Publications by authors named "Brian M Suzuki"

25 Publications

  • Page 1 of 1

A multi-dimensional, time-lapse, high content screening platform applied to schistosomiasis drug discovery.

Commun Biol 2020 12 21;3(1):747. Epub 2020 Dec 21.

Center for Discovery and Innovation in Parasitic Diseases, Department of Pathology, University of California, San Francisco, CA, 94158, USA.

Approximately 10% of the world's population is at risk of schistosomiasis, a disease of poverty caused by the Schistosoma parasite. To facilitate drug discovery for this complex flatworm, we developed an automated high-content screen to quantify the multidimensional responses of Schistosoma mansoni post-infective larvae (somules) to chemical insult. We describe an integrated platform to process worms at scale, collect time-lapsed, bright-field images, segment highly variable and touching worms, and then store, visualize, and query dynamic phenotypes. To demonstrate the methodology, we treated somules with seven drugs that generated diverse responses and evaluated 45 static and kinetic response descriptors relative to concentration and time. For compound screening, we used the Mahalanobis distance to compare multidimensional phenotypic effects induced by 1323 approved drugs. Overall, we characterize both known anti-schistosomals and identify new bioactives. Apart from facilitating drug discovery, the multidimensional quantification provided by this platform will allow mapping of chemistry to phenotype.
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http://dx.doi.org/10.1038/s42003-020-01402-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752906PMC
December 2020

Tutuilamides A-C: Vinyl-Chloride-Containing Cyclodepsipeptides from Marine Cyanobacteria with Potent Elastase Inhibitory Properties.

ACS Chem Biol 2020 03 28;15(3):751-757. Epub 2020 Jan 28.

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093, United States.

Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A-C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A-C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency.
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http://dx.doi.org/10.1021/acschembio.9b00992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480958PMC
March 2020

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 1. Amide Linked Analogs.

ACS Infect Dis 2020 02 14;6(2):173-179. Epub 2020 Jan 14.

Anacor Pharmaceuticals, Inc. , 1020 E. Meadow Circle , Palo Alto , California 94303 , United States.

A series of benzimidazole-benzoxaborole hybrid molecules linked via an amide linker are described that exhibit good activity against , a filarial nematode responsible for the disease onchocerciasis, also known as river blindness. The lead identified in this series,  (AN8799), was found to have acceptable pharmacokinetic properties to enable evaluation in animal models of human filariasis. Compound was effective in killing , , and worms present in Mongolian gerbils when dosed subcutaneously as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 28 days. The measurement of plasma levels of at the end of the dosing period and at the time of sacrifice revealed an interesting dependence of activity on the extended exposure for both and the positive control, flubendazole.
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http://dx.doi.org/10.1021/acsinfecdis.9b00396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026885PMC
February 2020

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 2. Ketone Linked Analogs.

ACS Infect Dis 2020 02 28;6(2):180-185. Epub 2020 Jan 28.

Anacor Pharmaceuticals, Inc. , 1020 E. Meadow Circle , Palo Alto , California 94303 , United States.

The optimization of a series of benzimidazole-benzoxaborole hybrid molecules linked via a ketone that exhibit good activity against , a filarial nematode responsible for the disease onchocerciasis, also known as river blindness, is described. The lead identified in this series, (AN15470), was found to have acceptable pharmacokinetic properties to enable an evaluation following oral dosing in an animal model of onchocerciasis. Compound was effective in killing worms implanted in Mongolian gerbils when dosed orally as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 7 days.
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http://dx.doi.org/10.1021/acsinfecdis.9b00397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026882PMC
February 2020

Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery.

Parasit Vectors 2019 Oct 22;12(1):493. Epub 2019 Oct 22.

Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA.

Background: Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel.

Methods: Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis.

Results: For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline.

Conclusions: The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified .
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http://dx.doi.org/10.1186/s13071-019-3747-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805474PMC
October 2019

The Proteasome as a Drug Target in the Metazoan Pathogen, .

ACS Infect Dis 2019 10 12;5(10):1802-1812. Epub 2019 Aug 12.

