Biochemistry of mobile zinc and nitric oxide revealed by fluorescent sensors.

Biological mobile zinc and nitric oxide (NO) are two prominent examples of inorganic compounds involved in numerous signaling pathways in living systems. In the past decade, a synergy of regulation, signaling, and translocation of these two species has emerged in several areas of human physiology, providing additional incentive for developing adequate detection systems for Zn(II) ions and NO in biological specimens. Fluorescent probes for both of these bioinorganic analytes provide excellent tools for their detection, with high spatial and temporal resolution. We review the most widely used fluorescent sensors for biological zinc and nitric oxide, together with promising new developments and unmet needs of contemporary Zn(II) and NO biological imaging. The interplay between zinc and nitric oxide in the nervous, cardiovascular, and immune systems is highlighted to illustrate the contributions of selective fluorescent probes to the study of these two important bioinorganic analytes.

Aneuploidy increases resistance to chemotherapeutics by antagonizing cell division.

Aneuploidy, defined as whole chromosome gains and losses, is associated with poor patient prognosis in many cancer types. However, the condition causes cellular stress and cell cycle delays, foremost in G1 and S phase. Here, we investigate how aneuploidy causes both slow proliferation and poor disease outcome. We test the hypothesis that aneuploidy brings about resistance to chemotherapies because of a general feature of the aneuploid condition-G1 delays. We show that single chromosome gains lead to increased resistance to the frontline chemotherapeutics cisplatin and paclitaxel. Furthermore, G1 cell cycle delays are sufficient to increase chemotherapeutic resistance in euploid cells. Mechanistically, G1 delays increase drug resistance to cisplatin and paclitaxel by reducing their ability to damage DNA and microtubules, respectively. Finally, we show that our findings are clinically relevant. Aneuploidy correlates with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that a general and seemingly detrimental effect of aneuploidy, slowed proliferation, provides a selective benefit to cancer cells during chemotherapy treatment.

Native Zinc Catalyzes Selective and Traceless Release of Small Molecules in ß-Cells.

The loss of insulin-producing ß-cells is the central pathological event in type 1 and 2 diabetes, which has led to efforts to identify molecules to promote ß-cell proliferation, protection, and imaging. However, the lack of ß-cell specificity of these molecules jeopardizes their therapeutic potential. A general platform for selective release of small-molecule cargoes in ß-cells over other islet cells ex vivo or other cell-types in an organismal context will be immensely valuable in advancing diabetes research and therapeutic development. Here, we leverage the unusually high Zn(II) concentration in ß-cells to develop a Zn(II)-based prodrug system to selectively and tracelessly deliver bioactive small molecules and fluorophores to ß-cells. The Zn(II)-targeting mechanism enriches the inactive cargo in ß-cells as compared to other pancreatic cells; importantly, Zn(II)-mediated hydrolysis triggers cargo activation. This prodrug system, with modular components that allow for fine-tuning selectivity, should enable the safer and more effective targeting of ß-cells.

Repair shielding of platinum-DNA lesions in testicular germ cell tumors by high-mobility group box protein 4 imparts cisplatin hypersensitivity.

Cisplatin is the most commonly used anticancer drug for the treatment of testicular germ cell tumors (TGCTs). The hypersensitivity of TGCTs to cisplatin is a subject of widespread interest. Here, we show that high-mobility group box protein 4 (HMGB4), a protein preferentially expressed in testes, uniquely blocks excision repair of cisplatin-DNA adducts, 1,2-intrastrand cross-links, to potentiate the sensitivity of TGCTs to cisplatin therapy. We used CRISPR/Cas9-mediated gene editing to knockout the HMGB4 gene in a testicular human embryonic carcinoma and examined cellular responses. We find that loss of HMGB4 elicits resistance to cisplatin as evidenced by cell proliferation and apoptosis assays. We demonstrate that HMGB4 specifically inhibits repair of the major cisplatin-DNA adducts in TGCT cells by using the human TGCT excision repair system. Our findings also reveal characteristic HMGB4-dependent differences in cell cycle progression following cisplatin treatment. Collectively, these data provide convincing evidence that HMGB4 plays a major role in sensitizing TGCTs to cisplatin, consistent with shielding of platinum-DNA adducts from excision repair.

Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration.

Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn2+) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn2+ accumulation in amacrine cell processes involves the Zn2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn2+ chelation extends for several days after nerve injury. These results show that retinal Zn2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

DNA Intercalation Facilitates Efficient DNA-Targeted Covalent Binding of Phenanthriplatin.

Phenanthriplatin, a monofunctional anticancer agent derived from cisplatin, shows significantly more rapid DNA covalent-binding activity compared to its parent complex. To understand the underlying molecular mechanism, we used single-molecule studies with optical tweezers to probe the kinetics of DNA-phenanthriplatin binding as well as DNA binding to several control complexes. The time-dependent extensions of single λ-DNA molecules were monitored at constant applied forces and compound concentrations, followed by rinsing with a compound-free solution. DNA-phenanthriplatin association consisted of fast and reversible DNA lengthening with time constant τ ≈ 10 s, followed by slow and irreversible DNA elongation that reached equilibrium in ∼30 min. In contrast, only reversible fast DNA elongation occured for its stereoisomer  trans-phenanthriplatin, suggesting that the distinct two-rate kinetics of phenanthriplatin is sensitive to the geometric conformation of the complex. Furthermore, no DNA unwinding was observed for pyriplatin, in which the phenanthridine ligand of phenanthriplatin is replaced by the smaller pyridine molecule, indicating that the size of the aromatic group is responsible for the rapid DNA elongation. These findings suggest that the mechanism of binding of phenanthriplatin to DNA involves rapid, partial intercalation of the phenanthridine ring followed by slower substitution of the adjacent chloride ligand by, most likely, the N7 atom of a purine base. The cis isomer affords the proper stereochemistry at the metal center to facilitate essentially irreversible DNA covalent binding, a geometric advantage not afforded by trans-phenanthriplatin. This study demonstrates that reversible DNA intercalation provides a robust transition state that is efficiently converted to an irreversible DNA-Pt bound state.

Control of substrate access to the active site in methane monooxygenase.

Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH-MMOB (hereafter termed H-B) complex. Here we remedy this deficiency by providing a crystal structure of H-B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α(2)ß(2) interface of α(2)ß(2)γ(2) MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.

Glutathione-Scavenging Poly(disulfide amide) Nanoparticles for the Effective Delivery of Pt(IV) Prodrugs and Reversal of Cisplatin Resistance.

Despite the broad antitumor spectrum of cisplatin, its therapeutic efficacy in cancer treatment is compromised by the development of drug resistance in tumor cells and systemic side effects. A close correlation has been drawn between cisplatin resistance in tumor cells and increased levels of intracellular thiol-containing species, especially glutathione (GSH). The construction of a unique nanoparticle (NP) platform composed of poly(disulfide amide) polymers with a high disulfide density for the effective delivery of Pt(IV) prodrugs capable of reversing cisplatin resistance through the disulfide-group-based GSH-scavenging process, as described herein, is a promising route by which to overcome limitations associated with tumor resistance. Following systematic screening, the optimized NPs (referred to as CP5 NPs) showed a small particle size (76.2 nm), high loading of Pt(IV) prodrugs (15.50% Pt), a sharp response to GSH, the rapid release of platinum (Pt) ions, and notable apoptosis of cisplatin-resistant A2780cis cells. CP5 NPs also exhibited long blood circulation and high tumor accumulation after intravenous injection. Moreover, in vivo efficacy and safety results showed that CP5 NPs effectively inhibited the growth of cisplatin-resistant xenograft tumors with an inhibition rate of 83.32% while alleviating serious side effects associated with cisplatin. The GSH-scavenging nanoplatform is therefore a promising route by which to enhance the therapeutic index of Pt drugs used currently in cancer treatment.

A Sensitive, Nonradioactive Assay for Zn(II) Uptake into Metazoan Cells.

Sensitive measurements of cellular Zn(II) uptake currently rely on quantitating radioactive emissions from cells treated with 65Zn(II). Here, we describe a straightforward and reliable method employing a stable isotope to sensitively measure Zn(II) uptake by metazoan cells. First, biological medium selectively depleted of natural abundance Zn(II) using A12-resin [Richardson, C. E. R., et al. (2018) J. Am. Chem. Soc. 140, 2413] is restored to physiological levels of Zn(II) by addition of a non-natural Zn(II) isotope distribution comprising 70% 70Zn(II). The resulting 70Zn(II)-enriched medium facilitates quantitation of Zn(II) uptake using inductively coupled plasma-mass spectrometry (ICP-MS). This sensitive and reliable assay assesses Zn(II)-uptake kinetics at early time points and can be used to delineate how chemical and genetic perturbations influence Zn(II) uptake. Further, the use of ICP-MS in a Zn(II)-uptake assay permits simultaneous measurement of multiple metal ion concentrations. We used this capability to show that, across three cell lines, Zn(II) deficiency enhances selectivity for Zn(II) over Cd(II) uptake.

Speciation of Phenanthriplatin and Its Analogs in the Core of Tobacco Mosaic Virus.

Efficient loading of drugs in novel delivery agents has the potential to substantially improve therapy by targeting the diseased tissue while avoiding unwanted side effects. Here we report the first systematic study of the loading mechanism of phenanthriplatin and its analogs into tobacco mosaic virus (TMV), previously used by our group as an efficient carrier for anticancer drug delivery. A detailed investigation of the preferential uptake of phenanthriplatin in its aquated form (∼2000 molecules per TMV particle versus ∼1000 for the chlorido form) is provided. Whereas the net charge of phenanthriplatin analogs and their ionic mobilities have no effect on loading, the reactivity of aqua phenanthriplatin with the glutamates, lining the interior walls of the channel of TMV, has a pronounced effect on its loading. MALDI-MS analysis along with NMR spectroscopic studies of a model reaction of hydroxy-phenanthriplatin with acetate establish the formation of stable covalent adducts. The increased number of heteroaromatic rings on the platinum ligand appears to enhance loading, possibly by stabilizing hydrophobic stacking interactions with TMV core components, specifically Pro102 and Thr103 residues neighboring Glu97 and Glu106 in the channel. Electron transfer dissociation MS/MS fragmentation, a technique that can prevent mass-condition-vulnerable modification of proteins, reveals that Glu97 preferentially participates over Glu106 in covalent bond formation to the platinum center.

A Method for Selective Depletion of Zn(II) Ions from Complex Biological Media and Evaluation of Cellular Consequences of Zn(II) Deficiency.

We describe the preparation, evaluation, and application of an S100A12 protein-conjugated solid support, hereafter the "A12-resin", that can remove 99% of Zn(II) from complex biological solutions without significantly perturbing the concentrations of other metal ions. The A12-resin can be applied to selectively deplete Zn(II) from diverse tissue culture media and from other biological fluids, including human serum. To further demonstrate the utility of this approach, we investigated metabolic, transcriptomic, and metallomic responses of HEK293 cells cultured in medium depleted of Zn(II) using S100A12. The resulting data provide insight into how cells respond to acute Zn(II) deficiency. We expect that the A12-resin will facilitate interrogation of disrupted Zn(II) homeostasis in biological settings, uncovering novel roles for Zn(II) in biology.

Photoactivatable Sensors for Detecting Mobile Zinc.

Fluorescent sensors for mobile zinc are valuable for studying complex biological systems. Because these sensors typically bind zinc rapidly and tightly, there has been little temporal control over the activity of the probe after its application to a sample. The ability to control the activity of a zinc sensor in vivo during imaging experiments would greatly improve the time resolution of the measurement. Here, we describe photoactivatable zinc sensors that can be triggered with short pulses of UV light. These probes are prepared by functionalizing a zinc sensor with protecting groups that render the probe insensitive to metal ions. Photoinduced removal of the protecting groups restores the binding site, allowing for zinc-responsive changes in fluorescence that can be observed in live cells and tissues.

