Discovery of Novel Retigabine Derivatives as Potent KCNQ4 and KCNQ5 Channel Agonists with Improved Specificity.

Recent research suggests that KCNQ isoforms, particularly the KCNQ4 and KCNQ5 subtypes expressed in smooth muscle cells, are involved in both establishing and maintaining resting membrane potentials and regulating smooth muscle contractility. Retigabine (RTG) is a first-in-class antiepileptic drug that potentiates neuronal KCNQ potassium channels, but poor subtype selectivity limits its further application as a pharmacological tool. In this study, we improved the subtype specificity of retigabine by altering the N-1/3 substituents and discovered several compounds that show better selectivity for KCNQ4 and KCNQ5 channels. Among these compounds, 10g is highly selective for KCNQ4 and KCNQ5 channels without potentiating KCNQ1 and KCNQ2 channels. These results are an advance in the exploration of small molecule modifiers that selectively activate different KCNQ isoforms. The developed compounds could also serve as new pharmacological tools for elucidating the function of KCNQ channels natively expressed in various tissues.

Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection.

Congenital human cytomegalovirus (HCMV) infection is a leading infectious cause of birth defects. Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells (MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multi-organ formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry, replication initiation, viral protein expression, and infectious virion release using western blotting, immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques.

AIM: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. METHODS: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. RESULTS: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 µmol/L and 1.37±0.06 µmol/L, respectively. CONCLUSION: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators.

Identification and BAC construction of Han, the first characterized HCMV clinical strain in China.

Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects, and may lead to severe or lethal diseases in immunocompromised individuals. Several HCMV strains have been identified and widely applied in research, but no isolate from China has been characterized. In the present study, we isolated, characterized and sequenced the first Chinese HCMV clinical strain Han, and constructed the novel and functional HCMV infectious clone Han-BAC-2311. HCMV Han was isolated from the urine sample of a Chinese infant with multiple developmental disorders. It expresses HCMV specific proteins and contains a representative HCMV genome with minor differences compared to other strains. By homologous recombination using mini-F derived BAC vector pUS-F6, the infectious clone Han-BAC-2311 was constructed containing representative viral genes across the HCMV genome. The insertion site and orientation of BAC sequence were confirmed by restriction enzyme digestion and Southern blotting. The reconstituted recombinant virus HanBAC-2311 expresses typical viral proteins with the same pattern as that of wild-type Han, and also displayed a similar growth kinetics to wild-type Han. The identification of the first clinical HCMV strain in China and the construction of its infectious clone will greatly facilitate the pathogenesis studies and vaccine development in China.

MicroRNA miR-21 attenuates human cytomegalovirus replication in neural cells by targeting Cdc25a.

UNLABELLED: Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products-IE1, pp71, and UL26-were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS. IMPORTANCE: Human cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

Later passages of neural progenitor cells from neonatal brain are more permissive for human cytomegalovirus infection.

Congenital human cytomegalovirus (HCMV) infection is the most frequent infectious cause of birth defects, primarily neurological disorders. Neural progenitor/stem cells (NPCs) are the major cell type in the subventricular zone and are susceptible to HCMV infection. In culture, the differentiation status of NPCs may change with passage, which in turn may alter susceptibility to virus infection. Previously, only early-passage (i.e., prior to passage 9) NPCs were studied and shown to be permissive to HCMV infection. In this study, NPC cultures derived at different gestational ages were evaluated after short (passages 3 to 6) and extended (passages 11 to 20) in vitro passages for biological and virological parameters (i.e., cell morphology, expression of NPC markers and HCMV receptors, viral entry efficiency, viral gene expression, virus-induced cytopathic effect, and release of infectious progeny). These parameters were not significantly influenced by the gestational age of the source tissues. However, extended-passage cultures showed evidence of initiation of differentiation, increased viral entry, and more efficient production of infectious progeny. These results confirm that NPCs are fully permissive for HCMV infection and that extended-passage NPCs initiate differentiation and are more permissive for HCMV infection. Later-passage NPCs being differentiated and more permissive for HCMV infection suggest that HCMV infection in fetal brain may cause more neural cell loss and give rise to severe neurological disabilities with advancing brain development.