VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy.

Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.

Multifunctional Pt(II) Reagents: Covalent Modifications of Pt Complexes Enable Diverse Structural Variation and In-Cell Detection.

To enhance the functionality of Pt-based reagents, several strategies have been developed that utilize Pt compounds modified with small, reactive handles. This Account encapsulates work done by us and other groups regarding the use of Pt(II) compounds with reactive handles for subsequent elaboration with fluorophores or other functional moieties. Described strategies include the incorporation of substituents for well-known condensation or nucleophilic displacement-type reactions and their use, for example, to tether spectroscopic handles to Pt reagents for in vivo investigation. Other chief uses of displacement-type reactions have included tethering various small molecules exhibiting pharmacological activity directly to Pt, thus adding synergistic effects. Click chemistry-based ligation techniques have also been applied, primarily with azide- and alkyne-appended Pt complexes. Orthogonally reactive click chemistry reactions have proven invaluable when more traditional nucleophilic displacement reactions induce side-reactivity with the Pt center or when systematic functionalization of a larger number of Pt complexes is desired. Additionally, a diverse assortment of Pt-fluorophore conjugates have been tethered via click chemistry conjugation. In addition to providing a convenient synthetic path for diversifying Pt compounds, the use of click-capable Pt complexes has proved a powerful strategy for postbinding covalent modification and detection with fluorescent probes. This strategy bypasses undesirable influences of the fluorophore camouflaged as reactivity due to Pt that may be present when detecting preattached Pt-fluorophore conjugates. Using postbinding strategies, Pt reagent distributions in HeLa and lung carcinoma (NCI-H460) cell cultures were observed with two different azide-modified Pt compounds, a monofunctional Pt(II)-acridine type and a difunctional Pt(II)-neutral complex. In addition, cellular distribution was observed with an alkyne-appended difunctional Pt(II)-neutral complex analogous in structure to the aforementioned difunctional azide-Pt(II) reagent. In all cases, significant accumulation of Pt in the nucleolus of cells was observed, in addition to broader localization in the nucleus and cytoplasm of the cell. Using the same strategy of postbinding click modification with fluorescent probes, Pt adducts were detected and roughly quantified on rRNA and tRNA from Pt-treated Saccharomyces cerevisiae; rRNA adducts were found to be relatively long-lived and not targeted for immediate degradation. Finally, the utility and feasibility of the alkyne-appended Pt(II) compound has been further demonstrated with a turn-on fluorophore, dansyl azide, in fluorescent detection of DNA in vitro. In all, these modifications utilizing reactive handles have allowed for the diversification of new Pt reagents, as well as providing cellular localization information on the modified Pt compounds.

Azide vs Alkyne Functionalization in Pt(II) Complexes for Post-treatment Click Modification: Solid-State Structure, Fluorescent Labeling, and Cellular Fate.

Tracking of Pt(II) complexes is of crucial importance toward understanding Pt interactions with cellular biomolecules. Post-treatment fluorescent labeling of functionalized Pt(II)-based agents using the bioorthogonal Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has recently been reported as a promising approach. Here we describe an azide-functionalized Pt(II) complex, cis-[Pt(2-azidobutyl)amido-1,3-propanediamine)Cl2] (1), containing the cis geometry and difunctional reactivity of cisplatin, and present a comparative study with its previously described alkyne-functionalized congener. Single-crystal X-ray diffraction reveals a dramatic change in the solid-state arrangement with exchange of the alkyne for an azide moiety wherein 1 is dominated by a pseudo-chain of Pt-Pt dimers and antiparallel alignment of the azide substituents, in comparison with a circular arrangement supported by CH/π(C≡C) interactions in the alkyne version. In vitro studies indicate similar DNA binding and click reactivity of both congeners observed by fluorescent labeling. Interestingly, complex 1 shows in vitro enhanced click reactivity in comparison to a previously reported azide-appended Pt(II) complex. Despite their similar behavior in vitro, preliminary in cellulo HeLa studies indicate a superior imaging potential of azide-functionalized 1. Post-treatment fluorescent labeling of 1 observed by confocal fluorescence microscopy shows nuclear and intense nucleolar localization. These results demonstrate the potential of 1 in different cell line localization studies and for future isolation and purification of Pt-bound targets.

An alkyne-appended, click-ready Pt(II) complex with an unusual arrangement in the solid state.

To better understand the range of cellular interactions of Pt(II) -based chemotherapeutics, robust and efficient methods to track and analyze Pt targets are needed. A powerful approach is to functionalize Pt(II) compounds with alkyne or azide moieties for post-treatment conjugation through the azide-alkyne cycloaddition (click) reaction. Herein, we report an alkyne-appended cis-diamine Pt(II) compound, cis-[Pt(2-(5-hexynyl)amido-1,3-propanediamine)Cl2] (1), the X-ray crystal structure of which exhibits a combination of unusual radially distributed CH/π(C≡C) interactions, Pt-Pt bonding, and NH:O/NH:Cl hydrogen bonds. In solution, 1 exhibits no Pt-alkyne interactions and binds readily to DNA. Subsequent click reactivity with nonfluorescent dansyl azide results in a 70-fold fluorescence increase. This result demonstrates the potential for this new class of alkyne-modified Pt compound for the comprehensive detection and isolation of Pt-bound biomolecules.

Convenient detection of metal-DNA, metal-RNA, and metal-protein adducts with a click-modified Pt(II) complex.

cis-[Pt(2-azido-1,3-propanediamine)Cl2] is a reagent for high-yield post-treatment fluorescent labelling of Pt(II) biomolecular targets using click chemistry and exhibits a bias in conformational isomers in the context of duplex DNA. Pt-protein adducts are detected using BSA as a model. Following in vivo treatment, long-lived Pt-RNA adducts are detected on ribosomal RNA.

Platinum-RNA modifications following drug treatment in S. cerevisiae identified by click chemistry and enzymatic mapping.

With the importance of RNA-based regulatory pathways, the potential for targeting noncoding and coding RNAs by small molecule therapeutics is of great interest. Platinum(II) complexes including cisplatin (cis-diamminedichloroplatinum(II)) are widely prescribed anticancer compounds that form stable adducts on nucleic acids. In tumors, DNA damage from Pt(II) initiates apoptotic signaling, but this activity is not necessary for cytotoxicity (e.g., Yu et al., 2008), suggesting accumulation and consequences of Pt(II) lesions on non-DNA targets. We previously reported an azide-functionalized compound, picazoplatin, designed for post-treatment click labeling that enables detection of Pt complexes (White et al., 2013). Here, we report in-gel fluorescent detection of Pt-bound rRNA and tRNA extracted from picazoplatin-treated S. cerevisiae and labeled using Cu-free click chemistry. These data provide the first evidence that cellular tRNA is a platinum drug substrate. We assess Pt(II) binding sites within rRNA from cisplatin-treated S. cerevisiae, in regions where damage is linked to significant downstream consequences including the sarcin-ricin loop (SRL) Helix 95. Pt-RNA adducts occur on the nucleotide substrates of ribosome-inactivating proteins, as well as on the bulged-G motif critical for elongation factor recognition of the loop. At therapeutically relevant concentrations, Pt(II) also binds robustly within conserved cation-binding pockets in Domains V and VI rRNA at the peptidyl transferase center. Taken together, these results demonstrate a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets and suggest a ribotoxic mechanism for cisplatin cytotoxicity.

Picazoplatin, an azide-containing platinum(II) derivative for target analysis by click chemistry.

