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For 2017, the estimated lifetime risk of developing colorectal cancer was 1 in 22. Even though preventative colonoscopy screening and standard-of-care surgery, radiation, and chemotherapy have decreased the death rate from colorectal cancer, new therapies are needed for metastatic colorectal cancer. Here, we developed a novel small molecule, compound 2, that inhibited proliferation and viability of human colorectal cancer cells (HCT-116, DLD-1, SW480, and 10.1). Compound 2 inhibited cell migration, invasion, and epithelial–mesenchymal transition processes and potently increased cell apoptosis in human colorectal cancer cells. Compound 2 also modulated mitotic stress signaling, leading to both inhibition of Wnt responsiveness and stabilization and activation of p53 to cause cell-cycle arrest. In mouse xenografts, treatment with compound 2 (20 mg/kg/day, i.p.) induced cell death and inhibited tumor growth more than four-fold compared with vehicle at day 34. Neither acute cytotoxicity nor toxicity in animals (up to 1,000 mg/kg, i.p.) were observed for compound 2. To our knowledge, compound 2 is the first reported potent small molecule that inhibits Wnt/β-catenin signaling, activates p53 signaling regardless of p53 mutation status, and binds microtubules without detectable toxicity. Thus, compound 2 offers a novel mechanism of action and a new strategy to treat colorectal cancer.Significance: These findings identify a potent small molecule that may be therapeutically useful for colon cancer that works by inhibiting Wnt/β-catenin signaling, activating p53, and binding microtubules without detectable toxicity. Cancer Res; 78(17); 5072–83. ©2018 AACR.

Therapeutic peptides have revolutionized treatment for a number of human diseases. In particular, the past two decades have witnessed rapid progress of stapled helical peptides in drug discovery. Stapled helical peptides are chemically modified and constrained in their bioactive α-helical conformation. Compared to unstabilized linear peptides, stapled helical peptides exhibit superior binding affinity and selectivity, enhanced membrane permeability, and improved metabolic stability, presenting exciting promise for targeting otherwise challenging protein-protein interfaces. In this Perspective, we summarize recent applications of high-throughput screening technologies for identification of potent stapled helical peptides with optimized binding properties. We expect to provide a broad reference to accelerate the development of stapled helical peptides as the next generation of therapeutic peptides for various human diseases.

Despite considerable research effort, there is a significant need for safe agents that stimulate bone formation. Treatment of large or complex bone defects remains a challenge. Implantation of small molecule-induced human bone marrow-derived mesenchymal stromal cells (hBMSCs) on an appropriate tricalcium phosphate (TCP) scaffold offers a robust system for noninvasive therapy for spinal fusion. To show the efficacy of this approach, we identified a small molecule curcuminoid that when combined with TCP ceramic in the presence of hBMSCs selectively induced growth of bone cells: after 8- or 25-day incubations, alkaline phosphatase was elevated. Treatment of hBMSCs with curcuminoid 1 and TCP ceramic increased osteogenic target gene expression (i.e., Runx2, BMP2, Osteopontin, and Osteocalcin) over time. In the presence of curcuminoid 1 and TCP ceramic, osteogenesis of hBMSCs, including proliferation, differentiation, and mineralization, was observed. No evidence of chondrogenic or adipogenic potential using this protocol was observed. Transplantation of curcuminoid 1-treated hBMSC/TCP mixtures into the spine of immunodeficient rats showed that it achieved spinal fusion and provided greater stability of the spinal column than untreated hBMSC-TCP implants or TCP alone implants. On the basis of histological analysis, greater bone formation was associated with curcuminoid 1-treated hBMSC implants manifested as contiguous growth plates with extensive hematopoietic territories. Stimulation of hBMSCs by administration of small molecule curcuminoid 1 in the presence of TCP ceramic afforded an effective noninvasive strategy that increased spinal fusion repair and provided greater stability of the spinal column after 8 weeks in immunodeficient rats. IMPACT STATEMENT: Bone defects only slowly regenerate themselves in humans. Current procedures to restore spinal defects are not always effective. Some have side effects. In this article, a new method to produce bone growth within 8 weeks in rats is presented. In the presence of tricalcium phosphate ceramic, curcuminoid-1 small molecule-stimulated human bone marrow-derived mesenchymal stromal cells showed robust bone cell growth in vitro. Transplantation of this mixture into the spine showed efficient spinal fusion in rats. The approach presented herein provides an efficient biocompatible scaffold for delivery of a potentially clinically useful system that could be applicable in patients.

