Project description:Combination of reverse- and chemical genetic screens reveals a network of novel angiogenesis inhibitors and targets Drug target identification and validation are bottlenecks in the drug discovery process. Accordingly there is a need to develop new methods to facilitate the development of much-needed innovative drugs. We have combined reverse- and chemical genetics to identify new targets modulating blood vessel development. Through mRNA expression profiling in mice we identified 155 drugable gene products that were enriched in the microvasculature. Orthologs of 50 of these candidates were knocked down in a reverse genetic screen in zebrafish. 16 of the 50 genes encoded products that affected angiogenesis. In parallel, screening of 300 known drugs and pharmacologically active compounds in a human cell-based angiogenesis assay identified 11 angiogenesis inhibitors. Strikingly the reverse- and chemical genetic screens identified an overlap of three gene products of the same superfamily of serine/threonine protein phosphatases and two compounds targeting that family. Furthermore, the gene products identified in the reverse genetic screen comprise an interacting network with the targets of the chemical genetic screen. Thus, combining reverse- and chemical genetic screens is a powerful approach to identify novel biological processes and drug targets in vertebrates. Keywords: Cell-type comparison 24 samples were analyzed, representing 8 sample groups. In every sample group, three biological replicates were hybridized separately. The microarrays were hybridized with a Cy3-labeled sample and Cy-5 labeled Common Reference (Universal Mouse Reference RNA, cat. No.: 740100, Stratagene) simultaneously.
Project description:Combination of reverse- and chemical genetic screens reveals a network of novel angiogenesis inhibitors and targets Drug target identification and validation are bottlenecks in the drug discovery process. Accordingly there is a need to develop new methods to facilitate the development of much-needed innovative drugs. We have combined reverse- and chemical genetics to identify new targets modulating blood vessel development. Through mRNA expression profiling in mice we identified 155 drugable gene products that were enriched in the microvasculature. Orthologs of 50 of these candidates were knocked down in a reverse genetic screen in zebrafish. 16 of the 50 genes encoded products that affected angiogenesis. In parallel, screening of 300 known drugs and pharmacologically active compounds in a human cell-based angiogenesis assay identified 11 angiogenesis inhibitors. Strikingly the reverse- and chemical genetic screens identified an overlap of three gene products of the same superfamily of serine/threonine protein phosphatases and two compounds targeting that family. Furthermore, the gene products identified in the reverse genetic screen comprise an interacting network with the targets of the chemical genetic screen. Thus, combining reverse- and chemical genetic screens is a powerful approach to identify novel biological processes and drug targets in vertebrates. Keywords: Cell-type comparison
Project description:Alzheimer’s disease (AD) drug discovery has rarely been addressed in the context of aging even though sporadic AD accounts for 99% of the cases. Previous phenotypic screens based upon old age-associated brain toxicities identified the potent AD drug candidates CMS121 and J147, whose mechanisms were investigated through an integrative multi-omics approach (PMID: 31742554; GSE101112). This study aimed to further reveal drug J147's role involved in liver metabolism during disease progression.
Project description:CRISPR knockout screens have accelerated the discovery of important cancer genetic dependencies. However, traditional CRISPR-Cas9 screens are limited in their ability to assay the function of redundant or duplicated genes. Paralogs in multi-gene families constitute two-thirds of the protein-coding genome, so this blind spot is the rule, not the exception. To overcome the limitations of single gene CRISPR knockout screens, we developed paired guide RNAs for Paralog gENetic interaction mapping (pgPEN), a pooled CRISPR/Cas9 approach which targets over a thousand duplicated human paralogs in single knockout and double knockout configurations. We applied pgPEN to two cell lineages and discovered that over 10% of human paralogs exhibit synthetic lethality in at least one cellular context. We recovered known synthetic lethal paralogs such as MAP2K1/MAP2K2, important drug targets such as CDK4/CDK6, and numerous other synthetic lethal pairs such as CCNL1/CCNL2. In addition, we identified ten tumor suppressive paralog pairs whose compound loss promotes cell growth. These findings identify a large number of previously unidentified essential gene families and nominate new druggable targets for oncology drug discovery.
Project description:iJN1462 (i) incorporates several hundred additional genes and associated reactions resulting in new predictive capabilities, including new nutrients supporting growth; (ii) was validated by in vivo growth screens that included previously untested carbon (48) and nitrogen (41) sources; (iii) yielded gene essentiality predictions showing large accuracy when compared with a knock-out library and Bar-seq data; and (iv) allowed mapping of its network to 82 P. putida sequenced strains revealing functional core that reflect the large metabolic versatility of this species, including aromatic compounds derived from lignin.
Project description:Cardiovascular disease remains a significant global health concern, yet the cardiotoxic effects of environmental chemicals are largely unexplored. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-Cardiomyocytes) are well-established as valuable high-throughput cardiotoxicity testing model based on the analysis of various beating parameters; however, additional adverse effects that may not be directly related to ion channel activity are difficult to ascertain from traditional assays. Transcriptomic data may provide a more comprehensive assessment for additional molecular responses and serve as a basis for dose-response analysis. We hypothesized that transcriptomic data and functional phenotypic measurements in iPSC-Cardiomyocytes could be used to effectively screen for both hazard and risk associated with chemical exposures. To test this hypothesis, we performed concentration-response analysis of 464 chemicals from 12 diverse chemical classes. Functional effects (beat frequency, QT-prolongation, and asystole) and cytotoxicity were measured. In addition, whole transcriptome RNA-seq was used to investigate global gene expression profiles. Points of departure (PODs) were derived from both phenotypic endpoints and transcriptomic data. Margin of safety (MOS) for drugs and margin of exposure (MOE) for non-drug chemicals were calculated using PODs and the exposure estimates. The phenotypic assays revealed that a varying proportion (10% to 44%) of chemicals in each class had effects on iPSC-Cardiomyocytes; positive chronotrope was the most sensitive phenotype. Overall, 244 (53%) chemicals were active in at least one functional phenotype. As expected, drugs with known adverse effects on the heart were most active. Transcriptomic data showed 69 (15%) had significant effects on the biological pathways, the pathways that were significantly affected by some tested compounds were highly relevant to cardiomyocyte function. Specifically, the enriched pathways aligned well with key characteristics of known cardiovascular toxicants, with mitochondrial dysfunction being the most affected key characteristic. Using exposure and hazard (transcriptomic and phenotypic PODs from this study), we derived MOS and MOE and found that drugs tend to have lower MOS than MOE for the environmental chemicals. Thirteen and 10 drugs had MOS < 1 using phenotypic and transcriptomic data, respectively, suggesting that further investigation may be needed for these substances to test for potential cardiovascular risk. Overall, our findings demonstrate how the integrative use of in vitro transcriptomic data and phenotypic assays in iPSC-Cardiomyocytes not only offers a unique and complementary approach for hazard and risk prioritization, but also offers comprehensive mechanistic support for in vitro test results.