Diarylethene moiety as an enthalpy-entropy switch: photoisomerizable stapled peptides for modulating p53/MDM2 interaction.

Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein-protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the "open" configuration, but up to eight times larger for the corresponding "closed" isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the "closed" isomers is mostly enthalpy-driven, whereas the "open" photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.

A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide.

The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.

Water-soluble, stable and azide-reactive strained dialkynes for biocompatible double strain-promoted click chemistry.

The Sondheimer dialkyne is extensively used in double strain-promoted azide-alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.

Macrocyclized Extended Peptides: Inhibiting the Substrate-Recognition Domain of Tankyrase.

We report a double-click macrocyclization approach for the design of constrained peptide inhibitors having non-helical or extended conformations. Our targets are the tankyrase proteins (TNKS), poly(ADP-ribose) polymerases (PARP) that regulate Wnt signaling by targeting Axin for degradation. TNKS are deregulated in many different cancer types, and inhibition of TNKS therefore represents an attractive therapeutic strategy. However, clinical development of TNKS-specific PARP catalytic inhibitors is challenging due to off-target effects and cellular toxicity. We instead targeted the substrate-recognition domain of TNKS, as it is unique among PARP family members. We employed a two-component strategy, allowing peptide and linker to be separately engineered and then assembled in a combinatorial fashion via click chemistry. Using the consensus substrate-peptide sequence as a starting point, we optimized the length and rigidity of the linker and its position along the peptide. Optimization was further guided by high-resolution crystal structures of two of the macrocyclized peptides in complex with TNKS. This approach led to macrocyclized peptides with submicromolar affinities for TNKS and high proteolytic stability that are able to disrupt the interaction between TNKS and Axin substrate and to inhibit Wnt signaling in a dose-dependent manner. The peptides therefore represent a promising starting point for a new class of substrate-competitive inhibitors of TNKS with potential for suppressing Wnt signaling in cancer. Moreover, by demonstrating the application of the double-click macrocyclization approach to non-helical, extended, or irregularly structured peptides, we greatly extend its potential and scope, especially given the frequency with which such motifs mediate protein-protein interactions.

A New Methodology for Incorporating Chiral Linkers into Stapled Peptides.

Stapled peptides have arisen as a new class of chemical probe and potential therapeutic agents for modulating protein-protein interactions. Here, we report the first two-component i,i+7 stapling methodology that makes use of two orthogonal, on-resin stapling reactions to incorporate linkers bearing a chiral centre into a p53-derived stapled peptide. Post-stapling modifications to the chain were performed on-resin and enabled rapid access to various peptide derivatives from a single staple. The stapled peptides have increased helicity, protease stability and in vitro binding affinities to MDM2 compared to the equivalent unstapled peptide. This approach can be used to generate a library of diverse stapled peptides with different properties starting from a single stapled peptide, with scope for much greater functional diversity than that provided by existing stapling methodologies.

Development of Cell-Permeable, Non-Helical Constrained Peptides to Target a Key Protein-Protein Interaction in Ovarian Cancer.

There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA-mediated knockdown of the target protein, HNF1ß, in five high- and low-HNF1ß-expressing CCC lines. To inhibit the protein function, cell-permeable, non-helical constrained proteomimetics to target the HNF1ß-importin α protein-protein interaction were designed, guided by X-ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors.

The bone-protective benefits of kaempferol combined with metformin by regulation of osteogenesis-angiogenesis coupling in OVX rats.

This study was to investigate the potential mechanisms of treatment with metformin (Met) combined with kaempferol (Kae) against postmenopausal osteoporosis. Experiments were conducted in both ovariectomy (OVX)-induced osteoporosis rats and in vitro using RAW264.7 cells, MC3T3-E1 cells, and HUVECs. Results demonstrated the therapeutic effect of Met combined with Kae on osteoporosis. In vivo, Kae alone and in combination with Met treatments enhanced tibial trabecular microstructure, bone mineral density (BMD), and mechanical properties in OVX rats without causing hepatotoxicity and nephrotoxicity. It also reduced bone resorption markers (CTX-1 and TRAP) and increased the bone formation marker (PINP) level in the serum of OVX rats. The expression of bone resorption marker TRAP was reduced, while bone formation markers Runx2 and ALP were enhanced in the bone tissue of OVX rats. Furthermore, Met combined with Kae also promoted the expression of angiogenesis-related markers CD31 and VEGF in OVX rats. In vitro, MC3T3-E1s cells treated with Met combined with Kae showed higher expression of ALP, Runx2, and VEGF. Interestingly, the treatment did not directly promote HUVECs migration and angiogenesis, but enhanced osteoblast-mediated angiogenesis by upregulating VEGF levels. Additionally, Met combined with Kae treatment promoted VEGF secretion in MC3T3-E1, and activated the Notch intracelluar pathway by upregulating HES1 and HEY1 in HUVECs. Meantime, their stimulation on CD31 expression were inhibited by DAPT, a Notch signaling inhibitor. Overall, this study demonstrates the positive effects of Met combined with Kae on osteoporotic rats by promoting osteogenesis-angiogenesis coupling, suggesting their potential application in postmenopausal osteoporosis.

Copper-catalyzed carboarylation of alkynes via vinyl cations.

Copper-catalyzed arylation of electron rich alkynes reveals stabilized trisubstituted vinyl cation equivalents that react with pendant arene nucleophiles to form all carbon tetrasubstituted alkenes. The new process streamlines the synthesis of important medicinally relevant molecules.

The mechanism of metformin combined with total flavonoids of Rhizoma Drynariae on ovariectomy-induced osteoporotic rats.

The present study evaluated the in vitro effect of metformin (Met) and total flavonoids of Rhizoma Drynariae (TFRD) on osteoclasts, osteocytes, and osteoblasts at different stages. We also assessed the effect and mechanism of treatment with Met combined with TFRD on ovariectomy (OVX)-induced osteoporosis in rats. The results showed that Met combined with TFRD significantly promoted the migration of osteoprogenitor cells and stimulated the differentiation and maturation of osteoblast precursor cells. Furthermore, Met combined with TFRD treatment significantly inhibited the osteogenic inhibitor sclerostin (SOST)/dickkopf 1 (DKK1) protein expression and the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio in osteocytes. In the in vivo study, Met combined with TFRD effectively reduced bone resorption markers levels, including type-I collagen carboxy-terminal peptide (CTX-1) and tartrate-resistant acid phosphatase (TRAP), and remarkably increased the bone formation marker propeptide of type I procollagen (PINP) level in the serum of rats with osteoporosis. Met combined with TFRD treatment improved bone mineral density (BMD), trabecular microstructure, and mechanical properties of osteoporotic rats. Mechanistically, Met combined with TFRD downregulated SOST and DKK1 levels, and upregulated Wnt10b, ß-catenin, runt-related transcription factor 2 (Runx2) et al. Meanwhile, Met combined with TFRD treatment reduced the RANKL/OPG ratio, and reduced the receptor activator of nuclear factor-κB (RANK), nuclear factor of activated T cells c1 (NFATC1), and TRAP levels. In conclusion, Met combined with TFRD ameliorated bone mass in osteoporotic rats through regulating Wnt/ß-catenin signaling pathway and OPG/RANKL/RANK axis.

Role of TGF-ß1 in Fluoride-Treated Osteoblasts at Different Stages.

