Melissa Sykes

Identification of Compounds with Anti-Proliferative Activity against Trypanosoma brucei brucei Strain 427 by a Whole Cell Viability Based HTS Campaign

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC50 value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC50 values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.

Antitrypanosomal Cyclic Polyketide Peroxides from the Australian Marine Sponge Plakortis sp.

Bioassay-guided fractionation of the crude extract from the Australian marine sponge Plakortis sp. led to the isolation of two new cyclic polyketide peroxides, 11,12-didehydro-13-oxo-plakortide Q (1) and 10-carboxy-11,12,13,14-tetranor-plakortide Q (2). Antitrypanosomal studies showed that compound 1 had an IC(50) value of 49 nM against Trypanosoma brucei brucei, and compound 2, where a carboxylic acid is present in the side chain, had a 20-fold reduction of activity. 11,12-Didehydro-13-oxo-plakortide Q (1) is the most active peroxide isolated so far against T. b. brucei, and it indicates the potential therapeutic value of this class of compounds.

Antiparasitic activity of alkaloids from plant species of Papua New Guinea and Australia.

New drugs are needed to help overcome the increasing problem of drug resistance in parasites that cause diseases such as malaria and trypanosomiasis. In this study, alkaloid compounds isolated from extracts of the plants Flindersia amboinensis, Stephania zippeliana and Voacanga papuana from Papua New Guinea and Flindersia acuminata from Australia were examined for their antiparasitic activity against Plasmodium falciparum strains and Trypanosoma brucei brucei as well as their cytotoxicity against the mammalian cell lines HEK 293 and HeLa. The most active compound, dimethylisoborreverine (DMIB), showed submicromolar activity, with 50% inhibitory concentration (IC(50)) values between 20 nM and 810 nM both against drug-sensitive and drug-resistant P. falciparum strains, along with moderate selectivity against T. b. brucei and mammalian cells. Stage specificity studies revealed that P. falciparum trophozoite-stage parasites were more susceptible to DMIB than ring- or schizont-stage parasites. DMIB-treated trophozoites showed changes in food vacuole morphology, with an apparent reduction in haemozoin formation that does not appear to be inhibited via the direct binding of haem. These findings suggest a potential for indole alkaloids from Flindersia spp. as new antiparasitic agents.

Pseudoceramines A–D, new antibacterial bromotyrosine alkaloids from the marine sponge Pseudoceratina sp.

Bioassay-guided fractionation of the CH(2)Cl(2)/MeOH extract of the Australian marine sponge Pseudoceratina sp. resulted in the purification of four new bromotyrosine alkaloids, pseudoceramines A-D (1-4), along with a known natural product, spermatinamine (5). The structures of 1-5 were determined by spectroscopic methods. Pseudoceramines A (1) and B (2) feature a rare bromotyrosyl-spermine-bromotyrosyl sequence, and pseudoceramine C (3) is the first example of bromotyrosine coupled with an N-methyl derivative of spermidine. Compounds 1-5 were screened for inhibition of toxin secretion by the type III secretion (T3S) pathway in Yersinia pseudotuberculosis. Compounds 2 and 5 inhibited secretion of the Yersinia outer protein YopE (IC(50) = 19 and 6 μM, respectively) and the enzyme activity of YopH (IC(50) = 33 and 6 μM, respectively).

Ianthelliformisamines A–C, Antibacterial Bromotyrosine-Derived Metabolites from the Marine Sponge Suberea ianthelliformis

A high-throughput screening campaign using a prefractionated natural product library and an in vitro Pseudomonas aeruginosa (PAO200 strain) assay identified two antibacterial fractions derived from the marine sponge Suberea ianthelliformis. Mass-directed isolation of the CH(2)Cl(2)/CH(3)OH extract from S. ianthelliformis resulted in the purification of three new bromotyrosine-derived metabolites, ianthelliformisamines A-C (1-3), together with the known natural products aplysamine 1 (4) and araplysillin I (5). The structures of 1-3 were determined following analysis of 1D and 2D NMR and MS spectroscopic data. This is the first report of chemistry from the marine sponge S. ianthelliformis. Ianthelliformisamine A (1) showed inhibitory activity against the Gram-negative bacterium P. aeruginosa with an IC(50) value of 6.8 μM (MIC = 35 μM).

