An adaptable, fit-for-purpose screening approach with high-throughput capability to determine speed of action and stage specificity of anti-malarial compounds.

A revamped in vitro compound identification and activity profiling approach is required to meet the large unmet need for new anti-malarial drugs to combat parasite drug resistance. Although compound hit identification utilizing high-throughput screening of large compound libraries is well established, the ability to rapidly prioritize such large numbers for further development is limited. Determining the speed of action of anti-malarial drug candidates is a vital component of malaria drug discovery, which currently occurs predominantly in lead optimization and development. This is due in part to the capacity of current methods which have low throughput due to the complexity and labor intensity of the approaches. Here, we provide an adaptable screening paradigm utilizing automated high content imaging, including the development of an automated schizont maturation assay, which collectively can identify anti-malarial compounds, classify activity into fast and slow acting, and provide an indication of the parasite stage specificity, with high-throughput capability. By frontloading these critical biological parameters much earlier in the drug discovery pipeline, it has the potential to reduce lead compound attrition rates later in the development process. The capability of the approach in its alternative formats is demonstrated using three Medicines for Malaria Venture open access compound "boxes," namely Pathogen Box (malaria set-125 compounds), Global Health Priority Box [Malaria Box 2 (80 compounds) and zoonotic neglected diseases (80 compounds)], and the Pandemic Response Box (400 compounds). From a total of 685 compounds tested, 79 were identified as having fast ring-stage-specific activity comparable to that of artemisinin and therefore of high priority for further consideration and development.

Chemical and Antiplasmodial Investigations on Eremophila-Derived Alkaloids and Semisynthetic Ether Analogues.

Microthecaline A (1), the known antiplasmodial quinoline serrulatane alkaloid from the roots of Eremophila microtheca F. Muell. ex Benth. (Scrophulariaceae), was targeted for isolation and subsequent use in the generation of a semisynthetic ether library. A large-scale extraction and isolation yielded the previously undescribed quinoline serrulatane microthecaline B (2), along with crystalline 1 that enabled the first X-ray crystallographic analysis to be undertaken on this rare alkaloid structure class. The X-ray diffraction analysis of 1 supported the absolute configuration assignment of microthecaline A, which was originally assigned by ECD data analysis. Microthecaline A (1) was converted into 10 new semisynthetic ether derivatives (3-12) using a diverse series of commercially available alkyl halides. Chemical structures of the new serrulatane alkaloid and semisynthetic ether analogues were assigned by spectroscopic and spectrometric analyses. Antiplasmodial evaluations of 1-12 showed that the semisynthetic derivative 5 elicited the most potent activity with an IC50 value of 7.2 µM against Plasmodium falciparum 3D7 (drug-sensitive) strain.

Synthesis and Antimalarial Evaluation of Halogenated Analogues of Thiaplakortone A.

The incorporation of bromine, iodine or fluorine into the tricyclic core structure of thiaplakortone A (1), a potent antimalarial marine natural product, is reported. Although yields were low, it was possible to synthesise a small nine-membered library using the previously synthesised Boc-protected thiaplakortone A (2) as a scaffold for late-stage functionalisation. The new thiaplakortone A analogues (3-11) were generated using N-bromosuccinimide, N-iodosuccinimide or a Diversinate™ reagent. The chemical structures of all new analogues were fully characterised by 1D/2D NMR, UV, IR and MS data analyses. All compounds were evaluated for their antimalarial activity against Plasmodium falciparum 3D7 (drug-sensitive) and Dd2 (drug-resistant) strains. Incorporation of halogens at positions 2 and 7 of the thiaplakortone A scaffold was shown to reduce antimalarial activity compared to the natural product. Of the new compounds, the mono-brominated analogue (compound 5) displayed the best antimalarial activity with IC50 values of 0.559 and 0.058 µM against P. falciparum 3D7 and Dd2, respectively, with minimal toxicity against a human cell line (HEK293) observed at 80 µM. Of note, the majority of the halogenated compounds showed greater efficacy against the P. falciparum drug-resistant strain.

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Property activity refinement of 2-anilino 4-amino substituted quinazolines as antimalarials with fast acting asexual parasite activity.

Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.

Orthoscuticellines A-E, ß-Carboline Alkaloids from the Bryozoan Orthoscuticella ventricosa Collected in Australia.

Antiplasmodial high-throughput screening of extracts derived from marine invertebrates collected from northern NSW, Australia, resulted in the methanol extract of the bryozoan Orthoscuticella ventricosa being identified as inhibitory toward the 3D7 strain of Plasmodium falciparum. Purification of this extract resulted in two new bis-ß-carbolines that possess a cyclobutane moiety, orthoscuticellines A and B (1 and 2), three new ß-carboline alkaloids, orthoscuticellines C-E (3-5), and six known compounds, 1-ethyl-4-methylsulfone-ß-carboline (6), 1-ethyl-ß-carboline (7), 1-acetyl-ß-carboline (8) 1-(1'-hydroxyethyl)-ß-carboline (9), 1-methoxycarbonyl-ß-carboline (10), and 1-vinyl-ß-carboline (11). The structures of all compounds were determined from analysis of MS and 1D and 2D NMR data. The compounds showed modest antiplasmodial activity against P. falciparum in the range of 12-21 µM.

A Meroisoprenoid, Heptenolides, and C-Benzylated Flavonoids from Sphaerocoryne gracilis ssp. gracilis.

A new meroisoprenoid (1), two heptenolides (2 and 3), two C-benzylated flavonoids (4 and 5), and 11 known compounds (6-16) were isolated from leaf, stem bark, and root bark extracts of Sphaerocoryne gracilis ssp. gracilis by chromatographic separation. The structures of the new metabolites 1-5 were established by NMR, IR, and UV spectroscopic and mass spectrometric data analysis. (Z)-Sphaerodiol (7), (Z)-acetylmelodorinol (8), 7-hydroxy-6-hydromelodienone (10), and dichamanetin (15) inhibited the proliferation of Plasmodium falciparum (3D7, Dd2) with IC50 values of 1.4-10.5 µM, although these compounds also showed cytotoxicity against human embryonic kidney HEK-293 cells. None of the compounds exhibited significant disruption in protein translation when assayed in vitro.

Prenylated Flavonoids from the Roots of Tephrosia rhodesica.

Five new compounds-rhodimer (1), rhodiflavan A (2), rhodiflavan B (3), rhodiflavan C (4), and rhodacarpin (5)-along with 16 known secondary metabolites, were isolated from the CH2Cl2-CH3OH (1:1) extract of the roots of Tephrosia rhodesica. They were identified by NMR spectroscopic, mass spectrometric, X-ray crystallographic, and ECD spectroscopic analyses. The crude extract and the isolated compounds 2-5, 9, 15, and 21 showed activity (100% at 10 µg and IC50 = 5-15 µM) against the chloroquine-sensitive (3D7) strain of Plasmodium falciparum.

Secoiridoids and Iridoids from Morinda asteroscepa.

The new 2,3-secoiridoids morisecoiridoic acids A (1) and B (2), the new iridoid 8-acetoxyepishanzilactone (3), and four additional known iridoids (4-7) were isolated from the leaf and stem bark methanol extracts of Morinda asteroscepa using chromatographic methods. The structure of shanzilactone (4) was revised. The purified metabolites were identified using NMR spectroscopic and mass spectrometric techniques, with the absolute configuration of 1 having been established by single-crystal X-ray diffraction analysis. The crude leaf extract (10 µg/mL) and compounds 1-3 and 5 (10 µM) showed mild antiplasmodial activities against the chloroquine-sensitive malaria parasite Plasmodium falciparum (3D7).

Acrotrione: An Oxidized Xanthene from the Roots of Acronychia pubescens.

A new oxidized xanthene, acrotrione (1), and two known acetophenones (2 and 3) were isolated from a methanol extract of the roots of Acronychia pubescens. The structure of 1 was elucidated on the basis of its (+)-HRESIMS, 2D NMR, and ECD data. Acritrione (1) contains an unusual oxidized furo[2,3- c]xanthene moiety that has not been previously reported. Moderate antiplasmodial activity for these natural products against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum was determined with IC50 values ranging from 1.7 to 4.7 µM.

