A Potent Cancer Vaccine Adjuvant System for Particleization of Short, Synthetic CD8+ T Cell Epitopes.

Short major histocompatibility complex (MHC) class I (MHC-I)-restricted peptides contain the minimal biochemical information to induce antigen (Ag)-specific CD8+ cytotoxic T cell responses but are generally ineffective in doing so. To address this, we developed a cobalt-porphyrin (CoPoP) liposome vaccine adjuvant system that induces rapid particleization of conventional, short synthetic MHC-I epitopes, leading to strong cellular immune responses at nanogram dosing. Along with CoPoP (to induce particle formation of peptides), synthetic monophosphoryl lipid A (PHAD) and QS-21 immunostimulatory molecules were included in the liposome bilayer to generate the "CPQ" adjuvant system. In mice, immunization with a short MHC-I-restricted peptide, derived from glycoprotein 70 (gp70), admixed with CPQ safely generated functional, Ag-specific CD8+ T cells, resulting in the rejection of multiple tumor cell lines, with durable immunity. When cobalt was omitted, the otherwise identical peptide and adjuvant components did not result in peptide binding and were incapable of inducing immune responses, demonstrating the importance of stable particle formation. Immunization with the liposomal vaccine was well-tolerated and could control local and metastatic disease in a therapeutic setting. Mechanistic studies showed that particle-based peptides were better taken up by antigen-presenting cells, where they were putatively released within endosomes and phagosomes for display on MHC-I surfaces. On the basis of the potency of the approach, the platform was demonstrated as a tool for in vivo epitope screening of peptide microlibraries comprising a hundred peptides.

Transmembrane Cu(I) P-type ATPase pumps are electrogenic uniporters.

Cu(i) P-type ATPases are transmembrane primary active ion pumps that catalyze the extrusion of copper ions across cellular membranes. Their activity is critical in controlling copper levels in all kingdoms of life. Biochemical and structural characterization established the structural framework by which Cu-pumps perform their function. However, the details of the overall mechanism of transport (uniporter vs. cotransporter) and electrogenicity still remain elusive. In this work, we developed a platform to reconstitute the model Cu(i)-pump from E. coli (EcCopA) in artificial lipid bilayer small unilamellar vesicles (SUVs) to quantitatively characterize the metal substrate, putative counter-ions and charge translocation. By encapsulating in the liposome lumen fluorescence detector probes (CTAP-3, pyranine and oxonol VI) responsive to diverse stimuli (Cu(i), pH and membrane potential), we correlated substrate, secondary-ion translocation and charge movement events in EcCopA proteoliposomes. This platform centered on multiple fluorescence reporters allowed study of the mechanism and translocation kinetic parameters in real-time for wild-type EcCopA and inactive mutants. The maximal initial Cu(i) transport rate of 165 nmol Cu(i) mg-1 min-1 and KM, Cu(I) = 0.15 ± 0.07 µM was determined with this analysis. We reveal that Cu(i) pumps are primary-active uniporters and electrogenic. The Cu(i) translocation cycle does not require proton counter-transport resulting in electrogenic generation of transmembrane potential upon translocation of one Cu(i) per ATP hydrolysis cycle. Thus, mechanistic differences between Cu(i) pumps and other better characterized P-type ATPases are discussed. The platform opens the venue to study translocation events and mechanisms of transport in other transition metal P-type ATPase pumps.

Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells.

Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2'-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.

Preorganized PSP Ligands Yield Monomeric Cu(I) Complexes with Subzeptomolar Cu(I) Dissociation Constants.

Unraveling the function of biological copper (Cu) requires tools that can selectively recognize and manipulate this trace nutrient within the complex chemical environment of biological systems. Increasing evidence suggests that cells maintain an exchangeable pool of Cu(I) that is buffered in the high zeptomolar to low attomolar range. While mixed amine-thioether donors have been commonly employed for the design of Cu(I)-selective ligands and probes, their dissociation constants are limited to the pico- to femtomolar range. To address this challenge, we combined our previously devised phosphine sulfide-stabilized phosphine donor motifs with a rigid 1,2-phenylene or 1,8-naphthylene ligand backbone. The resulting ligands, phenPS and naphPS, bind Cu(I) with a 1:1 complex stoichiometry and offer dissociation constants of 0.6 and 0.8 zM, respectively. Concluding from the crystal structures of the free and Cu(I)-bound ligands, the 1,2-phenylene-bridged ligand phenPS provides a high degree of structural preorganization to accommodate the Cu(I) center without large conformational changes, while the 1,8-naphthylene-bridged ligand revealed significant out-of-plane distortions in both the free and Cu(I)-bound states. Both ligands were accessed by palladium-catalyzed cross-coupling reactions from the corresponding arylhalides under mild conditions, an approach that could be readily expanded toward the design of other ligands and probes.

Stabilization of Aliphatic Phosphines by Auxiliary Phosphine Sulfides Offers Zeptomolar Affinity and Unprecedented Selectivity for Probing Biological CuI.

Full elucidation of the functions and homeostatic pathways of biological copper requires tools that can selectively recognize and manipulate this trace nutrient within living cells and tissues, where it exists primarily as CuI . Buffered at attomolar concentrations, intracellular CuI is, however, not readily accessible to commonly employed amine and thioether-based chelators. Herein, we reveal a chelator design strategy in which phosphine sulfides aid in CuI coordination while simultaneously stabilizing aliphatic phosphine donors, producing a charge-neutral ligand with low-zeptomolar dissociation constant and 1017 -fold selectivity for CuI over ZnII , FeII , and MnII . As illustrated by reversing ATP7A trafficking in cells and blocking long-term potentiation of neurons in mouse hippocampal brain tissue, the ligand is capable of intercepting copper-dependent processes. The phosphine sulfide-stabilized phosphine (PSP) design approach, which confers resistance towards protonation, dioxygen, and disulfides, could be readily expanded towards ligands and probes with tailored properties for exploring CuI in a broad range of biological systems.

Chromis-1, a Ratiometric Fluorescent Probe Optimized for Two-Photon Microscopy Reveals Dynamic Changes in Labile Zn(II) in Differentiating Oligodendrocytes.

Despite the significant advantages of two-photon excitation microscopy (TPEM) over traditional confocal fluorescence microscopy in live-cell imaging applications, including reduced phototoxicity and photobleaching, increased depth penetration, and minimized autofluorescence, only a few metal ion-selective fluorescent probes have been designed and optimized specifically for this technique. Building upon a donor-acceptor fluorophore architecture, we developed a membrane-permeant, Zn(II)-selective fluorescent probe, chromis-1, that exhibits a balanced two-photon cross section between its free and Zn(II)-bound form and responds with a large spectral shift suitable for emission-ratiometric imaging. With a Kd of 1.5 nM and wide dynamic range, the probe is well suited for visualizing temporal changes in buffered Zn(II) levels in live cells as demonstrated with mouse fibroblast cell cultures. Moreover, given the importance of zinc in the physiology and pathophysiology of the brain, we employed chromis-1 to monitor cytoplasmic concentrations of labile Zn(II) in oligodendrocytes, an important cellular constituent of the brain, at different stages of development in cell culture. These studies revealed a decrease in probe saturation upon differentiation to mature oligodendrocytes, implying significant changes to cellular zinc homeostasis during maturation with an overall reduction in cellular zinc availability. Optimized for TPEM, chromis-1 is especially well-suited for exploring the role of labile zinc pools in live cells under a broad range of physiological and pathological conditions.

Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster.

The tripeptide glutathione (GSH) is a crucial intracellular reductant and radical scavenger, but it may also coordinate the soft Cu(I) cation and thereby yield pro-oxidant species. The GSH-Cu(I) interaction is thus a key consideration for both redox and copper homeostasis in cells. However, even after nearly four decades of investigation, the nature and stability of the GSH-Cu(I) complexes formed under biologically relevant conditions remain controversial. Here, we revealed the unexpected predominance of a tetranuclear [Cu4(GS)6] cluster that is sufficiently stable to limit the effective free aquacopper(I) concentration to the sub-femtomolar regime. Combined spectrophotometric-potentiometric titrations at biologically realistic GSH/Cu(I) ratios, enabled by our recently developed Cu(I) affinity standards and corroborated by low-temperature phosphorescence studies, established cooperative assembly of [Cu4(GS)6] as the dominant species over a wide pH range, from 5.5 to 7.5. Our robust model for the glutathione-Cu(I) equilibrium system sets a firm upper limit on the thermodynamic availability of intracellular copper that is 3 orders of magnitude lower than previously estimated. Taking into account their ability to catalyze the production of deleterious superoxide, the formation of Cu(I)-glutathione complexes might be avoided under normal physiological conditions. The actual intracellular Cu(I) availability may thus be regulated a further 3 orders of magnitude below the GSH/Cu(I) affinity limit, consistent with the most recent affinity determinations of Cu(I) chaperones.