Center for Discovery and Innovation in Parasitic Diseases , University of California, San Diego , 9500 Gilman Drive , La Jolla , California 92093 , United States.

Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult were incubated with 1 μM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (β1), trypsin-type (β2), and chymotrypsin-type (β5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the β5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S β2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 μM decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.
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http://dx.doi.org/10.1021/acsinfecdis.9b00237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283364PMC
October 2019

Bioactivity of Farnesyltransferase Inhibitors Against and .

Front Cell Infect Microbiol 2019 29;9:180. Epub 2019 May 29.

Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States.

The protozoan parasite can induce amebic colitis and amebic liver abscess. First-line drugs for the treatment of amebiasis are nitroimidazoles, particularly metronidazole. Metronidazole has side effects and potential drug resistance is a concern. Schistosomiasis, a chronic and painful infection, is caused by various species of the flatworm. There is only one partially effective drug, praziquantel, a worrisome situation should drug resistance emerge. As many essential metabolic pathways and enzymes are shared between eukaryotic organisms, it is possible to conceive of small molecule interventions that target more than one organism or target, particularly when chemical matter is already available. Farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway, is vital for diverse functions, including cell differentiation and growth. Both and genomes encode FT genes. In this study, we phenotypically screened and with the established FT inhibitors, lonafarnib and tipifarnib, and with 125 tipifarnib analogs previously screened against both the whole organism and/or the FT of and . For , we also explored whether synergy arises by combining lonafarnib and metronidazole or lonafarnib with statins that modulate protein prenylation. We demonstrate the anti-amebic and anti-schistosomal activities of lonafarnib and tipifarnib, and identify 17 tipifarnib analogs with more than 75% growth inhibition at 50 μM against . Apart from five analogs of tipifarnib exhibiting activity against both and , 10 additional analogs demonstrated anti-schistosomal activity (severe degenerative changes at 10 μM after 24 h). Analysis of the structure-activity relationship available for the FT suggests that FT may not be the relevant target in and . For , combination of metronidazole and lonafarnib resulted in synergism for growth inhibition. Also, of a number of statins tested, simvastatin exhibited moderate anti-amebic activity which, when combined with lonafarnib, resulted in slight synergism. Even in the absence of a definitive molecular target, identification of potent anti-parasitic tipifarnib analogs encourages further exploration while the synergistic combination of metronidazole and lonafarnib offers a promising treatment strategy for amebiasis.
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http://dx.doi.org/10.3389/fcimb.2019.00180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548881PMC
January 2020

TPT sulfonate, a single, oral dose schistosomicidal prodrug: In vivo efficacy, disposition and metabolic profiling.

Int J Parasitol Drugs Drug Resist 2018 12 20;8(3):571-586. Epub 2018 Nov 20.

Center for Discovery and Innovation in Parasitic Diseases, University of California, 1700 4th St, San Francisco, CA, 94158, USA; Department of Pathology, University of California, 1700 4th St, San Francisco, CA, 94158, USA. Electronic address:

Treatment of schistosomiasis relies precariously on just one drug, praziquantel (PZQ). In the search for alternatives, 15 S-[2-(alkylamino)alkane] thiosulfuric acids were obtained from a previous research program and profiled in mice for efficacy against both mature (>42-day-old) and juvenile (21-day-old) Schistosoma mansoni using a screening dose of 100 mg/kg PO QDx4. One compound, S-[2-(tert-butylamino)-1-phenylethane] thiosulfuric acid (TPT sulfonate), was the most effective by decreasing female and male worm burdens by ≥ 90% and ≥46% (mature), and ≥89% and ≥79% (juvenile), respectively. In contrast, PZQ decreased mature female and male worm burdens by 95% and 94%, respectively, but was ineffective against juvenile stages. Against 7-day-old lung-stage worms, TPT sulfonate was only effective at twice the dose decreasing female and male burdens by 95 and 80%, respectively. Single oral doses at 400 and/or 600 mg/kg across various developmental time-points (1-, 7-, 15-, 21- and/or 42 day-old) were consistent with the QD x4 data; efficacy was strongest once the parasites had completed lung migration, and female and male burdens were decreased by at least 90% and 80%, respectively. In vitro, TPT sulfonate is inactive against the parasite suggesting a pro-drug mechanism of action. In mice, TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism that is initiated by desulfation and yields a series of metabolites. The initially-formed free thiol-containing metabolite, termed TP thiol, was chemically synthesized; it dose-dependently decreased S. mansoni and Schistosoma haematobium motility in vitro. Also, when administered as a single 50 mg/kg IP dose, TP thiol decreased 33-day-old S. mansoni female and male burdens by 35% and 44%, with less severe organomegaly. Overall, TPT sulfonate's efficacy profile is competitive with that of PZQ. Also, the characterization of a parasiticidal metabolite facilitates an understanding and improvement of the chemistry, and identification of the mechanism of action and/or target.
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http://dx.doi.org/10.1016/j.ijpddr.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287543PMC
December 2018

