Ana Lisa Valenciano

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. 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.

Xylopic acid (15β-acetyloxy-kaur-16-en-19-oic acid) is a kaurene diterpene that can be obtained from various Xylopia spp. Xylopic acid has demonstrated several pharmacological activities in vitro and in vivo. The compound has shown promising effect as a potent analgesic, anti-inflammatory and anti-allergic agent. Xylopic acid is a CNS depressant and was able to ameliorate anxiety-like symptoms in mice in addition to its neuroprotective effects. Deleterious effects of xylopic acid on the reproductive system of mice have been well documented but extensive toxicity study detailing effect of the acid upon chronic exposure needs to be determined. Due to the heavy consumption of X. aethiopica fruits, it is recommended that the pharmacokinetics of xylopic acid be determined to ascertain the possible food-drug interaction that may occur when conventional drugs are taken together with foods containing xylopic acid.

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.

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.

The shikimate pathway, a metabolic pathway absent in humans, is responsible for the production of chorismate, a branch point metabolite. In the malaria parasite, chorismate is postulated to be a direct precursor in the synthesis of p -aminobenzoic acid (folate biosynthesis), p -hydroxybenzoic acid (ubiquinone biosynthesis), menaquinone, and aromatic amino acids. While the potential value of the shikimate pathway as a drug target is debatable, the metabolic dependency of chorismate in P. falciparum remains unclear. Current evidence suggests that the main role of chorismate is folate biosynthesis despite ubiquinone biosynthesis being active and essential in the malaria parasite. Our goal in the present work was to expand our knowledge of the ubiquinone head group biosynthesis and its potential metabolic dependency on chorismate in P. falciparum. These data led us to further characterize the mechanism of action of MMV688345, a compound from the open-access “Pathogen Box” collection from Medicine for Malaria Venture. We systematically assessed the development of both asexual and sexual stages of P. falciparum in a defined medium in the absence of an exogenous supply of chorismate end-products and present biochemical evidence suggesting that the benzoquinone ring of ubiquinones in this parasite may be synthesized through a yet unidentified route.

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.

An extract of Malleastrum sp. (Meliaceae) collected in Madagascar by the Madagascar International Cooperative Biodiversity Group was found to have antimalarial activity, with an IC₅₀ value between 2.5 and 5 μg ml⁻¹. After purification by liquid‐liquid partition, chromatography on a Diaion open column, C₁₈ SPE and C₁₈ reversed phase HPLC, the new butanolide, malleastrumolide A, was isolated. The structure of malleastrumolide A was determined by mass spectrometry, NMR, and ECD. The double bond position was determined by cross‐metathesis and mass spectrometry. The compound has antiproliferative activity against the A2780 ovarian cancer cell line with an IC₅₀ value of 17.4 μm and antiplasmodial activity against the drug‐resistant Dd2 strain of Plasmodium falciparum with an IC₅₀ value of 2.74 μm.

The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC₅₀ for TbERK8 that is several hundred times higher than its reported IC₅₀ for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite.