Supplementary Materials

Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target

Mahmood M. Alam, Ana Sanchez-Azqueta, Omar Janha, Erika L. Flannery, Amit Mahindra, Kopano Mapesa, Aditya B. Char, Dev Sriranganadane, Nicolas M. B. Brancucci, Yevgeniya Antonova-Koch, Kathryn Crouch, Nelson Victor Simwela, Scott B. Millar, Jude Akinwale, Deborah Mitcheson, Lev Solyakov, Kate Dudek, Carolyn Jones, Cleofé Zapatero, Christian Doerig, Davis C. Nwakanma, Maria Jesús Vázquez, Gonzalo Colmenarejo, Maria Jose Lafuente-Monasterio, Maria Luisa Leon, Paulo H. C. Godoi, Jon M. Elkins, Andrew P. Waters, Andrew G. Jamieson, Elena Fernández Álvaro, Lisa C. Ranford-Cartwright, Matthias Marti, Elizabeth A. Winzeler, Francisco Javier Gamo, Andrew B. Tobin

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S13
  • Captions for tables S1 to S5
  • References
Table S1
Compound Screen. Hits from the primary single dose HTS (2838) and compounds contained in the MMV-malaria box (259) were used to generate concentration inhibitor curves. Shown are pIC50 values against PfCLK3, PfCLK1, PvCLK3 and hCLK2 (sheet 1). These data are divided into data associated with TCAMs/GSK compounds (sheet 2), PKIS compounds (sheet 3), MRCT compounds (sheet 4) and MMV compounds (sheet 5).
Table S2
Transcription changes in parent P. falciparum strain Dd2 following treatment with TCMDC-135051. Sheet 1 - Statistically significant changes (t-test n=4) in transcript levels following treatment of Dd2 parasites at trophozoite stage with TCMDC-135051 (1μM for 60 mins). Shown are the probes used and changes associated with each of the probes. Sheet 2 - Summary of the probes that reveal significantly down-regulated genes following TCMDC-135051 treatment. Sheet 3 - Summary of the genes that are significantly down-regulated following TCMDC- 135051 treatment. Sheet 4 - Summary of the probes that reveal significantly up-regulated genes following TCMDC-135051 treatment. Sheet 5- Summary of the genes that are significantly up-regulated following TCMDC-135051 treatment.
Table S3
Transcription changes in the resistant parasite strain TM051C following treatment with TCMDC-135051. Sheet 1 - Statistically significant changes (t-test n=4) in transcript levels following treatment of TM051C parasites at trophozoite stage with TCMDC-135051 (1μM for 60 mins). Shown are the probes used and changes associated with each of the probes. Sheet 2 - Summary of the probes that reveal significantly down-regulated genes following TCMDC-135051 treatment. Sheet 3 - Summary of the genes that are significantly down-regulated following TCMDC-135051 treatment. Sheet 4 - Summary of the probes that reveal significantly upregulated genes following TCMDC-135051 treatment. Sheet 5- Summary of the genes that are significantly up-regulated following TCMDC-135051 treatment.
Table S4
Transcription changes designated as a result of on-target inhibition of PfCLK3 and a comparison with essential Plasmodium falciparum genes. Subtracting the genes where transcription was significantly changed following treatment with TCMDC-135051 (1μM for 60 mins) in TM051C parasites with those changed in Dd2 parasites revealed the "on target" transcriptional changes associated with inhibition of PfCLK3. Sheet 1-On target up-regulated genes. Sheet-2 Identification (highlighted) of the on target up regulated genes that are essential for parasite blood stage survival. Sheet 3-On target downregulated genes. Sheet 4-Identification (high-lighted) of the on target down regulated genes that are essential for parasite blood stage survival.
Table S5
Gene ontology analysis of on-target down regulated genes following inhibition of PfCLK3 in Dd2 parasites. Gene ontology enrichment analysis was carried out on genes that were significantly downregulated by PfCLK3 inhibition. GO terms with P < 0.05 (derived from the Fisher's Exact test) and with FDR values <0.05 (calculated using the Benjamini Hochberg method) are shown on sheet 1, whilst all Go terms with a P<0.05 are shown on sheet 2. From left to right, columns show: the GO term id, the GO term name, the number of genes in the background annotated with this GO term, the number of genes significantly downregulated by PfCLK3 inhibition annotated with this GO term, a list of the downregulated genes annotated with this GO term, the percentage of genes in the background annotated with this GO term that also appear in the list of downregulated genes, the fold enrichment of the GO term in the downregulated gene list, an odds ratio, a p-value and the FDR.