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome.

Biol Chem 2018 11;399(12):1375-1388

Department of Molecular Microbiology and Immunology, Brown University, 171 Meeting Street, Providence, RI 02912, USA.

Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.
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http://dx.doi.org/10.1515/hsz-2018-0240DOI Listing
November 2018

Effect of Phenotypic Screening of Extracts and Fractions of Leaf and Stem Bark on Immature and Adult Stages of .

J Parasitol Res 2018 7;2018:9431467. Epub 2018 Jun 7.

Center for Discovery and Innovation in Parasitic Diseases (CDIPD), Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA.

Schistosomiasis is a disease caused by a flatworm parasite that infects people in tropical and subtropical regions of Sub-Saharan Africa, South America, China, and Southeast Asia. The reliance on just one drug for current treatment emphasizes the need for new chemotherapeutic strategies. The aim of this study was to determine the phenotypic effects of extracts and fractions of leaf and stem bark of (family Euphorbiaceae), a tree that grows in tropical parts of Africa, on two developmental stages of , namely, postinfective larvae (schistosomula or somules) and adults. Methanol leaf and stem bark extracts of were successively fractionated with acetone, petroleum ether, ethyl acetate, and methanol. These fractions were then incubated with somules at 0.3125 to 100 g/mL and with adults at 1.25 g/mL. The acetone fractions of both the methanol leaf and bark of were most active against the somules whereas the petroleum ether fractions showed least activity. For adult parasites, the acetone fraction of methanol bark extract also elicited phenotypic changes. The data arising provide the first step in the discovery of new treatments for an endemic infectious disease using locally sourced African medicinal plants.
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http://dx.doi.org/10.1155/2018/9431467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011076PMC
June 2018

Sertraline, Paroxetine, and Chlorpromazine Are Rapidly Acting Anthelmintic Drugs Capable of Clinical Repurposing.

Sci Rep 2018 01 17;8(1):975. Epub 2018 Jan 17.

Geriatrics Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, WA, 98108, USA.

Parasitic helminths infect over 1 billion people worldwide, while current treatments rely on a limited arsenal of drugs. To expedite drug discovery, we screened a small-molecule library of compounds with histories of use in human clinical trials for anthelmintic activity against the soil nematode Caenorhabditis elegans. From this screen, we found that the neuromodulatory drugs sertraline, paroxetine, and chlorpromazine kill C. elegans at multiple life stages including embryos, developing larvae and gravid adults. These drugs act rapidly to inhibit C. elegans feeding within minutes of exposure. Sertraline, paroxetine, and chlorpromazine also decrease motility of adult Trichuris muris whipworms, prevent hatching and development of Ancylostoma caninum hookworms and kill Schistosoma mansoni flatworms, three widely divergent parasitic helminth species. C. elegans mutants with resistance to known anthelmintic drugs such as ivermectin are equally or more susceptible to these three drugs, suggesting that they may act on novel targets to kill worms. Sertraline, paroxetine, and chlorpromazine have long histories of use clinically as antidepressant or antipsychotic medicines. They may represent new classes of anthelmintic drug that could be used in combination with existing front-line drugs to boost effectiveness of anti-parasite treatment as well as offset the development of parasite drug resistance.
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http://dx.doi.org/10.1038/s41598-017-18457-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772060PMC
January 2018

Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

PLoS Negl Trop Dis 2016 08 8;10(8):e0004893. Epub 2016 Aug 8.

