Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein, thus repressing translation. This mechanism is supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.
Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that acts as a ligand for five G-protein coupled receptors (S1P1–5) whose downstream effects are implicated in a variety of important pathologies including sickle cell disease, cancer, inflammation, and fibrosis. The synthesis of S1P is catalyzed by sphingosine kinase (SphK) isoforms 1 and 2, and hence, inhibitors of this phosphorylation step are pivotal in understanding the physiological functions of SphKs. To date, SphK1 and 2 inhibitors with the potency, selectivity, and in vivo stability necessary to determine the potential of these kinases as therapeutic targets are lacking. Herein, we report the design, synthesis, and structure–activity relationship studies of guanidine-based SphK inhibitors bearing an oxadiazole ring in the scaffold. Our studies demonstrate that SLP120701, a SphK2-selective inhibitor (Ki = 1 μM), decreases S1P levels in histiocytic lymphoma (U937) cells. Surprisingly, homologation with a single methylene unit between the oxadiazole and heterocyclic ring afforded a SphK1-selective inhibitor in SLP7111228 (Ki = 48 nM), which also decreased S1P levels in cultured U937 cells. In vivo application of both compounds, however, resulted in contrasting effect in circulating levels of S1P. Administration of SLP7111228 depressed blood S1P levels while SLP120701 increased levels of S1P. Taken together, these compounds provide an in vivo chemical toolkit to interrogate the effect of increasing or decreasing S1P levels and whether such a maneuver can have implications in disease states.
The bioactive lipid lysophosphatidate (LPA) is produced mainly by the secreted enzyme autotaxin (ATX) ( 1-3 ). The ATX gene is among the 40 most upregulated genes in metastatic cancers ( 4 ). LPA signals through at least six G-proteincoupled receptors to increase cell division, survival, migration, and angiogenesis ( 1-3 ). Overexpression of ATX, LPA 1 , LPA 2 , or LPA 3 receptors in mammary cells causes spontaneous development of mammary tumors in mice ( 5 ). Women with breast carcinomas that express high levels of LPA 3 receptors in cancer epithelial cells, or ATX in stromal cells, have larger tumors, nodal involvement, and higher stage disease ( 6 ). LPA produces resistance to the cytotoxic effects of paclitaxel ( 2, 7-9 ), carboplatin ( 10 ), and radiation-induced cell death ( 2,11,12 ). Inhibiting ATX activity and lowering LPA concentrations in plasma and tumors decreases the initial phase of breast tumor growth and subsequent lung metastasis by ف 60% in a mouse model ( 13 ). These combined results provide strong evidence that increased LPA signaling in cancer cells promotes the growth and metastasis of breast tumors.The present studies focus on a different approach to attenuating LPA signaling. This involves increasing the expression of lipid phosphate phosphatase-1 (LPP1), which is a member of a family of three phosphatases that dephosphorylate bioactive lipid phosphates and pyrophosphates ( 14-16 ). Despite this broad specifi city for lipid phosphates that is observed with cell-free systems, changing the expression of the different LPPs in animal models produces different
Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of therapeutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative modifications at the C(5)- and C(6)- positions yielded derivative 24, which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift mapping was performed using the 2D SOFAST- [1H-13C] HMQC NMR method, which allowed for facile and rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to allow unprecedented evaluation of small molecule:RNA interactions.
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