Despite the broad use of platinum-based chemotherapeutics, identification of their full range of cellular targets remains a significant challenge. In order to identify, visualize, and isolate cellular targets of Pt(II) complexes, we have modified the chemotherapeutic drug picoplatin with an azide moiety for subsequent click reactivity. The new compound picazoplatin readily binds DNA and RNA oligonucleotides and undergoes facile post-labeling click reactions to alkyne-fluorophore conjugates. Pt-fluorophore click reactions in rRNA purified from drug-treated Saccharomyces cerevisiae demonstrate its potential for future in vivo efforts.

Combination treatment for menstrual migraine and dysmenorrhea using sumatriptan-naproxen: two randomized controlled trials.

OBJECTIVE: To evaluate the efficacy and tolerability of sumatriptan-naproxen during the mild pain phase of a single menstrual migraine attack associated with dysmenorrhea. METHODS: Two replicate randomized, multicenter, double-blind, placebo-controlled, trials of adults with menstrual migraine and dysmenorrhea were conducted. Participants treated their menstrual migraine attack during the mild pain phase (within 1 hour of onset) with sumatriptan 85 mg and naproxen sodium 500 mg in a single fixed-dose formulation (sumatriptan-naproxen) or placebo. The primary endpoint was 2-hour pain-free response. RESULTS: Sumatriptan-naproxen was statistically superior to placebo in both studies (n=311, Study 1; n=310, Study 2) for 2-hour and, 2- to 24-hour sustained pain-free response, use of headache and menstrual rescue medications, and several nonpain menstrual symptom categories. Two-hour pain-free rates were Study 1, 42% compared with 23%, and Study 2, 52% compared with 22%, P<.001. Two- to 24-hour sustained pain-free rates were Study 1, 29% compared with 18%, P=.022; Study 2, 38% compared with 10%, P<.001. Headache and menstrual medication rates were Study 1, 37% compared with 53%, P=.005; Study 2, 31% compared with 69%, P<.001. Women treated with sumatriptan-naproxen continued to be pain free through 48 hours compared with placebo: Study 1, 26% compared with 17%, P=.040; Study 2, 28% compared with 8%, P<.001. No serious adverse events were reported in either study; nausea and dizziness were the most frequently reported adverse events. CONCLUSION: Sumatriptan-naproxen provided an effective pain-free response at 2 hours, which was maintained up to 48 hours in menstrual migraineurs with dysmenorrhea. Sumatriptan-naproxen was well-tolerated and resulted in decreased rescue medication use and relief of nonpainful menstrual symptoms. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00329459 and NCT00329355 LEVEL OF EVIDENCE: I.

Effects of gender on relapse prevention in smokers treated with bupropion SR.

BACKGROUND: Recent data suggest that women smokers respond differently than men to cessation pharmacotherapies, particularly nicotine replacement therapy (NRT). Lower abstinence and higher relapse rates are often reported for women treated with NRT. Gender effects for those treated with non-nicotinic, bupropion-hydrochloride sustained release for relapse prevention have not been studied. METHODS: Data from a multicenter relapse-prevention (RP) trial of bupropion (November 1995-June 1998) were analyzed for gender differences. Men and women smokers (N=784) were treated with open-label bupropion for 7 weeks. Those abstinent at Week 7 (n=432) were enrolled in the double-blind relapse-prevention phase and randomized to placebo or continued bupropion for 45 additional weeks. RESULTS: Differences in point-prevalence abstinence rates between men (61.8%) and women (55.6%) in open-label bupropion (Week 7) were not significant. In the RP-phase Week 52, continuous abstinence rates for men and women were 37.8% and 36.4% (bupropion) and 36.6% and 29.9% (placebo), respectively; point-prevalence abstinence rates for men and women were 54.1% and 55.9% (bupropion) and 42.9% and 41.3% (placebo), respectively. Abstinence rates and time to relapse were superior for both men and women who received longer treatment. Gender differences within treatment groups were not significant. Median time to relapse was equal for men and women within each treatment group: Week 32 for bupropion and Week 20 for placebo. CONCLUSIONS: Our data suggest that bupropion is a promising pharmacotherapy for preventing relapse, particularly for women.