Our exciting journey to ACT-451840

We describe our work resulting in the selection of ACT-451840 ( 38 ) as a novel antimalarial drug with a novel mode of action. The compound was broadly characterized in vitro as well as in vivo in rat PK experiments as well as two different mouse malaria models. In the P. berghei infected mouse model cure could be achieved at oral doses of 300 mg/kg over 3 consecutive days. ACT-451840 was clinically investigated up to an experimental human malaria infection model, where therapeutic effects could be shown

Anti-malarial ozonides OZ439 and OZ609 tested at clinically relevant compound exposure parameters in a novel ring-stage survival assay

BACKGROUND: Drug efficacy against kelch 13 mutant malaria parasites can be determined in vitro with the ring-stage survival assay (RSA). The conventional assay protocol reflects the exposure profile of dihydroartemisinin. METHODS: Taking into account that other anti-malarial peroxides, such as the synthetic ozonides OZ439 (artefenomel) and OZ609, have different pharmacokinetics, the RSA was adjusted to the concentration-time profile of these ozonides in humans and a novel, semi-automated readout was introduced. RESULTS: When tested at clinically relevant parameters, it was shown that OZ439 and OZ609 are active against the Plasmodium falciparum clinical isolate Cam3.I(R539T). CONCLUSION: If the in vitro RSA does indeed predict the potency of compounds against parasites with increased tolerance to artemisinin and its derivatives, then the herein presented data suggest that following drug-pulses of at least 48 h, OZ439 and OZ609 will be highly potent against kelch 13 mutant isolates, such as P. falciparum Cam3.I(R539T)

Ancient Chinese methods are remarkably effective for the preparation of artemisinin-rich extracts of Qing Hao with potent antimalarial activity.

yesAncient Chinese herbal texts as far back as the 4th Century Zhou hou bei ji fang describe methods for the use of Qing Hao (Artemisia annua) for the treatment of intermittent fevers. Today, the A. annua constituent artemisinin is an important antimalarial drug and the herb itself is being grown and used locally for malaria treatment although this practice is controversial. Here we show that the ancient Chinese methods that involved either soaking, (followed by wringing) or pounding, (followed by squeezing) the fresh herb are more effective in producing artemisinin-rich extracts than the usual current method of preparing herbal teas from the dried herb. The concentrations of artemisinin in the extracts was up to 20-fold higher than that in a herbal tea prepared from the dried herb, but the amount of total artemisinin extracted by the Chinese methods was much less than that removed in the herbal tea. While both extracts exhibited potent in vitro activities against Plasmodium falciparum, only the pounded juice contained sufficient artemisinin to suppress parasitaemia in P. berghei infected mice. The implications of these results are discussed in the context of malaria treatment using A. annua infusions

Discovery of FNDR-20123, a histone deacetylase inhibitor for the treatment of Plasmodium falciparum malaria

BACKGROUND: Emergence of anti-malarial drug resistance and perpetual increase in malaria incidence necessitates the development of novel anti-malarials. Histone deacetylases (HDAC) has been shown to be a promising target for malaria, despite this, there are no HDAC inhibitors in clinical trials for malaria treatment. This can be attributed to the poor pharmacokinetics, bioavailability and selectivity of the HDAC inhibitors. METHODS: A collection of HDAC inhibitors were screened for anti-malarial activity, and the best candidate was profiled in parasite-killing kinetics, growth inhibition of sensitive and multi-drug resistant (MDR) strains and against gametocytes. Absorption, distribution, metabolism and excretion pharmacokinetics (ADME-PK) parameters of FNDR-20123 were determined, and in vivo efficacy was studied in a mouse model for Plasmodium falciparum infection. RESULTS: A compound library of HDAC inhibitors (180 in number) was screened for anti-malarial activity, of which FNDR-20123 was the most potent candidate. The compound had been shown to inhibit Plasmodium HDAC with IC50 of 31 nM and human HDAC with IC50 of 3 nM. The IC50 obtained for P. falciparum in asexual blood-stage assay was 42 nM. When compared to atovaquone and pyrimethamine, the killing profiles of FNDR-20123 were better than atovaquone and comparable to pyrimethamine. The IC50 values for the growth inhibition of sensitive and MDR strains were similar, indicating that there is no cross-resistance and a low risk of resistance development. The selected compound was also active against gametocytes, indicating a potential for transmission control: IC50 values being 190 nM for male and > 5 microM for female gametocytes. FNDR-20123 is a stable candidate in human/mouse/rat liver microsomes (> 75% remaining post 2-h incubation), exhibits low plasma protein binding (57% in humans) with no human Ether-a-go-go-Related Gene (hERG) liability (> 100 microM), and does not inhibit any of the cytochrome P450 (CYP) isoforms tested (IC50 > 25 microM). It also shows negligible cytotoxicity to HepG-2 and THP-1 cell lines. The oral pharmacokinetics in rats at 100 mg/kg body weight shows good exposures (Cmax = 1.1 microM) and half-life (T1/2 = 5.5 h). Furthermore, a 14-day toxicokinetic study at 100 mg/kg daily dose did not show any abnormality in body weight or gross organ pathology. FNDR-20123 is also able to reduce parasitaemia significantly in a mouse model for P. falciparum infection when dosed orally and subcutaneously. CONCLUSION: FNDR-20123 may be a suitable candidate for the treatment of malaria, which can be further developed

