Croton megalobotrys Müll Arg. and Vitex doniana (Sweet): Traditional medicinal plants in a three-step treatment regimen that inhibit in vitro replication of HIV-1.

ETHNOPHARMACOLOGICAL RELEVANCE: Human Immunodeficiency Virus (HIV) strains resistant to licensed anti-retroviral drugs (ARVs) continue to emerge. On the African continent, uneven access to ARVs combined with occurrence of side-effects after prolonged ARV therapy have led to searches for traditional medicines as alternative or complementary remedies to conventional HIV/AIDS management. AIM OF THE STUDY: Here we characterize a specific three-step traditional HIV/AIDS treatment regimen consisting of Cassia sieberiana root, Vitex doniana root, and Croton megalobotrys bark by combining qualitative interviews of traditional medical knowledge users in Botswana with in vitro HIV replication studies. MATERIALS AND METHODS: Crude extracts from a total of seven medicinal plants were tested for in vitro cytotoxicity and inhibition of wild-type (NL4.3) and ARV-resistant HIV-1 replication in an immortalized GFP-reporter CD4+ T-cell line. RESULTS: C. sieberiana root, V. doniana root, and C. megalobotrys bark extracts inhibited HIV-1NL4.3 replication with dose-dependence and without concomitant cytotoxicity. C. sieberiana and V. doniana extracts inhibited HIV-1 replication by 50% at 84.8µg/mL and at 25µg/mL, respectively, while C. megalobotrys extracts inhibited HIV-1 replication by a maximum of 45% at concentrations as low as 0.05µg/mL. Extracts did not interfere with antiviral activities of licensed ARVs when applied in combination and exhibited comparable efficacies against viruses harboring major resistance mutations to licensed protease, reverse-transcriptase, or integrase inhibitors. CONCLUSIONS: We report for the first time a three-step traditional HIV/AIDS regimen, used alone or in combination with standard ARV regimens, where each step exhibited more potent ability to inhibit HIV replication in vitro. Our observations support the "reverse pharmacology" model where documented clinical experiences are used to identify natural products of therapeutic value.

Screening of the Pan-African natural product library identifies ixoratannin A-2 and boldine as novel HIV-1 inhibitors.

The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.

The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis.

The transport of calcium ions (Ca(2+)) to the cytosol is essential for immunoreceptor signaling, regulating lymphocyte differentiation, activation, and effector function. Increases in cytosolic-free Ca(2+) concentrations are thought to be mediated through two interconnected and complementary mechanisms: the release of endoplasmic reticulum Ca(2+) "stores" and "store-operated" Ca(2+) entry via plasma membrane channels. However, the identity of molecular components conducting Ca(2+) currents within developing and mature T cells is unclear. Here, we have demonstrated that the L-type "voltage-dependent" Ca(2+) channel Ca(V)1.4 plays a cell-intrinsic role in the function, development, and survival of naive T cells. Plasma membrane Ca(V)1.4 was found to be essential for modulation of intracellular Ca(2+) stores and T cell receptor (TCR)-induced rises in cytosolic-free Ca(2+), impacting activation of Ras-extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) pathways. Collectively, these studies revealed that Ca(V)1.4 functions in controlling naive T cell homeostasis and antigen-driven T cell immune responses.

Functional characterization of the LQT2-causing mutation R582C and the associated voltage-dependent fluorescence signal.

BACKGROUND: The R582C mutation is one of many Long-QT Syndrome type 2 (LQT2)-causing mutations localized to the human ether-a-go-go related gene (hERG) channel's S5-P linker subdomain, yet its specific mechanism of dysfunction has not been examined. OBJECTIVE: This study sought to characterize the biophysical properties of the congenital LQT2-causing mutation, R582C, and utilize this mutation to provide the first report of voltage-dependent fluorescence from the S5-P linker. METHODS: Properties of the R582C channels were characterized by heterologous expression in both HEK293 cells and Xenopus oocytes using a combination of patch-clamp, 2-electrode voltage-clamp, immunoblot assay, and voltage-clamp fluorimetry. RESULTS: Expression of hERG R582C was found to be deficient in HEK293 cells, yet was amenable to rescue by incubation at reduced temperature or by treatment with dofetilide. Rescued channels expressed at levels comparable to wild type (WT) channels. Kinetic differences result in decreased outward repolarizing current evoked by an action potential clamp protocol. Voltage-clamp fluorimetry experiments utilized the introduced cysteine to covalently attach a fluorescent probe (tetramethylrhodamine-5-maleimide) to the S5-P linker to directly observe conformational changes occurring due to inactivation. CONCLUSION: The major mechanism underlying pathogenicity of the R582C mutation is a trafficking deficiency, although channels also exhibit kinetic deficiencies, perhaps reflecting the position of the mutation in the pore turret. Voltage clamp fluorescence signals from R582C channels provide evidence that the hERG turret undergoes distinct conformational changes during inactivation.

The mechanism of atrial antiarrhythmic action of RSD1235.

INTRODUCTION: RSD1235 is a novel drug recently shown to convert AF rapidly and safely in patients.(1) Its mechanism of action has been investigated in a rat model of ischemic arrhythmia, along with changes in action potential (AP) morphology in isolated rat ventricular myocytes and effects on cloned channels. METHODS AND RESULTS: Ischemic arrhythmias were inhibited with an ED50 of 1.5 micromol/kg/min, and repolarization times increased with non-significant effects on PR and QRS durations. AP prolongation was observed in rat myocytes at low doses, with plateau elevation and a reduction in the AP overshoot at higher doses. RSD1235 showed selectivity for voltage-gated K+ channels with IC50 values of 13 microM on hKv1.5 (1 Hz) versus 38 and 30 microM on Kv4.2 and Kv4.3, respectively, and 21 microM on hERG channels. RSD1235 did not block IK1 (IC50 > 1 mM) nor ICa,L (IC50= 220 microM) at 1 Hz in guinea pig ventricular myocytes (n = 4-5). The drug displayed mild (IC50= 43 microM at 1 Hz) open-channel blockade of Nav1.5 with rapid recovery kinetics after rate reduction (10-->1 Hz, 75% recovery with tau= 320 msec). Nav1.5 blocking potency increased with stimulus frequency from an IC50= 40 microM at 0.25 Hz, to an IC50= 9 microM at 20 Hz, and with depolarization increasing from 107 microM at -120 mV to 31 microM at -60 mV (1 Hz). CONCLUSIONS: These data suggest that RSD1235's clinical selectivity and AF conversion efficacy result from block of potassium channels combined with frequency- and voltage-dependent block of INa.

Flux assays in high throughput screening of ion channels in drug discovery.

Ion channels have been identified as therapeutic targets in various disorders, such as cardiovascular disease, neurological disease, and cystic fibrosis. Flux assays to detect functional ionic flux through ion channels are becoming increasingly popular as tools for screening compounds. In an optimized flux assay, modulation of ion channel activity may produce readily detectable changes in radiolabeled or nonradiolabeled ionic flux. Technologies based on flux assays are currently available in a fully automated high throughput format for efficient screening. This application offers sensitive, precise, and reproducible measurements giving accurate drug rank orders matching those of patch clamp data. Conveniently, the flux assay is amenable to adaptation for different ion channels, such as potassium, sodium, calcium, and chloride channels, by using suitable tracer ions. The nonradiolabeled rubidium-based flux assay coupled with the ion channel reader (ICR) technology has become very successful in ion channel activity analysis and is emerging as a popular technique in modern drug discovery.