Challenges and Opportunities in Brain Bioinorganic Chemistry.

Metal ions play critical roles in neurotransmission, memory formation, and sensory perception. Understanding the molecular details of these processes is the Holy Grail of metalloneurochemistry. Here we describe five challenges for collaborative teams of chemists, biologists, and neuroscientists to help make this dream a reality.

The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs.

The platinum drugs, cisplatin, carboplatin, and oxaliplatin, prevail in the treatment of cancer, but new platinum agents have been very slow to enter the clinic. Recently, however, there has been a surge of activity, based on a great deal of mechanistic information, aimed at developing nonclassical platinum complexes that operate via mechanisms of action distinct from those of the approved drugs. The use of nanodelivery devices has also grown, and many different strategies have been explored to incorporate platinum warheads into nanomedicine constructs. In this Review, we discuss these efforts to create the next generation of platinum anticancer drugs. The introduction provides the reader with a brief overview of the use, development, and mechanism of action of the approved platinum drugs to provide the context in which more recent research has flourished. We then describe approaches that explore nonclassical platinum(II) complexes with trans geometry or with a monofunctional coordination mode, polynuclear platinum(II) compounds, platinum(IV) prodrugs, dual-threat agents, and photoactivatable platinum(IV) complexes. Nanoparticles designed to deliver platinum(IV) complexes will also be discussed, including carbon nanotubes, carbon nanoparticles, gold nanoparticles, quantum dots, upconversion nanoparticles, and polymeric micelles. Additional nanoformulations, including supramolecular self-assembled structures, proteins, peptides, metal-organic frameworks, and coordination polymers, will then be described. Finally, the significant clinical progress made by nanoparticle formulations of platinum(II) agents will be reviewed. We anticipate that such a synthesis of disparate research efforts will not only help to generate new drug development ideas and strategies, but also will reflect our optimism that the next generation of approved platinum cancer drugs is about to arrive.

AMPA receptor inhibition by synaptically released zinc.

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.

Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc.

Many excitatory synapses contain high levels of mobile zinc within glutamatergic vesicles. Although synaptic zinc and glutamate are coreleased, it is controversial whether zinc diffuses away from the release site or whether it remains bound to presynaptic membranes or proteins after its release. To study zinc transmission and quantify zinc levels, we required a high-affinity rapid zinc chelator as well as an extracellular ratiometric fluorescent zinc sensor. We demonstrate that tricine, considered a preferred chelator for studying the role of synaptic zinc, is unable to efficiently prevent zinc from binding low-nanomolar zinc-binding sites, such as the high-affinity zinc-binding site found in NMDA receptors (NMDARs). Here, we used ZX1, which has a 1 nM zinc dissociation constant and second-order rate constant for binding zinc that is 200-fold higher than those for tricine and CaEDTA. We find that synaptic zinc is phasically released during action potentials. In response to short trains of presynaptic stimulation, synaptic zinc diffuses beyond the synaptic cleft where it inhibits extrasynaptic NMDARs. During higher rates of presynaptic stimulation, released glutamate activates additional extrasynaptic NMDARs that are not reached by synaptically released zinc, but which are inhibited by ambient, tonic levels of nonsynaptic zinc. By performing a ratiometric evaluation of extracellular zinc levels in the dorsal cochlear nucleus, we determined the tonic zinc levels to be low nanomolar. These results demonstrate a physiological role for endogenous synaptic as well as tonic zinc in inhibiting extrasynaptic NMDARs and thereby fine tuning neuronal excitability and signaling.

Chronic cisplatin treatment promotes enhanced damage repair and tumor progression in a mouse model of lung cancer.