Complex microwave conductivity of Na-DNA powders

We report the complex microwave conductivity, $\sigma=\sigma_1-i\sigma_2$, of Na-DNA powders, which was measured from 80 K to 300 K by using a microwave cavity perturbation technique. We found that the magnitude of $\sigma_1$ near room temperature was much larger than the contribution of the surrounding water molecules, and that the decrease of $\sigma_1$ with decreasing temperature was sufficiently stronger than that of the conduction of counterions. These results clearly suggest that the electrical conduction of Na-DNA is intrinsically semiconductive.Comment: 16 pages, 7 figure

The plasmodium lactate/H+ transporter PfFNT is essential and druggable in vivo

Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H(+) from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target. Small, drug-like FNT inhibitors potently block lactate transport and kill Plasmodium falciparum parasites in culture. The protein structure of Plasmodium falciparum FNT (PfFNT) in complex with the inhibitor has been resolved and confirms its previously predicted binding site and its mode of action as a substrate analog. Here, we investigated the mutational plasticity and essentiality of the PfFNT target on a genetic level, and established its in vivo druggability using mouse malaria models. We found that, besides a previously identified PfFNT G107S resistance mutation, selection of parasites at 3 x IC(50) (50% inhibitory concentration) gave rise to two new point mutations affecting inhibitor binding: G21E and V196L. Conditional knockout and mutation of the PfFNT gene showed essentiality in the blood stage, whereas no phenotypic defects in sexual development were observed. PfFNT inhibitors mainly targeted the trophozoite stage and exhibited high potency in P. berghei- and P. falciparum-infected mice. Their in vivo activity profiles were comparable to that of artesunate, demonstrating strong potential for the further development of PfFNT inhibitors as novel antimalarials

The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis

Casein kinase 2 (CK2) is a pleiotropic kinase phosphorylating substrates in different cellular compartments in eukaryotes. In the malaria parasite Plasmodium falciparum, PfCK2 is vital for asexual proliferation of blood-stage parasites. Here, we applied CRISPR/Cas9-based gene editing to investigate the function of the PfCK2alpha catalytic subunit in gametocytes, the sexual forms of the parasite that are essential for malaria transmission. We show that PfCK2alpha localizes to the nucleus and cytoplasm in asexual and sexual parasites alike. Conditional knockdown of PfCK2alpha expression prevented the transition of stage IV into transmission-competent stage V gametocytes, whereas the conditional knockout of pfck2a completely blocked gametocyte maturation already at an earlier stage of sexual differentiation. In summary, our results demonstrate that PfCK2alpha is not only essential for asexual but also sexual development of P. falciparum blood-stage parasites and encourage studies exploring PfCK2alpha as a potential target for dual-active antimalarial drugs

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target

The anomaly of the oxygen bond-bending mode at 320 cm−1^{-1} and the additional absorption peak in the c-axis infrared conductivity of underdoped YBa2_{2}Cu3_{3}O7−δ_{7-\delta} single crystals revisited by ellipsometricmeasurements

We have performed ellipsometric measurements of the far-infrared c-axis dielectric response of underdoped YBa$_{2}$Cu$_{3}$O$_{7-\delta}$ single crystals. Here we report a detailed analysis of the temperature-dependent renormalization of the oxygen bending phonon mode at 320 cm$^{-1}$ and the formation of the additional absorption peak around 400-500 cm$^{-1}$. For a strongly underdoped YBa$_{2}$Cu$_{3}$O$_{6.5}$ crystal with T$_{c}$=52 K we find that, in agreement with previous reports based on conventional reflection measurements, the gradual onset of both features occurs well above T$_{c}$ at T*$\sim$150 K. Contrary to some of these reports, however, our data establish that the phonon anomaly and the formation of the additional peak exhibit very pronounced and steep changes right at T$_{c}$. For a less underdoped YBa$_{2}$Cu$_{3}$O$_{6.75}$ crystal with T$_{c}$=80 K, the onset temperature of the phonon anomaly almost coincides with T$_{c}$. Also in contrast to some previous reports, we find for both crystals that a sizeable fraction of the spectral weight of the additional absorption peak cannot be accounted for by the spectral-weight loss of the phonon modes but instead arises from a redistribution of the electronic continuum. Our ellipsometric data are consistent with a model where the bilayer cuprate compounds are treated as a superlattice of intra- and inter-bilayer Josephson-junctions

The Parasite Reduction Ratio (PRR) assay version 2: standardized assessment of Plasmodium falciparum viability after antimalarial treatment in vitro

With artemisinin-resistant Plasmodium falciparum parasites emerging in Africa, the need for new antimalarial chemotypes is persistently high. The ideal pharmacodynamic parameters of a candidate drug are a rapid onset of action and a fast rate of parasite killing or clearance. To determine these parameters, it is essential to discriminate viable from nonviable parasites, which is complicated by the fact that viable parasites can be metabolically inactive, whilst dying parasites can still be metabolically active and morphologically unaffected. Standard growth inhibition assays, read out via microscopy or [3H] hypoxanthine incorporation, cannot reliably discriminate between viable and nonviable parasites. Conversely, the in vitro parasite reduction ratio (PRR) assay is able to measure viable parasites with high sensitivity. It provides valuable pharmacodynamic parameters, such as PRR, 99.9% parasite clearance time (PCT99.9%) and lag phase. Here we report the development of the PRR assay version 2 (V2), which comes with a shorter assay duration, optimized quality controls and an objective, automated analysis pipeline that systematically estimates PRR, PCT99.9% and lag time and returns meaningful secondary parameters such as the maximal killing rate of a drug (Emax) at the assayed concentration. These parameters can be fed directly into pharmacokinetic/pharmacodynamic models, hence aiding and standardizing lead selection, optimization, and dose prediction. © 2023 by the authors