Cell experiment:

A clonal population of P. falciparum Dd2 parasites is used to initiate two or three independent parasite cultures under the initial selection pressure of 12 nM KAI407, 1 nM KAI715 or 40 nM BQR695. Stepwise drug evolution continues until the final concentration is at least 3-fold higher than the initial concentration (typically 80 to 120 days). For each of the ten resistant strains, copy number variations (CNVs) and single nucleotide variations (SNVs) are detected using a whole-genome tiling array and analyzed with PfGenominator. The susceptibility of each resistant strain to KAI407, KAI715, KDU691 and BQR695 is determined by the 72-hr SYBR Green cell proliferation assay with four independent experiments assayed in duplicate[2].

BQR-695 is a PI4KIIIβ inhibitor with IC50s of 80 and 3.5 nM for human PI4KIIIβ and Plasmodium variant of PI4KIIIβ, respectively.

Treatment with 0.5 μM of either KAI407 or BQR695 causes GFP-PHOsh2 to redistribute to the parasite plasma membrane, consistent with depletion of intracellular PI4P upon inhibition of PfPI4K function. BQR695 shows no evidence of toxicity against mature red blood cells (RBCs), induces a schizont-stage arrest indistinguishable from that observed in imidazopyrazine-treated parasites and exhibits cross-resistance with the imidazopyrazine-resistant lines[2].

[1]. Fowler ML, et al. Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11. Protein Sci. 2016 Apr;25(4):826-39. [2]. McNamara CW, et al. Targeting Plasmodium PI(4)K to eliminate malaria. Nature. 2013 Dec 12;504(7479):248-253.