Sequencing and microarray results summary.

<p>n/a: not applicable, SNV: single nucleotide variant, Mb: Mega base,</p>*<p>SNVs in parenthesis were within 15 kb from a chromosome end and could not be confirmed by WGS. ctr: control parasite lines without drug pressure, G1, G2: generation 1 and 2, R: resistant, S: sensitive.</p

Location and evolutionary relationship of genetic changes following long-term culturing of <i>P. falciparum</i> parasites.

<p>Genetic changes in each individual clone were detected by microarray analysis as well as by WGS. A. Genetic differences were detected by WGS between our parental 3D7 clone and the available 3D7 reference genome from PlasmoDB v9.1. The 14 chromosomes are indicated in grey, with their relative length on the y-axis. The mitochondrial (Mit) and apicoplast (Api) genomes are shown in the inset. B. Genetic changes identified by microarray and WGS in each individual clone compared to the parental 3D7 clone. SNVs are indicated with circles, large-scale deletions (>1000 bp) with diamonds, and CNVs with arrowheads. For annotations of genes harboring SNVs or that are part of a structural variant, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003293#pgen.1003293.s005" target="_blank">Table S2</a>. C. Cladogram showing derived evolutionary relationship in B, computed by a heuristic algorithm to find the tree of minimum complexity starting with the parental clone.</p

A Chemical Genomic Analysis of Decoquinate, a <i>Plasmodium falciparum</i> Cytochrome <i>b</i> Inhibitor

Decoquinate has single-digit nanomolar activity against <i>in vitro</i> blood stage <i>Plasmodium falciparum</i> parasites, the causative agent of human malaria. <i>In vitro</i> evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome <i>b</i>. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome <i>b</i>, an essential subunit of the cytochrome <i>bc</i>₁ complex. As with other cytochrome <i>bc</i>₁ inhibitors, such as atovaquone, decoquinate has low nanomolar activity against <i>in vitro</i> liver stage <i>P. yoelii</i> and provides partial prophylaxis protection when administered to infected mice at 50 mg kg^–1. In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite’s mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome <i>b</i>. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome <i>b</i>

Genomic changes and drug sensitivities of atovaquone (ATQ)-selected clones.

<p>A. Amino acid change (nucleotide change) and codon position in cytochrome b of each clone. B. Location of the amplification event detected on chromosome 1 for R5a (423,658–643,292 bp). The location of the multidrug resistance associated protein 1 (<i>Pf</i>mrp1, PFA0590w) is shown. The log2 ratio of the intensity of each unique probe in R5a relative to that in S1c is plotted and colored by the moving average over a 500-base pair window. C. The EC₅₀ values for four tested drugs are shown for the 3D7 parent, the sensitive clones and the ATQ-resistant clones of the first generation (G1). Statistically significant differences between the EC₅₀ values of the parental 3D7 line and the ATQ-resistant clones were calculated by a one-way ANOVA followed by a Dunnett posttest (*, p<0.0001). EC₅₀ values are means ± SD of three independent experiments performed in quadruplicate.</p

Mitotic recombination events detected by WGS.

<p>Paired-end reads next to deletions as well as their read pair mates that mapped to a different chromosome were extracted. <i>De novo</i> assembly of these reads, starting with a single seed that mapped next to the deletion, generated new contigs. Hypothetical scenarios of recombination events that created gene conversions are shown on the left; the sequence alignments of the contigs (center sequences), where the two sequences from different chromosomes joined, are on the right. The chromosomal position (with orientation) and the <i>var</i> gene ID are indicated when applicable.</p

Detection of deletions in subtelomeric regions by WGS and microarray.

<p>A. Microarray and WGS detection of deletion events. The top two panels show the number of WGS paired-end reads mapping to 16 kb of chromosome 2 for 3D7 and S1a. The third panel shows the same region but with data from the microarray. The log2 ratio of the intensity of each unique probe for S1a relative to 3D7 parent is indicated and colored by the moving average over a 500-base pair window. B. Southern blot analysis. The gDNA of the 3D7 parent and S1a was cut with restriction enzymes <i>Hpa</i>I and <i>Fsp</i>I and analyzed by pulsed field gel electrophoresis using a probe to the <i>rifin</i> gene PFB0015c adjacent to the <i>var</i> gene containing the deletion (schematic on the left, southern blot on the right). Arrows indicate the expected sizes for the fragments of the full-length 3D7 and the truncated S1a <i>var</i> gene (PFB0010w). Stars show nonspecific bands.</p

Distribution of deleted genes.

<p>Schematic of the location of structural variants detected in individual clones compared to the 3D7 parent. The chromosomal location is indicated on the left, followed by the name of the clone harboring the structural variant (grey box). Dotted boxes indicate regions with low read coverage and absence of unique probes, which mask the exact size of deletions. Different members of gene families are color labeled. Deletions in clones marked with an asterisk are associated with recombination events shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003293#pgen-1003293-g006" target="_blank">Figure 6</a>.</p

Generation of atovaquone (ATQ)-resistant parasites.

<p>A. Selection schematic. An initial parasite clone (3D7) was split into three lines after 55 days, and 20 nM or 50 nM ATQ pressure was applied to lines R4 and R5, respectively. The parental line was kept drug-free. After 24 days, the parental line was split again into four lines, and 2 nM ATQ pressure was applied to three lines (R1, R2, and R3) while line S1 was kept drug free. R2 was again split after 74 days, and 20 nM ATQ was applied repeatedly to line R2b. After the indicated time in culture, all lines were cloned by limiting dilution. Four ATQ-resistant clones were kept in culture (Generation 1 (G1): R1a and b G1 and R2a and b G1) and recloned, resulting in a second generation of clones (Generation 2 (G2): R1a and b G2 and R2a and b G2). The number of days (d) in culture between splits is indicated above each flask. B. ATQ structure and growth inhibition assay. EC₅₀ values for 3D7 parent, the sensitive clones, and the ATQ-resistant clones are the means ± SD of three independent experiments performed in quadruplicate. Statistically significant differences between EC₅₀ values of the parental 3D7 line and the ATQ-resistant clones were calculated by a one-way ANOVA followed by a Dunnett posttest (*, p<0.0001).</p

(<i>R</i>)‑2-Phenylpyrrolidine Substituted Imidazopyridazines: A New Class of Potent and Selective Pan-TRK Inhibitors

Deregulated kinase activities of tropomyosin receptor kinase (TRK) family members have been shown to be associated with tumorigenesis and poor prognosis in a variety of cancer types. In particular, several chromosomal rearrangements involving TRKA have been reported in colorectal, papillary thyroid, glioblastoma, melanoma, and lung tissue that are believed to be the key oncogenic driver in these tumors. By screening the Novartis compound collection, a novel imidazopyridazine TRK inhibitor was identified that served as a launching point for drug optimization. Structure guided drug design led to the identification of (<i>R</i>)-2-phenylpyrrolidine substituted imidazopyridazines as a series of potent, selective, orally bioavailable pan-TRK inhibitors achieving tumor regression in rats bearing KM12 xenografts. From this work the (<i>R</i>)-2-phenylpyrrolidine has emerged as an ideal moiety to incorporate in bicyclic TRK inhibitors by virtue of its shape complementarity to the hydrophobic pocket of TRKs