Today, pancreatic cancer (PC) is a major health problem in the United States. It remains a challenge to develop efficacious clinically useful PC therapies. New avenues, based on translational approaches and innovative validated biomarkers could be a preclinical option to evaluate PC drug candidates or drug combinations before clinical trials. Herein, we describe evaluation of combination therapies by incorporating a novel pathway modulator, p53-Activator Wnt Inhibitor-2 (PAWI-2) with other FDA-approved cancer drugs that have been used in PC clinical trials. PAWI-2 is a potent inhibitor of drug-resistant PC cells that has been shown to selectively ameliorate human pancreatic cancer stem cells (i.e., hPCSCs, FGβ3 cells). In the present study, we showed PAWI-2 produced therapeutic synergism with certain types of anti-cancer drugs. These drugs themselves oftentimes do not ameliorate PC cells (especially PCSCs) due to high levels of drug-resistance. PAWI-2 has the ability to rescue the potency of drugs (i.e., erlotinib, trametinib) and inhibit PC cell growth. Key molecular regulators of PAWI-2 could be used to predict synergistic/antagonistic effects between PAWI-2 and other anti-cancer drugs. Anti-cancer results showed potency could be quite accurately correlated to phosphorylation of optineurin (OPTN) in PC cells. Synergism/antagonism was also associated with inhibition of PCSC marker SOX2 that was observed in FGβ3 cells. Synergism broadens the potential use of PAWI-2 as an adjunct chemotherapy in patients with PC that have developed resistance to first-line targeted therapies or chemotherapies.

Cancer is a major worldwide public health problem and is still the leading cause of death in the United States. There are many types of cancer treatment but completely successful results are oftentimes not attained. It remains a challenge to develop efficacious clinically useful cancer therapies. Therapies targeting dysregulated signal transduction pathways in cancer can be efficacious anti-cancer therapies with minimal adverse effects. In this study, we focus on novel small molecule p53 Activator Wnt Inhibitor-2 (PAWI-2) that was developed by optimizing potency and pharmaceutical properties. PAWI-2 is a nontoxic DNA-damage pathway inhibitor that shows a broad spectrum of potency and significant efficacy in vitro and in vivo. This study focuses on the application of PAWI-2 to four major types of cancers including colorectal cancer (CRC), breast cancer (BC), prostate cancer (PCa), and pancreatic cancer (PC). PAWI-2 shows a novel mechanism of action (MOA) by modulating two mechanisms of cancer invasion. In cancer with unimpaired p53, PAWI-2 activates DNA-damage checkpoint and mitochondrial p53-dependent apoptotic signaling. Consistently observed in most cancer types, PAWI-2 induces phosphorylation of optineurin (OPTN) to cause G2/M cell cycle arrest. These two mechanisms operate regardless of p53 variants and/or KRAS mutation status and also manipulate the effect of PAWI-2 to overcome tumor stemness and drug resistance in PC stem cells (PCSCs). This study summarizes the development of PAWI-2 as an attractive targeted therapeutic for mechanism-driven anti-cancer drug discovery.

Today, pancreatic cancer (PC) remains a major health problem in the US. The fact that cancer stem cells (CSCs) become enriched in humans following anti-cancer therapy implicates CSCs as key contributors to tumor dormancy, metastasis, and relapse in PC. A highly validated CSC model (FGβ3 cells) was used to test a novel compound (PAWI-2) to eradicate CSCs. Compared to parental bulk FG cells, PAWI-2 showed greater potency to inhibit cell viability and self-renewal capacity of FGβ3 cells. For FGβ3 cells, dysregulated integrin β3-KRAS signaling drives tumor progression. PAWI-2 inhibited β3-KRAS signaling independent of KRAS. This is clinically relevant. PAWI-2 targeted the downstream TBK1 phosphorylation cascade that was negatively regulated by optineurin phosphorylation via a feedback mechanism. This was confirmed by TBK1 genetic knockdown or co-treatment with TBK1-specific inhibitor (MRT67307). PAWI-2 also overcame erlotinib (an EGFR inhibitor) resistance in FGβ3 cells more potently than bortezomib. In the proposed working model, optineurin acts as a key regulator to link inhibition of KRAS signaling and cell cycle arrest (G2/M). The findings show PAWI-2 is a new approach to reverse tumor stemness that resensitizes CSC tumors to drug inhibition.

For 2017, the estimated lifetime risk of developing colorectal cancer was 1 in 22. Even though preventative colonoscopy screening and standard-of-care surgery, radiation, and chemotherapy have decreased the death rate from colorectal cancer, new therapies are needed for metastatic colorectal cancer. Here, we developed a novel small molecule, compound 2, that inhibited proliferation and viability of human colorectal cancer cells (HCT-116, DLD-1, SW480, and 10.1). Compound 2 inhibited cell migration, invasion, and epithelial–mesenchymal transition processes and potently increased cell apoptosis in human colorectal cancer cells. Compound 2 also modulated mitotic stress signaling, leading to both inhibition of Wnt responsiveness and stabilization and activation of p53 to cause cell-cycle arrest. In mouse xenografts, treatment with compound 2 (20 mg/kg/day, i.p.) induced cell death and inhibited tumor growth more than four-fold compared with vehicle at day 34. Neither acute cytotoxicity nor toxicity in animals (up to 1,000 mg/kg, i.p.) were observed for compound 2. To our knowledge, compound 2 is the first reported potent small molecule that inhibits Wnt/β-catenin signaling, activates p53 signaling regardless of p53 mutation status, and binds microtubules without detectable toxicity. Thus, compound 2 offers a novel mechanism of action and a new strategy to treat colorectal cancer. Significance: These findings identify a potent small molecule that may be therapeutically useful for colon cancer that works by inhibiting Wnt/β-catenin signaling, activating p53, and binding microtubules without detectable toxicity. Cancer Res; 78(17); 5072–83. ©2018 AACR.

