Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ∼3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function.DOI: http://dx.doi.org/10.7554/eLife.07369.001

Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far-and near-UV circular dichroism spectroscopy, onedimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) mimetic that induces the same “closed” conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.

SUMMARY Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, “S1QELs”). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQ is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.

SignificanceThe ReFRAME collection of 12,000 compounds is a best-in-class drug repurposing library containing nearly all small molecules that have reached clinical development or undergone significant preclinical profiling. The purpose of such a screening collection is to enable rapid testing of compounds with demonstrated safety profiles in new indications, such as neglected or rare diseases, where there is less commercial motivation for expensive research and development. Providing the academic and nonprofit research community access to a high-value compound collection and related screening data in an open-access platform should provide new tool compounds for biomedical research, as well as accelerate drug-discovery and/or development programs aimed at developing new therapies for diverse unmet medical needs.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species (ROS), which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies but its role remains controversial. Using high-throughput screening we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

The objective of this study was to evaluate the suitability of the WW domain as a desirable model system to understand the folding and stability of an isolated three-stranded antiparallel b-sheet structure. The WW domain was subjected to thermal and chaotropic denaturation0reconstitution utilizing a variety of biophysical methods. This three-stranded sheet folds reversibly and cooperatively utilizing both urea and GdnHCl as denaturants; however, the denatured state retains structure in the form of a hydrophobic cluster involving at least one aromatic side chain. In contrast to chaotropic denaturation, thermal denaturation appears to be more complete and may be a two state process. The suitability of the WW domain for future studies aimed at understanding the kinetics and thermodynamics of antiparallel b-sheet folding clearly emerges from this initial study. The most exciting and significant result in this manuscript is the finding that the chaotropic denatured state of WW has a hydrophobic cluster as discerned by near-UV CD evidence. The role that the denatured state plays in the folding and stability of a three-stranded b-sheets, and its capacity for preventing aggregation may be particularly important and is the subject of ongoing studies.Keywords: b-sheet folding; hydrophobic cluster; reversible folding; WW The WW domain is a recently discovered protein module consisting of a three-stranded antiparallel b-sheet structure that is named for the two highly conserved Trp residues separated in the sequence by

Starting with the published 2.0 Å X-ray crystal structure of the transthyretin‚(flufenamic acid) 2 complex, a simple structure-based ligand design strategy was employed to conceive of N-phenyl phenoxazine transthyretin (TTR) amyloid fibril inhibitors. Fifteen N-phenyl phenoxazines were chemically synthesized and evaluated using a quantitative amyloid fibril assay in vitro. The structure of one of the two most active phenoxazines, 4, bound to TTR was solved to a resolution of 1.9 Å to understand the structural basis of its efficacy. N-phenyl phenoxazine 4 binds similar to the orientation anticipated, although not as deeply into the channel as expected. Like flufenamic acid, 4 mediates binding-induced conformational changes that enable intersubunit H-bonding in tetrameric TTR which may be important for preventing fibril formation. Analytical ultracentrifugation analysis demonstrates that 4 blocks the first step of TTR amyloid fibril formation, that is, tetramer dissociation to the alternatively folded amyloidogenic monomer. Isothermal titration calorimetry was used to determine the binding constants of 4 to TTR and to dissect the enthalpy and entropy contributions associated with ligand binding. Phenoxazine 4 exhibits binding and inhibitor efficacy against WT TTR that is very similar to that of flufenamic acid, unlike the situation with the inhibition of L55P fibril formation where 4 is superior to Flu as an inhibitor but not as a binder. It is clear that 4 functions in part by stabilizing the normally folded tetramer through formation of the TTR‚(4) 2 complex, which in turn increases the activation energy for tetramer dissociation. The data also suggest that 4 destabilizes the transition state associated with TTR dissociation to the monomeric amyloidogenic intermediate. Future biophysical studies, including kinetic measurements, are needed to understand the exact mechanism(s) of the action of 4.