Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding.

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

Evaluation of fendiline treatment in VP40 system with nucleation-elongation process: a computational model of Ebola virus matrix protein assembly.

Ebola virus (EBOV) infection is threatening human health, especially in Central and West Africa. Limited clinical trials and the requirement of biosafety level-4 laboratories hinder experimental work to advance our understanding of EBOV and the evaluation of treatment. In this work, we use a computational model to study the assembly and budding process of EBOV and evaluate the effect of fendiline on these processes in the context of fluctuating host membrane lipid levels. Our results demonstrate for the first time that the assembly of VP40 filaments may follow the nucleation-elongation theory, as this mechanism is critical to maintaining a pool of VP40 dimers for the maturation and production of virus-like particles (VLPs). We further find that this nucleation-elongation process is likely influenced by fluctuating phosphatidylserine (PS), which can complicate the efficacy of lipid-targeted therapies like fendiline, a drug that lowers cellular PS levels. Our results indicate that fendiline-induced PS reduction may actually increase VLP production at earlier time points (24 h) and under low fendiline concentrations (≤2 µM). However, this effect is transient and does not change the conclusion that fendiline generally decreases VLP production. In the context of fluctuating PS levels, we also conclude that fendiline can be more efficient at the late stage of VLP budding relative to earlier phases. Combination therapy with a VLP budding step-targeted drug may therefore further increase the treatment efficiency of fendiline. Finally, we also show that fendiline-induced PS reduction more effectively lowers VLP production when VP40 expression is high. Taken together, our results provide critical quantitative information on how fluctuating lipid levels (PS) affect EBOV assembly and egress and how this mechanism can be disrupted by lipid-targeting molecules like fendiline. IMPORTANCE: Ebola virus (EBOV) infection can cause deadly hemorrhagic fever, which has a mortality rate of ~50%-90% without treatment. The recent outbreaks in Uganda and the Democratic Republic of the Congo illustrate its threat to human health. Though two antibody-based treatments were approved, mortality rates in the last outbreak were still higher than 30%. This can partly be due to the requirement of advanced medical facilities for current treatments. As a result, it is very important to develop and evaluate new therapies for EBOV infection, especially those that can be easily applied in the developing world. The significance of our research is that we evaluate the potential of lipid-targeted treatments in reducing EBOV assembly and egress. We achieved this goal using the VP40 system combined with a computational approach, which both saves time and lowers cost compared to traditional experimental studies and provides innovative new tools to study viral protein dynamics.

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

Calcium sensing receptors as integrators of multiple metabolic signals.

Calcium sensing receptors are critical to maintenance of organismal Ca2+ homeostasis, translating small changes in serum Ca2+ into changes in PTH secretion by the parathyroid glands and Ca2+ excretion by the kidneys. Calcium sensing receptors are also expressed in many cells and tissues not directly involved in Ca2+ homeostasis where their role(s) are less defined. Recent studies have demonstrated that calcium sensing receptors integrate a variety of metabolic signals, including polyvalent cations, pH, ionic strength, amino acids, and polypeptides, making CaR uniquely capable of generating cell- and tissue-specific responses, sensing not only Ca2+, but the local metabolic environment. The challenge for future studies is to define CaR responsiveness in each varied physiological context.

Single dose pharmacokinetics of fendiline in humans.

Fendiline was administered intravenously (3 mg) and orally (50 mg and 75 mg) in a cross-over study to six healthy volunteers. The plasma levels of unchanged fendiline and of total radioactivity were measured. Fendiline was absorbed well and its concentration declined biexponentially with mean terminal half-lives of 20-35 h. Since the drug is extensively metabolized, only 12% of total radioactivity in plasma corresponded to fendiline in the case of intravenous administration as compared to less than 2% after oral administration. 56-65% of the administered dose are excreted via the urine and 18-25% with the feces within five days.

Tolerance and pharmacokinetics of oral fendiline.

Two studies with healthy volunteers were carried out to correlate safety with pharmacokinetics of the calcium antagonistic drug N-(3,3-diphenylpropyl)-(1-phenylethyl)-amine (fendiline, Sensit) after single and multiple oral doses. In the first study single doses of 200, 400, 600, 800, 1000, and 1200 mg of fendiline hydrochloride were administered to 6 subjects per dose level. 3 additional subjects per dose level received placebo. No significant objective or subjective effects were noted in the dose range studied. The pharmacokinetic analysis revealed that doses higher than 800 mg were absorbed incompletely. In the second study initially 400 mg twice daily was given to 9 subjects. 3 additional subjects received placebo. Due to subjective intolerability (trembling, dizziness) after 5 days, the dose was reduced stepwise to 2 X 200 mg and was then continued for another 19 days. The pharmacokinetic evaluation revealed manifold interindividual differences in plasma levels for maximal concentrations (9-170 ng/ml) as well as for minimal concentrations (4-96 ng/ml). The absorption profile in both studies has linear and nonlinear components. Maximal plasma levels were reached after about 4 h. Terminal elimination half-lives were about 20 h.

Intracellular localization of the calcium- and calmodulin antagonist fendiline.

Microautoradiograpic studies of mouse heart sections were performed to evaluate intracellular localization of the antianginal drug fendiline (Sensit). 15 microCi 3H-fendiline/g b. w. (0.5 mg/kg b.w.) were injected intravenously. 10 min p.a. the heart was removed and either frozen in acetone/dry ice or, after perfusion in situ with dextran 40 and formaldehyde, fixed in formaldehyde. Frozen, paraffin-embedded, and semithin epoxy resin sections were prepared and coated with Ilford K2 emulsion. After 7 to 30 days exposure and development in amidol silver grains were counted above cells, nuclei and extracellular space. The results show that fendiline is able to enter myocardial cells.

Crystallization and preliminary diffraction analysis of Ca(2+)-calmodulin-drug and apocalmodulin-drug complexes.

Ca(2+)-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3"-(beta-chloroethyl)-2",4"-dioxo-3,5"- spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4- di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 A and differ in unit cell parameters from each other as well as from crystals of Ca(2+)-calmodulin or Ca(2+)-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca(2+)-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca(2+)-free calmodulin crystals diffracting-beyond 2.5 A resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca(2+)-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies.