A miniaturized mode-of-action profiling platform enables high throughput characterization of the molecular and cellular dynamics of EZH2 inhibition.

The market approval of Tazemetostat (TAZVERIK) for the treatment of follicular lymphoma and epithelioid sarcoma has established "enhancer of zeste homolog 2" (EZH2) as therapeutic target in oncology. Despite their structural similarities and common mode of inhibition, Tazemetostat and other EZH2 inhibitors display differentiated pharmacological profiles based on their target residence time. Here we established high throughput screening methods based on time-resolved fluorescence energy transfer, scintillation proximity and high content analysis microscopy to quantify the biochemical and cellular binding of a chemically diverse collection of EZH2 inhibitors. These assays allowed to further characterize the interplay between EZH2 allosteric modulation by methylated histone tails (H3K27me3) and inhibitor binding, and to evaluate the impact of EZH2's clinically relevant mutant Y641N on drug target residence times. While all compounds in this study exhibited slower off-rates, those with clinical candidate status display significantly slower target residence times in wild type EZH2 and disease-related mutants. These inhibitors interact in a more entropy-driven fashion and show the most persistent effects in cellular washout and antiproliferative efficacy experiments. Our work provides mechanistic insights for the largest cohort of EZH2 inhibitors reported to date, demonstrating that-among several other binding parameters-target residence time is the best predictor of cellular efficacy.

Novel Class of Potent and Cellularly Active Inhibitors Devalidates MTH1 as Broad-Spectrum Cancer Target.

MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.

Direct visualization of newly synthesized target proteins in situ.

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.

Correlative light- and electron microscopy with chemical tags.

Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.

Role of N-cadherin cis and trans interfaces in the dynamics of adherens junctions in living cells.

Cadherins, Ca(2+)-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca(2+)-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca(2+)-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca(2+)-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.

Single cysteines in the extracellular and transmembrane regions modulate pannexin 1 channel function.

Pannexins form high-conductance ion channels in the membranes of many vertebrate cells. Functionally, they have been associated with multiple functional pathways like the propagation of calcium waves, ATP release, responses to ischemic conditions and apoptosis. In contrast to accumulating details which uncovered their functions, the molecular mechanisms for pannexin channel regulation and activation are hardly understood. To further elucidate regulatory mechanisms, we substituted cysteine residues, expected key elements for channel function, in extracellular and transmembrane regions of Pannexin 1 (Panx1). Most apparently, substitution of the transmembrane cysteine C40 resulted in constitutively open channels with profoundly increased activity. Hence, Xenopus laevis oocytes injected with corresponding cRNA showed strongly impaired viability, anomalous dye uptake and greatly increased whole-cell conductivity. All changes induced by C40 substitution were significantly reduced by the Panx1 channel blocker carbenoxolone, indicating that channel activity of the mutated Panx1 had been affected. In contrast, no changes occurred after substitution of the two other transmembrane cysteines, C215 and C227, in terms of channel conductivity. Finally, substitution of any of the four extracellular cysteines resulted in complete loss of channel function in both X. laevis oocytes and transfected N2A cells. From this, we conclude that cysteine residues of Panx1 reveal differential functional profiles for channel activation and drug sensitivity.

Intracellular cysteine 346 is essentially involved in regulating Panx1 channel activity.

Pannexins constitute a family of proteins exhibiting predominantly hemichannel activity. Pannexin channels have been suggested to participate in a wide spectrum of biological functions such as propagation of calcium waves, release of IL-1ß, and responses to ischemic conditions. At present, the molecular mechanisms regulating pannexin hemichannel activity are essentially unknown. Because cysteines have been shown to constitute key elements in regulating hemichannel properties of the connexin-type we performed site-directed mutagenesis of intracellular cysteine residues of Panx1. Cysteine to serine exchange (Cys → Ser) at the C-terminal position amino acid 346 led to a constitutively leaky hemichannel and subsequently to cell death. Increased channel activity was demonstrated by dye uptake and electrophysiological profiling in injected Xenopus laevis oocytes and transfected N2A cells. Mutations of the remaining intracellular cysteines did not result in major changes of Panx1 channel properties. From these data we conclude that the Cys-346 residue is important for proper functioning of the Panx1 channel.

The potassium channel subunit Kvbeta3 interacts with pannexin 1 and attenuates its sensitivity to changes in redox potentials.

Pannexin 1 (Panx1), a member of the second gap junction protein family identified in vertebrates, appears to preferentially form non-junctional membrane channels. A candidate regulatory protein of Panx1 is the potassium channel subunit Kvbeta3, previously identified by bacterial two-hybrid strategies. Here, we report on the physical association of Panx1 with Kvbeta3 by immunoprecipitation when co-expressed in a neuroblastoma cell line (Neuro2A). Furthermore, in vivo co-expression of Panx1 and Kvbeta3 was shown to occur in murine hippocampus and cerebellum. Kvbeta3 is known to accelerate inactivation of otherwise slowly inactivating potassium channels under reducing conditions. We subsequently found that Panx1 channel currents exhibit a significant reduction when exposed to reducing agents, and that this effect is attenuated in the presence of Kvbeta3. Apparently, Kvbeta3 is involved in regulating the susceptibility of Panx1 channels to redox potential. Furthermore, the Panx1 channel blockers carbenoxolone and Probenecid were less effective in inhibiting Panx1 currents when Kvbeta3 was co-expressed. The influence of Kvbeta3 on Panx1 is the first example of modulation of Panx1 channel function(s) by interacting proteins, and suggests the physiological importance of sensing changes in redox potentials.

Molecular diversity of connexin and pannexin genes in the retina of the zebrafish Danio rerio.

Gap junctions are among the most widely distributed cell structures involved in cell-to-cell communication. Recently completed genome sequencing projects including species from all major phyla have demonstrated the existence of three distinct gene families, the connexins, pannexins, and innexins, as molecular building blocks of gap junctional communication. In the present study, the authors have addressed the molecular complexity of gap junction gene expression in the zebrafish retina, a remarkably complex sensory organ built by diverse neuronal subtypes. Using a combination of cDNA library and genomic DNA library screening and/or RACE technology, the authors have cloned, in addition to the four previously reported connexins, seven novel connexins and four pannexin transcripts resembling two pannexin genes. This result demonstrates the presence of two distinct gap junction type gene families and indicates a remarkable molecular and functional diversity of gap junction-mediated coupling in the fish retina.

Identification of a potential regulator of the gap junction protein pannexin1.

Recent studies have revealed a second class of gap-junction-forming proteins in vertebrates. These genes are termed pannexins, and it has been suggested that they perform similar functions as connexins. Pannexin1 is expressed in diverse tissues including the central nervous system and seems to form gap junction channels in the Xenopus oocyte expression system. Since protein interacting partners have frequently been described for connexins, the most prominent family of gap junction forming proteins, we thus started to search for candidate genes of pannexin interacting partners. Kvbeta3, a protein belonging to the family of regulatory beta-subunits of the voltage-dependent potassium channels, was identified as a binding partner of pannexin1 in an E. coli two-hybrid system. This result was verified by confocal laser scanning microscopy using double transfected Neuro2A cells. The colocalization of both proteins at the plasma membrane is suggestive of functional interaction.