For adult women in the United States, breast cancer is the most prevalent form of cancer. Compounds that target dysregulated signal transduction can be efficacious anti-cancer therapies. A prominent signaling pathway frequently dysregulated in breast cancer cells is the Wingless-related integration site (Wnt) pathway. The purpose of the work was to optimize a “hit” from a screening campaign. 76,000 compounds were tested in a Wnt transcription assay and revealed potent and reproducible “hit,” compound 1. Medicinal chemistry optimization of 1 led to more potent and drug-like molecules, 19, 24 and 25 (i.e., Wnt pathway IC50 values = 11, 18 and 7 nM, respectively). The principal results showed compounds 19, 24 and 25 were potent anti-proliferative agents in breast cancer cell lines, MCF-7 (i.e., IC50 values = 10, 7 and 4 nM, respectively) and MDA-MB 231 (i.e., IC50 values = 13, 13 and 16 nM, respectively). Compound 19 synergized anti-proliferation with chemotherapeutic Doxorubicin in vitro. A major conclusion was that compound 19 enhanced anti-proliferation of Doxorubicin in vitro and in a xenograft animal model of breast cancer.

Prostate cancer (PCa) is the second leading cause of cancer-related death for men in the United States. Approximately 35% of PCa recurs and is often transformed to castration-resistant prostate cancer (CRPCa), the most deadly and aggressive form of PCa. However, the CRPCa standard-of-care treatment (enzalutamide with abiraterone) usually has limited efficacy. Herein, we report a novel molecule (PAWI-2) that inhibits cellular proliferation of androgen-sensitive and androgen-insensitive cells (LNCaP and PC-3, respectively). In vivo studies in a PC-3 xenograft model showed that PAWI-2 (20 mg/kg per day i.p., 21 days) inhibited tumor growth by 49% compared with vehicle-treated mice. PAWI-2 synergized currently clinically used enzalutamide in in vitro inhibition of PCa cell viability and resensitized inhibition of in vivo PC-3 tumor growth. Compared with vehicle-treated mice, PC-3 xenograft studies also showed that PAWI-2 (20 mg/kg per day i.p., 21 days) and enzalutamide (5 mg/kg per day i.p., 21 days) inhibited tumor growth by 63%. Synergism was mainly controlled by the imbalance of prosurvival factors (e.g., Bcl-2, Bcl-xL, Mcl-1) and antisurvival factors (e.g., Bax, Bak) induced by affecting mitochondrial membrane potential/mitochondria dynamics. Thus, PAWI-2 utilizes a distinct mechanism of action to inhibit PCa growth independently of androgen receptor signaling and overcomes enzalutamide-resistant CRPCa. SIGNIFICANCE STATEMENT: Castration-resistant prostate cancer (CRPCa) is the most aggressive human prostate cancer (PCa) but standard chemotherapies for CRPCa are largely ineffective. PAWI-2 potently inhibits PCa proliferation in vitro and in vivo regardless of androgen receptor status and uses a distinct mechanism of action. PAWI-2 has greater utility in treating CRPCa than standard-of-care therapy. PAWI-2 possesses promising therapeutic potency in low-dose combination therapy with a clinically used drug (e.g., enzalutamide). This study describes a new approach to address the overarching challenge in clinical treatment of CRPCa.

Summary Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through β-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Today, pancreatic cancer (PC) remains a major health problem in the US and soon will be the second most common cause of mortality due to cancer. However, most of the front-line PC chemotherapies cause serious side effects and a majority of patients with PC are often resistant to clinical therapies. The fact that cancer stem cells (CSCs) become enriched after anti-cancer therapy implicates CSCs as key contributors to tumor dormancy, metastasis, and relapse in PC. Given the important role of human pancreatic cancer stem cells (hPCSCs) in PC, development of a novel treatment strategy that targets hPCSCs or their extrinsic and intrinsic regulators could be of great clinical utility to treat PC. Herein, we used a highly validated hPCSCs model (FGβ3 cells) to test a novel compound (PAWI-2) to target CSCs. In this hPCSC model, integrin αvβ3 expression was shown to be a marker/functional contributor to CSC progression and drug resistance attributed to its ability to trigger anchorage-independent cell survival and tumor metastasis. Specifically, integrin αvβ3 recruits KRAS and RalB to tumor cell plasma membranes to activate NF-κB signaling that triggers dysregulated KRAS-RalB-NF-κB pathways. This was shown to be a pharmacological target to reverse CSC-like properties or re-sensitize drug resistance (e.g., EGFR inhibitors, erlotinib) of established FGβ3 tumors. Compared to parental FG cells, PAWI-2 showed greater potency to inhibit cell viability and self-renewal capacity of FGβ3 cells. PAWI-2 inhibited dysregulated integrin β3-KRAS-RalB-NF-κB signaling independent of KRAS and Ral. This is clinically relevant because >90% of KRAS is activated by mutations in PC and RAS or Ral inhibitors have not proven effective clinically. PAWI-2 targeted downstream of KRAS signaling and was negatively regulated by other feedback mechanisms. This was further shown by genetic knockdown of key regulator and co-treatment of specific inhibitors. PAWI-2 overcomes erlotinib resistance in FGβ3 cells with greater potency than bortezomib. In the proposed mechanism of PAWI-2 action, key regulators link the inhibition of KRAS signaling and cell cycle arrest (G2/M). The great utility and pharmacological potency of PAWI-2 in FGβ3 cells (that possess stem-like properties) compared to FG cells provide a basis for further development. PAWI-2 as an efficient therapeutic to reverse tumor stemness and resensitize CSC tumors to drug inhibition. This work was supported by Inception Award from California Institute for Regenerative Medicine (CIRM) (DISC1-10583; J.R. Cashman) and by funds from the Human BioMolecular Research Institute. Citation Format: Jiongjia Cheng, John Cashman, Jorge Gomez-Galeno. PAWI-2 overcomes tumor stemness and drug resistance in pancreatic cancer stem cells [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 694.

