Cripowellins Pause Plasmodium falciparum Intraerythrocytic Development at the Ring Stage.

Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B "pauses" the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.

Aptamer-Based Imaging of Polyisoprenoids in the Malaria Parasite.

Dolichols are isoprenoid end-products of the mevalonate and 2C-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of several molecules of isopentenyl diphosphate to farnesyl diphosphate. This reaction is catalyzed by a cis-prenyltransferase and leads to the formation of polyprenyl diphosphate. Subsequent steps involve the dephosphorylation and reduction of the α-isoprene unit by a polyprenol reductase, resulting in the generation of dolichol. The size of the dolichol varies, depending on the number of isoprene units incorporated. In eukaryotes, dolichols are synthesized as a mixture of four or more different lengths. Their biosynthesis is predicted to occur in the endoplasmic reticulum, where dolichols play an essential role in protein glycosylation. In this study, we have developed a selection of aptamers targeting dolichols and enhanced their specificity by incorporating fatty acids for negative selection. One aptamer showed high enrichment and specificity for linear polyisoprenoids containing at least one oxygen atom, such as an alcohol or aldehyde, in the α-isoprene unit. The selected aptamer proved to be a valuable tool for the subcellular localization of polyisoprenoids in the malaria parasite. To the best of our knowledge, this is the first time that polyisoprenoids have been localized within a cell using aptamer-based imaging techniques.

In Vitro Diagnostic Assay to Detect SARS-CoV-2-Neutralizing Antibody in Patient Sera Using Engineered ACE-2 Mini-Protein.

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.

Cephalotane-type C20 diterpenoids from Cephalotaxus fortunei var. alpina.

Seventeen new cephalotane-type diterpenoids, fortalides A-Q (1-17), along with five known analogues, were isolated from the seeds of Cephalotaxus fortunei var. alpina. Their structures were determined by extensive spectroscopic methods, as well as electronic circular dichroism (ECD) and X-ray crystallographic data analyses. Some isolates exhibited unusual structural features that were first found in cephalotane-type diterpenoids, such as the occurrence of the 7-oxabicyclo[4.1.1]octane moiety in 14 and 15 and the cis-arrangement of 3-OH and Me-19 in 9. Besides, the antiplasmodial activity of these compounds was evaluated in this study.

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp.

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite's ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesised that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalysed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

Enantiopure Benzofuran-2-carboxamides of 1-Aryltetrahydro-ß-carbolines Are Potent Antimalarials In Vitro.

The tetrahydro-ß-carboline scaffold has proven fertile ground for the discovery of antimalarial agents (e.g., MMV008138 (1) and cipargamin (2)). Similarity searching of a publicly disclosed collection of antimalarial hits for molecules resembling 1 drew our attention to N2-acyl tetrahydro-ß-carboline GNF-Pf-5009 ((±)-3b). Compound purchase, "analog by catalog", and independent synthesis of hits indicated the benzofuran-2-yl amide portion was required for in vitro efficacy against P. falciparum. Preparation of pure enantiomers demonstrated the pharmacological superiority of (R)-3b. Synthesis and evaluation of D- and F-ring substitution variants and benzofuran isosteres indicated a clear structure-activity relationship. Ultimately (R)-3b was tested in Plasmodium berghei-infected mice; unfavorable physicochemical properties may be responsible for the lack of oral efficacy.

Malaria Box-Inspired Discovery of N-Aminoalkyl-ß-carboline-3-carboxamides, a Novel Orally Active Class of Antimalarials.

Virtual ligand screening of a publicly available database of antimalarial hits using a pharmacophore derived from antimalarial MMV008138 identified TCMDC-140230, a tetrahydro-ß-carboline amide, as worthy of exploration. All four stereoisomers of this structure were synthesized, but none potently inhibited growth of the malaria parasite Plasmodium falciparum. Interestingly, 7e, a minor byproduct of these syntheses, proved to be potent in vitro against P. falciparum and was orally efficacious (40 mg/kg) in an in vivo mouse model of malaria.

Lysosomal cholesterol accumulation contributes to the movement phenotypes associated with NUS1 haploinsufficiency.

PURPOSE: Variants in NUS1 are associated with a congenital disorder of glycosylation, developmental and epileptic encephalopathies, and are possible contributors to Parkinson disease pathogenesis. How the diverse functions of the NUS1-encoded Nogo B receptor (NgBR) relate to these different phenotypes is largely unknown. We present three patients with de novo heterozygous variants in NUS1 that cause a complex movement disorder, define pathogenic mechanisms in cells and zebrafish, and identify possible therapy. METHODS: Comprehensive functional studies were performed using patient fibroblasts, and a zebrafish model mimicking NUS1 haploinsufficiency. RESULTS: We show that de novo NUS1 variants reduce NgBR and Niemann-Pick type C2 (NPC2) protein amount, impair dolichol biosynthesis, and cause lysosomal cholesterol accumulation. Reducing nus1 expression 50% in zebrafish embryos causes abnormal swim behaviors, cholesterol accumulation in the nervous system, and impaired turnover of lysosomal membrane proteins. Reduction of cholesterol buildup with 2-hydroxypropyl-ß-cyclodextrin significantly alleviates lysosomal proteolysis and motility defects. CONCLUSION: Our results demonstrate that these NUS1 variants cause multiple lysosomal phenotypes in cells. We show that the movement deficits associated with nus1 reduction in zebrafish arise in part from defective efflux of cholesterol from lysosomes, suggesting that treatments targeting cholesterol accumulation could be therapeutic.

Probing the B- & C-rings of the antimalarial tetrahydro-ß-carboline MMV008138 for steric and conformational constraints.

The antimalarial candidate MMV008138 (1a) is of particular interest because its target enzyme (IspD) is absent in human. To achieve higher potency, and to probe for steric demand, a series of analogs of 1a were prepared that featured methyl-substitution of the B- and C-rings, as well as ring-chain transformations. X-ray crystallography, NMR spectroscopy and calculation were used to study the effects of these modifications on the conformation of the C-ring and orientation of the D-ring. Unfortunately, all the B- and C-ring analogs explored lost in vitro antimalarial activity. The possible role of steric effects and conformational changes on target engagement are discussed.

Resistance to Some But Not Other Dimeric Lindenane Sesquiterpenoid Esters Is Mediated by Mutations in a Plasmodium falciparum Esterase.

Unique lindenane sesquiterpenoid dimers from Chloranthecae spp. were recently identified with promising in vitro antiplasmodial activity and potentially novel mechanisms of action. To gain mechanistic insights to this new class of natural products, in vitro selection of Plasmodium falciparum resistance to the most active antiplasmodial compound, chlorajaponilide C, was explored. In all selected resistant clones, the half-maximal effective concentration (EC50) of chlorajaponilide C increased >250-fold, and whole genome sequencing revealed mutations in the recently discovered P. falciparum prodrug activation and resistance esterase (PfPARE). Chlorajaponilide C was highly potent (mean EC50 = 1.6 nM, n = 34) against fresh Ugandan P. falciparum isolates. The analysis of the structure-resistance relationships revealed that in vitro potency of a subset of lindenane sesquiterpenoid dimers was not mediated by PfPARE mutations. Thus, chlorajaponilide C, but not some related compounds, required parasite esterase activity for in vitro potency, and those compounds serve as the foundation for development of potent and selective antimalarials.

Flavanones from the Twigs and Barks of Artocarpus lakoocha Having Antiplasmodial and Anti-TB Activities.

Chromatographic separation of the acetone extracts from the twigs and barks of Artocarpus lakoocha led to the isolation of the one new flavanone, lakoochanone (1), together with eleven known compounds (2-12). Lakoochanone (1) and moracin C (4) exhibited weak antiplasmodial activity against Plasmodium falciparum Dd2 with IC50 values of 36.7 and 33.9 µM, respectively. Moreover, moracin C (4) and sanggenofuran B (5) showed cytotoxic activity against A2780 cell line with the respective IC50 values of 15.0 and 57.1 µM. In addition, cyclocommunin (7) displayed strong antimycobacterial activity against Mycobacterium tuberculosis H37Ra with the minimum inhibitory concentration (MIC) value of 12.3 µM.

Limonoids from Cipadessa baccifera.

Eighteen new limonoids, including eight methyl angolensates (1-8) and 10 cipadesins (9-18), were isolated from the leaves of Cipadessa baccifera. Their structures were characterized by means of spectroscopic data analyses, single-crystal X-ray diffraction, and quantum chemistry computational methods. The C-6 configurations in those compounds possessing a C-6 hydroxy group were all assigned as S regardless of the magnitude of J5,6, and the C-2' configuration in those bearing a 2-methylbutyryl residue was defined by single-crystal X-ray diffraction and NMR data. Compounds 1, 5, 6, 7, 11, and 12 showed moderate antimalarial activities with IC50 values ranging from 12 to 28 µM.

Antimalarial diterpenoids from Vitex rotundifolia: Isolation, structure elucidation, and in vitro antiplasmodial activity.

Vitex rotundifolia is an important medicinal plant frequently employed in traditional medicines for the treatment of various ailments. Although this plant species has been under exploration for its constituents by various research groups including our own group, no reports were found regarding the antimalarial potential of this plant or of its purified phytochemicals. Phytochemical investigation of this plant yielded three new (1-3) and five known (4-8) diterpenoids. These compounds were purified by modern chromatographic techniques and their structures were determined by advanced spectroscopic techniques such as nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The in vitro antiplasmodial activities were encouraging, as compounds 2, 6, and 8 were found to have significant IC50 values of 1.2, 1.3 and 11.0 µM, respectively against Plasmodium falciparum.

Galtonosides A-E: Antiproliferative and Antiplasmodial Cholestane Glycosides from Galtonia regalis.

An extract of Galtonia regalis from the Natural Products Discovery Institute showed moderate antiplasmodial activity, with an IC50 value less than 1.25 µg/mL. The two known cholestane glycosides 1 and 2 and the five new cholestane glycosides galtonosides A-E (3-7) were isolated after bioassay-directed fractionation. The structures of the new compounds were determined by interpretation of their NMR and mass spectra. Among these compounds, galtonoside B (4) displayed the most potent antiplasmodial activity, with an IC50 value of 0.214 µM against the drug-resistant Dd2 strain of Plasmodium falciparum.

Anibamine and Its Analogues: Potent Antiplasmodial Agents from Aniba citrifolia.

In our continuing search for novel natural products with antiplasmodial activity, an extract of Aniba citrifolia was found to have good activity, with an IC50 value less than 1.25 µg/mL. After bioassay-directed fractionation, the known indolizinium alkaloid anibamine (1) and the new indolizinium alkaloid anibamine B (2) were isolated as the major bioactive constituents, with antiplasmodial IC50 values of 0.170 and 0.244 µM against the drug-resistant Dd2 strain of Plasmodium falciparum. The new coumarin anibomarin A (3), the new norneolignan anibignan A (5), and six known neolignans (7-12) were also obtained. The structures of all the isolated compounds were determined based on analyses of 1D and 2D NMR spectroscopic and mass spectrometric data, and the absolute configuration of anibignan A (5) was assigned from its ECD spectrum. Evaluation of a library of 28 anibamine analogues (13-40) indicated that quaternary charged analogues had IC50 values as low as 58 nM, while uncharged analogues were inactive or significantly less active. Assessment of the potential effects of anibamine and its analogues on the intraerythrocytic stages and morphological development of P. falciparum revealed substantial activity against ring stages for compounds with two C-10 side chains, while those with only one C-10 side chain exhibited substantial activity against trophozoite stages, suggesting different mechanisms of action.