Skaggs School of Pharmacy and Pharmaceutical Chemistry, University of California, San Diego, La Jolla, California, United States of America.

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.
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http://dx.doi.org/10.1371/journal.pntd.0004893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976874PMC
August 2016

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

PLoS Pathog 2016 07 28;12(7):e1005763. Epub 2016 Jul 28.

Department of Biochemistry and Molecular Biology and Huck Center for Malaria Research, Pennsylvania State University, University Park, Pennsylvania, United States of America.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.
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http://dx.doi.org/10.1371/journal.ppat.1005763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965013PMC
July 2016

Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo.

Pharmaceuticals (Basel) 2016 Jul 4;9(3). Epub 2016 Jul 4.

Center for Discovery and Innovation in Parasitic Diseases, Department of Pathology, University of California San Francisco, San Francisco, CA 94158, USA.

Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite's resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck's cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite's CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN's somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039492PMC
http://dx.doi.org/10.3390/ph9030039DOI Listing
July 2016

Evaluation of the CCA Immuno-Chromatographic Test to Diagnose Schistosoma mansoni in Minas Gerais State, Brazil.

PLoS Negl Trop Dis 2016 Jan 11;10(1):e0004357. Epub 2016 Jan 11.

Center for Discovery and Innovation in Parasitic Diseases and the Department of Pathology, University of California San Francisco, San Francisco, California, United States of America.

Background: The Kato-Katz (KK) stool smear is the standard test for the diagnosis of Schistosoma mansoni infection, but suffers from low sensitivity when infections intensities are moderate to low. Thus, misdiagnosed individuals remain untreated and contribute to the disease transmission, thereby forestalling public health efforts to move from a modality of disease control to one of elimination. As an alternative, the urine-based diagnosis of schistosomiasis mansoni via the circulating cathodic antigen immuno-chromatographic test (CCA-ICT) has been extensively evaluated in Africa with the conclusion that it may replace the KK test in areas where prevalences are moderate or high.

Methods And Findings: The objective was to measure the performance of the CCA-ICT in a sample study population composed of residents from non-endemic and endemic areas for schistosomiasis mansoni in two municipalities of Minas Gerais state, Brazil. Volunteers (130) were classified into three infection status groups based on duplicate Kato-Katz thick smears from one stool sample (2KK test): 41 negative individuals from non-endemic areas, 41 negative individuals from endemic areas and 48 infected individuals from endemic areas. Infection status was also determined by the CCA-ICT and infection exposure by antibody ELISA (enzyme-linked immunosorbent assay) to S. mansoni soluble egg antigen (SEA) and soluble (adult) worm antigen preparation (SWAP). Sensitivity and specificity were influenced by whether the trace score visually adjudicated in the CCA-ICT was characterized as positive or negative for S. mansoni infection. An analysis of a two-graph receiver operating characteristic was performed to change the cutoff point. When the trace score was interpreted as a positive rather than as a negative result, the specificity decreased from 97.6% to 78.0% whereas sensitivity increased from 68.7% to 85.4%. A significantly positive correlation between the CCA-ICT scores and egg counts was identified (r = 0.6252, p = 0.0001). However, the CCA-ICT misdiagnosed as negative 14.6% of 2KK positive individuals, predominantly those with light infections (fewer than 100 eggs/g feces). Considering 2KK as reference test, the discriminating power of the CCA-ICT (the area under the curve [AUC] = 0.817) was greater than the SEA-ELISA (AUC = 0.744) and SWAP-ELISA (AUC = 0.704).

Conclusion: Our data for the performance of the CCA-ICT in the Brazilian communities endemic for schistosomiasis mansoni support those from Africa, i.e., in areas with greater infection prevalence and intensities, the CCA-ICT may be useful as a tool to indicate community-based preventative chemotherapy without individual diagnosis. However, because of the Brazilian Ministry of Health's recommendation for individual diagnosis in areas where prevalence is less than 15%, i.e., those areas in which infection intensities are likely to be lowest, the CCA-ICT lacks the sensitivity to be used as standalone diagnostic tool.
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http://dx.doi.org/10.1371/journal.pntd.0004357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709075PMC
January 2016

Structure-Bioactivity Relationship for Benzimidazole Thiophene Inhibitors of Polo-Like Kinase 1 (PLK1), a Potential Drug Target in Schistosoma mansoni.