Pancreatic ductal adenocarcinoma (PDAC), constitutes >90% of pancreatic cancers (PC) and is one of the most aggressive human tumors. Standard chemotherapies for PDAC (e.g., gemcitabine, FOLFIRINOX, etc.) has proven to be largely ineffective. Herein, we report a novel molecule (i.e., compound 1) that potently inhibits proliferation and induces apoptosis of PDAC cells. As we observed in other cancer types (i.e., colorectal, breast cancer), the effect of 1 against PDAC cells is also related to microtubule destabilization and DNA damage checkpoint activation. However, in PDAC cells, the inhibitory effect of 1 was mainly controlled by mitochondrial p53-dependent apoptosis. Compound 1 worked with cells of different p53 mutant status and affected p53 activation/phosphorylation not simply by stabilizing p53 protein but through antagonizing anti-apoptotic effects of Bcl-xL and restoring p53 to activate mitochondrial-apoptotic pathways (i.e., cytochrome c release, caspase activation and PARP cleavage). Compound 1 was more efficient than a typical PDAC combination therapy (i.e., gemcitabine with paclitaxel) and showed synergism in inhibiting PDAC cell proliferation with gemcitabine (or gemcitabine with paclitaxel). This synergism varied between different types of PDAC cells and was partially controlled by the phosphorylation of p53 on Serine15 (phospho-Ser15-p53). In vivo studies in an orthotopic syngeneic murine model showed that 1 (20 mg/kg/day, 28 days, i.p.) inhibited tumor growth by 65% compared to vehicle-treated mice. No apparent acute or chronic toxicity was observed. Thus, compound 1 utilizes a distinct mechanism of action to inhibit PC growth in vitro and in vivo and is a novel anti-PDAC compound.

The Notch signaling pathway is involved in cell proliferation and differentiation, and has been recognized as an active pathway in regenerating tissue and cancerous cells. Notch signaling inhibition is considered a viable approach to the treatment of a variety of conditions including colorectal cancer, pancreatic cancer, breast cancer and metastatic melanoma. The discovery that the b-annulated dihydropyridine FLI-06 (1) is an inhibitor of the Notch pathway with an EC50 ≈ 2.5 μM prompted us to screen a library of related analogs. After structure activity studies were conducted, racemic compound 7 was identified with an EC50 = 0.36 μM. Synthesis of individual enantiomers provided (+)-7 enantiomer with an EC50 = 0.13 μM, or about 20-fold the potency of 1.

Peripheral membrane proteins can be targeted to specific organelles or the plasma membrane by differential recognition of phospholipid headgroups. Although molecular determinants of specificity for several headgroups, including phosphatidylserine and phosphoinositides are well defined, specific recognition of the headgroup of the zwitterionic phosphatidylcholine (PC) is less well understood. In cytosolic proteins the cation-π box provides a suitable receptor for choline recognition and binding through the trimethylammonium moiety. In PC, this moiety might provide a sufficient handle to bind to peripheral proteins via a cation-π cage, where the π systems of two or more aromatic residues are within 4–5 A of the quaternary amine. We prove this hypothesis by engineering the cation-π box into secreted phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, which lacks specific PC recognition. The N254Y/H258Y variant selectively binds PC-enriched vesicles, and x-ray crystallography reveals N254Y/H258Y binds choline and dibutyroylphosphatidylcholine within the cation-π motif. Such simple PC recognition motifs could be engineered into a wide variety of secondary structures providing a generally applicable method for specific recognition of PC.

The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro(245) and Pro(254), are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro(245) disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro(245) variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro(254) appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding.

