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Western Analysis and Pull-down assay data.

p28 is an anionic cell-penetrating peptide of 28 amino acids that activates wild-type and mutated p53, leading subsequently to selective inhibition of CDK2 and cyclin A expression and G2–M cell-cycle arrest. In this study, we investigated the cytotoxic effects of p28 treatment alone and in combination with DNA-damaging and antimitotic agents on human cancer cells. p28 enhanced the cytotoxic activity of lower concentrations (IC20-50) of DNA-damaging drugs (doxorubicin, dacarbazine, temozolamide) or antimitotic drugs (paclitaxel and docetaxel) in a variety of cancer cells expressing wild-type or mutated p53. Mechanistic investigations revealed that p28 induced a post-translational increase in the expression of wild-type or mutant p53 and p21, resulting in cell-cycle inhibition at the G2–M phase. The enhanced activity of these anticancer agents in combination with p28 was facilitated through the p53/p21/CDK2 pathway. Taken together, these results highlight a new approach to maximize the efficacy of chemotherapeutic agents while reducing dose-related toxicity. Cancer Res; 76(8); 2354–65. ©2016 AACR.

Precise identification of the tumor margins during breast-conserving surgery (BCS) remains a challenge given the lack of visual discrepancy between malignant and surrounding normal tissues. Therefore, we developed a fluorescent imaging agent, ICG-p28, for intraoperative imaging guidance to better aid surgeons in achieving negative margins in BCS. Here, we determined the pharmacokinetics (PK), biodistribution, and preclinical toxicity of ICG-p28. The PK and biodistribution of ICG-p28 indicated rapid tissue uptake and localization at tumor lesions. There were no dose-related effect and no significant toxicity in any of the breast cancer and normal cell lines tested. Furthermore, ICG-p28 was evaluated in clinically relevant settings with transgenic mice that spontaneously developed invasive mammary tumors. Intraoperative imaging with ICG-p28 showed a significant reduction in the tumor recurrence rate. This simple, nontoxic, and cost-effective method can offer a new approach that enables surgeons to intraoperatively identify tumor margins and potentially improves overall outcomes by reducing recurrence rates.

A Lewis acid-mediated decarboxylative 1,2-rearrangement reaction of cyclic carbonates was developed. The selectivity of the migration in the decarboxylative 1,2-rearrangement of cyclic carbonates was opposite to that of the corresponding 1,2-diols under the same reaction conditions. This contrasting selectivity of the migration was confirmed in a variety of substrates.

Background Brain metastases (BMs), the most common tumors of the central nervous system, are life-threatening with a dismal prognosis. The major challenges to developing effective treatments for BMs are the limited abilities of drugs to target tumors and to cross the blood-brain barrier (BBB). We aimed to investigate the efficacy of our therapeutic approach against BMs in mouse models that recapitulate the clinical manifestations of BMs. Methods BMs mouse models were constructed by injecting human breast, lung cancer, and melanoma intracardially, which allowed the BBB to remain intact. We investigated the ability of the cell-penetrating peptide p28 to cross the BBB in an in vitro 3D model and in the BMs animal models. The therapeutic effects of p28 in combination with DNA-damaging agents (radiation and temozolomide) on BMs were also evaluated. Results p28 crossed the intact BBB more efficiently than the standard chemotherapeutic agent, temozolomide. Upon crossing the BBB, p28 localized preferentially to tumor lesions and enhanced the efficacy of DNA-damaging agents by activating the p53-p21 axis. In the BMs animal models, radiation in combination with p28 significantly reduced the tumor burden of BMs. Conclusions The cell-cycle inhibitor p28 can cross the BBB localize to tumor lesions in the brain and enhance the inhibitory effects of DNA-damaging agents on BMs, suggesting the potential therapeutic benefits of this molecule in BMs.

Cell-Penetrating Peptides (CPPs) are short peptides consisting of

A silver-catalyzed carbon dioxide fixation reaction into 2-alkynylindole derivatives was developed to afford tricyclic indoles. Carbon dioxide was selectively fixed on the N atom of the indole, and only

Decarboxylative homo-Nazarov cyclization catalyzed by a Lewis acid was achieved using a cyclic enol carbonate bearing a cyclopropane moiety as a substrate. Various substrates were converted into the corresponding multi-substituted cyclohexenones in good yields via decarboxylation, followed by 6-membered ring formation involving cyclopropane-ring-opening.

The synthesis of polycyclic indene derivatives via silver-catalyzed carbon dioxide fixation on 2-alkynylindene derivatives was achieved by nucleophilic addition of the indenyl anion to carbon dioxide involving carbon-carbon bond formation and subsequent intramolecular cyclization to the alkyne part activated by a silver catalyst. This cascade process could be applied to various substrates to obtain the corresponding products in high yields. The endo/exo selectivity of the cyclization could be controlled by the steric or electronic effect of the substituents on the substrates, and 6-endo-selective cyclization was realized to afford α-pyrone-fused indenes.

Decarboxylative Nazarov cyclization of chiral cyclic enol carbonates proceeded to afford chiral 2-cyclopentenones with excellent chirality transfer under thermal conditions without any catalyst. In...

Precise surgical resection directly influences the prognosis and survival of patients with solid tumors. However, it is often difficult to distinguish tumor from normal tissue during resection without any intraoperative imaging guidance. Image-guided surgery particularly when coupled with a near-infrared (NIR) fluorescent agent may improve positive-margin rate thereby improving the overall prognosis. We have developed a unique tumor-targeting fluorescence imaging agent that can aid in the accurate localization of human cancer cells in preclinical settings. The NIR imaging agent, ICG-p28, a water-soluble, nontoxic, and pan-tumor targeting probe consisting of a cell-penetrating peptide (p28) conjugated to indocyanine green (ICG), can accurately localize tumors in vivo. Development of the noninvasive, targeted imaging agent can potentially improve in the resections of tumors by enabling the localization of lesions that are currently difficult or impossible to detect by visual observation or palpation. Here, we describe the methods of preclinical animal imaging models by using NIR fluorescence imager coupled with a new tumor-targeting agent.

Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death

A copper-catalyzed Nazarov cyclization was carried out under conventional heating conditions and microwave conditions. Under strictly temperature-controlled conditions, the use of microwave irradia...

The stereoselective synthesis of aminovinyloxazolidinones based on the electrophilic amination of a vinyl-silver intermediate, generated by silver-catalyzed carbon dioxide incorporation on a propargyl amine, was achieved. The geometry of the aminated product was determined by a single crystal X-ray analysis and NOE measurement and it was elucidated that the geometry was proved to be opposite to the geometry predicted from the previous silver-catalyzed carbon dioxide fixation on a propargyl amine derivative. This unexpected stereoselectivity could be successfully explained by a radical mechanism.

Highly substituted 2-cyclopentenones were stereospecifically and regioselectively constructed with high catalytic efficiency through Lewis-acid catalyzed decarboxylative Nazarov cyclization of the cyclic carbonate derivative, which is prepared by reacting the propargyl alcohol with carbon dioxide in the presence of a silver catalyst. The stereochemistry of the 2-cyclopentenone is strictly controlled by the geometry of the alkene in the starting material. This method is applicable for various substrates.

Facile and versatile access to highly functionalized tetronic acids has been successfully achieved through the reaction of conjugated ynones with carbon dioxide. In the presence of a base, the enolates generated from the ynones capture CO2 via a carbon–carbon bond-forming reaction, accompanied by a 5-exo-dig cyclization reaction of the resulting carboxylate to the alkyne, activated by a silver catalyst. The present method should be applicable to the synthesis of a wide variety of tetronic acids.

The amidation reaction of carboxylic acid derivatives was developed using sulfur trioxide pyridine complex (SO3·py) as a commercially available and easily handled oxidant. This method could be applied to the reaction of various aromatic and aliphatic carboxylic acids, including optically active ones, with formamide derivatives to afford the corresponding amides in good to high yields.

Asymmetric synthesis of 2-cyclopentenones was achieved by chirality transfer based on Lewis acid catalyzed decarboxylative Nazarov cyclization of optically active cyclic enol carbonates, which are prepared by silver-catalyzed carbon dioxide incorporation into optically pure propargyl alcohols. The stereochemistry at the 4,5-positions of the 2-cyclopentenones was cleanly constructed by reflecting the stereochemistry of the starting materials. This method could be applied to various substrates to obtain the corresponding products in high yields with highly efficient chirality transfer.

The combined use of an organic base for resorcinols realized a Kolbe–Schmitt type reaction under ambient conditions. When resorcinols (3-hydroxyphenol derivatives) were treated with DBU under a carbon dioxide atmosphere, nucleophilic addition to carbon dioxide proceeded to afford the corresponding salicylic acid derivatives in high yields.

p28 is an anionic cell-penetrating peptide of 28 amino acids that activates wild-type and mutated p53, leading subsequently to selective inhibition of CDK2 and cyclin A expression and G2–M cell-cycle arrest. In this study, we investigated the cytotoxic effects of p28 treatment alone and in combination with DNA-damaging and antimitotic agents on human cancer cells. p28 enhanced the cytotoxic activity of lower concentrations (IC20-50) of DNA-damaging drugs (doxorubicin, dacarbazine, temozolamide) or antimitotic drugs (paclitaxel and docetaxel) in a variety of cancer cells expressing wild-type or mutated p53. Mechanistic investigations revealed that p28 induced a post-translational increase in the expression of wild-type or mutant p53 and p21, resulting in cell-cycle inhibition at the G2–M phase. The enhanced activity of these anticancer agents in combination with p28 was facilitated through the p53/p21/CDK2 pathway. Taken together, these results highlight a new approach to maximize the efficacy of chemotherapeutic agents while reducing dose-related toxicity. Cancer Res; 76(8); 2354–65. ©2016 AACR.

