Understanding the Antiplasmodial Action of Resistance-Refractory Xanthoquinodin A1

Abstract

Our previous work identified a series of 12 xanthoquinodin analogues and 2 emodin-dianthrones with broad-spectrum activities against Trichomonas vaginalis, Mycoplasma genitalium, Cryptosporidium parvum, and Plasmodium falciparum. Analyses conducted in this study revealed that the most active analogue, xanthoquinodin A1, also inhibits Toxoplasma gondii tachyzoites and the liver stage of Plasmodium berghei, with no cross-resistance to the known antimalarial targets PfACS, PfCARL, PfPI4K, or DHODH. In Plasmodium, inhibition occurs prior to multinucleation and induces parasite death following 12 h of compound exposure. This moderately fast activity has impeded resistance line generation, with xanthoquinodin A1 demonstrating an irresistible phenotype in both T. gondii and P. falciparum.

Keywords: Xanthoquinodin, Fungal derived, Plasmodium, malaria, antiplasmodial.

Figure 1.

Xanthoquinodin A1 apicomplexan activity and Plasmodium killing profile. (A) Structure of xanthoquinodin A1 and activity against P. falciparum, P. berghei liver stages, and T. gondii. P. falciparum Dd2 activity was previously reported in (PMID: 37276438). (B) EC₅₀ ratios obtained against parasite lines carrying resistance to known antiplasmodial targets: PfACS (control: MMV084978), PfCARL (control: GNF179), and PfPI4K (control: KDU691), along with Dd2/3D7 activity comparison (control: chloroquine). Activity in Pf Dd2 ScDHODH line with and without attb and with and without proguanil (PG). (C) The in vitro parasite killing rate obtained for xanthoquinodin A1 and controls atovaquone (slow acting) and dihydroartemisinin (DHA, fast acting). Lag phase: time needed to achieve the maximum rate of killing; log(PRR): log₁₀ of the parasite reduction ratio, corresponds to the decrease of parasite number over 48 h (excluding the lag phase); 99.9% PCT: time needed to reduce the initial parasite population by 99.9%. (D) Killing effect of xanthoquinodin A1 (10 × EC₅₀) following a 12 h incubation. Graph represents the percentage of viable parasites over time determined by SYBR⁺MTR⁺ gating. Data represent the mean and SEM of three biological replicates.

Figure 2.

The intraerythrocytic stage specificity of xanthoquinodin A1 shows that maximum inhibition occurs prior to 30 HPI. Samples were collected for thin blood smears and flow cytometry with YOYO-1 every 12 h after a 5 × EC₅₀ treatment at 6, 18, 30, and 42 HPI. (A) Histogram plots of the control and treated cultures. Results are representative of 3 biological replicates. (B) Giemsa staining of the control and treated cultures. (C) Total fluorescence signal across all 3 replicates. (D) Median fluorescence signal across all 3 replicates. Significance was determined using a two-way ANOVA against the vehicle-treated control per each collection point. For the final time point (treatment at 42 HPI), significance determined by paired t test.

Figure 3.

RNASeq differential expression of mRNA with the incubation of xanthoquinodin A1. (A) Heatmap of mRNA transcripts (p-value < 0.1, log2 fc > |1|) differentially expressed at the gene level following 1 h exposure to xanthoquinodin A1 (XanQ A1) at EC₅₀ concentration. Results from three biological replicates. Transcripts validated via RT-qPCR are shown in bold and underlined. GO-term enrichment for individual gene clusters is shown to the right of the heatmap. (B) Heatmap of mRNA transcripts (p-value <0.1, log2 fc > |1|) differentially expressed at the transcript level. GO-term enrichment for individual gene clusters shown to the right of heatmap. (C) RT-qPCR validation of differentially expressed transcripts. Data represent the mean and SEM of three biological replicates using housekeeping genes serine tRNA ligase (PF3D7_0717700) and 60S ribosomal protein L18–2 (PF3D7_1341300).

Figure 4.

RNASeq differential expression of lncRNA with the incubation of xanthoquinodin A1. (A) Heatmap of lncRNA transcripts (p-value <0.1, log2 fc > |1|) differentially expressed following 1 h exposure to xanthoquinodin A1 (XanQ A1) at EC₅₀ concentration. Results from three biological replicates. Transcripts validated via RT-qPCR shown in bold and underlined. Predicted cis-targets of lncRNA per cluster are shown to the right of the heatmap, along with target-enriched GO-terms. (B) RT-qPCR validation of differentially expressed lncRNA. Data represent the mean and SEM of three biological replicates using housekeeping genes serine tRNA ligase (PF3D7_0717700) and 60S ribosomal protein L18–2 (PF3D7_1341300). Published on NCBI SRA Accession # PRJNA1073928.

Figure 5.

STRING analysis of the xanthoquinodin A1-shared transcriptome. (A) Correlation graph of the pEC₅₀ activity of xanthoquinodin A1 and its analogues across distinct phyla. Inhibition against these organisms originally communicated in the DOI: 10.1021/acs.jnatprod.3c00283. (B) Gene cooccurrence of differentially expressed transcripts (p < 0.05) or predicted cis targets of differentially expressed lncRNA (p < 0.05) identified following xanthoquinodin A1 incubation in P. falciparum. Transcripts shown are those with genome occurrence pattern similarity in five or more of the six organisms shown to be inhibited by xanthoquinodin A1, as determined by STRING. (C) Interaction network of transcripts with genome similarity. The top six functional enrichments based on STRING strength score are shown by varying node colors. Edge color is reflective of the interaction source.