Phosphatidylinositol-specific phospholipase C (PI-PLC) is a virulence factor produced and secreted by many Gram positive bacteria (1). When secreted by extracellular pathogens (e.g., Bacillus and Staphylococcus strains (2–5) this enzyme catalyzes the cleavage of glycan-phosphatidylinositol (GPI) anchored proteins. PI-PLC removal of the protective GPI-anchored proteins on the exterior surface of mammalian cells allows cellular damage to occur, and the diacylglycerol, translocating across the bilayer (6), can interfere with cellular signaling processes (7). Phosphatidylinositol molecules are also good substrates for this enzyme and provide an easier system to explore mechanistic details of these enzymes (1). PI-PLC cleaves its substrate via an intramolecular phosphotransferase reaction on the PI moiety to form a cyclic inositol phosphate molecule and diacylglycerol (8). Subsequent hydrolysis of the cyclic phosphodiester produces inositol 1-phosphate if PI is the substrate (9, 10). The PI-PLC from S. aureus is unusual in exhibiting significant activity towards PI at acidic pH (4) – a property that may contribute to the virulence of the organism, which is often in an acidic milieu (11). Crystal structures of the PI-PLC enzymes from Bacillus species (12, 13) and Listeria monocytogenes (14), an intracellular pathogen, show very similar distorted TIM barrels with an active site close to the barrel rim. Each has a short helix at the rim that contains at least one lysine, important for binding to negatively charged interfaces, and a tryptophan that, in the case of the Bacillus PI-PLC, has been proposed to insert into target membranes (15–17). Another key structural feature is a rim loop near the active site that contains one large hydrophobic residue (either a tryptophan or phenylalanine) amid many small flexible residues. This surface-exposed hydrophobic residue is also thought to aid in anchoring the protein to membranes (15, 16). Several of the bacterial PI-PLC enzymes exhibit kinetic activation by non-substrate phospholipids. Specifically, inclusion of PC in vesicles containing PI lead to large increases in the specific activity of these secreted PI-PLC enzymes (9, 18, 19). The detailed mechanism for PC activation is not the same for the different enzymes. B. thuringiensis PI-PLC has a discrete site for PC binding (20), while the L. monocytogenes PI-PLC requires PC to dilute cationic enzyme/anionic lipid aggregates that trap the enzyme in a nonproductive state (19). The PI-PLC from S. aureus can also exhibit a significant increase in specific activity towards PI in vesicles containing PC (vide infra). We have solved the structure of S. aureus PI-PLC strain FPR3757 at two different pH values: (i) pH 4.6, where the enzyme shows low activity (but significantly higher than other bacterial PI-PLC enzymes), and (ii) pH 7.5, where it is very active towards PI/PC (but not pure PI) vesicles. A large conformational change in the rim mobile loop between the acidic and basic pH structures depends on a titratable π-histidine cation interaction. π-Cation interactions are well known in proteins, however these interactions commonly exist in protein-protein interfaces, enzyme-drug interactions, helix stabilization and protein-DNA interactions (21–23). π-Cation complexes with protonated histidine as the cation are also known (24). Most of these interactions result in subtle structural changes; typically they mediate ligand binding or helix stabilization (25, 26). However, a recent structure of the transferrin receptor showed a large change in structure in the dimer interface region upon transferrin binding whereby a protonated surface histidine flipped into the protein to engage in π-stacking with a phenylalanine and tyrosine (27). Observation of the titratable intramolecular π-cation interaction in S. aureus PI-PLC suggests higher activity at acidic pH is connected with easier release of water-soluble product inositol 1,2-(cyclic)phosphate (cIP). This structural feature also provides an explanation for how PC may activate this bacterial PI-PLC. The presence of that zwitterionic phospholipid in a substrate-containing interface provides cationic choline moieties that can compete with cationic His258 for interactions with the π system of Phe249 allowing the latter to partition into membranes. The higher activity once PC is added indicates that this membrane conformation must differ from the one where only PI is present.

The interaction between double-strand calf thymus gland DNA (ds-DNA) and Al(III) was studied by using differential pulse voltammetry (DPV) at a hanging mercury drop electrode (HMDE), Raman spectrometry and circular dichroism (CD) spectra. It was shown that at neutral pH ds-DNA did not produce any cathodic peak at the HMDE in the potential window from -550 to -2000 mV vs. SCE. However, in the presence of Al(III), a cathodic peak was generated at about -1660 mV, which is ascribed to a reduction of adenine and cytosine residues of single denatured DNA (sd-DNA). It was concluded that ds-DNA was completely denatured to sd-DNA by Al(III) at a neutral pH. The apparent denaturing kinetic velocity constants of ds-DNA by Al(III) were derived from linear increases of the cathodic peak currents with time. When [Al(III)]x[OH(-)](3) > or = 2 x 10(-26), the precipitation of Al(OH)(3) was observed and identified by the Raman spectrum, and inductively coupled plasma atomic emission spectrometry (ICP-AES). CD spectra showed that the B-type of structure conformations of ds-DNA and related sd-DNA did not change with the increment of Al(III) from 5.0 x 10(-7) to 1.0 x 10(-5) M, but the corresponding absorption strengths increased. The related physiological significances and possible applications of the observations were considered.

This paper reports the preparation of dodecanethiol self-assembled monolayers (C 12 SH-SAMs) on polycrystalline gold electrodes in a magnetic field. The qualities of C 12 SH-SAMs were characterized by both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results show that highly dense and well-ordered SAMs could form in a relatively short time (3 h), indicating that the rate of SAMs formation increased under an external magnetic field compared with the natural self-assembly process. Moreover, the results of CV and EIS measurements also suggested that the presence of a magnetic field had influenced the qualities of the SAMs; the stronger magnetic intensity can help to obtain much denser and well-ordered SAMs.