A New Benzopyranyl Cadenane Sesquiterpene and Other Antiplasmodial and Cytotoxic Metabolites from Cleistochlamys kirkii.

Phytochemical investigations of ethanol root bark and stem bark extracts of Cleistochlamys kirkii (Benth.) Oliv. (Annonaceae) yielded a new benzopyranyl cadinane-type sesquiterpene (cleistonol, 1) alongside 12 known compounds (2-13). The structures of the isolated compounds were established from NMR spectroscopic and mass spectrometric analyses. Structures of compounds 5 and 10 were further confirmed by single crystal X-ray crystallographic analyses, which also established their absolute stereochemical configuration. The ethanolic crude extract of C. kirkii root bark gave 72% inhibition against the chloroquine-sensitive 3D7-strain malaria parasite Plasmodium falciparum at 0.01 µg/mL. The isolated metabolites dichamanetin, (E)-acetylmelodorinol, and cleistenolide showed IC50 = 9.3, 7.6 and 15.2 µM, respectively, against P. falciparum 3D7. Both the crude extract and the isolated compounds exhibited cytotoxicity against the triple-negative, aggressive breast cancer cell line, MDA-MB-231, with IC50 = 42.0 µg/mL (crude extract) and 9.6-30.7 µM (isolated compounds). Our findings demonstrate the potential applicability of C. kirkii as a source of antimalarial and anticancer agents.

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

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.

Organometallic Conjugates of the Drug Sulfadoxine for Combatting Antimicrobial Resistance.

Fourteen novel arene RuII , and cyclopentadienyl (Cpx ) RhIII and IrIII complexes containing an N,N'-chelated pyridylimino- or quinolylimino ligand functionalized with the antimalarial drug sulfadoxine have been synthesized and characterized, including three by X-ray crystallography. The rhodium and iridium complexes exhibited potent antiplasmodial activity with IC50 values of 0.10-2.0 µm in either all, or one of the three Plasmodium falciparum assays (3D7 chloroquine sensitive, Dd2 chloroquine resistant and NF54 sexual late stage gametocytes) but were only moderately active towards Trichomonas vaginalis. They were active in both the asexual blood stage and the sexual late stage gametocyte assays, whereas the clinical parent drug, sulfadoxine, was inactive. Five complexes were moderately active against Mycobacterium tuberculosis (IC50 <6.3 µm), while sulfadoxine showed no antitubercular activity. An increase in the size of both the Cpx ligand and the aromatic imino substituent increased hydrophobicity, which resulted in an increase in antiplasmodial activity.

Microthecaline A, a Quinoline Serrulatane Alkaloid from the Roots of the Australian Desert Plant Eremophila microtheca.

Chemical investigation of the roots of the Australian desert plant Eremophila microtheca yielded microthecaline A (1), a novel quinoline-serrulatane natural product. The structure of 1 was determined by spectroscopic analysis, and the absolute configuration was assigned by ECD. Compound 1 exhibited moderate antimalarial activity against Plasmodium falciparum (3D7 strain), with an IC50 of 7.7 µM. Microthecaline A represents the first quinoline-serrulatane alkaloid to be isolated from Nature.

Antiplasmodial ß-Triketone-Flavanone Hybrids from the Flowers of the Australian Tree Corymbia torelliana.

The methanol extract of the flowers of the Australian eucalypt tree Corymbia torelliana yielded six new ß-triketone-flavanone hybrids, torellianones A-F (1-6), the tetrahydroxycyclohexane torellianol A (7), and known ß-triketones (4 S)-ficifolidione (8) and (4 R)-ficifolidione (9), and ß-triketone-flavanones kunzeanone A (10) and kunzeanone B (11). Torellianones A and B, C and D, and E and F were each isolated as inseparable diastereomeric mixtures. Exchange correlations observed in a ROESY spectrum indicated that 5 and 6 also interconverted between stable conformers. The structures of 1-7 were elucidated from the analysis of 1D/2D NMR and MS data. Relative configurations of torellianones C-F and torrellianol A were determined from analysis of ROESY data. Compounds 1-10 were tested for antiplasmodial activity against a drug-sensitive (3D7) strain of Plasmodium falciparum, with 3-6 and 8-10 showing limited antiplasmodial activity, with IC50 values ranging from 3.2 to 16.6 µM.

