2,8-Disubstituted-1,5-naphthyridines as Dual Inhibitors of Plasmodium falciparum Phosphatidylinositol-4-kinase and Hemozoin Formation with In Vivo Efficacy.

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase ß (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.

Exploring the modulatory influence on the antimalarial activity of amodiaquine using scaffold hybridisation with ferrocene integration.

Amodiaquine (AQ) is a potent antimalarial drug used in combination with artesunate as part of artemisinin-based combination therapies (ACTs) for malarial treatment. Due to the rising emergence of resistant malaria parasites, some of which have been reported for ACT, the usefulness of AQ as an efficacious therapeutic drug is threatened. Employing the organometallic hybridisation approach, which has been shown to restore the antimalarial activity of chloroquine in the form of an organometallic hybrid clinical candidate ferroquine (FQ), the present study utilises this strategy to modulate the biological performance of AQ by incorporating ferrocene. Presently, we have conceptualised ferrocenyl AQ derivatives and have developed facile, practical routes for their synthesis. A tailored library of AQ derivatives was assembled and their antimalarial activity evaluated against chemosensitive (NF54) and multidrug-resistant (K1) strains of the malaria parasite, Plasmodium falciparum. The compounds generally showed enhanced or comparable activities to those of the reference clinical drugs chloroquine and AQ, against both strains, with higher selectivity for the sensitive phenotype, mostly in the double-digit nanomolar IC50 range. Moreover, representative compounds from this series show the potential to block malaria transmission by inhibiting the growth of stage II/III and V gametocytes in vitro. Preliminary mechanistic insights also revealed hemozoin inhibition as a potential mode of action.

Novel 3-Trifluoromethyl-1,2,4-oxadiazole Analogues of Astemizole with Multi-stage Antiplasmodium Activity and In Vivo Efficacy in a Plasmodium berghei Mouse Malaria Infection Model.

Iterative medicinal chemistry optimization of an ester-containing astemizole (AST) analogue 1 with an associated metabolic instability liability led to the identification of a highly potent 3-trifluoromethyl-1,2,4-oxadiazole analogue 23 (PfNF54 IC50 = 0.012 µM; PfK1 IC50 = 0.040 µM) displaying high microsomal metabolic stability (HLM CLint < 11.6 µL·min-1·mg-1) and > 1000-fold higher selectivity over hERG compared to AST. In addition to asexual blood stage activity, the compound also shows activity against liver and gametocyte life cycle stages and demonstrates in vivo efficacy in Plasmodium berghei-infected mice at 4 × 50 mg·kg-1 oral dose. Preliminary interrogation of the mode of action using live-cell microscopy and cellular heme speciation revealed that 23 could be affecting multiple processes in the parasitic digestive vacuole, with the possibility of a novel target at play in the organelles associated with it.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

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.

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.

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.

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.

Semi-Synthetic Analogues of Cryptolepine as a Potential Source of Sustainable Drugs for the Treatment of Malaria, Human African Trypanosomiasis, and Cancer.

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3-6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3-6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3-6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

Adsorption to the Surface of Hemozoin Crystals: Structure-Based Design and Synthesis of Amino-Phenoxazine ß-Hematin Inhibitors.

In silico adsorption of eight antimalarials that inhibit ß-hematin (synthetic hemozoin) formation identified a primary binding site on the (001) face, which accommodates inhibitors via formation of predominantly π-π interactions. A good correlation (r2 =0.64, P=0.017) between adsorption energies and the logarithm of ß-hematin inhibitory activity was found for this face. Of 53 monocyclic, bicyclic and tricyclic scaffolds, the latter yielded the most favorable adsorption energies. Five new amino-phenoxazine compounds were pursued as ß-hematin inhibitors based on adsorption behaviour. The 2-substituted phenoxazines show good to moderate ß-hematin inhibitory activity (<100 µM) and Plasmodium falciparum blood stage activity against the 3D7 strain. N1 ,N1 -diethyl-N4 -(10H-phenoxazin-2-yl)pentane-1,4-diamine (P2a) is the most promising hit with IC50 values of 4.7±0.6 and 0.64±0.05 µM, respectively. Adsorption energies are predictive of ß-hematin inhibitory activity, and thus the in silico approach is a beneficial tool for structure-based development of new non-quinoline inhibitors.