Silver-catalyzed reactions are some of the important methodologies in organic chemistry. Since 2007, a new application of silver catalysts has been emerging. For the sequential carboxylation and cyclization of alkyne derivatives, such as propargyl alcohols and amines, using carbon dioxide, silver catalysts show significant reactivity under mild conditions unlike other transition metals. These developments have received much attention for the effective utilization of carbon dioxide in organic chemistry to synthesize heterocyclic compounds. Related silver-catalyzed C-C bond forming reactions with carbon dioxide have also provided the synthetic methods of the corresponding carboxylic acid derivatives. In this review, the recent studies of the silver-catalyzed carboxylation reactions using carbon dioxide are described.

Microwave irradiation was applied to a sterically demanding ring-closing metathesis catalyzed by a second-generation Grubbs catalyst. Under strict temperature control, reaction acceleration by microwave irradiation was observed. The observed acceleration effect was estimated to be equivalent to a rising temperature of 2.5–5 °C range under conventional heating conditions.

Breast cancer is one of the most common types of cancer diagnosis in women of all ages. The primary treatment of breast cancer is surgical excision of the tumor with the margin of normal breast tissue surrounding the tumor. However, current clinical goals include complete resection of the tumor at the initial surgery, there is often a need for further surgery and removal of more tissue due to the difficulties inherent in getting negative margins. Therefore, there is a clinical need to optimize the excision by precisely identifying tumor margins significantly benefitting patients with breast cancer. Image-guided surgery with fluorescent agents provides surgeons numerous advantages such as real-time detection with a high-resolution image and relatively flexible instrument. We have developed a unique tumor-targeting fluorescence imaging agent. We identified a cell-penetrating peptide (CPP) p28, a fragment of azurin isolated from opportunistic pathogen Pseudomonas pathogen. p28 was chemically conjugated with Indocyanine green (ICG), a near-infrared red (NIR) fluorescent agent. ICG has been approved by the FDA for clinical applications with an excellent safety record. When p28 was conjugated to ICG, there was no significant alternation of fluorescence quantum yield (ΦF) of ICG-p28 compared to ICG alone. Triple-negative (ER, PR, HER2) human breast cancer PDX mouse model in NSG mice was used to evaluate the image-guided surgical procedure with ICG-p28. After 24h of ICG-p28 injection i.v. opportunistic 0.5 mg/kg b.w., NIR-fluorescence positive mammary tumors with a 2-mm safe margin were resected under real-time guidance of the PDE imaging unit (Mitaka USA, Hamamatsu Photonics). NIR-fluorescence negative surrounding tissues of at least two different sites (superior and inferior) were also collected separately. To validate the accuracy of tumor margin identification by the image guidance, Alu-based real-time PCR analysis was used for the quantitative detection of human cancer cells. Five ng of genomic DNA obtained from tumors and surrounding tissues were subjected to RT-PCR. Ct values were normalized by the housekeeping gene, mouse GAPDH. Our results showed that preclinical intraoperative imaging with our imaging agent, ICG-p28 at 0.5 mg/kg, significantly reduced positive margin as compared to control groups (e.g. ICG alone, p Citation Format: Masahide Goto, Ingeun Ryoo, Samer Naffouje, Konstantin Christov, Jing Wang, Albert Green, Anne Shilkaitis, Tapas K. Das Gupta, Tohru Yamada. Real-time intraoperative tumor imaging in a breast cancer PDX model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-022.

The microwave specific effect was investigated on the catalytic enantioselective reactions; atropo-enantiveselective ring-opening reaction of biaryl lactones and enantioselective Claisen rearrangement reaction. These reactions were efficiently enhanced with microwave irradiation under reaction temperature control, thought any loss of the enantioselectivities were not detected. It was also found that the racemization rate of the atropo-optically active biaryl lactone was enhanced by microwave irradiation. As these observations could not be explained by the simple thermal effect, microwave specific effect should be considered.

C-C bond-forming carboxylation and cyclization of trimethyl(2-methylenebut-3-yn-1-yl)silane derivatives and carbon dioxide was developed. Silver catalysts and CsF promoted the reaction to afford the corresponding 2-furanone and 2-pyrone derivatives in good-to-high yields. The structure of the 2-furanone was confirmed by single-crystal X-ray crystallography, which revealed that the geometry of the exo-olefin was that of a Z-isomer. When an aromatic ring-substituted alkyne was used, 2-furanone derivatives were selectively obtained via 5-exo-dig cyclization, whereas the reaction of alkyl-substituted alkynes produced 2-pyrone derivatives with high selectivity.

