Levoketoconazole treatment in endogenous Cushing's syndrome: extended evaluation of clinical, biochemical, and radiologic outcomes.

Objective: This extended evaluation (EE) of the SONICS study assessed the effects of levoketoconazole for an additional 6 months following open-label, 6-month maintenance treatment in endogenous Cushing's syndrome. Design/Methods: SONICS included dose-titration (150-600 mg BID), 6-month maintenance, and 6-month EE phases. Exploratory efficacy assessments were performed at months 9 and 12 (relative to the start of maintenance). For pituitary MRI in patients with Cushing's disease, a threshold of ≥2 mm denoted change from baseline in the largest tumor diameter. Results: Sixty patients entered EE at month 6; 61% (33/54 with data) exhibited normal mean urinary free cortisol (mUFC). At months 9 and 12, respectively, 55% (27/49) and 41% (18/44) of patients with data had normal mUFC. Mean fasting glucose, total and LDL-cholesterol, body weight, BMI, abdominal girth, hirsutism, CushingQoL, and Beck Depression Inventory-II scores improved from the study baseline at months 9 and 12. Forty-six patients completed month 12; four (6.7%) discontinued during EE due to adverse events. The most common adverse events in EE were arthralgia, headache, hypokalemia, and QT prolongation (6.7% each). No patient experienced alanine aminotransferase or aspartate aminotransferase >3× upper limit of normal, Fridericia-corrected QT interval >460 ms, or adrenal insufficiency during EE. Of 31 patients with tumor measurements at baseline and month 12 or follow-up, the largest tumor diameter was stable in 27 (87%) patients, decreased in one, and increased in three (largest increase 4 mm). Conclusion: In the first long-term levoketoconazole study, continued treatment through a 12-month maintenance period sustained the early clinical and biochemical benefits in most patients completing EE, without new adverse effects.

The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion.

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins-a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion-the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.

Ginkgolic acid inhibits fusion of enveloped viruses.

Ginkgolic acids (GA) are alkylphenol constituents of the leaves and fruits of Ginkgo biloba. GA has shown pleiotropic effects in vitro, including: antitumor effects through inhibition of lipogenesis; decreased expression of invasion associated proteins through AMPK activation; and potential rescue of amyloid-ß (Aß) induced synaptic impairment. GA was also reported to have activity against Escherichia coli and Staphylococcus aureus. Several mechanisms for this activity have been suggested including: SUMOylation inhibition; blocking formation of the E1-SUMO intermediate; inhibition of fatty acid synthase; non-specific SIRT inhibition; and activation of protein phosphatase type-2C. Here we report that GA inhibits Herpes simplex virus type 1 (HSV-1) by inhibition of both fusion and viral protein synthesis. Additionally, we report that GA inhibits human cytomegalovirus (HCMV) genome replication and Zika virus (ZIKV) infection of normal human astrocytes (NHA). We show a broad spectrum of fusion inhibition by GA of all three classes of fusion proteins including HIV, Ebola virus (EBOV), influenza A virus (IAV) and Epstein Barr virus (EBV). In addition, we show inhibition of a non-enveloped adenovirus. Our experiments suggest that GA inhibits virion entry by blocking the initial fusion event. Data showing inhibition of HSV-1 and CMV replication, when GA is administered post-infection, suggest a possible secondary mechanism targeting protein and DNA synthesis. Thus, in light of the strong effect of GA on viral infection, even after the infection begins, it may potentially be used to treat acute infections (e.g. Coronavirus, EBOV, ZIKV, IAV and measles), and also topically for the successful treatment of active lesions (e.g. HSV-1, HSV-2 and varicella-zoster virus (VZV)).

Efficacy and safety of levoketoconazole in the treatment of endogenous Cushing's syndrome (SONICS): a phase 3, multicentre, open-label, single-arm trial.

