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.

The p53 pathway is disrupted in virtually every human tumor. In ϳ50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. Tumorigenesis is a multistep process that involves dysregulation of several pathways that are crucial for cell growth and survival (1). The p53 pathway regulates cell survival in response to cellular stress (e.g. DNA damage) or oncogenic stress (e.g. Rb pathway dysregulation) (2, 3) and is suppressed in virtually every human cancer by genetic lesions in the p53 gene or other components of the pathway (4). Approximately half of all cancers express wild-type p53, and considerable research over the past decade has focused on inducing p53-mediated cell death in these tumors (4, 5). Most efforts to date have focused on inhibiting MDM2, a negative regulator of p53 (6 -14).Another key regulator of the p53 pathway is a protein related to MDM2 called MDMX (15-17). MDM2 and MDMX share homology in their p53-binding domains, but MDMX is believed to regulate p53 through distinct mechanisms. Specifically, MDM2 primarily regulates p53 stability and subcellular localization, whereas MDMX may directly regulate p53 transcription (17-21). MDMX is genetically amplified in 19% of breast carcinomas, 19% of colon carcinomas, 18% of lung carcinomas, and a smaller percentage of gliomas (17). One of the best characterized tumors with an MDMX amplification is retinoblastoma. Approximately 65% of human retinoblastomas have increased MDMX copy number, which correlates with increased MDMX mRNA and protein (22). Previous studies have demonstrated that the MDMX amplification suppresses p53-mediated cell death in Rb pathway-deficient retinoblasts (22).A general consensus is emerging that to efficiently induce a p53 response in tumor cells that express wild-type p53, it may be necessary to inactivate both MDM2 and MDMX (18,23,24). To date, no screens to identify small molecule inhibitors of MDMX have been reported, and MDM2 inhibitors probably do not bind as efficiently to MDMX because of structural differences in the p53-binding pockets of the two proteins (25-27). Consistent with this theory, nutlin-3a binds MDMX with at least a 40-fold weaker equilibrium binding constant than for MDM2 (22). Therefore, to identify small molecules that bind MDMX and prevent its interaction with p53, we developed biochemical and cell-based assays suitable for high throughput screening (HTS)

The development of an automated, high-throughput fractionation procedure to prepare and analyze natural product libraries for drug discovery screening is described. Natural products obtained from plant materials worldwide were extracted and first prefractionated on polyamide solid-phase extraction cartridges to remove polyphenols, followed by high-throughput automated fractionation, drying, weighing, and reformatting for screening and storage. The analysis of fractions with UPLC coupled with MS, PDA and ELSD detectors provides information that facilitates characterization of compounds in active fractions. Screening of a portion of fractions yielded multiple assay-specific hits in several high-throughput cellular screening assays. This procedure modernizes the traditional natural product fractionation paradigm by seamlessly integrating automation, informatics, and multimodal analytical interrogation capabilities.Natural products are a vast resource of compounds with seemingly unlimited chemical and functional diversity, and have been a rich source for lead molecules in drug discovery programs. 1-4 Sixty percent of new drugs for cancer and 75% of those for infectious diseases have originated from natural sources. 5,6 Between 2001 and 2005, 23 natural product based drugs were launched in Europe, Japan, and the United States for treating various disorders such as cancer, diabetes, dyslipidemia, atopic dermatitis, Alzheimer's disease, bacterial and fungal infections, genetic diseases such as tyrosinemia, and Gaucher's disease. 7 However, during the last two decades, research efforts in the discovery of therapeutic natural products have waned because of the complications and significant time requirements inherent in compound isolation. Primary screening of crude plant extracts or microbial fermentations, followed by bioassay-guided fractionation, purification, and structure elucidation of novel bioactive compounds can take several months. 8 The required scale of isolation has been too large to be implemented effectively in an automated, high-throughput fashion. The combination of these and other factors has led to a lagging emphasis in natural product discovery. However, recent advances in high-throughput screening (HTS) technology have