The ecdysone receptor regulates several key physiological factors in Anopheles funestus.

BACKGROUND: Malaria is a devastating disease, transmitted by female Anopheles mosquitoes infected with Plasmodium parasites. Current insecticide-based strategies exist to control the spread of malaria by targeting vectors. However, the increase in insecticide resistance in vector populations hinder the efficacy of these methods. It is, therefore, essential to develop novel vector control methods that efficiently target transmission reducing factors such as vector density and competence. A possible vector control candidate gene, the ecdysone receptor, regulates longevity, reproduction, immunity and other physiological processes in several insects, including malaria vectors. Anopheles funestus is a prominent vector in sub-Saharan Africa, however, the function of the ecdysone receptor in this mosquito has not previously been studied. This study aimed to determine if the ecdysone receptor depletion impacts An. funestus longevity, reproduction and susceptibility to Plasmodium falciparum infection. METHODS: RNA interference was used to reduce ecdysone receptor expression levels in An. funestus females and investigate how the above-mentioned phenotypes are influenced. Additionally, the expression levels of the ecdysone receptor, and reproduction genes lipophorin and vitellogenin receptor as well as the immune gene, leucine rich immune molecule 9 were determined in ecdysone receptor-depleted mosquitoes using quantitative polymerase chain reaction. RESULTS: Ecdysone receptor-depleted mosquitoes had a shorter lifespan, impaired oogenesis, were less fertile, and had reduced P. falciparum infection intensity. CONCLUSIONS: Overall, this study provides the first experimental evidence that supports ecdysone receptor as a potential target in the development of vector control measures targeting An. funestus.

A Dynamic and Combinatorial Histone Code Drives Malaria Parasite Asexual and Sexual Development.

Histone posttranslational modifications (PTMs) frequently co-occur on the same chromatin domains or even in the same molecule. It is now established that these "histone codes" are the result of cross talk between enzymes that catalyze multiple PTMs with univocal readout as compared with these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 77 PTMs on H3 and H3.3, of which 34 are newly identified in P. falciparum. Coexisting PTMs with unique stage distinctions were identified, indicating that many of these combinatorial PTMs are associated with specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2, which we tied to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with coexisting partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher-order gene regulation for distinct proliferation and differentiation processes.

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.

Chemogenomic Fingerprints Associated with Stage-Specific Gametocytocidal Compound Action against Human Malaria Parasites.

Kinase-focused inhibitors previously revealed compounds with differential activity against different stages of Plasmodium falciparum gametocytes. MMV666810, a 2-aminopyrazine, is more active on late-stage gametocytes, while a pyrazolopyridine, MMV674850, preferentially targets early-stage gametocytes. Here, we probe the biological mechanisms underpinning this differential stage-specific killing using in-depth transcriptome fingerprinting. Compound-specific chemogenomic profiles were observed with MMV674850 treatment associated with biological processes shared between asexual blood stage parasites and early-stage gametocytes but not late-stage gametocytes. MMV666810 has a distinct profile with clustered gene sets associated primarily with late-stage gametocyte development, including Ca2+-dependent protein kinases (CDPK1 and 5) and serine/threonine protein kinases (FIKK). Chemogenomic profiling therefore highlights essential processes in late-stage gametocytes, on a transcriptional level. This information is important to prioritize compounds that preferentially compromise late-stage gametocytes for further development as transmission-blocking antimalarials.

Structure-Activity Relationship Studies Reveal New Astemizole Analogues Active against Plasmodium falciparum In Vitro.

In the context of drug repositioning and expanding the existing structure-activity relationship around astemizole (AST), a new series of analogues were designed, synthesized, and evaluated for their antiplasmodium activity. Among 46 analogues tested, compounds 21, 30, and 33 displayed high activities against asexual blood stage parasites (PfNF54 IC50 = 0.025-0.043 µM), whereas amide compound 46 additionally showed activity against late-stage gametocytes (stage IV/V; PfLG IC50 = 0.6 ± 0.1 µM) and 860-fold higher selectivity over hERG (46, SI = 43) compared to AST. Several analogues displaying high solubility (Sol > 100 µM) and low cytoxicity in the Chinese hamster ovary (SI > 148) cell line have also been identified.

H3K36 methylation reprograms gene expression to drive early gametocyte development in Plasmodium falciparum.

BACKGROUND: The Plasmodium sexual gametocyte stages are the only transmissible form of the malaria parasite and are thus responsible for the continued transmission of the disease. Gametocytes undergo extensive functional and morphological changes from commitment to maturity, directed by an equally extensive control program. However, the processes that drive the differentiation and development of the gametocyte post-commitment, remain largely unexplored. A previous study reported enrichment of H3K36 di- and tri-methylated (H3K36me2&3) histones in early-stage gametocytes. Using chromatin immunoprecipitation followed by high-throughput sequencing, we identify a stage-specific association between these repressive histone modifications and transcriptional reprogramming that define a stage II gametocyte transition point. RESULTS: Here, we show that H3K36me2 and H3K36me3 from stage II gametocytes are associated with repression of genes involved in asexual proliferation and sexual commitment, indicating that H3K36me2&3-mediated repression of such genes is essential to the transition from early gametocyte differentiation to intermediate development. Importantly, we show that the gene encoding the transcription factor AP2-G as commitment master regulator is enriched with H3K36me2&3 and actively repressed in stage II gametocytes, providing the first evidence of ap2-g gene repression in post-commitment gametocytes. Lastly, we associate the enhanced potency of the pan-selective Jumonji inhibitor JIB-04 in gametocytes with the inhibition of histone demethylation including H3K36me2&3 and a disruption of normal transcriptional programs. CONCLUSIONS: Taken together, our results provide the first description of an association between global gene expression reprogramming and histone post-translational modifications during P. falciparum early sexual development. The stage II gametocyte-specific abundance of H3K36me2&3 manifests predominantly as an independent regulatory mechanism targeted towards genes that are repressed post-commitment. H3K36me2&3-associated repression of genes is therefore involved in key transcriptional shifts that accompany the transition from early gametocyte differentiation to intermediate development.

Antimalarial Benzimidazole Derivatives Incorporating Phenolic Mannich Base Side Chains Inhibit Microtubule and Hemozoin Formation: Structure-Activity Relationship and In Vivo Oral Efficacy Studies.

A novel series of antimalarial benzimidazole derivatives incorporating phenolic Mannich base side chains at the C2 position, which possess dual asexual blood and sexual stage activities, is presented. Structure-activity relationship studies revealed that the 1-benzylbenzimidazole analogues possessed submicromolar asexual blood and sexual stage activities in contrast to the 1H-benzimidazole analogues, which were only active against asexual blood stage (ABS) parasites. Further, the former demonstrated microtubule inhibitory activity in ABS parasites but more significantly in stage II/III gametocytes. In addition to being bona fide inhibitors of hemozoin formation, the 1H-benzimidazole analogues also showed inhibitory effects on microtubules. In vivo efficacy studies in Plasmodium berghei-infected mice revealed that the frontrunner compound 41 exhibited high efficacy (98% reduction in parasitemia) when dosed orally at 4 × 50 mg/kg. Generally, the compounds were noncytotoxic to mammalian cells.

Benzimidazole Derivatives Are Potent against Multiple Life Cycle Stages of Plasmodium falciparum Malaria Parasites.

The continued emergence of resistance to front-line antimalarial treatments is of great concern. Therefore, new compounds that potentially have a novel target in various developmental stages of Plasmodium parasites are needed to treat patients and halt the spread of malaria. Here, several benzimidazole derivatives were screened for activity against the symptom-causing intraerythrocytic asexual blood stages and the transmissible gametocyte stages of P. falciparum. Submicromolar activity was obtained for 54 compounds against asexual blood stage parasites with 6 potent at IC50 < 100 nM while not displaying any marked toxicity against mammalian cells. Nanomolar potency was also observed against gametocytes with two compounds active against early stage gametocytes and two compounds active against late-stage gametocytes. The transmission-blocking potential of the latter was confirmed as they could prevent male gamete exflagellation and the lead compound reduced transmission by 72% in an in vivo mosquito feeding model. These compounds therefore have activity against multiple stages of Plasmodium parasites with potential for differential targets.

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.