Here, we demonstrate proof-of-principle for a novel and inexpensive assay for ALK inhibitor activity and identification in zebrafish. We demonstrate that the human oncogenic ALK fusion, NPM-ALK, drives overproduction of iridophores, a highly visible, shiny pigment cell-type in zebrafish. Treatment with the potent ALK inhibitor, TAE684, fully inhibits production Pacritinib FLT3 of ALK-dependent iridophores. Using our assay, we test multiple properties of TAE684 in vivo, including efficacy, specificity, and toxicity. We note that TAE684 also inhibits the closely related leukocyte tyrosine kinase (Ltk) that is required for endogenous iridophore development. Similar Inhibitors,Modulators,Libraries effects are observed with an independent inhibitor, Crizotinib. Our assay can thus be utilized to identify ALK or LTK inhibitors.

Importantly, the natural reflectivity of iridophores lends itself to automation for high throughput assessment of ALK and LTK inhibitor compounds in vivo.
Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions, including lymphocyte trafficking and vascular development, by activating Inhibitors,Modulators,Libraries G protein-coupled receptors for S1P, namely, S1P(1) through S1P(5). Here, we map the S1P(3) binding pocket with a novel allosteric agonist (CYM-5541), an orthosteric agonist (S1P), and a novel bitopic antagonist (SPM-242). With a combination of site-directed mutagenesis, ligand competition assay, and molecular modeling, we concluded that S1P and CYM-5541 occupy different chemical Inhibitors,Modulators,Libraries spaces in the ligand binding pocket of S1P(3).

CYM-5541 allowed us to identify an allosteric site where Phe263 is a key gate-keeper residue for its affinity and efficacy. This ligand lacks a polar moiety, and the novel allosteric hydrophobic pocket permits S1P(3) selectivity of CYM-5541 within the highly similar S1P receptor family. However, a Inhibitors,Modulators,Libraries novel S1P(3)-selective antagonist, SPM-242, in the S1P(3) pocket occupies the ligand binding spaces of both SIP and CYM-5541, showing its bitopic mode of binding. Therefore, our coordinated approach with biochemical data and molecular GSK-3 modeling, based on our recently published S1P(1) crystal structure data in a highly conserved set of related receptors with a shared ligand, provides a strong basis for the successful optimization of orthosteric, allosteric, and bitopic modulators of S1P(3).

Myotonic dystrophy type selleck chem 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)(exp)) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6′-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)(exp). However, these parent compounds are not appreciably active in cell-based models of DM1.