Highly Z-selective syntheses of oxazolidin-2-ones from propargylic alcohols containing internal alkynes and phenyl isocyanate were achieved by using a combination of silver acetate and N,N-dimethylaminopyridine. The catalytic system was applied to propargylic alcohols containing alkyl-substituted alkyne groups. By considering the results in the presence and absence of an electron-withdrawing group on the aromatics, it was shown that the silver catalyst effectively activates the C≡C triple bond by acting as a π-Lewis acid to produce the corresponding oxazolidinones with high Z-selectivities.

The optically active 1-chlorovinyl cobalt(III) complex was prepared from the ketoiminatocobalt(II) complex upon treatment with sodium borohydride in the presence of 1,1,1-trichloroethane. The obtained complex was found to catalyze the enantioselective Michael addition of various malonates on cyclic or acyclic enones to afford the corresponding adduct. In this reaction, the use of sodium alkoxide as a base with 1-chlorovinyl cobalt(III) complex catalyst was crucial to improving both the reactivity and enantioselectivity.

Multiple substitution of d- for l-amino acids decreases the intracellular uptake of cationic cell penetrating peptides (CPP) in a cell line-dependent manner. We show here that a single d-amino acid substitution can decrease the overall uptake of the anionic, amphipathic CPP, p28, into cancer and histologically matched normal cell lines, while not altering the preferential uptake of p28 into cancer cells. The decrease appears dependent on the position of the d-substitution within the peptide and the ability of the substituted d-amino acid to alter chirality. We also suggest that when d-substitution alters the ratio of α-helix to β-sheet content of an anionic CPP, its translocation across the cell membrane is altered, reducing overall entry. These observations may have a significant effect on the design of future d-substituted analogues of cell penetrating peptides.

The microwave specific effect on the catalytic atropo-enantioselective ring-opening reaction of biaryl lactones was investigated. Under strictly controlled temperature conditions, the reaction was accelerated by microwave irradiation without any loss of the enantioselectivity. Also, the racemization rate of the atropo-optically active biaryl lactone was enhanced by the microwave irradiation.

Microwave irradiation was applied to the enantioselective Conia-ene reaction catalyzed by Cu(OTf)2 with a chiral amine. Under strictly controlled temperature conditions, microwave irradiation impro...

Tetramic acid derivatives have been studied as biologically active heterocycle structures for pharmaceutical or agricultural chemicals. Conventional preparative approaches often require highly functionalized starting materials and harsh heating conditions in basic media. The present report provides a conceptually new synthetic strategy for the synthesis of tetramic acid derivatives from easily available propargylic amines and carbon dioxide with a silver salt and DBU under mild reaction conditions.

Emilia Coppari,1 Tohru Yamada,2 Anna Rita Bizzarri,1 Craig W Beattie,2 Salvatore Cannistraro1 1Biophysics and Nanoscience Centre, CNISM-DEB, Università della Tuscia, Viterbo, Italy; 2Division of Surgical Oncology, Department of Surgery, University of Illinois College of Medicine, Chicago, IL, USA Abstract: p28 is an anionic, amphipathic, cell-penetrating peptide derived from the cupredoxin azurin that binds to the DNA-binding domain (DBD) of the tumor suppressor protein, p53, and induces a post-translational increase in the level of wild type and mutated p53 in a wide variety of human cancer cells. As p63 and p73, additional members of the p53 superfamily of proteins, also appear to be involved in the cellular response to cancer therapy and are reportedly required for p53-induced apoptosis, we asked whether p28 also binds to p63 and p73. Atomic force spectroscopy demonstrates that p28 forms a stable, high-affinity complex with full-length p63, the DBD of p63, and full-length p73. Exposure to p28 decreased the level of TAp63α and ΔNp63α, the truncated form of p63, in p53 wild type and mutated human breast cancer cells, respectively. p28 increased the level of TAp73α, but not ΔNp73α, in the same breast cancer cell lines. In contrast, p28 increased the level of the TA and ΔN isoforms of p63 in p53 wild type, but not in p53 mutated melanoma cells, while decreasing TA p73α in p53 wild type and mutated human melanoma cells. All changes were mirrored by an associated change in the expression of the HECT E3 ligases Itch/AIP4, AIP5, and the RING E3 ligase Pirh2, but not in the receptor for activated C kinase or the RING E3 ligases Mdm2 and Cop1. Collectively, the data suggest that molecules such as p28 bind with high affinity to the DBD of p63 and p73 and alter their expression independent of the Mdm2 and Cop1 pathways. Keywords: molecular interaction, anticancer peptide, p53 superfamily of proteins