BACKGROUND: Levoketoconazole is a ketoconazole stereoisomer in development for treatment of Cushing's syndrome and has not been assessed previously in a clinical trial in patients with Cushing's syndrome. We aimed to investigate the efficacy and safety of levoketoconazole in patients with endogenous Cushing's syndrome. METHODS: SONICS is a phase 3, multicentre, open-label, non-randomised, single-arm study in which we recruited adults (≥18 years) with confirmed Cushing's syndrome and a mean 24-h urinary free cortisol (mUFC) of at least 1·5 times the upper limit of normal from 60 hospital and community sites in 19 countries (15 countries in Europe, and Canada, Israel, Turkey, and the USA). Patients were treated with oral levoketoconazole in a 2-21 week incremental dose-titration phase starting at 150 mg twice daily (150 mg increments until mUFC normalisation, maximum 600 mg twice daily) and a 6-month maintenance phase. The primary outcome was the proportion of patients with mUFC normalisation at end of maintenance, without dose increase during the maintenance phase (in the intention-to-treat population). Prespecified adverse events of special interest were potential liver toxicity, corrected QT prolongation, and adrenal insufficiency. This trial is registered with ClinicalTrials.gov, NCT01838551. FINDINGS: Between July 30, 2014, and June 30, 2017, 201 individuals were screened and 94 patients were enrolled and received at least one dose of study medication. Of the 94 patients, 80 (85%) had pituitary Cushing's syndrome. Mean mUFC at baseline was 671·4 nmol/24 h (243·3 µg/24 h), which is 4·9 times the upper limit of normal. Of the 77 patients who advanced to the maintenance phase, 62 (81%) had mUFC normalisation by end-of-dose titration. At the end of the 6-month maintenance phase, 29 (31%) of 94 patients were responders; the least-squares mean estimate of the proportion of responders was 0·30 (95% CI 0·21-0·40; p=0·0154 vs null hypothesis of ≤0·20). The most common adverse events in the 94 patients were nausea (30 [32%]) and headache (26 [28%]). Adverse events led to study discontinuation in 12 (13%) of 94 patients. Two patients had a QT interval (Fridericia corrected) of more than 500 ms, and three patients had suspected adrenal insufficiency. Alanine aminotransferase reversibly increased to more than three times the upper limit of normal in ten (11%) patients. Four patients had serious adverse events that were considered probably or definitely related to the study drug: abnormal liver function test results (n=1), prolonged QT interval (n=2), and adrenal insufficiency (n=1). One person died from colon carcinoma unrelated to study medication. INTERPRETATION: Twice-daily oral levoketoconazole treatment led to sustained improvements in urinary free cortisol, with an acceptable safety and tolerability profile. Levoketoconazole might represent a useful therapeutic option for the medical treatment of Cushing's syndrome. FUNDING: Strongbridge Biopharma.

The Chemical Potential of Plasma Membrane Cholesterol: Implications for Cell Biology.

Cholesterol is abundant in plasma membranes and exhibits a variety of interactions throughout the membrane. Chemical potential accounts for thermodynamic consequences of molecular interactions, and quantifies the effective concentration (i.e., activity) of any substance participating in a process. We have developed, to our knowledge, the first method to measure cholesterol chemical potential in plasma membranes. This was accomplished by complexing methyl-ß-cyclodextrin with cholesterol in an aqueous solution and equilibrating it with an organic solvent containing dissolved cholesterol. The chemical potential of cholesterol was thereby equalized in the two phases. Because cholesterol is dilute in the organic phase, here activity and concentration were equivalent. This equivalence allowed the amount of cholesterol bound to methyl-ß-cyclodextrin to be converted to cholesterol chemical potential. Our method was used to determine the chemical potential of cholesterol in erythrocytes and in plasma membranes of nucleated cells in culture. For erythrocytes, the chemical potential did not vary when the concentration was below a critical value. Above this value, the chemical potential progressively increased with concentration. We used standard cancer lines to characterize cholesterol chemical potential in plasma membranes of nucleated cells. This chemical potential was significantly greater for highly metastatic breast cancer cells than for nonmetastatic breast cancer cells. Chemical potential depended on density of the cancer cells. A method to alter and fix the cholesterol chemical potential to any value (i.e., a cholesterol chemical potential clamp) was also developed. Cholesterol content did not change when cells were clamped for 24-48 h. It was found that the level of activation of the transcription factor STAT3 increased with increasing cholesterol chemical potential. The cholesterol chemical potential may regulate signaling pathways.

The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.

Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a 'lipidic junction'. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture-insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.

Safety and efficacy of prolonged levofloxacin inhalation solution (APT-1026) treatment for cystic fibrosis and chronic Pseudomonas aeruginosa airway infection.

BACKGROUND: Levofloxacin inhalation solution (LIS) is the first aerosolized fluoroquinolone licensed for treatment of patients with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa lung infection. This study evaluated the safety and efficacy of extended LIS treatment. METHODS: Patients completing a multinational, randomized study comparing LIS and tobramycin inhalation solution (TIS) were enrolled in an open-label extension in which all patients received three additional cycles of 28days of LIS 240mg twice daily followed by 28days off drug. Endpoints included mean relative change in percent predicted forced expiratory volume in 1s (FEV1), time to pulmonary exacerbation, and patient-reported quality of life. RESULTS: Extended treatment with LIS in 88 patients was well tolerated with no new safety signals and evidence of positive effects on FEV1 and quality of life. CONCLUSION: Patients receiving extended LIS treatment continued to show favorable efficacy with no additional safety concerns.