This paper reports the study of the solvent (tetrachloromethane, toluene, tetrahydrofuran, acetone, N , N -dimethylformamide, acetonitrile, ethanol, ethanol–water mixture (1:1, v:v) and neat dodecanethiol liquid) effect on the quality of self-assembled monolayers of dodecanethiol (C 12 SH-SAMs) on polycrystalline gold. The quality of the C 12 SH-SAMs was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and the results show that highly dense and well-ordered C 12 SH-SAMs can be prepared from ethanol–water mixture and neat dodecanethiol liquid. The property parameters of the solvents, such as polarity, solubility parameter, molecular diameter, octanol–water partition coefficients and viscosity were correlated with their ability to influence the quality of the C 12 SH-SAMs.

The effects of nanometer-sized tridecameric aluminum polycation (nano-Al13) on lactate dehydrogenase (LDH) activity have been investigated using differential pulse voltammetry (DPV) at the molecular level. The effects of nano-Al13 on LDH activity at different temperature and pH values were investigated. The results showed that these factors had quite different influences. The characteristic parameters of Michaelis constant Km and maximum velocity vmax were evaluated by monitoring the DPV reduction current of NAD+ (β-nicotinamide adenine dinucleotide) and compared with the other known inhibitors of LDH. The proposed method might be hypothesized to be a simple and convenient approach to evaluate the toxic and biological effects of nanoparticles on the environment.

The promotion effect of titania nanoparticles (nano-TiO(2)) on the direct electron transfer between lactate dehydrogenase (LDH) and the silica sol-gel modified gold electrode was investigated by adding nano-TiO(2) (50 nm) in the modification process. This nano-TiO(2)-LDH electrode showed a pair of quasi-reversible cyclic voltammetry peaks with the formal potential of 70 mV (vs. SCE). Compared to the previous result of LDH modified electrode with only an irreversible cathodic peak, an anodic peak appeared and the cathodic peak potential shifted to the positive direction on this nano-TiO(2)-LDH electrode, which demonstrated that the direct electrochemistry of LDH was enhanced by nano-TiO(2). We supposed that the direct electrochemistry of LDH may be due to the redox reaction of some electroactive amino acids in the LDH molecule. The surface morphologies of electrodes characterized by SEM indicated that LDH was successfully immobilized on the sol-gel matrix and also had some interactions with nano-TiO(2). This electrode can be used as a biosensor for the determination of lactic acid. The calibration range of lactic acid was from 1.0 to 20 micromolL(-1) and the detection limit was 0.4 micromolL(-1). Meanwhile, the small K(m)(app) value (2.2 micromolL(-1)) suggested that LDH possessed high enzymatic activity and good affinity to lactic acid owing to the promotion effect of nano-TiO(2).

This paper reports a new solvent, room-temperature ionic liquid (RTIL), for the preparation of dodecanethiol self-assembled monolayers (C12SH-SAMs) on polycrystalline gold. The quality of C12SH-SAMs was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). From CV experiments, we find that the differential capacitance Cd values of the C12SH-SAM prepared in RTIL, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6) containing 10 μL neat C12SH for 24 h (C12SH-SAMs[BMIM]PF6,10μL,24h) are independent of the scan rate, the effective thickness deff value and the average cant angle φ value of this monolayer are 18 ± 1 Å and 27 ± 4°, respectively. The difference value of the current density at −0.2 and 0.5 V (Δip) is only 0.73 ± 0.18 μA cm−2. EIS experiments show that the phase angle value at 1 Hz Φ1Hz, the charge transfer resistance Rct value and surface coverage θ value of this C12SH-SAM are 88.2 ± 0.7°, 3.44 ± 1.91 MΩ cm2 and 99.998 ± 0.001%, respectively. These results indicate that high-quality C12SH-SAMs can be formed in [BMIM]PF6. In addition, the rate of formations of high-quality C12SH-SAMs in RTIL can be substantially improved by ultrasound. Keywords: Self-assembled monolayer, Dodecanethiol, Room-temperature ionic liquids, Cyclic voltammetry, Electrochemical impedance spectroscopy

This article reports a rapid method of preparing self-assembled monolayers of dodecanethiol (C12SH-SAMs) on polycrystalline gold by microwave irradiation (MWI, 650 W, duty cycle is 10%). The qualities of C12SH-SAMs were characterized by both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results show that the C12SH-SAMs formed by MWI in 120 s (C12SH-SAMsMWI,120 s) have low ionic permeability (the differential capacitance Cd values are independent of the scan rate and phase angle at 1 Hz Φ1 Hz = 89 ± 0.9°), excellent electrochemical blocking ability towards the redox probe (the current iMWI,120 s obtained from CV is lowest when compared to other SAMs and charge transfer resistance Rct = (1.15 ± 0.19) × 106 Ω cm2), and high surface coverage (99.996 ± 0.001%). Keywords: Rapid formation, Self-assembled monolayer, Dodecanethiol, Microwave irradiation, Cyclic voltammetry, Electrochemical impedance spectroscopy