Probing ternary complex equilibria of crown ether ligands by time-resolved fluorescence spectroscopy.

Ternary complex formation with solvent molecules and other adventitious ligands may compromise the performance of metal-ion-selective fluorescent probes. As Ca(II) can accommodate more than 6 donors in the first coordination sphere, commonly used crown ether ligands are prone to ternary complex formation with this cation. The steric strain imposed by auxiliary ligands, however, may result in an ensemble of rapidly equilibrating coordination species with varying degrees of interaction between the cation and the specific donor atoms mediating the fluorescence response, thus diminishing the change in fluorescence properties upon Ca(II) binding. To explore the influence of ligand architecture on these equilibria, we tethered two structurally distinct aza-15-crown-5 ligands to pyrazoline fluorophores as reporters. Due to ultrafast photoinduced electron-transfer (PET) quenching of the fluorophore by the ligand moiety, the fluorescence decay profile directly reflects the species composition in the ground state. By adjusting the PET driving force through electronic tuning of the pyrazoline fluorophores, we were able to differentiate between species with only subtle variations in PET donor abilities. Concluding from a global analysis of the corresponding fluorescence decay profiles, the coordination species composition was indeed strongly dependent on the ligand architecture. Altogether, the combination of time-resolved fluorescence spectroscopy with selective tuning of the PET driving force represents an effective analytical tool to study dynamic coordination equilibria and thus to optimize ligand architectures for the design of high-contrast cation-responsive fluorescence switches.

Robust affinity standards for Cu(I) biochemistry.

The measurement of reliable Cu(I) protein binding affinities requires competing reference ligands with similar binding strengths; however, the literature on such reference ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-soluble monovalent copper ligands, MCL-1, MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu(I) in aqueous solution. X-ray structural data, electrochemical measurements, and an extensive network of equilibrium titrations showed that all three ligands form discrete Cu(I) complexes with 1:1 stoichiometry and are capable of buffering Cu(I) concentrations between 10(-10) and 10(-17) M. As most Cu(I) protein affinities have been obtained from competition experiments with bathocuproine disulfonate or 2,2'-bicinchoninic acid, we further calibrated their Cu(I) stability constants against the MCL series. To demonstrate the application of these reagents, we determined the Cu(I) binding affinity of CusF (log K = 14.3 ± 0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in Escherichia coli . Altogether, this interconnected set of affinity standards establishes a reliable foundation that will facilitate the precise determination of Cu(I) binding affinities of proteins and small-molecule ligands.

High-contrast fluorescence sensing of aqueous Cu(I) with triarylpyrazoline probes: dissecting the roles of ligand donor strength and excited state proton transfer.

Cu(I)-responsive fluorescent probes based on a photoinduced electron transfer (PET) mechanism generally show incomplete fluorescence recovery relative to the intrinsic quantum yield of the fluorescence reporter. Previous studies on probes with an N-aryl thiazacrown Cu(I)-receptor revealed that the recovery is compromised by incomplete Cu(I)-N coordination and resultant ternary complex formation with solvent molecules. Building upon a strategy that successfully increased the fluorescence contrast and quantum yield of Cu(I) probes in methanol, we integrated the arylamine PET donor into the backbone of a hydrophilic thiazacrown ligand with a sulfonated triarylpyrazoline as a water-soluble fluorescence reporter. This approach was not only expected to disfavor ternary complex formation in aqueous solution but also to maximize PET switching through a synergistic Cu(I)-induced conformational change. The resulting water-soluble probe 1 gave a strong 57-fold fluorescence enhancement upon saturation with Cu(I) with high selectivity over other cations, including Cu(II), Hg(II), and Cd(II); however, the recovery quantum yield did not improve over probes with the original N-aryl thiazacrown design. Concluding from detailed photophysical data, including responses to acidification, solvent isotope effects, quantum yields, and time-resolved fluorescence decay profiles, the fluorescence contrast of 1 is compromised by inadequate coordination of Cu(I) to the weakly basic arylamine nitrogen of the PET donor and by fluorescence quenching via two distinct excited state proton transfer pathways operating under neutral and acidic conditions.

