Fluorescence Cross-correlation Spectroscopy Research Articles

Combining Orthogonal Reactive Groups in Block Copolymers for Functional Nanoparticle Synthesis in a Single Step.

We report on the synthesis of polysarcosine-block-poly(S-alkylsulfonyl)-l-cysteine block copolymers, which combine three orthogonal addressable groups enabling site-specific conversion of all reactive entities in a single step. The polymers are readily obtained by ring-opening polymerization (ROP) of corresponding α-amino acid N-carboxyanhydrides (NCAs) combining azide and amine chain ends, with a thiol-reactive S-alkylsulfonyl cysteine. Functional group interconversion of chain ends using strain-promoted azide-alkyne cycloaddition (SPAAC) and activated ester chemistry with NHS- and DBCO-containing fluorescent dyes could be readily performed without affecting the cross-linking reaction between thiols and S-alkylsulfonyl protective groups. Eventually, all three functionalities can be combined in the formation of multifunctional disulfide core cross-linked nanoparticles bearing spatially separated functionalities. The simultaneous attachment of dyes in core and corona during the formation of core-cross-linked nanostructures with controlled morphology is confirmed by fluorescence cross-correlation spectroscopy (FCCS).

Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

Ribonucleoprotein purification and characterization using RNA Mango.

The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.

Lateral Organization of Host Heterogeneous Raft‐like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c‐Src

AbstractMembrane‐bound c‐Src non‐receptor tyrosine kinase, unlike other acyl‐modified lipid‐anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale‐clusters upon membrane binding of the Myr‐Src (2–9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr‐Src (2–9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.

Lateral Organization of Host Heterogeneous Raft-like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c-Src.

Membrane-bound c-Src non-receptor tyrosine kinase, unlike other acyl-modified lipid-anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale-clusters upon membrane binding of the Myr-Src (2-9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr-Src (2-9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.

Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes for apoptosis regulation

The Bcl-2 proteins form a complex interaction network that controls mitochondrial permeabilization and apoptosis. The relative importance of different Bcl-2 complexes and their spatio-temporal regulation is debated. Using fluorescence cross-correlation spectroscopy to quantify the interactions within a minimal Bcl-2 network, comprised by cBid, Bax, and Bcl-xL, we show that membrane insertion drastically alters the pattern of Bcl-2 complexes, and that the C-terminal helix of Bcl-xL determines its binding preferences. At physiological temperature, Bax can spontaneously activate in a self-amplifying process. Strikingly, Bax also recruits Bcl-xL to membranes, which is sufficient to retrotranslocate Bax back into solution to secure membrane integrity. Our study disentangles the hierarchy of Bcl-2 complex formation in relation to their environment: Bcl-xL association with cBid occurs in solution and in membranes, where the complex is stabilized, whereas Bcl-xL binding to Bax occurs only in membranes and with lower affinity than to cBid, leading instead to Bax retrotranslocation.

Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy

Fluorescence correlation spectroscopy has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners, however, comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding, and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color fluorescence cross-correlation spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants, KD. The total protein concentration, but not the fraction of labeled protein, needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds “major-minor-mix” liposomes. A Langmuir isotherm model yields KD=(2.1±1.1)μM as the single-site dissociation constant. The dcFCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.

A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell

Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid response element (GRE), to activate a specific gene as a homodimer. It is still a puzzle, however, whether GRα forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GRα, we constructed the spectrally different fluorescent protein tagged hGRα and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (Kd) of mCherry2-fused hGRα or EGFP-fused hGRα was determined in vitro. Then, Kd of wild-type hGRα was found to be 3.00 μM in the nucleus, which was higher than that in vitro. Kd of a DNA-binding-deficient mutant was 3.51 μM in the nucleus. This similarity indicated that GRα homodimerization was not necessary for DNA binding but could take place on GRE by means of GRE as a scaffold. Moreover, cytoplasmic homodimerization was also observed using GRα mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRα function both in the cytoplasm and nucleus.

