cancer driver genes that may have utility for early detection applications.

Restoration of MARCKS enhances chemosensitivity in cancer.

PURPOSE: Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1. METHODS: CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU). RESULTS: We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU. CONCLUSIONS: Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

Direct Activation of TRPC3 Channels by the Antimalarial Agent Artemisinin.

(1) Background: Members of the TRPC3/TRPC6/TRPC7 subfamily of canonical transient receptor potential (TRP) channels share an amino acid similarity of more than 80% and can form heteromeric channel complexes. They are directly gated by diacylglycerols in a protein kinase C-independent manner. To assess TRPC3 channel functions without concomitant protein kinase C activation, direct activators are highly desirable. (2) Methods: By screening 2000 bioactive compounds in a Ca2+ influx assay, we identified artemisinin as a TRPC3 activator. Validation and characterization of the hit was performed by applying fluorometric Ca2+ influx assays and electrophysiological patch-clamp experiments in heterologously or endogenously TRPC3-expressing cells. (3) Results: Artemisinin elicited Ca2+ entry through TRPC3 or heteromeric TRPC3:TRPC6 channels, but did not or only weakly activated TRPC6 and TRPC7. Electrophysiological recordings confirmed the reversible and repeatable TRPC3 activation by artemisinin that was inhibited by established TRPC3 channel blockers. Rectification properties and reversal potentials were similar to those observed after stimulation with a diacylglycerol mimic, indicating that artemisinin induces a similar active state as the physiological activator. In rat pheochromocytoma PC12 cells that endogenously express TRPC3, artemisinin induced a Ca2+ influx and TRPC3-like currents. (4) Conclusions: Our findings identify artemisinin as a new biologically active entity to activate recombinant or native TRPC3-bearing channel complexes in a membrane-confined fashion.

Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors.

The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration-response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC50 values of 0.76 µM, 22 µM, 763 µM or 118 µM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 µM ATA did not compete with [3H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration-response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αß-methylene-ATP-sensitive P2X1 and P2X3 receptors.

Artifact-free objective-type multicolor total internal reflection fluorescence microscopy with light-emitting diode light sources-Part I.

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective-type or prism-less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light-emitting diode (LED)-based implementation of objective-type TIRF (LED-TIRF). The proposed LED-TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF-epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED-TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non-coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser-TIRF. Unlike azimuthal spinning laser-TIRF, LED-TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short-lived, localized translocation events of a Ca2+ -sensitive protein kinase C α fusion protein are demonstrated.