Little attention has been paid to the tolerance of osteoblasts to fluoride in distinct differentiation stages, and the role of TGF-ß1 in fluoride-treated osteoblast differentiation of progenitors and precursors was rarely mentioned in previous studies. The present study aimed to clarify how fluoride affected different differentiation stages of osteoblasts, and to elucidate the role of TGF-ß1 in this process. We assessed cell migration, proliferation, DNA damage, and apoptosis of early-differentiated osteoblasts derived from bone marrow stem cells (BMSCs) exposed to fluoride with or without TGF-ß1. Subsequently, MC3T3-E1 cells cultured with mineral induction medium were treated with fluoride to test fluoride's effect on late-differentiated osteoblasts. The specific fluoride concentrations and treatment times were chosen to evaluate the role of TGF-ß1 in fluoride-induced osteoblastic differentiation and function. Results showed early-differentiated osteoblasts treated with a low dose of fluoride grew and moved more rapidly. TGF-ß1 promoted cell proliferation and inhibited cell apoptosis in early-differentiated osteoblasts exposed to a low fluoride dose, but enhanced apoptosis at higher fluoride conditions. In the late-differentiated osteoblasts, the fluorine dose range with anabolic effects was narrowed, and the fluoride range with catabolic effects was widened. Treatment with a low fluoride dose stimulated the alkaline phosphatase (ALP) expression. TGF-ß1 treatment inhibited Runx2 expression but increased RANKL expression in late-differentiated osteoblasts exposed to fluoride. Meanwhile, TGF-ß1 treatments activated Smad3 phosphorylation but blocked Wnt10b expression in osteoblasts. We conclude that TGF-ß1 plays an essential role in fluoride-induced differentiation and osteoblast function via activation of Smad3 instead of Wnt10 signaling.

Effect of flavonoids from Rhizoma Drynariae on osteoporosis rats and osteocytes.

In this experimental study, we evaluated the protective effects and the safety of main flavanones derived from Rhizoma Drynariae (Gusuibu) in vitro and in vivo. The MTT assay showed that compared with vehicle treatment, treatment with such flavanones as neoeriocitrin, naringin, and naringenin significantly promoted the viability of osteocyte-like cells. Quantitative real-time PCR showed that neoeriocitrin and naringin significantly attenuated mRNA expressions of RANKL and SOST in osteocyte-like cells. In rats with retinoic acid-induced osteoporosis, total flavonoid of Rhizoma Drynariae (TFRD), naringin, and naringenin significantly increased the number of trabeculae and improved trabecular bone structure compared with no treatment, without affecting liver and renal function. In addition, naringenin and naringin administration significantly increased bone mineral density of femur neck and femur shaft compared with the osteoporotic model rats. Western blot analysis showed that naringenin and naringin significantly attenuated protein expressions of bone resorption-related factors (TRAP, RANKL and RANK), and inhibited sclerostin expression compared with the osteoporotic model rats. On the other hand, naringin markedly increased protein expressions of ALP and PTH1R, and TFRD and naringenin also promoted PTH1R expression compared with the model rats. In conclusion, such flavanones as naringenin and naringin exhibited antiresorptive properties, and naringin particularly showed potential benefits for osteoporosis treatment.

Human growth hormone supplement promotes human lymphohematopoietic cell reconstitution in immunodeficient mice.

Aim: To investigate the potential of human growth hormone (hGH) to improve human hematopoietic reconstitution in humanized mice. Materials & methods: Immunodeficient mice were conditioned by total body irradiation and transplanted with human CD34+ fetal liver cells. Peripheral blood, spleen and bone marrow were harvested, and levels of human lymphohematopoietic cells were determined by flow cytometry. Results: Supplementation with hGH elevated human lymphohematopoietic chimerism by more than twofold. Treatment with hGH resulted in significantly increased reconstitution of human B cells and myeloid cells in lymphoid organs, enhanced human erythropoiesis in the bone morrow, and improved engraftment of human hematopoietic stem cells. Conclusion: hGH supplementation promotes human lymphohematopoietic reconstitution in humanized mice.

Humanized mice generated by human hematopoietic stem cell transplantation play crucial roles in biomedical investigations. One of the factors hindering the efficacy of their construction is the lack of or insufficient interaction of human cells to mouse cytokines and growth hormones (GHs) that are crucial for hematopoiesis and immune cell differentiation. In this study, we show that injection of human GH significantly improved human hematopoietic stem cell engraftment and function, as well as immune cell reconstitution in humanized mice. Our findings indicate that human cells may not efficiently respond to mouse GH, and generation of immunodeficient mice producing human GH may improve the efficacy of humanized mouse construction.

