Avinoam O et al. (2011) Science "Conserved eukaryotic fusogens can fuse viral envelopes to cells."

Valansi C, Zeev-Ben-Mordehai T, Grunewald K, White JM, Maurer UE, Avinoam O, Abutbul I, Fridman K, Podbilewicz B, Danino D, Sapir A

[Science, 2011]

Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Abong RA et al. (2020) BMC Infect Dis "Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment."

Fru-Cho J, Amambo GN, Wanji S, Poole CB, Chounna Ndongmo PW, Hoerauf A, Nji TM, Carlow CKS, Fombad FF, Deribe K, Pfarr K, Manuel R, Beng AA, Abong RA, Njouendou AJ, Enyong PI, Esum ME

[BMC Infect Dis, 2020]

BACKGROUND: Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques. METHOD: Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6months using microscopy, actin real-time PCR (actin-qPCR) and O-150 LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of actin-qPCR and LAMP. RESULTS: In Bafia with over 20years of IVM-MDA, 11/51 (21.6%) direct observe treated microfilaridemic participants were still positive for skin microfilariae after 1month. In Melong, with 10years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia. In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity. CONCLUSION: Patterns in skin mirofilariae clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA displayed a lower clearance and higher repopulation rate after treatment with ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

Sjoberg HT et al. (2019) PLoS Negl Trop Dis "Short-course, oral flubendazole does not mediate significant efficacy against Onchocerca adult male worms or Brugia microfilariae in murine infection models."

Engelen M, Taylor MJ, Pionnier N, Lachaud S, Tayong DB, Fombad FF, Chounna PWN, Turner JD, Quirynen L, Njouendou AJ, Wanji S, Steven A, Gandjui NVT, Ward SA, Chunda VC, Tekle F, Ndzeshang BL, Baeten B, Aljayyoussi G, Akumtoh DN, Sjoberg HT, Metuge HM

[PLoS Negl Trop Dis, 2019]

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.

Smurova K et al. (2016) Small GTPases "Endocytosis regulates membrane localization and function of the fusogen EFF-1."

Podbilewicz B, Smurova K

[Small GTPases, 2016]

Cell fusion is essential for sexual reproduction and formation of muscles, bones, and placenta. Two families of cell fusion proteins (Syncytins and FFs) have been identified in eukaryotes. Syncytins have been shown to form the giant syncytial trophoblasts in the placenta. The FFs are essential to fuse cells in the skin, reproductive, excretory, digestive and nervous systems in nematodes. EFF-1 (Epithelial Fusion Failure 1), a member of the FF family, is a type I membrane glycoprotein that is essential for most cell fusions in C. elegans. The crystal structure of EFF-1 ectodomain reveals striking structural similarity to class II fusion glycoproteins from enveloped viruses (e.g. dengue and rubella) that mediate virus to cell fusion. We found EFF-1 to be present on the plasma membrane and in RAB-5-positive early endosomes, with EFF-1 recycling between these two cell compartments. Only when EFF-1 proteins transiently arrive to the surfaces of two adjacent cells do they dynamically interact in trans and mediate membrane fusion. EFF-1 is continuously internalized by receptor-mediated endocytosis via the activity of two small GTPases: RAB-5 and Dynamin. Here we propose a model that explains how EFF-1 endocytosis together with interactions in trans can control cell-cell fusion. Kontani etal. showed that vacuolar ATPase (vATPase) mutations result in EFF1dependent hyperfusion. (1) We propose that vATPase is required for normal degradation of EFF1. Failure to degrade EFF-1 results in delayed hyperfusion and mislocalization to organelles that appear to be recycling endosomes. EFF-1 is also required to fuse neurons as part of the repair mechanism following injury and to prune dendrites. We speculate that EFF-1 may regulate neuronal tree like structures via endocytosis. Thus, endocytosis of cell-cell fusion proteins functions to prevent merging of cells and to sculpt organs and neurons.

Taylor MJ et al. (2019) Sci Transl Med "Preclinical development of an oral anti-Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis."

Turner JD, von Geldern TW, Hubner MP, Carr R, Ehrens A, Murphy E, Kempf DJ, Metuge HM, Aljayyoussi G, Ward SA, Taylor MJ, Johnston KL, Chunda VC, Wanji S, Specht S, Marsh K, Lenz F, Clare RH, Morton HE, Steven A, Ndongmo Chounna PW, Hoerauf A, Ford L, Sjoberg HT, Fombad FF, Koschel M, Archer J, Bloemker D, Njouendou AJ, Cook DAN, Pionnier N, Tyrer HE

[Sci Transl Med, 2019]

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against <i>Wolbachia</i> This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis (<i>Brugia malayi</i> and <i>Litomosoides sigmodontis</i>) and onchocerciasis (<i>Onchocerca ochengi</i>). A 1- or 2-week course of oral A-1535469 or A-1574083 provided &gt;90% <i>Wolbachia</i> depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations &gt;10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti-<i>Wolbachia</i> macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.

Ubuka T et al. (2018) Front Neurosci "Comparative and Evolutionary Aspects of Gonadotropin-Inhibitory Hormone and FMRFamide-Like Peptide Systems."

Ubuka T, Tsutsui K

[Front Neurosci, 2018]

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that was found in the brain of Japanese quail when investigating the existence of RFamide peptides in birds. GnIH was named because it decreased gonadotropin release from cultured anterior pituitary, which was located in the hypothalamo-hypophysial system. GnIH and GnIH precursor gene related peptides have a characteristic C-terminal LPXRFamide (X = L or Q) motif that is conserved in jawed vertebrates. Orthologous peptides to GnIH are also named RFamide related peptide or LPXRFamide peptide from their structure. A G-protein coupled receptor GPR147 is the primary receptor for GnIH. Similarity-based clustering of neuropeptide precursors in metazoan species indicates that GnIH precursor of vertebrates is evolutionarily related to FMRFamide precursor of mollusk and nematode. FMRFamide peptide is the first RFamide peptide that was identified from the ganglia of the venus clam. In order to infer the evolutionary history of the GnIH-GnIH receptor system we investigate the structural similarities between GnIH and its receptor and well-studied nematode <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) FMRFamide-like peptides (FLPs) and their receptors. We also compare the functions of FLPs of nematode with GnIH of chordates. A multiple sequence alignment and phylogenetic analyses of GnIH, neuropeptide FF (NPFF), a paralogous peptide of GnIH, and FLP precursors have shown that GnIH and NPFF precursors belong to different clades and some FLP precursors have structural similarities to either precursor. The peptide coding regions of FLP precursors in the same clade align well with those of GnIH or NPFF precursors. Alignment of GnIH (LPXRFa) peptides of chordates and FLPs of <i>C. elegans</i> grouped the peptides into five groups according to the last C-terminal amino acid sequences, which were MRFa, LRFa, VRFa, IRFa, and PQRFa. Phylogenetic analysis of receptors suggested that GPR147 has evolutionary relationships with FLP receptors, which regulate reproduction, aggression, locomotion, and feeding. GnIH and some FLPs mediate the effect of stress on reproduction and behavior, which may also be a conserved property of these peptide systems. Future studies are needed to investigate the mechanism of how neuropeptide precursor genes are mutated to evolve new neuropeptides and their inheritance.

Bakowski MA et al. (2019) Sci Transl Med "Discovery of short-course antiwolbachial quinazolines for elimination of filarial worm infections."

Yang B, Hull MV, Hubner MP, Chu XJ, Ehrens A, Gandjui NV, Metuge HM, Murphy E, Chounna PW, Archer J, Hoerauf A, Kuhen KL, Xiong W, Tremblay MS, McNamara CW, Akumtoh DN, Wanji S, Chappell L, Pionnier N, Sjoberg H, Frohberger SJ, Steven A, Liu R, Gagaring K, Petrassi HM, Bakowski MA, Guo H, Olejniczak J, Landmann F, Woods AK, Turner JD, Joseph SB, Kwenti TDB, Schultz PG, Ndzeshang BL, Lenz F, Fombad FF, Struever D, Dubben B, Roland J, Njouendou AJ, Chatterjee AK, Chunda VC, Shiroodi RK, Sullivan W, White PM, Taylor MJ, Debec A

[Sci Transl Med, 2019]

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets <i>Wolbachia</i>, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve &gt;99% elimination of <i>Wolbachia</i> in the in vivo <i>Litomosoides sigmodontis</i> filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify <i>Wolbachia</i> elimination in <i>Brugia pahangi</i> filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in <i>L. sigmodontis</i>, <i>Brugia malayi</i>, and <i>Onchocerca ochengi</i> in vivo preclinical models of filarial disease and in vitro selectivity against <i>Loa loa</i> (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.