Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery.

Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes [1][2][3][4][5][6][7][8][9] . Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore-microtubule attachment using quantitative fluorescence microscopy of GFPtagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone 1 , the copy number ranges from one or two for inner kinetochore proteins such as Mif2p 2 , to 16 for the 9 at the kinetochoremicrotubule interface. These counts allow us to visualize the overall arrangement of a kinetochoremicrotubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments.Accurate segregation of sister chromosomes during mitosis depends on the assembly of structural proteins at the kinetochore that link spindle microtubule plus-ends to centromeric DNA (CEN DNA). The structural arrangement of these proteins within the kinetochore underlies its function in force generation. It may also influence how the spindle assembly checkpoint senses kinetochore-microtubule attachment, and how errors in attachment are corrected to prevent chromosome mis-segregation. Although serial-section transmission electron microscopy has revealed the overall three-dimensional architecture of vertebrate kinetochores, the structure of individual kinetochore-microtubule attachment remains poorly characterized. Consequently, a mechanistic model of kinetochore function that integrates the details of its structure cannot currently be constructed. To understand the molecular architecture of a kinetochore-microtubule attachment site, we focused on counting the copy number for the core structural kinetochore proteins and protein complexes that are necessary for stable kinetochore-microtubule attachment. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Localization of antibodies to Ndc80 in vertebrate cells suggests that the Ndc80p-Nuf2p end of the NDC80 complex localizes proximal to the microtubule attachment site, whereas the other end localizes proximal to the inner centromere7 , 15. In budding yeast, the NDC80 complex and the microtubule associated protein complex, DAM-DASH, are both necessary for microtubule attachment10 , 11. The DAM-DASH complex is a heterodecamer and contains the protein Ask1p. Purified DAM-DASH complexes assemble into rings aro...

The C loop conformation reveals the structural basis for sister-kinetochore clustering in budding yeast and for kinetochore biorientation and thus resolves the paradox of maximal interstrand separation in regions of highest cohesin concentration.

Summary During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.

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