Self-assembled monolayers of dodecanethiol (C 12 SH-SAMs) on polycrystalline gold were prepared under ultrasonic irradiation at 100 W (the actual ultrasonic power intensity is about 0.1 W cm −2 including the heat loss) for different time and investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). CV experiments show that the differential capacitance C d values of the C 12 SH-SAM prepared under ultrasonic irradiation at 100 W (0.1 W cm −2 ) for 15 min are independent of the scan rate, the thickness d value of this monolayer is 17.5 A, the tilt angle φ value of the molecules in this monolayer from the gold surface normal was calculated to be 30° and the difference value of the current density at −0.2 and 0.5 V (Δ i p ) is only 0.69 μA cm −2 . From the EIS experiments, we find that the phase angle value at 1 Hz Φ 1 Hz of the C 12 SH-SAM prepared under ultrasonic irradiation at 100 W (0.1 W cm −2 ) for 15 min is 89°, the charge transfer resistance R ct value of this monolayer is 1.40 × 10 6 Ω cm 2 and the surface coverage θ value of this monolayer was calculated to be 99.997% from R ct . These results indicate that the C 12 SH-SAM of almost defect-free structure and very low ionic permeability can be formed under ultrasonic irradiation at 100 W (0.1 W cm −2 ) in a short time (15 min).

We present a novel method for the study of the aggregation of protein induced by metal ion aluminum(III) using resonance Rayleigh scattering (RRS) technique. In neutral Tris–HCl medium, the effect of this aggregation of protein results in the enhancement of RRS intensity and the relationship between the enhancement of the RRS signal and the Al concentration is nonlinear. On this basis, we established a new method for the determination of the critical induced-aggregation concentrations ( C CIAC ) of metal ion Al(III) inducing the protein aggregation. Our results show that many factors, such as, pH value, anions, salts, temperature and solvents have obvious effects. We also studied the extent of aggregation and structural changes using ultra-violet spectrometry, protein intrinsic fluorescence and circular dichroism to further understand the exact mechanisms of the aggregation characteristics of proteins induced by metal ion Al(III) at the molecular level, to help us to develop effective methods to investigate the toxicity of metal ion Al, and to provide theoretical and quantitative evidences for the development of appropriate treatments for neurodementia such as Parkinson's disease, Alzheimer's disease and dementia related to dialysis.

In this paper, we firstly report the direct voltammetric recognition and determination of dopamine (DA) by using Al(III)-DA complexes at the hanging mercury drop electrode (HMDE). A new sensitive cathodic peak of Al(III)-DA can be detected at -900 mV (vs. SCE) in 0.1 M NH(4)Cl-NH(3).H(2)O-0.1 M KCl buffer solution at pH 8.5. This unique -900 mV cathodic peak arises from the specific interaction between Al(III) and DA on the HMDE, whereas other substances with similar structures, such as L-dopa, epinephrine (EP), norepinephrine (NE), catechols, caffeic acid (CA), trihydric phenols and tiron, do not yield any new peak on the voltammograms in the potential range from -100 to -1200 mV when Al(III) is added. The distinct voltammetric characteristic of the recognition of DA can effectively inhibit the interferences of both ascorbic acid and uric acid in the DA determination by the direct electrochemistry, which is a major difficulty when a solid electrode is used. The proposed method can be anticipated as an effective means for the recognition of DA in the elucidation of the mechanisms of Parkinson's disease (PD) and Alzheimer's disease (AD) in the presence of Al(III).

A novel and reliable high-performance liquid chromatographic method for trace short-chain organic acids measurement in wheat root exudates under aluminum stress has been optimized and validated. The chromatographic separation of the short-chain organic acids (citric, oxalic, malonic, succinic, tartaric, malic, and acetic acids) was achieved with Bio-rad Aminex HPX-87H cation exchange resin column. These seven organic acids were identified and quantified in 25 min. Well-shaped peaks were obtained for trace organic acids using dilute sulfuric acid as mobile phase. Under optimum conditions, Bio-rad Aminex HPX-87H column showed distinct advantages of the ability to well separate different short-chain organic acids (especially for tartaric and malic acids, as well as malonic and acetic acids) in wheat root exudates under aluminum stress, and offered accurate and precise results for the analysis of these organic acids. This HPLC method can efficiently eliminate the aluminum’s interference and is quite suitable to the trace detection of trace organic acids in wheat root exudates under aluminum stress.

Through extensive and systematic reviewing of literatures, this paper presents some thoughts on the existence of ion and water channels in highly dense and well-ordered CH(3)-terminated alkanethiol self-assembled monolayers (C(n)SH-SAMs) on gold: (1) based on the combinational effect of "the van der Waals interaction between the neighbor hydrocarbon chains", "the framework size of C(n)SH molecule" and "the surface lattice structure of C(n)SH-SAMs on gold", a simple ideal model of the close-packed C(n)SH-SAMs is proposed for the first time. The channel with an appropriate diameter of about 3A ( approximately 3A) existing in SAMs on Au is deduced, which is found large enough for ions and water molecules to permeate; (2) through summarizing the literature reports for various experiments (e.g. scan microscopy techniques and electrochemical methods, etc.), the existence of the ion and water channels ( approximately 3A) in close-packed C(n)SH-SAMs is verified; (3) furthermore, the effect of the ions and water molecules permeation on the studies of the SAMs' electron tunneling process is discussed. This simple ideal model of the close-packed C(n)SH-SAMs established by us may clarify the controversies about the permeation mechanism of ions and water molecules in C(n)SH-SAMs.