Effect of fluoride on osteocyte-driven osteoclastic differentiation.

Excessive systemic uptake of inorganic fluorides causes disturbances of bone homeostasis. The mechanism of skeletal fluorosis is still uncertain. This study aimed to study the effect of fluoride on osteocyte-driven osteoclastogenesis and probe into the role of PTH in this process. IDG-SW3 cells seeded in collagen-coated constructs were developed into osteocyte-like cells through induction of mineral agents. Then, osteocyte-like cells were exposed to fluoride in the presence or absence of parathyroid hormone (PTH). Cell viability and their capacity to produce receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and sclerostin (SOST) were detected by MTT and Western blot assays, respectively. Finally, a transwell coculture system using osteocyte-like cells seeded in the low compartment, and osteoclast precursors added in the inserts was developed to observe the osteocyte-driven osteoclasogenesis response to fluoride with or without PTH, and the expression of molecules involved in this mechanism were measure by real time RT-PCR. Results showed that osteocytes withstood a toxic dose of fluoride, and yet PTH administration significantly reduced osteocytes viability. PTH amplified the effect of fluoride on the expression of osteoclastogenesis-related molecules in osteocyte, but did not enlarged the stimulating effect of fluoride on osteoclastogenesis drove by osteocyte coculture. Gene expression levels of TRAP, RANK, JNK and NFAtc1 significantly increased in fluoride affected osteoclast precursor cocultured with osteocyte-like cells. The impact of fluoride on osteocyte-driven osteoclast differentiation was stronger than that of PTH. In conclusion, osteocyte played a pivotal role on the mechanism underlying fluoride-affected osteoclastogenesis in which RANK-JNK-NFATc1 signaling pathway was involved, and PTH had a significant impact in this process.

Surface design of photon-upconversion nanoparticles for high-contrast immunocytochemistry.

Immunohistochemistry (IHC) and immunocytochemistry (ICC) are routinely employed for the microscopic identification and diagnosis of cancerous cells in histological tissues and cell cultures. The maximally attainable contrast of conventional histological staining techniques, however, is low. While the anti-Stokes emission of photon-upconversion nanoparticles (UCNP) can efficiently eliminate optical background interference, excluding non-specific interactions of the label with the histological sample is equally important for specific immunolabeling. To address both requirements, we have designed and characterized several UCNP-based nanoconjugates as labels for the highly specific detection of the cancer biomarker HER2 on various breast cancer cell lines. An optimized streptavidin-PEG-neridronate-UCNP conjugate provided an unsurpassed signal-to-background ratio of 319, which was 50-fold better than conventional fluorescent labeling under the same experimental conditions. In combination, the absence of optical interference and non-specific binding lays the foundation for computer-based data evaluation in digital pathology.

Functionalized Double Strain-Promoted Stapled Peptides for Inhibiting the p53-MDM2 Interaction.

The Sondheimer dialkyne reagent has previously been employed in strain-promoted double-click cycloadditions with bis-azide peptides to generate stapled peptide inhibitors of protein-protein interactions. The substituted variants of the Sondheimer dialkyne can be used to generate functionalized stapled peptide inhibitors with improved biological properties; however, this remains a relatively underdeveloped field. Herein, we report the synthesis of new substituted variants of Sondheimer dialkyne and their application in the stapling of p53-based diazido peptides to generate potent stapled peptide-based inhibitors of the oncogenic p53-MDM2 interaction. The functionalized stapled peptide formed from a meta-fluoro-substituted Sondheimer dialkyne was found to be the most potent inhibitor. Furthermore, through experimental studies and density functional theory calculations, we investigated the impact of the substituent on the strain-promoted double-click reactivity of Sondheimer dialkyne.

Toolbox of Diverse Linkers for Navigating the Cellular Efficacy Landscape of Stapled Peptides.

Stapled peptides have great potential as modulators of protein-protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a "toolbox" of diversified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure-activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.

Single-molecule FRET reveals hidden complexity in a protein energy landscape.

Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.