Isolation and characterization of antiplasmodial constituents from the marine sponge Coscinoderma sp.

Six known compounds, namely two halisulfates 1 and 2 and four epidioxy sterols 3-6, were isolated from the marine sponge Coscinoderma sp. The structures of these compounds were confirmed by nuclear magnetic resonance (1H and 13C NMR) spectroscopy, and their antiplasmodial activities were determined against the chloroquine-resistant Dd2 strain of Plasmodium falciparum. The epidioxy steroids 3-6 all showed moderate to weak antiplasmodial activity, with IC50 values of 2.7 µM for (24S)-5α,8α-epidioxy-24-methylcholesta-6-en-3ß-ol (3), 11.6 µM for 5α,8α-epidioxycholesta-6,24(28)-dien-3ß-o1 (4), 2.33 µM for 5α,8α-epidioxy-24-methylcholesta-6,9(11)-24(28)-trien-3ß-ol (5), and between 12 and 24 µM for 5α,8α-epidioxycholesta-6-en-3ß-ol (6). In contrast, halisulfate 2 (1) was inactive, and halisulfate 1 (2) had an of IC50 value of about 24 µM.

Phloroglucinols from the Roots of Garcinia dauphinensis and Their Antiproliferative and Antiplasmodial Activities.

Garcinia dauphinensis is a previously uninvestigated endemic plant species of Madagascar. The new phloroglucinols dauphinols A-F and 3'-methylhyperjovoinol B (1-7) and six known phloroglucinols (8-13) together with tocotrienol 14 and the three triterpenoids 15-17 were isolated from an ethanolic extract of G. dauphinensis roots using various chromatographic techniques. The structures of the isolated compounds were elucidated by NMR, MS, optical rotation, and ECD data. Theoretical ECD spectra and specific rotations for 2 were calculated and compared to experimental data in order to assign its absolute configuration. Among the compounds tested, 1 showed the most promising growth inhibitory activity against A2870 ovarian cancer cells, with IC50 = 4.5 ± 0.9 µM, while 2 had good antiplasmodial activity against the Dd2 drug-resistant strain of Plasmodium falciparum, with IC50 = 0.8 ± 0.1 µM.

Antiplasmodial flavanones and a stilbene from Carpha glomerata.

Bioassay-guided fractionation of an extract of Carpha glomerata (Cyperaceae) led to the isolation of seven compounds. Compounds 1 (carphorin A), 3 (carphorin C), 4 (carphorin D), and 5 (carphabene) are new compounds, and compound 2 (8-(3″-hydroxyisoamyl)-naringenin) was isolated for the first time as a natural product. All structures were elucidated based on analyses of their HR-ESIMS and 1D and 2D NMR data. Compounds 1, 2, and 6, which have prenyl or hydroxyprenyl side chains, exhibited antiplasmodial activities with IC50 values of 5.2 ±â€¯0.6, 3.4 ±â€¯0.4, and 6.7 ±â€¯0.8 µM against the drug-resistant Dd2 strain of Plasmodium falciparum. In addition the prenylated stilbene 5 also showed good activity, with IC50 5.8 ±â€¯0.7 µM.

Antiplasmodial Diterpenoids and a Benzotropolone from Petradoria pumila.

An extract of Petradoria pumila from the Natural Products Discovery Institute was found to have moderate antiplasmodial activity, with an IC50 value between 5 and 10 µg/mL. The four new diterpenoids petradoriolides A-D (1-4), the new benzotropolone petradoriolone (5), and the two known lignans 6 and 7 were isolated after bioassay-directed fractionation. The structures and stereochemistries of the new compounds were determined by interpretation of NMR spectroscopy, mass spectrometry, and ECD spectra. Among these compounds, petradoriolide C (3) displayed the most potent antiplasmodial activity, with an IC50 value of 7.3 µM.