PLoS Negl Trop Dis 2016 Jan 11;10(1):e0004356. Epub 2016 Jan 11.

Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, San Francisco, California, United States of America.

Background: Schistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control. However, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and there is a concern of drug resistance. Thus, there is a need to identify new drugs and drug targets.

Methodology/principal Findings: We show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits a deleterious phenotypic alteration in post-infective larvae (schistosomula or somules). Phenotypic screening and analysis of schistosomula and adult S. mansoni with small molecule inhibitors of huPLK1 identified a number of potent anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor that has completed Phase I clinical trials for treatment of solid tumor malignancies. We then obtained GSKs Published Kinase Inhibitor Sets (PKIS) 1 and 2, and phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a distinctive phenotype distribution. Using principal component analysis (PCA), the phenotypes exhibited by these populations were mapped, visualized and analyzed through projection to a low-dimensional space. The phenotype distribution was found to have a distinct shape and topology, which could be elicited using cluster analysis. A structure-activity relationship (SAR) was identified for the benzimidazole thiophenes that held for both somules and adult parasites. The most potent inhibitors produced marked phenotypic alterations at 1-2 μM within 1 h. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays.

Conclusions/significance: The reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals.
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http://dx.doi.org/10.1371/journal.pntd.0004356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709140PMC
January 2016

Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles.

Biochimie 2016 Mar 26;122:99-109. Epub 2015 Sep 26.

Dept. of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USA. Electronic address:

Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates.
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http://dx.doi.org/10.1016/j.biochi.2015.09.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747843PMC
March 2016

Anti-Schistosomal Activity of Cinnamic Acid Esters: Eugenyl and Thymyl Cinnamate Induce Cytoplasmic Vacuoles and Death in Schistosomula of Schistosoma mansoni.

Molecules 2015 Jun 12;20(6):10873-83. Epub 2015 Jun 12.

Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany.

Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1-30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy.
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http://dx.doi.org/10.3390/molecules200610873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272620PMC
June 2015

Synthesis of a sugar-based thiosemicarbazone series and structure-activity relationship versus the parasite cysteine proteases rhodesain, cruzain, and Schistosoma mansoni cathepsin B1.

Antimicrob Agents Chemother 2015 May 23;59(5):2666-77. Epub 2015 Feb 23.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

The pressing need for better drugs against Chagas disease, African sleeping sickness, and schistosomiasis motivates the search for inhibitors of cruzain, rhodesain, and Schistosoma mansoni CB1 (SmCB1), the major cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni, respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was synthesized and evaluated against cruzain, rhodesain, and SmCB1 through biochemical assays to determine their potency and structure-activity relationships (SAR). This approach led to the discovery of 6 rhodesain, 4 cruzain, and 5 SmCB1 inhibitors with 50% inhibitory concentrations (IC50s) of ≤ 10 μM. Among the compounds tested, the thiosemicarbazone derivative of peracetylated galactoside (compound 4i) was discovered to be a potent rhodesain inhibitor (IC50 = 1.2 ± 1.0 μM). The impact of a range of modifications was determined; removal of thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds, and modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the parasites T. cruzi, T. brucei, and S. mansoni, revealing active compounds among this series.
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http://dx.doi.org/10.1128/AAC.04601-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394791PMC
May 2015

The QDREC web server: determining dose-response characteristics of complex macroparasites in phenotypic drug screens.

Bioinformatics 2015 May 24;31(9):1515-8. Epub 2014 Dec 24.

Department of Computer Science, San Francisco State University, San Francisco, CA, USA, Department of Pathology and Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, San Francisco, CA, USA Department of Computer Science, San Francisco State University, San Francisco, CA, USA, Department of Pathology and Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, San Francisco, CA, USA.

