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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The human pregnane X receptor (hPXR) regulates the expression of critical drug metabolism enzymes. One of such enzymes, cytochrome P450 3A4 (CYP3A4), plays critical roles in drug metabolism in hepatocytes that are either quiescent or passing through the cell cycle. It has been well established that the expression of P450, such as CYP3A4, is markedly reduced during liver development or regeneration. Numerous studies have implicated cellular signaling pathways in modulating the functions of nuclear receptors, including hPXR. Here we report that inhibition of cyclin-dependent kinases (Cdks) by kenpaullone and roscovitine (two small molecule inhibitors of Cdks that we identified in a screen for compounds that activate hPXR) leads to activation of hPXR-mediated CYP3A4 gene expression in HepG2 human liver carcinoma cells. Consistent with this finding, activation of Cdk2 attenuates the activation of CYP3A4 gene expression. In vitro kinase assays revealed that Cdk2 directly phosphorylates hPXR. A phosphomimetic mutation of a putative Cdk phosphorylation site, Ser 350 , significantly impairs the function of hPXR, whereas a phosphorylation-deficient mutation confers resistance to Cdk2. Using HepG2 that has been stably transfected with hPXR and the CYP3A4-luciferase reporter, enriched in different phases of the cell cycle, we found that hPXR-mediated CYP3A4 expression is greatly reduced in the S phase. Our results indicate for the first time that Cdk2 negatively regulates the activity of hPXR, and suggest an important role for Cdk2 in regulating hPXR activity and CYP3A4 expression in hepatocytes passing through the cell cycle, such as those in fetal or regenerating adult liver.

The increase in spindle length upon histone repression and restoration of wild-type spindle length by the loss of plus-end-directed motors suggests that during metaphase, centromere separation and spindle length are governed in part by the stretching of pericentric chromatin. Chromatin is an elastic molecule that is stretched in direct opposition to the outward force generators Cin8p and Kip1p. Thus, we assign a new role to chromatin packaging as an integral biophysical component of the mitotic apparatus.

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G 2 -M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G 2 phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924. Cancer Res; 73(1); 225-34. Ó2012 AACR.