Salinipostins A–K, Long-Chain Bicyclic Phosphotriesters as a Potent and Selective Antimalarial Chemotype

Despite significant advances in antimalarial chemotherapy over the past 30 years, development of resistance to frontline drugs remains a significant challenge that limits efforts to eradicate the disease. We now report the discovery of a new class of antimalarials, salinipostins A–K, with low nanomolar potencies and high selectivity indices against mammalian cells (salinipostin A: <i>Plasmodium falciparum</i> EC₅₀ 50 nM, HEK293T cytotoxicity EC₅₀ > 50 μM). These compounds were isolated from a marine-derived <i>Salinospora</i> sp. bacterium and contain a bicyclic phosphotriester core structure, which is a rare motif among natural products. This scaffold differs significantly from the structures of known antimalarial compounds and represents a new lead structure for the development of therapeutic targets in malaria. Examination of the growth stage specificity of salinipostin A indicates that it exhibits growth stage-specific effects that differ from compounds that inhibit heme polymerization, while resistance selection experiments were unable to identify parasite populations that exhibited significant resistance against this compound class

Sequencing results.

<p>Sanger and NGS sequencing coverage of targeted CRISPR mutations at the <i>pfatp4</i> locus for ACP-B6-L350H and ACP-B6-P412T with clonal wild type parent strain ACP-B6. Red bars delineate the respective 20 nt guide RNA target sites and PAM sites required for each edit. NGS coverage at each location is indicated by blue columns. (<b>a</b>) Sequencing data of targeted locus 1002–1072 in <i>pfatp4</i> from strain ACP-B6-L350H showing SJ733 resistance-conferring SNPs in L350 and four other synonymous mutations introduced by CRISPR. Sequences of wild type <i>pfatp4</i> and repair template ssODN L350H are shown in alignment. The two silent mutations in ssODN L350H located 39 and 42 nt away were not incorporated into ACP-B6-L350H. (<b>b</b>) Sequencing data of targeted locus 1206–1276 in <i>pfatp4</i> from strain ACP-B6-P412T showing the SJ733 resistance-conferring SNP and silent mutations introduced by CRISPR. Sequences of wild type <i>pfatp4</i> and repair template ssODN P412T are shown in alignment.</p

Strategy for introducing plasmid-free CRISPR/Cas9 edits to the <i>Plasmodium falciparum</i> gene <i>pfatp4</i>.

<p>Synchronized ring-stage parasites at 17% parasitemia in fresh donor RBCs were nucleofected with Cas9 protein, guide RNA, and template ssODN. Cultures were kept under drug pressure with 500 nM SJ733 starting on day two post transfection. After drug-resistant parasites emerged from culture, genomic DNA was isolated with standard phenol-chloroform extraction methods for library preparation. The presence and penetrance of the targeted CRISPR edits were confirmed using Sanger sequencing and whole genome NGS.</p

Plasmid-free CRISPR/Cas9 genome editing in <i>Plasmodium falciparum</i> confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733

<div><p>Genetic manipulation of the deadly malaria parasite <i>Plasmodium falciparum</i> remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite’s genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in <i>P</i>. <i>falciparum</i> without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN) repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in <i>P</i>. <i>falciparum</i> could greatly simplify future editing attempts regardless of the nuclease used or the delivery method.</p></div

Characterization of drug resistance.

<p>Dose-response curves and EC₅₀ values for the antimalarial SJ733 on the parent strain ACP-B6 and the mutants ACP-B6-L350H and ACP-B6-P412T. The growth inhibition assay was conducted by seeding synchronized ring-stage parasites from each strain at 0.8% parasitemia in media supplemented with SJ733 at concentrations ranging from 3.16 nM to 100 μM and allowing for growth over 72 hours. Parasites were fixed with 1% paraformaldehyde and stained with 50 nM YOYO-1. Final parasitemia was assessed by flow cytometry and values were normalized to DMSO-only controls. Values reported are mean ± standard error (n = 3). The inset shows parasitemia of each culture after 72 hours of growth in the presence of DMSO only.</p

Association Mapping and the Genomic Consequences of Selection in Sunflower

<div><p>The combination of large-scale population genomic analyses and trait-based mapping approaches has the potential to provide novel insights into the evolutionary history and genome organization of crop plants. Here, we describe the detailed genotypic and phenotypic analysis of a sunflower (<i>Helianthus annuus</i> L.) association mapping population that captures nearly 90% of the allelic diversity present within the cultivated sunflower germplasm collection. We used these data to characterize overall patterns of genomic diversity and to perform association analyses on plant architecture (i.e., branching) and flowering time, successfully identifying numerous associations underlying these agronomically and evolutionarily important traits. Overall, we found variable levels of linkage disequilibrium (LD) across the genome. In general, islands of elevated LD correspond to genomic regions underlying traits that are known to have been targeted by selection during the evolution of cultivated sunflower. In many cases, these regions also showed significantly elevated levels of differentiation between the two major sunflower breeding groups, consistent with the occurrence of divergence due to strong selection. One of these regions, which harbors a major branching locus, spans a surprisingly long genetic interval (ca. 25 cM), indicating the occurrence of an extended selective sweep in an otherwise recombinogenic interval.</p> </div

Graphical genotypes from linkage group 10.

<p>Graphical genotypes from linkage group 10 plotted against a heat map of the branching data sorted by average level of branching across all three locations. Unbranched plants (red) are to the left whereas highly branched plants (green) are to the right. Note that the scale of the y-axis scale changes based on marker density, and white squares in the phenotype heat map represent missing data in an individual location.</p

Heat map of linkage disequilibrium across the sunflower genome.

<p>Individual data points reflect squared allele frequency correlations (<i>r²</i>) for all possible pairs of polymorphic SNP markers, MAF ≥10%. The x- and y-axes correspond to the 17 linkage groups in sunflower with marker orders based on the work of Bowers et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003378#pgen.1003378-Bowers1" target="_blank">[21]</a>. Note that the values above and below the diagonal are identical.</p

Summary of significant flowering time (DTF) associations.

<p>For each location, the results for three different models (K, P+K, and Q+K) are presented (see text for details). Associations are named based on their linkage group. When multiple associations were detected on a single linkage group (LG 13), the associations are lettered in order of their map position. Cases in which more than one model within a location supported an association are underlined.</p

Manhattan and quantile–quantile plots of flowering time associations.

<p>(A, B, C) Upper panels: Manhattan plots of flowering (DTF) associations in three locations (GA, IA, BC respectively) plotted for the three models tested: red = K, blue = P+K, dark grey = Q3+K. The dashed line indicates the significance threshold based on the multiple testing correction method of Gao et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003378#pgen.1003378-Gao1" target="_blank">[35]</a> (alpha = 0.05, <i>P</i> = 0.00025, log 1/<i>P</i> = 3.60). Lower panels: Quantile-quantile plots of flowering time associations in all three locations plotted for the three models tested.</p