Adjusting the size of multicompartmental containers made of anionic liposomes and polycations by introducing branching and PEO moieties

We propose a strategy to adjust the size of multiliposomal complexes made of small unilamellar anionic liposomes (lipid bilayer vesicles prepared from zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC and ionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine POPS with a typical diameter of 50 nm) with cationic polymers due to electrostatic attraction. Hereby, the use of quaternized poly(dimethylaminoethyl methacrylate), qPDMAEMA, allows the construction of multicompartmental carriers. Variation of the architecture and composition of various qPDMAEMA samples demonstrates the effect of introducing poly(ethylene oxide) PEO moieties and/or branching on the size of the hierarchical assemblies: both the PEO block (as seen for block copolymers and miktoarm stars) and the branching (as seen for star-shaped homopolymers and miktoarm stars) lead to smaller complexes compared to complexes with a linear qPDMAEMA homopolymer. Besides the use of cryogenic transmission electron microscopy (cryo-TEM), the formation of multicompartmental carriers composed of differently loaded liposomes is verified by two-color fluorescence cross-correlation spectroscopy (FCCS).

Fluorescence and Scattering Light Cross Correlation Spectroscopy and Its Applications in Homogeneous Immunoassay.

In this work, we propose fluorescence and scattering light cross-correlation spectroscopy (FSCCS) based on laser confocal configuration using silver nanoparticle (SNPs) and Alexa Fluor 488 (Alexa) as probe pairs. FSCCS is a single molecule (particle) method, and its principle is similar to that of fluorescence cross-correlation spectroscopy (FCCS). We established the setup of FSCCS using single wavelength laser and developed an immunoassay model of FSCCS. The reliability and adaptability of FSCCS method were evaluated by homogeneous sandwich immunoassay mode. In the study, liver cancer biomarker alpha-fetoprotein (AFP) was used as an assay model, two different antibodies were labeled with SNPs and fluorophore Alexa Fluor 488, respectively. In the optimal conditions, the linear range of AFP covers 5 pM to 580 pM and the detection limit is 3.1 pM. This method was successfully applied for direct determination of AFP levels in human serum samples, and the obtained results were in good agreement with data obtained via ELISAs. The advantage of this method lies in its simplicity, attractive SNPs probes, high sensitivity and selectivity and high efficiency. We believe that FSCCS method exhibits promising potential applications in homogeneous bioassays and study on the molecular interaction and nanoparticle-molecule interaction.

Membrane microdomains and the cytoskeleton constrain AtHIR1 dynamics and facilitate the formation of an AtHIR1‐associated immune complex

Arabidopsis hypersensitive-induced reaction (AtHIR) proteins function in plant innate immunity. However, the underlying mechanisms by which AtHIRs participate in plant immunity remain elusive. Here, using VA-TIRFM and FLIM-FRET, we revealed that AtHIR1 is present in membrane microdomains and co-localizes with the membrane microdomain marker REM1.3. Single-particle tracking analysis revealed that membrane microdomains and the cytoskeleton, especially microtubules, restrict the lateral mobility of AtHIR1 at the plasma membrane and facilitate its oligomerization. Furthermore, protein proximity index measurements, fluorescence cross-correlation spectroscopy, and biochemical experiments demonstrated that the formation of the AtHIR1 complex upon pathogen perception requires intact microdomains and cytoskeleton. Taken together, these findings suggest that microdomains and the cytoskeleton constrain AtHIR1 dynamics, promote AtHIR1 oligomerization, and increase the efficiency of the interactions of AtHIR1 with components of the AtHIR1 complex in response to pathogens, thus providing valuable insight into the mechanisms of defense-related responses in plants.

Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.

Enzyme-Loaded Polyion Complex Vesicles as in Vivo Nanoreactors Working Sustainably under the Blood Circulation: Characterization and Functional Evaluation.

Enzyme-loaded synthetic vesicles have attracted great attention for their feasibility to exert the efficient and prolonged functionality of loaded enzymes in harsh environments, such as in vivo. However, several issues remain regarding the optimization of their structures toward practical application. Herein, we fabricated polyion complex vesicles (PICsomes) loaded with l-asparaginase (ASNase@PICsomes) and conducted a detailed characterization to ensure their utility as nanoreactors functioning under the harsh in vivo environment of the bloodstream. ASNase@PICsomes showed 100 nm-sized monodispersed vesicular structures. Fluorescence cross-correlation spectroscopy revealed essentially no empty PICsome fraction in the product, indicating the quantitative formation of ASNase@PICsomes. Furthermore, fluorescence anisotropy measurement showed that the loaded enzymes were located essentially in the inner aqueous phase of PICsomes, being successfully segregated from the external environment. ASNase@PICsomes exhibited significantly prolonged enzymatic reaction compared with free ASNase after systemic injection into mice, corroborating their functionality as in vivo nanoreactors working under the blood circulation.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Forster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