HSQC-TOCSY Fingerprinting for Prioritization of Polyketide- and Peptide-Producing Microbial Isolates.

Microbial products are a promising source for drug leads as a result of their unique structural diversity. However, reisolation of already known natural products significantly hampers the discovery process, and it is therefore important to incorporate effective microbial isolate selection and dereplication protocols early in microbial natural product studies. We have developed a systematic approach for prioritization of microbial isolates for natural product discovery based on heteronuclear single-quantum correlation-total correlation spectroscopy (HSQC-TOCSY) nuclear magnetic resonance profiles in combination with antiplasmodial activity of extracts. The HSQC-TOCSY experiments allowed for unfractionated microbial extracts containing polyketide and peptidic natural products to be rapidly identified. Here, we highlight how this approach was used to prioritize extracts derived from a library of 119 ascidian-associated actinomycetes that possess a higher potential to produce bioactive polyketides and peptides.

ß-Triketone-Monoterpene Hybrids from the Flowers of the Australian Tree Corymbia intermedia.

Four new ß-triketone monoterpene hybrids, intermediones A-D (1-4), have been identified from the flowers of the Australian eucalypt tree Corymbia intermedia. Intermediones A-D are ß-triketones that incorporate a pinene moiety attached via a benzyl group to a syncarpic acid. The structures of 1-4, including relative configurations, were elucidated from the analysis of 1D/2D NMR and MS data. The absolute configurations of intermediones A and B were determined by comparison of experimental and predicted ECD spectra. Intermedione D possesses a tetracyclic ring system that is related to that found in the meroterpenes, guadials B and C. Low to moderate antiplasmodial activity toward the chloroquine-sensitive (3D7) strain of Plasmodium falciparum, with IC50 values ranging from 9.9 to 20.8 µM, was observed for intermediones A, B, and D.

Screening the Medicines for Malaria Venture Pathogen Box across Multiple Pathogens Reclassifies Starting Points for Open-Source Drug Discovery.

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.

Synthesis of antimalarial amide analogues based on the plant serrulatane diterpenoid 3,7,8-trihydroxyserrulat-14-en-19-oic acid.

A plant-derived natural product scaffold, 3,7,8-trihydroxyserrulat-14-en-19-oic acid (1) was isolated in high yield from the aerial parts of the endemic Australian desert plant Eremophila microtheca. This scaffold (1) was subsequently used in the generation of a series of new amide analogues via a one-pot mixed anhydride amidation using pivaloyl chloride. The structures of all analogues were characterized using MS, NMR, and UV data. The major serrulatane natural products (1-3), isolated from the plant extract, and all amide analogues (6-15) together with several pivaloylated derivatives of 3,7,8-trihydroxyserrulat-14-en-19-oic acid (16-18) were evaluated for their antimalarial activity against 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant) Plasmodium falciparum strains, and preliminary cytotoxicity data were also acquired using the human embryonic kidney cell line HEK293. The natural product scaffold (1) did not display any antimalarial activity at 10µM. Replacing the carboxylic acid of 1 with various amides resulted in moderate activity against the P. falciparum 3D7 strain with IC50 values ranging from 1.25 to 5.65µM.

Antiplasmodial ß-triketones from the flowers of the Australian tree Angophora woodsiana.

Chemical investigations of the MeOH extract of air dried flowers of the Australian tree Angophora woodsiana (Myrtaceae) yielded two new ß-triketones, woodsianones A and B (1, 2) and nine known ß-triketones (3-11). Woodsianone A is a ß-triketone-sesquiterpene adduct and woodsianone B is a ß-triketone epoxide derivative. The structures of the new and known compounds were elucidated from the analysis of 1D/2D NMR and MS data. The relative configurations of the compounds were determined from analysis of 1H-1H coupling constants and ROESY correlations. All compounds (1-11) had antiplasmodial activity against the chloroquine sensitive strain 3D7. The known compound rhodomyrtone (5) and new compound woodsianone B (2) showed moderate antiplasmodial activities against the 3D7 strain (1.84µM and 3.00µM, respectively) and chloroquine resistant strain Dd2 (4.00µM and 2.53µM, respectively).