Designed to dissolve: suppression of colloidal aggregation of Cu(I)-selective fluorescent probes in aqueous buffer and in-gel detection of a metallochaperone.

Due to the lipophilicity of the metal-ion receptor, previously reported Cu(I)-selective fluorescent probes form colloidal aggregates, as revealed by dynamic light scattering. To address this problem, we have developed a hydrophilic triarylpyrazoline-based fluorescent probe, CTAP-2, that dissolves directly in water and shows a rapid, reversible, and highly selective 65-fold fluorescence turn-on response to Cu(I) in aqueous solution. CTAP-2 proved to be sufficiently sensitive for direct in-gel detection of Cu(I) bound to the metallochaperone Atox1, demonstrating the potential for cation-selective fluorescent probes to serve as tools in metalloproteomics for identifying proteins with readily accessible metal-binding sites.

Electronically tuned 1,3,5-triarylpyrazolines as Cu(I)-selective fluorescent probes.

We have prepared and characterized a Cu(i)-responsive fluorescent probe, constructed using a large tetradentate, 16-membered thiazacrown ligand ([16]aneNS(3)) and 1,3,5-triaryl-substituted pyrazoline fluorophores. The fluorescence contrast ratio upon analyte binding, which is mainly governed by changes of the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 50-fold fluorescence enhancement upon saturation with Cu(i) in methanol and a greater than 300-fold enhancement upon protonation with trifluoroacetic acid. The observed fluorescence increase was selective towards Cu(i) over a broad range of mono- and divalent transition metal cations. Previously established Hammett LFERs proved to be a valuable tool to predict two of the PET key parameters, the acceptor potential (E(A/A(-)) and the excited state energy DeltaE(00), and thus to identify a set of pyrazolines that would best match the thermodynamic requirements imposed by the donor potential E(D(+)/D) of the thiazacrown receptor. The described approach should be applicable for rationally designing high-contrast pyrazoline-based PET probes selective towards other metal cations.

Kinetically controlled photoinduced electron transfer switching in Cu(I)-responsive fluorescent probes.

Copper(I)-responsive fluorescent probes based on photoinduced electron transfer (PET) switching consistently display incomplete recovery of emission upon Cu(I) binding compared to the corresponding isolated fluorophores, raising the question of whether Cu(I) might engage in adverse quenching pathways. To address this question, we performed detailed photophysical studies on a series of Cu(I)-responsive fluorescent probes that are based on a 16-membered thiazacrown receptor ([16]aneNS(3)) tethered to 1,3,5-triarylpyrazoline-fluorophores. The fluorescence enhancement upon Cu(I) binding, which is mainly governed by changes in the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 29-fold fluorescence enhancement upon saturation with Cu(I) in methanol and a greater than 500-fold enhancement upon protonation with trifluoroacetic acid. Time-resolved fluorescence decay data for the Cu(I)-saturated probe indicated the presence of three distinct emissive species in methanol. Contrary to the notion that Cu(I) might engage in reductive electron transfer quenching, femtosecond time-resolved pump-probe experiments provided no evidence for formation of a transient Cu(II) species upon photoexcitation. Variable temperature (1)H NMR experiments revealed a dynamic equilibrium between the tetradentate NS(3)-coordinated Cu(I) complex and a ternary complex involving coordination of a solvent molecule, an observation that was further supported by quantum chemical calculations. The combined photophysical, electrochemical, and solution chemistry experiments demonstrate that electron transfer from Cu(I) does not compete with radiative deactivation of the excited fluorophore, and, hence, that the Cu(I)-induced fluorescence switching is kinetically controlled.