Total RNA-seq identifies pharmacological effects on specific stages of mRNA synthesis
Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis. Boswell SA, Snavely A, Landry HM, Churchman LS, Gray JM, Springer M. Nat Chem Biol. 2017 Mar 6. doi: 10.1038/nchembio.2317. [Epub ahead of print] PMID:28263964
- Abstract: Pharmacological perturbation is a powerful tool for understanding mRNA synthesis, but identification of the specific steps of this multi-step process that are targeted by small molecules remains challenging. Here we applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. We found unexpectedly that the natural product isoginkgetin, previously described as a splicing inhibitor, inhibits transcription elongation. Compared to well-characterized elongation inhibitors that target CDK9, isoginkgetin caused RNA polymerase accumulation within a broader promoter-proximal band, indicating that elongation inhibition by isoginkgetin occurs after release from promoter-proximal pause. RNA-seq distinguished isoginkgetin and CDK9 inhibitors from topoisomerase I inhibition, which alters elongation across gene bodies. We were able to detect these and other specific defects in mRNA synthesis at low sequencing depth using simple metagene-based metrics. These metrics now enable total-RNA-seq-based screening for high-throughput identification of pharmacological effects on individual stages of mRNA synthesis.
Cellular context matters: lessons from synovial fbroblasts and rheumatoid arthritis
Profiling drugs for rheumatoid arthritis that inhibit synovial fibroblast activation. Jones DS, Jenney AP, Swantek JL, Burke JM, Lauffenburger DA, Sorger PK. Nat Chem Biol. 2017 Jan;13(1):38-45. doi: 10.1038/nchembio.2211. PMID:27820799
- Abstract: Activation of synovial fibroblasts (SFs) contributes to rheumatoid arthritis (RA) by damaging synovial membranes and generating inflammatory cytokines that recruit immune cells to the joint. In this paper we profile cytokine secretion by primary human SFs from healthy tissues and from donors with RA and show that SF activation by TNF, IL-1α, and polyinosinic-polycytidylic acid (Poly(I:C)) cause secretion of multiple cytokines found at high levels in RA synovial fluids. We used interaction multiple linear regression to quantify therapeutic and countertherapeutic drug effects across activators and donors and found that the ability of drugs to block SF activation was strongly dependent on the identity of the activating cytokine. (5z)-7-oxozeaenol (5ZO), a preclinical drug that targets transforming growth factor-β-activated kinase 1 (TAK1), was more effective at blocking SF activation across all contexts than the approved drug tofacitinib, which supports the development of molecules similar to 5ZO for use as RA therapeutics.
Novel inhibitors of TAK1, a potential therapeutic target for the treatment of immune diseases and cancer
Studies of TAK1-centered polypharmacology with novel covalent TAK1 inhibitors. Tan L, Gurbani D, Weisberg EL, Jones DS, Rao S, Singer WD, Bernard FM, Mowafy S, Jenney A, Du G, Nonami A, Griffin JD, Lauffenburger DA, Westover KD, Sorger PK, Gray NS. Bioorg Med Chem. 2017 Feb 15;25(4):1320-1328. doi: 10.1016/j.bmc.2016.11.034. PMID:28038940
- Abstract: Targeted polypharmacology provides an efficient method of treating diseases such as cancer with complex, multigenic causes provided that compounds with advantageous activity profiles can be discovered. Novel covalent TAK1 inhibitors were validated in cellular contexts for their ability to inhibit the TAK1 kinase and for their polypharmacology. Several inhibitors phenocopied reported TAK1 inhibitor 5Z-7-oxozaenol with comparable efficacy and complementary kinase selectivity profiles. Compound 5 exhibited the greatest potency in RAS-mutated and wild-type RAS cell lines from various cancer types. A biotinylated derivative of 5, 27, was used to verify TAK1 binding in cells. The newly described inhibitors constitute useful tools for further development of multi-targeting TAK1-centered inhibitors for cancer and other diseases.
Highly multiplexed imaging at the single cell level
Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single-cell Imaging. Lin JR, Fallahi-Sichani M, Chen JY, Sorger PK. Curr Protoc Chem Biol. 2016 Dec 7;8(4):251-264. doi: 10.1002/cpch.14. PMID:27925668
- Abstract: Cyclic Immunofluorescence (CycIF) is a public-domain method for performing highly multiplexed immunofluorescence imaging using a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4- to 6-channel imaging followed by fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells with antibodies directly conjugated to Alexa Fluor dyes and imaging in four colors, inactivating fluorophores using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging. Cell morphology is preserved through multiple rounds of CycIF, and signal-to-noise ratios appear to increase. Unlike antibody-stripping methods, CycIF is gentle and optimized for monolayers of cultured cells. A second protocol involves indirect immunofluorescence and a third enables chemical inactivation of genetically encoded fluorescent proteins, allowing multiplex immunofluorescence to be combined with live-cell analysis of cells expressing fluorescent reporter proteins.
A better way to quantify sensitivity of different cells to cancer drugs
Growth rate inhibition metrics correct for confounders in measuring sensitivity to cancer drugs. Hafner M, Niepel M, Chung M, Sorger PK. Nat Methods. 2016 Jun;13(6):521-7. doi: 10.1038/nmeth.3853. PMID:27135972
- Abstract: Drug sensitivity and resistance are conventionally quantified by IC50 or Emax values, but these metrics are highly sensitive to the number of divisions taking place over the course of a response assay. The dependency of IC50 and Emax on division rate creates artefactual correlations between genotype and drug sensitivity, while obscuring valuable biological insights and interfering with biomarker discovery. We derive alternative small molecule drug-response metrics that are insensitive to division number. These are based on estimation of the magnitude of drug-induced growth rate inhibition (GR) using endpoint or time-course assays. We show that GR50 and GRmax are superior to conventional metrics for assessing the effects of small molecule drugs in dividing cells. Moreover, adopting GR metrics requires only modest changes in experimental protocols. We expect GR metrics to improve the study of cell signaling and growth using small molecules and biologics and to facilitate the discovery of drug-response biomarkers and the identification of drugs effective against specific patient-derived tumor cells.