A simple and sensitive nonchromatographic approach for indirect determination of sulfide at ultratrace levels in natural waters based on its selective precipitation with Hg2+ on the inner wall of a knotted reactor (KR) was developed for flow injection on-line sorption coupled with hydride generation atomic fluorescence spectrometry (HG-AFS). With the Hg2+ pH kept at 2.0, the HgS precipitation was formed in the KR after a reaction time of 120 s. A 10% (v/v) HCl was introduced to elute the remnant inorganic mercury and to merge with the KBH4 solution (0.05% m/v) for HG-AFS detection. Under the optimal experimental conditions, the sample throughputs were 20 h(-1). The detection limit was found to be 0.05 microg L(-1), and the relative standard deviation (RSD, n = 11) for determination of 2.0 microg L(-1) sulfide was 3.3%. The developed method was successfully applied to the determination of sulfide in a variety of natural water samples and wastewater samples with the gas-phase separation and sorption apparatus.

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol–gel film on gold electrode was investigated, and an obvious cathodic peak at about −200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient ( α ) of 0.79 and the apparent heterogeneous electron transfer rate constant ( K s ) of 3.2 s −1 . The activated voltammetric response and decreased charge-transfer resistance of Ru(NH 3 ) 6 2+/3+ on the LDH-modified electrode provided further evidence. The surface morphologies of silica sol–gel and the LDH embedded in silica sol–gel film were characterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated. The calibration range of lactic acid was from 2.0 × 10 −6 to 3.0 × 10 −5 mol L −1 and the detection limit was 8.0 × 10 −7 mol L −1 at a signal-to-noise ratio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimental results of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modified electrode can be applied as a biomarker to study the pollution effect in the environment.

Staphylococcus aureus secretes a phosphatidylinositol-specific phospholipase C (PIPLC) that contributes to bacterial virulence. We have determined the crystal structure of this enzyme at pH 4.6 and pH 7.5. When crystallized under slightly basic conditions (pH 7.5), the S. aureus PIPLC (SaPI-PLC) structure closely follows the conformation of other PI-PLCs. However, when crystallized under acidic conditions (pH 4.6), a large section of mobile loop at the αβ-barrel rim in the vicinity of the active site shows ∼10 A shift from the position of this loop in all other published PI-PLC structures. The cause of this loop shift under acidic conditions is the result of a titratable intramolecular π-cation interaction between His258 and Phe249. A structure of the mutant protein H258Y crystallized at pH 4.6 does not exhibit this interaction, and the mobile loop remains in the open position. The π-cation latched mobile loop, when in the closed position, can restrict substrate access to the active site. The pH profile for enzyme activity exhibits a maximum at pH 6.5 with steep decreases at pH 6 and 7. Studies of the enzyme binding to vesicles indicate the Kd is lower at acidic pH values (consistent with electrostatic interactions dominating binding) and increases dramatically above pH 7. Thus, rather than affect bulk binding of the protein to target vesicles, this titratable π-histidine cation interaction must affect access of the substrate in the active site.

This paper reports the electrochemical behavior of lactate dehydrogenase (LDH) immobilized in the silica sol-gel film on gold electrode after adding nanometre-sized tridecameric aluminium polycation (nano-Al13, also called nanopolynuclear Al13) as a promoter. A pair of surface controlled quasi-reversible cyclic voltammetry peaks with the formal potential (E0') of 154 mV (vs. SCE) was found in the presence of nano-Al13. A potential application of the nano-Al13-LDH electrode for the determination of resorcinol and p-xylene was also investigated. The experimental results showed that both resorcinol and p-xylene inhibited LDH activity, and the calibration ranges were 5.0 x 10(-6)-3.0 x 10(-4) mol L(-1) for resorcinol and 1.0 x 10(-6)-1.0 x 10(-5) mol L(-1) for p-xylene, respectively. The nano-Al13-LDH electrode can be anticipated to be applied to environmental toxic assessments.

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs) secreted by pathogenic bacteria are often virulence factors. For one well-studied example, Bacillus thuringiensis PI-PLC (BtPI-PLC), the presence of the non-substrate lipid phosphatidylcholine (PC) in membranes enhances both membrane binding and enzymatic activity towards phosphatidylinositol (PI). A strip of four surface-exposed Tyr residues near the rim of the α/β-barrel are involved in this enhancement, and it has been proposed that this strip contributes to a specific PC binding site. The related PI-PLC from Staphylococcus aureus (SaPI-PLC), has similar kinetic characteristics to BtPI-PLC, but has only two Tyr residues in the same region. While SaPI-PLC can also be specifically activated towards PI cleavage by incorporation of PC into assay systems, SaPI-PLC membrane binding as a function of PC content is considerably weaker than that of BtPI-PLC. Mutagenesis of the two SaPI-PLC Tyr residues (Y253S/Y255S) reduces interfacial activity of SaPI-PLC. Adding the two ‘missing’ Tyr residues (N254Y/H258Y) did not further enhance SaPI-PLC specific activity but it dramatically enhanced binding of the protein to PC-rich vesicles. This suggests that PC activation is more complicated than simply binding the protein to membranes. Comparing enzyme kinetics and the effects of mutations on BtPI-PLC, SaPI-PLC and the PI-PLC from an intracellular pathogen (Listeria monocytogenes) suggests a rationale for the acquisition of a discrete PC binding site in some, but not all, PI-PLC enzymes.