Membrane Binding and Remodelling by the COPII Coat

Dynamic remodelling of cellular membranes in conjunction with protein complex assembly and disassembly constitutes a key component of intracellular trafficking pathways. Reconstitution of selected steps outside the cell using artificial membranes facilitates the use of biophysical techniques for investigating structure and dynamics of protein-lipid assemblies. COPII-coated membrane vesicles are responsible for transporting cargo from the ER towards the Golgi apparatus in the secretory pathway. The small GTPase Sar1, which belongs to the Sar1/Arf family of GTPases and the Ras-superfamily, is an essential component in COPII-vesicle formation. Upon activation by GTP, Sar1 binds to membranes, embedding an amphipathic helix into the proximal leaflet of the bilayer. The role of GTP presence versus GTP hydrolysis in the fission of COPII vesicles is not completely clear. We study COPII vesicle formation in a bottom-up fashion on baker's yeast proteins and model membranes using cryo-electron microscopy techniques (cryo-EM), confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS). Cryo-EM shows strongly different COPII coated vesicle morphologies under GTP hydrolyzing versus non-hydrolyzing conditions. More subtle differences are observed by cryo-EM among reconstituted COPII samples under non-hydrolyzing conditions. Membrane binding of the small GTPases Sar1 and Arf1 is studied with biophysical techniques: By combining FCS with a Langmuir film balance, the protein's footprint in the proximal membrane leaflet is revealed. Dual-color fluorescence cross-correlation spectroscopy (FCCS) analysis has been developed into a method for obtaining binding curves and testing binding models of protein binding to freely diffusing vesicles. The higher specificity makes fluorescence cross-correlation advantageous compared to previous fluorescence autocorrelation analysis also for small vesicle binding studies.

Crowding Agents Direct Amyloid Beta into Membrane-Active Oligomers

Oligomeric Amyloid-β (1-42) peptides are considered to be the most toxic species connected to the occurrence of Alzheimer disease. However, not all aggregation conditions promote oligomer formation in vitro, raising the question whether oligomer formation in vivo also requires a specific suitable cellular environment. In this study we find that the presence of osmolytes such as glucose and sucrose Aβ (1-42) strongly favors oligomer formation. We characterized oligomers by dynamic light scattering, atomic force microscopy, immuno transmission electron microscopy and fluorescence cross correlation spectroscopy techniques. We found that Aβ (1-42) spontaneously and rapidly forms low molecular weight oligomers in the presence of sugars. Slightly acidic pH (6.7-7) greatly favors oligomer formation when compared to the extracellular physiological pH (7.4). Circular dichroism demonstrated that in presence of crowding agents Aβ (1-42) oligomers did not adopt a β-sheet structure, whereas in the absence of these agents Aβ (1-42) formed β-sheets. Unstructured oligomeric Aβ (1-42) interacted with membrane bilayers of giant unilamellar vesicles (GUV). We recently found that interaction with neuronal membranes initiates aggregation of Aβ (1-42) and neuronal uptake. Our data suggest that crowding agents in the extracellular space facilitate the formation of membrane-active Aβ (1-42) oligomers.

In Vitro Binding of 6S RNA Mango to RNA Polymerase by Two Photon Fluorescence Cross Correlation Spectroscopy

The assembly of RNA and protein complexes is a fundamental process for life but has been difficult to study both in vitro and in vivo. Recently a novel aptamer based method to fluorescently label RNA molecules has been reported(Dolgosheina et al. 2014). Here we report the successful utilization of the RNA Mango + thiazole orange derivative to measure the in vitro binding of 6S RNA to RNA Polymerase from E. coli. By two photon Fluorescence Cross Correlation Spectroscopy, we are able to independently measure the molecular brightness, diffusion coefficient and concentration of 6S RNA Mango, RNA Polymerase GFP and complex as we increase RNA Polymerase concentration giving us an estimate for the equilibrium binding constant of the complex in vitro. This experiment demonstrates the utility of RNA Mango for both in vitro and in vivo single molecule experiments.Dolgosheina, Elena V., Sunny C. Y. Jeng, Shanker Shyam S. Panchapakesan, Razvan Cojocaru, Patrick S. K. Chen, Peter D. Wilson, Nancy Hawkins, Paul A. Wiggins, and Peter J. Unrau. 2014. “RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking.” ACS Chemical Biology 9 (10): 2412-20.

Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.

Membrane proteins sense extracellular cues and transduce intracellular signaling to coordinate directionality and speed during cellular migration. They are often localized to specific regions, as with lipid rafts or tetraspanin-enriched microdomains; however, the dynamic interactions of tetraspanins with diverse receptors within tetraspanin-enriched microdomains on cellular surfaces remain largely unexplored. Here, we investigated effects of tetraspan(in) TM4SF5 (transmembrane 4 L6 family member 5)-enriched microdomains (T5ERMs) on the directionality of cell migration. Physical association of TM4SF5 with epidermal growth factor receptor (EGFR) and integrin α5 was visualized by live fluorescence cross-correlation spectroscopy and higher-resolution microscopy at the leading edge of migratory cells, presumably forming TM4SF5-enriched microdomains. Whereas TM4SF5 and EGFR colocalized at the migrating leading region more than at the rear, TM4SF5 and integrin α5 colocalized evenly throughout cells. Cholesterol depletion and disruption in TM4SF5 post-translational modifications, including N-glycosylation and palmitoylation, altered TM4SF5 interactions and cellular localization, which led to less cellular migration speed and directionality in 2- or 3-dimensional conditions. TM4SF5 controlled directional cell migration and invasion, and importantly, these TM4SF5 functions were dependent on cholesterol, TM4SF5 post-translational modifications, and EGFR and integrin α5 activity. Altogether, we showed that TM4SF5 dynamically interacted with EGFR and integrin α5 in migratory cells to control directionality and invasion.-Kim, H.-J., Kwon, S., Nam, S. H., Jung, J. W., Kang, M., Ryu, J., Kim, J. E., Cheong, J.-G., Cho, C. Y., Kim, S., Song, D.-G., Kim, Y.-N., Kim, T. Y., Jung, M.-K., Lee, K.-M., Pack, C.-G., Lee, J. W. Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.

Correlated fluorescence blinking in two-dimensional semiconductor heterostructures.

'Blinking', or 'fluorescence intermittency', refers to a random switching between 'ON' (bright) and 'OFF' (dark) states of an emitter; it has been studied widely in zero-dimensional quantum dots and molecules, and scarcely in one-dimensional systems. A generally accepted mechanism for blinking in quantum dots involves random switching between neutral and charged states (or is accompanied by fluctuations in charge-carrier traps), which substantially alters the dynamics of radiative and non-radiative decay. Here, we uncover a new type of blinking effect in vertically stacked, two-dimensional semiconductor heterostructures, which consist of two distinct monolayers of transition metal dichalcogenides (TMDs) that are weakly coupled by van der Waals forces. Unlike zero-dimensional or one-dimensional systems, two-dimensional TMD heterostructures show a correlated blinking effect, comprising randomly switching bright, neutral and dark states. Fluorescence cross-correlation spectroscopy analyses show that a bright state occurring in one monolayer will simultaneously lead to a dark state in the other monolayer, owing to an intermittent interlayer carrier-transfer process. Our findings suggest that bilayer van der Waals heterostructures provide unique platforms for the study of charge-transfer dynamics and non-equilibrium-state physics, and could see application as correlated light emitters in quantum technology.