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.

Cell-cell contact promotes Ebola virus GP-mediated infection.

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Here we provide evidence that cell-cell contact promotes infection mediated by the glycoprotein (GP) of EBOV. Interestingly, expression of EBOV GP alone, even in the absence of retroviral Gag-Pol, is sufficient to transfer a retroviral vector encoding Tet-off from cell to cell. Cell-to-cell infection mediated by EBOV GP is blocked by inhibitors of actin polymerization, but appears to be less sensitive to KZ52 neutralization. Treatment of co-cultured cells with cathepsin B/L inhibitors, or an entry inhibitor 3.47 that targets the receptor NPC1 for virus binding, also blocks cell-to-cell infection. Cell-cell contact also enhances spread of rVSV bearing GP in monocytes and macrophages, the primary targets of natural EBOV infection. Altogether, our study reveals that cell-cell contact promotes EBOV GP-mediated infection, and provides new insight into understanding EBOV spread and viral pathogenesis.

The transmembrane domain and acidic lipid flip-flop regulates voltage-dependent fusion mediated by class II and III viral proteins.

Voltage dependence of fusion induced by class II and class III viral fusion proteins was investigated. Class II proteins from Ross River and Sindbus virus and a mutant class III protein from Epstein Barr virus were found to induce cell-cell fusion that is voltage dependent. Combined with previous studies, in all, four class II and two class III protein have now been shown to exhibit voltage-dependent fusion, demonstrating that this is probably a general phenomenon for these two classes of viral fusion proteins. In the present study, monitoring fusion of pseudovirus expressing Vesicular Stomatitis virus (VSV) G within endosomes shows that here, too, fusion is voltage dependent. This supports the claim that voltage dependence of fusion is biologically relevant and that cell-cell fusion reliably models the voltage dependence. Fusion induced by class I viral proteins is independent of voltage; chimeras expressing the ectodomain of a class I fusion protein and the transmembrane domain of VSV G could therefore be used to explore the location within the protein responsible for voltage dependence. Results showed that the transmembrane domain is the region associated with voltage dependence. Experiments in which cells were enriched with acidic lipids led to the conclusion that it is the flip-flop of acidic lipids that carries the charge responsible for the observed voltage dependence of fusion. This flip-flop occurred downstream of hemifusion, in accord with previous findings that the voltage dependent steps of fusion occur at a stage subsequent to hemifusion.

IFITM proteins restrict viral membrane hemifusion.

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

Negative potentials across biological membranes promote fusion by class II and class III viral proteins.

Voltage was investigated as a factor in the fusion of virions. Virions, pseudotyped with a class II, SFV E1 or VEEV E, or a class III protein, VSV G, were prepared with GFP within the core and a fluorescent lipid. This allowed both hemifusion and fusion to be monitored. Voltage clamping the target cell showed that fusion is promoted by a negative potential and hindered by a positive potential. Hemifusion occurred independent of polarity. Lipid dye movement, in the absence of content mixing, ceased before complete transfer for positive potentials, indicating that reversion of hemifused membranes into two distinct membranes is responsible for voltage dependence and inhibition of fusion. Content mixing quickly followed lipid dye transfer for a negative potential, providing a direct demonstration that hemifusion induced by class II and class III viral proteins is a functional intermediate of fusion. In the hemifused state, virions that fused exhibited slower lipid transfer than did nonfusing virions. All viruses with class II or III fusion proteins may utilize voltage to achieve infection.

The six-helix bundle of human immunodeficiency virus Env controls pore formation and enlargement and is initiated at residues proximal to the hairpin turn.

Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.

Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell.

Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.

Time-resolved imaging of HIV-1 Env-mediated lipid and content mixing between a single virion and cell membrane.

A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily released upon permeabilization of the viral envelope, whereas the capsid was retained. The NC-GFP thus provides a relatively small and mobile aqueous marker to follow viral content transfer. In fusion experiments, virions were bound to cells at low temperature, and fusion was synchronously triggered by a temperature jump. DiD transferred from virions to cells without a significant lag after the temperature jump. Some virions released DiD but retained NC-GFP. Surprisingly, the fraction of lipid mixing events yielding NC-GFP transfer was dependent on the type of target cell: of three infectable cell lines, only one permitted NC-GFP transfer within minutes of raising temperature. NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge.

RWJ-241947 (MCC-555), a unique peroxisome proliferator-activated receptor-gamma ligand with antitumor activity against human prostate cancer in vitro and in beige/nude/ X-linked immunodeficient mice and enhancement of apoptosis in myeloma cells induced by arsenic trioxide.

PURPOSE: RWJ-241947 (MCC-555) is a novel peroxisome proliferator-activated receptor-gamma ligand of the thiazolidinedione class that was recently developed as an antidiabetic drug with unique properties. Some thiazolidinediones have anticancer activity against solid and hematological malignancies; the anticancer potency of RWJ-241947 has not been examined. We, therefore, investigated these effects in vitro and in vivo either alone or in combination with other compounds. EXPERIMENTAL DESIGN: Tumor growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony assay in vitro, and xenografts in nude mice. Its effects on cell cycle, differentiation, and apoptosis were examined. RESULTS: In vitro studies using various solid and hematological tumor cell lines showed that RWJ-241947 had antiproliferative activity against prostate cancer cells, with the strongest effect against the androgen-independent PC-3 prostate cancer cells. It increased expression of cyclin-dependent kinase inhibitor p21(WAF1), deceased cyclin E, and induced apoptosis in PC-3 cells. It increased E-cadherin and lowered protein expression of prostate-specific antigen without down-regulating the androgen receptor in androgen-dependent LNCaP prostate cancer cells. Reporter gene assays showed that this peroxisome proliferator-activated receptor-gamma ligand inhibited androgen activation of the androgen receptor response elements of the prostate-specific antigen gene. Remarkably, in vivo treatment of male beige/nude/X-linked immunodeficient (BNX) mice with RWJ-241947 profoundly suppressed growth of PC-3 prostate cancer xenografts with prominent apoptosis, as well as fibrosis, including inflammatory and giant cell reaction in the remaining tumor tissue. Notably, the experimented mice had a significantly decreased cholesterol. In addition, we studied the combination of arsenic trioxide (As2O3), which is used in the treatment of multiple myeloma, and RWJ-241947; these two reagents together prominently inhibited proliferation and caused apoptosis of multiple myeloma cells. CONCLUSIONS: RWJ-241947 has surprisingly potent antiproliferative effects against prostate cancer cells in vivo, and it enhances the antitumor activity of As2O3 against myeloma cells. Small, well-defined clinical studies using RWJ-241947 are in order for these cancers.

HIV-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation.

Fusion proteins of many viruses, including HIV-1 envelope protein (Env), fold into six-helix bundle structures. Fusion between individual Env-expressing cells and target cells was studied by fluorescence microscopy, and a temperature jump technique, to determine whether folding of Env into a bundle is complete by the time fusion pores have formed. Lowering temperature to 4 degrees C immediately after a pore opened halted pore growth, which quickly resumed when temperature was raised again. HIV gp41-derived peptides that inhibit bundle formation (C34 or N36) caused the cold-arrested pore to quickly and irreversibly close, demonstrating that bundle formation is not complete by the time a pore has formed. In contrast, lowering the temperature to an intermediate value also halted pore growth, but the pore was not closed by the bundle-inhibiting peptides, and it enlarged when temperature was again elevated. This latter result shows that bundle formation is definitely required for the fusion process, but surprisingly, some (if not all) bundle formation occurs after a pore has formed. It is concluded that an essential function of the bundle is to stabilize the pore against collapse and ensure its growth.

Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus.

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the trans side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C(2)-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C(2)-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.

The mechanism of inhibition of HIV-1 env-mediated cell-cell fusion by recombinant cores of gp41 ectodomain.

N36(L6)C34 is a recombinant protein that forms a six-helix bundle with high thermal stability. It consists of the N-terminal heptad-repeat region (N36 peptide) and the C-terminal heptad-repeat region (C34) of HIV-1 gp41, connected by six polar amino acids. The protein inhibits HIV-1 envelope-induced membrane fusion. Whether inhibition occurs while N36(L6)C34 is in its six-helix bundle configuration was investigated. Mutating a critical residue within the N36 region to promote dissociation of C34 from the grooves of the N36 coiled coil reduced bundle stability and increased the inhibition of fusion. In contrast, mutating a key residue within the C34 region to reduce bundle stability decreased inhibitory potency. The data provide strong evidence that the proteins inhibit fusion while they expose their C34 segments, rather than as six-helix bundles. Thus, despite high thermal stability of the bundle, the recombinants' less folded structures are present in sufficient concentration to inhibit fusion at physiological temperatures.