New Transmission-Selective Antimalarial Agents through Hit-to-Lead Optimization of 2-([1,1'-Biphenyl]-4-carboxamido)benzoic Acid Derivatives.

Malaria elimination requires multipronged approaches, including the application of antimalarial drugs able to block human-to-mosquito transmission of malaria parasites. The transmissible gametocytes of Plasmodium falciparum seem to be highly sensitive towards epidrugs, particularly those targeting demethylation of histone post-translational marks. Here, we report exploration of compounds from a chemical library generated during hit-to-lead optimization of inhibitors of the human histone lysine demethylase, KDM4B. Derivatives of 2-([1,1'-biphenyl]-4-carboxamido) benzoic acid, around either the amide or a sulfonamide linker backbone (2-(arylcarboxamido)benzoic acid, 2-carboxamide (arylsulfonamido)benzoic acid and N-(2-(1H-tetrazol-5-yl)phenyl)-arylcarboxamide), showed potent activity towards late-stage gametocytes (stage IV/V) of P. falciparum, with the most potent compound reaching single digit nanomolar activity. Structure-activity relationship trends were evident and frontrunner compounds also displayed microsomal stability and favourable solubility profiles. Simplified synthetic routes support further derivatization of these compounds for further development of these series as malaria transmission-blocking agents.

UCT943, a Next-Generation Plasmodium falciparum PI4K Inhibitor Preclinical Candidate for the Treatment of Malaria.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

Potent Plasmodium falciparum gametocytocidal compounds identified by exploring the kinase inhibitor chemical space for dual active antimalarials.

Objectives: Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. Methods: We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. Results: We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at <100 nM (8 were IMPs and 4 were 2-APs) and were also active against early stage gametocytes and asexual parasites, with >1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. Conclusions: This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.

Streamlined and Robust Stage-Specific Profiling of Gametocytocidal Compounds Against Plasmodium falciparum.

Malaria elimination is dependent on the ability to target both the pathogenic and transmissible stages of the human malaria parasite, Plasmodium falciparum. These forms of the parasite are differentiated by unique developmental stages, each with their own biological mechanisms and processes. These individual stages therefore also respond differently to inhibitory compounds, and this complicates the discovery of multistage active antimalarial agents. The search for compounds with transmission-blocking activity has focused on screening for activity on mature gametocytes, with only limited descriptions available for the activity of such compounds on immature stage gametocytes. This therefore poses a gap in the profiling of antimalarial agents for pan-reactive, multistage activity to antimalarial leads. Here, we optimized an effective and robust strategy for the simple and cost-effective description of the stage-specific action of gametocytocidal antimalarial compounds.

Adapt or Die: Targeting Unique Transmission-Stage Biology for Malaria Elimination.

Plasmodium parasites have a complex life cycle that includes development in the human host as well as the Anopheles vector. Successful transmission of the parasite between its host and vector therefore requires the parasite to balance its investments in asexual replication and sexual reproduction, varying the frequency of sexual commitment to persist within the human host and generate future opportunities for transmission. The transmission window is extended further by the ability of stage V gametocytes to circulate in peripheral blood for weeks, whereas immature stage I to IV gametocytes sequester in the bone marrow and spleen until final maturation. Due to the low gametocyte numbers in blood circulation and with the ease of targeting such life cycle bottlenecks, transmission represents an efficient target for therapeutic intervention. The biological process of Plasmodium transmission is a multistage, multifaceted process and the past decade has seen a much deeper understanding of the molecular mechanisms and regulators involved. Clearly, specific and divergent processes are used during transmission compared to asexual proliferation, which both poses challenges but also opportunities for discovery of transmission-blocking antimalarials. This review therefore presents an update of our molecular understanding of gametocyte and gamete biology as well as the status of transmission-blocking activities of current antimalarials and lead development compounds. By defining the biological components associated with transmission, considerations for the development of new transmission-blocking drugs to target such untapped but unique biology is suggested as an important, main driver for transmission-blocking drug discovery.

In vitro dual activity of Aloe marlothii roots and its chemical constituents against Plasmodium falciparum asexual and sexual stage parasites.

ETHNOPHARMACOLOGICAL RELEVANCE: Aloe marlothii A.Berger (Xanthorrhoeaceae) is indigenous to southern African countries where its aqueous preparations are used in traditional medicine to treat several ailments including hypertension, respiratory infections, venereal diseases, chest pain, sore throat and malaria. AIM OF THE STUDY: The aims of this study were as follows: (i) isolate and identify the antiplasmodial active compounds in A. marlothii roots. As the water extract was previously inactive, the dichloromethane:methanol (DCM:MeOH) (1:1) was used, (ii) examine the activity of the isolated compounds against Plasmodium falciparum asexual blood stage (ABS) parasites as well as for transmission-blocking activity against gametocytes and gametes, and (iii) to use in silico tools to predict the target(s) of the active molecules. MATERIALS AND METHODS: The crude DCM:MeOH (1:1) extract of A. marlothii roots was fractionated on a reverse phase C8 column, using a positive pressure solid-phase extraction (ppSPE) workstation to produce seven fractions. The resulting fractions and the crude DCM:MeOH extract were tested in vitro against P. falciparum (NF54) ABS parasites using the malaria SYBR Green I based-fluorescence assay. Flash silica chromatography and mass-directed preparative high-performance liquid chromatography were utilised to isolate the active compounds. The isolated compounds were evaluated in vitro against P. falciparum asexual (NF54 and K1 strains) and sexual (gametocytes and gametes) stage parasites. Molecular docking was then used for the in silico prediction of targets for the isolated active compounds in P. falciparum. RESULTS: The crude extract and two SPE fractions displayed good antiplasmodial activity with >97% and 100% inhibition of ABS parasites proliferation at 10 and 20 µg/mL, respectively. Following UPLC-MS analysis of these active fractions, a targeted purification resulted in the isolation of six compounds identified as aloesaponol I (1), aloesaponarin I (2), aloesaponol IV (3), ß-sorigenin-1-O-methylether (4), emodin (5), and chrysophanol (6). Aloesaponarin I (2) was the most bioactive, compared to other isolated constituents, against P. falciparum ABS parasites exhibiting equipotency against the drug-sensitive (NF54) (IC50 = 1.54 µg/mL (5 µM)) and multidrug-resistant (K1) (IC50 = 1.58 µg/mL (5 µM)) strains. Aloesaponol IV (3) showed pronounced activity against late-stage (>90% stage IV/V) gametocytes (IC50 = 6.53 µg/mL (22.6 µM)) demonstrating a 3-fold selective potency towards these sexual stages compared to asexual forms of the parasite (IC50 = 19.77 ± 6.835 µg/mL (68 µM)). Transmission-blocking potential of aloesaponol IV (3) was validated by in vitro inhibition of exflagellation of male gametes (94% inhibition at 20 µg/mL). In silico studies identified ß-hematin and DNA topoisomerase II as potential biological targets of compounds 2 and 3, respectively. CONCLUSION: The findings from our study substantiate the traditional use of A. marlothii to treat malaria. To our knowledge, this study has provided the first report on the isolation and identification of antiplasmodial compounds from A. marlothii roots. Furthermore, our study has provided the first report on the transmission-blocking potential of one of the compounds from the genus Aloe, motivating for the investigation of other species within this genus for their potential P. falciparum transmission-blocking activity.

The Artemiside-Artemisox-Artemisone-M1 Tetrad: Efficacies against Blood Stage P. falciparum Parasites, DMPK Properties, and the Case for Artemiside.

Because of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf) blood stage parasites (IC50 1.5-2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Naphthylisoquinoline alkaloids, validated as hit multistage antiplasmodial natural products.

The discovery and development of multistage antimalarial drugs targeting intra-erythrocytic asexual and sexual Plasmodium falciparum parasites is of utmost importance to achieve the ambitious goal of malaria elimination. Here, we report the validation of naphthylisoquinoline (NIQ) alkaloids and their synthetic analogues as multistage active antimalarial drug candidates. A total of 30 compounds were tested, of which 17 exhibited IC50 values <1 µM against drug-sensitive P. falciparum parasites (NF54 strain); 15 of these retained activity against a panel of drug-resistant strains. These compounds showed low in vitro cytotoxicity against HepG2 cells, with selectivity indices of >10. The tested compounds showed activity in vitro against both early- and late-stage P. falciparum gametocytes while blocking male gamete formation (>70% inhibition of exflagellation at 2 µM). Additionally, five selected compounds were found to have good solubility (≥170 µM in PBS at pH 6.5), while metabolic stability towards human, mouse, and rat microsomes ranged from >90% to >7% after 30 min. Dioncophylline C (2a) emerged as a front runner from the study, displaying activity against both asexual parasites and gametocytes, a lack of cross-resistance to chloroquine, good solubility, and microsomal stability. Overall, this is the first report on the multistage activity of NIQs and their synthetic analogues including gametocytocidal and gametocidal effects induced by this class of compounds.

Optimal 10-Aminoartemisinins With Potent Transmission-Blocking Capabilities for New Artemisinin Combination Therapies-Activities Against Blood Stage P. falciparum Including PfKI3 C580Y Mutants and Liver Stage P. berghei Parasites.

We have demonstrated previously that amino-artemisinins including artemiside and artemisone in which an amino group replaces the oxygen-bearing substituents attached to C-10 of the current clinical artemisinin derivatives dihydroartemisinin (DHA), artemether and artesunate, display potent activities in vitro against the asexual blood stages of Plasmodium falciparum (Pf). In particular, the compounds are active against late blood stage Pf gametocytes, and are strongly synergistic in combination with the redox active drug methylene blue. In order to fortify the eventual selection of optimum amino-artemisinins for development into new triple combination therapies also active against artemisinin-resistant Pf mutants, we have prepared new amino-artemisinins based on the easily accessible and inexpensive DHA-piperazine. The latter was converted into alkyl- and aryl sulfonamides, ureas and amides. These derivatives were screened together with the comparator drugs DHA and the hitherto most active amino-artemisinins artemiside and artemisone against asexual and sexual blood stages of Pf and liver stage P. berghei (Pb) sporozoites. Several of the new amino-artemisinins bearing aryl-urea and -amide groups are potently active against both asexual, and late blood stage gametocytes (IC50 0.4-1.0 nM). Although the activities are superior to those of artemiside (IC50 1.5 nM) and artemisone (IC50 42.4 nM), the latter are more active against the liver stage Pb sporozoites (IC50 artemisone 28 nM). In addition, early results indicate these compounds tend not to display reduced susceptibility against parasites bearing the Pf Kelch 13 propeller domain C580Y mutation characteristic of artemisinin-resistant Pf. Thus, the advent of the amino-artemisinins including artemiside and artemisone will enable the development of new combination therapies that by virtue of the amino-artemisinin component itself will possess intrinsic transmission-blocking capabilities and may be effective against artemisinin resistant falciparum malaria.

Activities of 11-Azaartemisinin and N-Sulfonyl Derivatives against Asexual and Transmissible Malaria Parasites.

Dihydroartemisinin (DHA), either used in its own right or as the active drug generated in vivo from the other artemisinins in current clinical use-artemether and artesunate-induces quiescence in ring-stage parasites of Plasmodium falciparum (Pf). This induction of quiescence is linked to artemisinin resistance. Thus, we have turned to structurally disparate artemisinins that are incapable of providing DHA on metabolism. Accordingly, 11-azaartemisinin 5 and selected N-sulfonyl derivatives were screened against intraerythrocytic asexual stages of drug-sensitive Pf NF54 and drug-resistant K1 and W2 parasites. Most displayed appreciable activities against all three strains, with IC50 values <10.5 nm. The p-trifluoromethylbenzenesulfonyl-11-azaartemisinin derivative 11 [(4'-trifluoromethyl)benzenesulfonylazaartemisinin] was the most active, with IC50 values between 2 and 3 nm. The compounds were screened against Pf NF54 early and transmissible late intraerythrocytic-stage gametocytes using luciferase and parasite lactate dehydrogenase (pLDH) assays. The 2'-thienylsulfonyl derivative 16 (2'-thiophenesulfonylazaartemisinin) was notably active against late-stage (IV-V) gametocytes with an IC50 value of 8.7 nm. All compounds were relatively nontoxic to human fetal lung WI-38 fibroblasts, showing selectivity indices of >2000 toward asexual parasites. Overall, the readily accessible 11-azaartemisinin 5 and the sulfonyl derivatives 11 and 16 represent potential candidates for further development, in particular for transmission blocking of artemisinin-resistant parasites.