XPO1 Target Occupancy Measurements Using Fluorescence Cross Correlation Spectroscopy Support the Selinexor Recommended Phase 2 Dose

Introduction:Selective Inhibitor of Nuclear Export (SINE) compounds are a new class of oral anti-cancers drugs that block nuclear export by inhibiting the exportin XPO1 (also known as CRM1). The most advanced compounds in this class are selinexor and KPT-8602, which are being evaluated in clinical trials for the treatment of hematological malignancies (clinicaltrials.gov). XPO1 is commonly overexpressed in cancer and high levels of XPO1 are correlated with poor prognosis. Based on Phase I efficacy and safety studies, the recommended Phase 2 dose (RP2D) of selinexor is 60 mg twice weekly on days 1 and 3. We have previously reported the development of a pharmacodynamics (PD) assay to measure XPO1 target occupancy by selinexor in cancer cells based on Fluorescence Cross Correlation Spectroscopy (FCCS). Here we report proof of concept studies to determine the level of XPO1 occupancy associated with the selinexor RP2D regimen.Methods:Z138 cells in culture were treated with 1 µM selinexor either continuously for up to 32 hours or for 6 hours followed by drug removal and culture for up to 72 hours post-wash out. Cell were treated in culture for 4 hours with either 0-10 µM selinexor, KPT-8602, or Leptomycin B (LMB). Cells were harvested, lysed, and cleared by ultracentrifugation. Total protein was measured from lysates for Western blot with α-XPO1, α-GAPDH, and α-β-tubulin and for FCCS analysis. In vivo, nude mice were inoculated in both flanks with Z138 cells. Once tumors reached approximately 250 mm3, mice were administered a single dose of 0, 5, 10, 15, or 20 mg/kg selinexor. Tumors were harvested at 6, 24, 48, 72, and 96 hours post-dose, lysed, and cleared by ultracentrifugation. Cell line or tumor supernatants were incubated with a fluorescently labeled antibody (Ab) recognizing XPO1 and fluorescently labeled LMB. To allow binding of labeled Ab and LMB to XPO1, samples were measured by FCCS after 15, 120, and 1200 minutes.Results:XPO1 protein dynamics were evaluated in Z138 cells treated with selinexor either continuously or after drug removal. Selinexor treatment resulted in the reduction of XPO1 protein by 26% as early as 4 hours of treatment and reduced XPO1 levels by 93% with continuous drug exposure up to 32 hours. However, cells treated with selinexor for only 6 hours followed by drug washout showed nearly full restoration of XPO1 expression by 72 hours, indicating that continuous presence of drug is required to sustain reduced XPO1 protein levels. Measurements by FCCS showed that brief exposure to selinexor (6 hours) resulted in rapid XPO1 occupancy, and drug washout resulted in sustained occupancy that was not relieved until 72 hours post-selinexor wash out. To determine whether the second generation SINE compound clinical candidate, KPT-8602, had a similar XPO1 occupancy profile as selinexor, a panel of hematologic cell lines were treated with either compound and analyzed by FCCS. FCCS was also measured from cells treated with LMB, a natural product inhibitor of XPO1. FCCS analysis confirmed that KPT-8602 treatment resulted in a dose-dependent increase in drug-target occupancy with increasing drug concentration similar to that of selinexor, while LMB showed complete occupancy at very low levels of compound. Lastly, to confirm the level of target engagement in the RP2D regimen for selinexor, drug-target occupancy was measured in Z138 xenograft tumors from mice treated with a single oral dose of 0, 5, 10, 15, or 20 mg/kg selinexor. Tumors were collected at 6, 24, 48, 72, and 96 hours post-single dose. The FCCS results showed that full occupancy occurred by 6 hours at all doses, but the duration of occupancy was dose-dependent. XPO1 occupancy was relieved by 24 hours at 5 and 10 mg/kg, 48 hours at 15 mg/kg, and 72 hours at 20 mg/kg selinexor. This finding supports the RP2D dosing regimen, where 10-15 mg/kg in mice (approximately equivalent to 50-75 mg flat dose in humans) is necessary to maintain XPO1 occupancy for up to 48 hours post-dose, thus requiring the twice weekly dosing for continuous target occupancy.Conclusions:Using an FCCS based PD assay developed to measure the dynamics of XPO1 target occupancy by selinexor, our studies support the selinexor RP2D dosing regimen for patients with heavily pretreated hematologic and solid tumors. Future studies are planned to measure XPO1 occupancy in CD138+ cells isolated from patients with advanced multiple myeloma following treatment with selinexor or KPT-8602. DisclosuresCrochiere:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Hannus:Intana Bioscience GmbH: Consultancy. Hansen:Intanat Bioscience GmbH: Consultancy. Becker:Intana Bioscience GmbH: Consultancy. Baloglu:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder.