Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo.

The use of receptor-ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

High-frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT3AB receptor.

The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB(Y129S) receptor are 20- and 10-fold slower, respectively, than those of the WT receptor. Single-channel measurements reveal a 7-fold-increased mean open time of 5-HT3AB(Y129S) receptors compared with WT receptors. The augmented signaling displayed by 5-HT3AB(Y129S) receptors may confer protection against the development of depression. The variant also may influence the development and/or treatment of nausea and other disorders involving 5-HT3 receptors. Thus, the impact of the high-frequency variant 5-HT3B(Y129S) on 5-HT3AB receptor signaling calls for a search for additional phenotypes, and the variant may thus aid in establishing the role of the 5-HT3AB receptor in pathophysiology.

3B but which 3B and that's just one of the questions: the heterogeneity of human 5-HT3 receptors.

The 5-hydroxytryptamine 3 (5-HT3) receptor is expressed widely in the central and peripheral nervous systems, where it mediates or modulates a wide range of physiological processes. The receptor is targeted by drugs administered for nausea and/or emesis and irritable bowel syndrome and has been proposed as a potential drug target in various psychiatric disorders. The 5-HT3 receptor is a pentameric ligand-gated ion channel and belongs to the Cys-loop receptor family. In contrast to the immense heterogeneity characterizing other Cysloop receptors, native 5-HT3 receptors historically have been considered a much more homogenous receptor population. However, the recent discovery of additional 5-HT3 subunits and the dawning realization that central and peripheral 5-HT3 receptor populations might comprise several subtypes characterized by distinct functional properties has emphasized the complexity of human 5-HT3 receptor signaling. In this review potential implications of these findings and of the entirely new layer of interindividual diversity introduced to the 5-HT3 receptor system by genetic variations will be outlined.

Novel Chemokine-Based Immunotoxins for Potent and Selective Targeting of Cytomegalovirus Infected Cells.

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX3CL1 specifically targeting US28 linked to the catalytic domain of Pseudomonas exotoxin A (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX3CR1, the endogenous receptor for CX3CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus may be targeted by FTPs.

Characterization of potassium channel modulators with QPatch automated patch-clamp technology: system characteristics and performance.

Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.

Characterization of the effects of four HTR3B polymorphisms on human 5-HT3AB receptor expression and signalling.

BACKGROUND: 5-Hydroxytryptamine 3 (5-HT3) receptors mediate the fast excitatory neurotransmission of serotonin. In this study, we have characterized the effects of four naturally occurring, nonsynonymous variants of the human 5-HT3B subunit on expression and signalling properties of heteromeric 5-HT3AB receptors. METHODS AND RESULTS: 5-HT3AB receptor signalling was studied in a fluorescence-based cell membrane potential assay and by electrophysiology. Expression levels of cotransfected epitope-tagged 5-HT3A and 5-HT3B subunits were determined using enzyme-linked immunosorbent assay and immunocytochemistry. In cells coexpressing 5-HT3A and 5-HT3B(I143T) subunits, cell surface expression levels of 5-HT3B in particular, and also 5-HT3A were markedly reduced compared with those of wild-type (WT) 5-HT3AB receptor-expressing cells. Electrophysiological recordings on cells coexpressing 5-HT3A and 5-HT3B(I143T) indicated cell surface expression of 5-HT3AB(I143T) receptors with macroscopic current kinetics similar to those of WT 5-HT3AB receptors but with 3-fold lower current densities. In the membrane potential assay, 5-HT3AB(I143T)-transfected cells exhibited signalling properties intermediate to those of WT 5-HT3AB and 5-HT3A receptors. Cotransfection of 5-HT3A, 5-HT3AB(I143T) and WT 5-HT3AB subunit cDNAs did not increase cell surface expression of the variant subunit nor did it restore WT 5-HT3AB receptor signalling completely in the membrane potential assay. In contrast to 5-HT3B(I143T), the 5-HT3B variants S156R, V183I and A223T did not give rise to significant changes in 5-HT3AB receptor expression or signalling properties. CONCLUSION: 5-HT3B(I143T)-containing 5-HT3AB receptors display significantly reduced cell surface expression and different signalling properties compared with WT 5-HT3AB receptors. In contrast, three other 5-HT3B variants, S156R, V183I and A223T, do not appear to alter 5-HT3AB receptor expression or signalling.

Naturally occurring variations in the human 5-HT3A gene profoundly impact 5-HT3 receptor function and expression.

BACKGROUND: The serotonin [5-hydroxytryptamine (5-HT)]-gated ion channel 5-HT3 is involved in the mediation of postoperative and radiotherapy/chemotherapy-induced nausea/emesis and in irritable bowel syndrome. It has also been suggested to play a role in various psychiatric diseases. Five naturally occurring single nucleotide polymorphisms leading to amino acid changes have been identified in the human 5-HT3A gene. METHODS AND RESULTS: We investigated the functional effects of these polymorphisms on the 5-HT3A receptor using fluorescence-based cellular assays. Notably, variants A33T, S253N, and M257I displayed 5-HT-induced maximal responses of 3-64% of the wild-type response, whereas R344H and P391R exhibited wild-type-like function. All variants displayed wild-type-like potencies of 5-HT and three 5-HT3 antagonists. Furthermore, all variants displayed Kd values similar to that of the wild-type receptor in a [H]GR65630-binding assay. The surface expression of A33T, M257I, and R344H was reduced 2-4-fold compared with the wild-type, despite similar total expression levels. Finally, coexpression of wild-type 5-HT3A or 5-HT3B subunits with 5-HT3A variants A33T, S253N, or M257I resulted in mixed or heteromeric receptors, characterized by significantly reduced maximal responses to 5-HT compared with the wild-type receptors. CONCLUSIONS: Three polymorphisms of the 5-HT3A gene gave rise to functionally impaired receptors whose function could not be rescued by either wild-type 5-HT3A or 5-HT3B. Three of the variant receptors were surface-expressed at reduced levels in spite of total expression levels similar to wild-type, indicating that these variants affect receptor biogenesis and/or trafficking. These severe single nucleotide polymorphism effects hold promise for identification of new 5-HT3A gene-disease causalities.

Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery.

Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.