Convolutamines I and J, antitrypanosomal alkaloids from the bryozoan Amathia tortusa

Mass-directed isolation of the CH(2)Cl(2)/CH(3)OH extract from the marine bryozoan Amathia tortusa resulted in the purification of two new brominated alkaloids, convolutamines I (1) and J (2). The structures of 1 and 2 were determined following spectroscopic data analysis. Both compounds were isolated during a drug discovery program aimed at identifying new antitrypanosomal leads from a prefractionated natural product library. Compounds 1 and 2 were shown to be active toward the parasite Trypanosoma brucei brucei with IC(50) values of 1.1 and 13.7 μM, respectively. Preliminary toxicity profiling was also performed on both 1 and 2 using the human embryonic kidney cell line, HEK293. Compound 1 was shown to exhibit cytotoxicity against HEK293 with an IC(50) of 22.0 μM whilst 2 was inactive at 41.0 μM.

3-(Oxazolo[4,5-b]pyridin-2-yl)anilides as a novel class of potent inhibitors for the kinetoplastid Trypanosoma brucei, the causative agent for human African trypanosomiasis

A whole organism high-throughput screen of approximately 87,000 compounds against Trypanosoma brucei brucei led to the recent discovery of several novel compound classes with low micromolar activity against this organism and without appreciable cytotoxicity to mammalian cells. Herein we report a structure-activity relationship (SAR) investigation around one of these hit classes, the 3-(oxazolo[4,5-b]pyridin-2-yl)anilides. Sharp SAR is revealed, with our most active compound (5) exhibiting an IC₅₀ of 91 nM against the human pathogenic strain T.b. rhodesiense and being more than 700 times less toxic towards the L6 mammalian cell line. Physicochemical properties are attractive for many compounds in this series. For the most potent representatives, we show that solubility and metabolic stability are key parameters to target during future optimisation.

(-)-Dibromophakellin : An α2B adrenoceptor agonist isolated from the Australian marine sponge, Acanthella costata

Bioassay-guided fractionation of the organic extract from the marine sponge Acanthella costata resulted in the isolation of the known natural product, (-)-dibromophakellin (1). Using a fluorescence imaging plate reader (FLIPR) based assay, compound 1 was identified as displaying agonist activity against the alpha(2B) adrenoceptor, with an EC(50) of 4.2muM. Debromination and Suzuki-Miyaura coupling reactions were undertaken in order to provide structure activity data about the pyrrole ring of this marine metabolite. These synthetic studies generated the known natural product analogues, (-)-phakellin (2), and (-)-monobromophakellin (3), along with the new synthetic derivatives (-)-4-bromo-5-phenylphakellin (5) and (-)-4,5-diphenylphakellin (6). Substitution of the C-5 Br of 1 with H (2 and 3) or phenyl (5 and 6) resulted in loss of activity indicating that Br at C-5 is required for agonist activity.

Iotrochamides A and B, antitrypanosomal compounds from the Australian marine sponge Iotrochota sp.

Bioassay-guided isolation of the CH(2)Cl(2)/MeOH extract from the Australian sponge Iotrochota sp. resulted in the purification of two new N-cinnamoyl-amino acids, iotrochamides A (1) and B (2). The chemical structures of 1 and 2 were determined by 1D/2D NMR and MS data analyses. Compounds 1 and 2 were shown to inhibit Trypanosoma brucei brucei with IC(50) values of 3.4 and 4.7 μM, respectively.

Screening the medicines for malaria venture pathogen box across multiple pathogens reclassifies starting points for open-source drug discovery

ABSTRACT Open-access drug discovery provides a substantial resource for diseases primarily affecting the poor and disadvantaged. The open-access Pathogen Box collection is comprised of compounds with demonstrated biological activity against specific pathogenic organisms. The supply of this resource by the Medicines for Malaria Venture has the potential to provide new chemical starting points for a number of tropical and neglected diseases, through repurposing of these compounds for use in drug discovery campaigns for these additional pathogens. We tested the Pathogen Box against kinetoplastid parasites and malaria life cycle stages in vitro. Consequently, chemical starting points for malaria, human African trypanosomiasis, Chagas disease, and leishmaniasis drug discovery efforts have been identified. Inclusive of this in vitro biological evaluation, outcomes from extensive literature reviews and database searches are provided. This information encompasses commercial availability, literature reference citations, other aliases and ChEMBL number with associated biological activity, where available. The release of this new data for the Pathogen Box collection into the public domain will aid the open-source model of drug discovery. Importantly, this will provide novel chemical starting points for drug discovery and target identification in tropical disease research.