Chemotherapy resistance is a major obstacle in cancer treatment, yet the mechanisms of response to specific therapies have been largely unexplored in vivo. Employing genetic, genomic, and imaging approaches, we examined the dynamics of response to a mainstay chemotherapeutic, cisplatin, in multiple mouse models of human non-small-cell lung cancer (NSCLC). We show that lung tumors initially respond to cisplatin by sensing DNA damage, undergoing cell cycle arrest, and inducing apoptosis-leading to a significant reduction in tumor burden. Importantly, we demonstrate that this response does not depend on the tumor suppressor p53 or its transcriptional target, p21. Prolonged cisplatin treatment promotes the emergence of resistant tumors with enhanced repair capacity that are cross-resistant to platinum analogs, exhibit advanced histopathology, and possess an increased frequency of genomic alterations. Cisplatin-resistant tumors express elevated levels of multiple DNA damage repair and cell cycle arrest-related genes, including p53-inducible protein with a death domain (Pidd). We demonstrate a novel role for PIDD as a regulator of chemotherapy response in human lung tumor cells.

CF2H, a Hydrogen Bond Donor.

The CF2H group, a potential surrogate for the OH group, can act as an unusual hydrogen bond donor, as confirmed by crystallographic, spectroscopic, and computational methods. Here, we demonstrate the bioisosterism of the OH and CF2H groups and the important roles of CF2-H···O hydrogen bonds in influencing intermolecular interactions and conformational preferences. Experimental evidence, corroborated by theory, reveals the distinctive nature of CF2H hydrogen bonding interactions relative to their normal OH hydrogen bonding counterparts.

Tuning the Diiron Core Geometry in Carboxylate-Bridged Macrocyclic Model Complexes Affects Their Redox Properties and Supports Oxidation Chemistry.

We introduce a novel platform to mimic the coordination environment of carboxylate-bridged diiron proteins by tethering a small, dangling internal carboxylate, (CH2)nCOOH, to phenol-imine macrocyclic ligands (H3PIMICn). In the presence of an external bulky carboxylic acid (RCO2H), the ligands react with [Fe2(Mes)4] (Mes = 2,4,6-trimethylphenyl) to afford dinuclear [Fe2(PIMICn)(RCO2)(MeCN)] (n = 4-6) complexes. X-ray diffraction studies revealed structural similarities between these complexes and the reduced diiron active sites of proteins such as Class I ribonucleotide reductase (RNR) R2 and soluble methane monooxygenase hydroxylase. The number of CH2 units of the internal carboxylate arm controls the diiron core geometry, affecting in turn the anodic peak potential of the complexes. As functional synthetic models, these complexes facilitate the oxidation of C-H bonds in the presence of peroxides and oxo transfer from O2 to an internal phosphine moiety.

Reaction-based fluorescent sensor for investigating mobile Zn2+ in mitochondria of healthy versus cancerous prostate cells.

Chelatable, mobile forms of divalent zinc, Zn(II), play essential signaling roles in mammalian biology. A complex network of zinc import and transport proteins has evolved to control zinc concentration and distribution on a subcellular level. Understanding the action of mobile zinc requires tools that can detect changes in Zn(II) concentrations at discrete cellular locales. We present here a zinc-responsive, reaction-based, targetable probe based on the diacetyled form of Zinpyr-1. The compound, (6-amidoethyl)triphenylphosphonium Zinpyr-1 diacetate (DA-ZP1-TPP), is essentially nonfluorescent in the metal-free state; however, exposure to Zn(II) triggers metal-mediated hydrolysis of the acetyl groups to afford a large, rapid, and zinc-induced fluorescence response. DA-ZP1-TPP is insensitive to intracellular esterases over a 2-h period and is impervious to proton-induced turn-on. A TPP unit is appended for targeting mitochondria, as demonstrated by live cell fluorescence imaging studies. The practical utility of DA-ZP1-TPP is demonstrated by experiments revealing that, in contrast to healthy epithelial prostate cells, tumorigenic cells are unable to accumulate mobile zinc within their mitochondria.