Summary: Neglected tropical diseases (NTDs) caused by helminths constitute some of the most common infections of the world's poorest people. The etiological agents are complex and recalcitrant to standard techniques of molecular biology. Drug screening against helminths has often been phenotypic and typically involves manual description of drug effect and efficacy. A key challenge is to develop automated, quantitative approaches to drug screening against helminth diseases. The quantal dose-response calculator (QDREC) constitutes a significant step in this direction. It can be used to automatically determine quantitative dose-response characteristics and half-maximal effective concentration (EC50) values using image-based readouts from phenotypic screens, thereby allowing rigorous comparisons of the efficacies of drug compounds. QDREC has been developed and validated in the context of drug screening for schistosomiasis, one of the most important NTDs. However, it is equally applicable to general phenotypic screening involving helminths and other complex parasites.

Availability And Implementation: QDREC is publically available at: http://haddock4.sfsu.edu/qdrec2/. Source code and datasets are at: http://tintin.sfsu.edu/projects/phenotypicAssays.html.

Contact: [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btu831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410654PMC
May 2015

Chemical and genetic validation of the statin drug target to treat the helminth disease, schistosomiasis.

PLoS One 2014 29;9(1):e87594. Epub 2014 Jan 29.

Center for Discovery and Innovation in Parasitic Diseases, Department of Pathology, University of California San Francisco, San Francisco, California, United States of America.

The mevalonate pathway is essential in eukaryotes and responsible for a diversity of fundamental synthetic activities. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the pathway and is targeted by the ubiquitous statin drugs to treat hypercholesterolemia. Independent reports have indicated the cidal effects of statins against the flatworm parasite, S. mansoni, and the possibility that SmHMGR is a useful drug target to develop new statin-based anti-schistosome therapies. For six commercially available statins, we demonstrate concentration- and time-dependent killing of immature (somule) and adult S. mansoni in vitro at sub-micromolar and micromolar concentrations, respectively. Cidal activity trends with statin lipophilicity whereby simvastatin and pravastatin are the most and least active, respectively. Worm death is preventable by excess mevalonate, the product of HMGR. Statin activity against somules was quantified both manually and automatically using a new, machine learning-based automated algorithm with congruent results. In addition, to chemical targeting, RNA interference (RNAi) of HMGR also kills somules in vitro and, again, lethality is blocked by excess mevalonate. Further, RNAi of HMGR of somules in vitro subsequently limits parasite survival in a mouse model of infection by up to 80%. Parasite death, either via statins or specific RNAi of HMGR, is associated with activation of apoptotic caspase activity. Together, our genetic and chemical data confirm that S. mansoni HMGR is an essential gene and the relevant target of statin drugs. We discuss our findings in context of a potential drug development program and the desired product profile for a new schistosomiasis drug.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087594PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906178PMC
October 2014

Quantification and clustering of phenotypic screening data using time-series analysis for chemotherapy of schistosomiasis.

BMC Genomics 2012 17;13 Suppl 1:S4. Epub 2012 Jan 17.

Department of Computer Science, San Francisco State University, San Francisco, CA 94132, USA.

Background: Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people. Development of techniques for automated, high-throughput drug screening against these diseases, especially in whole-organism settings, constitutes one of the great challenges of modern drug discovery.

Method: We present a method for enabling high-throughput phenotypic drug screening against diseases caused by helminths with a focus on schistosomiasis. The proposed method allows for a quantitative analysis of the systemic impact of a drug molecule on the pathogen as exhibited by the complex continuum of its phenotypic responses. This method consists of two key parts: first, biological image analysis is employed to automatically monitor and quantify shape-, appearance-, and motion-based phenotypes of the parasites. Next, we represent these phenotypes as time-series and show how to compare, cluster, and quantitatively reason about them using techniques of time-series analysis.

Results: We present results on a number of algorithmic issues pertinent to the time-series representation of phenotypes. These include results on appropriate representation of phenotypic time-series, analysis of different time-series similarity measures for comparing phenotypic responses over time, and techniques for clustering such responses by similarity. Finally, we show how these algorithmic techniques can be used for quantifying the complex continuum of phenotypic responses of parasites. An important corollary is the ability of our method to recognize and rigorously group parasites based on the variability of their phenotypic response to different drugs.

Conclusions: The methods and results presented in this paper enable automatic and quantitative scoring of high-throughput phenotypic screens focused on helmintic diseases. Furthermore, these methods allow us to analyze and stratify parasites based on their phenotypic response to drugs. Together, these advancements represent a significant breakthrough for the process of drug discovery against schistosomiasis in particular and can be extended to other helmintic diseases which together afflict a large part of humankind.
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http://dx.doi.org/10.1186/1471-2164-13-S1-S4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471343PMC
June 2012

Dimerizer regulation of AADC expression and behavioral response in AAV-transduced 6-OHDA lesioned rats.

Mol Ther 2006 Jan 26;13(1):167-74. Epub 2005 Aug 26.

Avigen, Inc., 1301 Harbor Bay Parkway, Alameda, CA 94502, USA.

Recombinant AAV vectors containing a dimerizer-inducible system of transcriptional activation provide a strategy for control of therapeutic gene expression in the CNS. Here we explored this system for regulated expression of human aromatic L-amino acid decarboxylase (hAADC) in a rodent model of Parkinson disease. Expression of hAADC, the enzyme that converts L-dopa to dopamine, was dependent on reconstitution of a functional transcription factor (TF) by the dimerizer rapamycin. Two vectors, AAV-CMV-TF and AAV-Z12-hAADC, were infused into striata of 6-OHDA-lesioned rats. Rapamycin-induced increases in expression of hAADC repeatedly produced robust rotational behavior in response to low doses of L-dopa. Seven weeks after vector infusion, AADC expression in brain was quantitated by both stereology and Western blot analysis following the final rapamycin treatment. While a low level of hAADC was observed in rats that were not induced with rapamycin, this basal expression was not significant enough to elicit a rotational response to L-dopa. This study demonstrated a robust behavioral response of parkinsonian rats to regulated hAADC expression. Recombinant AAV vectors controlled by rapamycin or its analogs show promise as candidates for CNS therapies in which regulation of the transgene is desired.
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http://dx.doi.org/10.1016/j.ymthe.2005.06.480DOI Listing
January 2006

AAV2-mediated gene delivery to monkey putamen: evaluation of an infusion device and delivery parameters.

Exp Neurol 2005 Aug;194(2):476-83

Avigen Inc., 1301 Harbor Bay Parkway, Alameda, CA 94502, USA.

In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic l-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinson's disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 muL/min to 1 muL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 muL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated.
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http://dx.doi.org/10.1016/j.expneurol.2005.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816113PMC
August 2005

Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV.

Mol Ther 2004 Mar;9(3):403-9

Avigen, Inc., Alameda, CA 94502, USA.

We tested the hypotheses that initial immunization of rats with rAAV might limit subsequent transduction by rAAV-hAADC when stereotaxically infused into the striatum and that the level of inhibition would correlate with AAV neutralizing antibody titers. Immunohistochemical detection of AADC and analysis by stereology revealed that the control group (no immunization) had the greatest volume of distribution of AADC (20.32 +/- 2.03 mm3) (+/-SD). There was a 58% decrease in spread (8.46 +/- 3.67 mm3, P < 0.008) in the high-dose immunization group (5 x 10(10) vg rAAV-null). Transduction weakly correlated with preexisting titer levels of neutralizing antibody at the time of intrastriatal rAAV-hAADC infusion. Only rats with neutralizing antibody titers of 1:1208 +/- 332 had significantly decreased AADC transgene expression compared to the unimmunized control group. Immunohistochemistry on serial sections for inflammatory markers including GFAP, CD11b, CD4, and CD8a revealed normal morphology and no cellular infiltration, suggesting little immune reaction in the CNS. We conclude that rAAV vectors can transduce brain tissue in the context of preexisting immunity, but that efficiency of transduction declines significantly in the presence of very high titers of neutralizing antibodies. These results have important implications for gene therapy for CNS disorders.
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http://dx.doi.org/10.1016/j.ymthe.2003.12.005DOI Listing
March 2004
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