Dendritic glycopeptides as Pseudomonas aeruginosa biofilm inhibitors, Oral presentation, 32nd European Peptide symposium, Athens, Greece, 2-7 September 2012

Glycopeptide dendrimers as Pseudomonas aeruginosa biofilm inhibitors. Glycopeptide dendrimers are being developed for inhibition of pathogen adhesion to host cells, a process mediated by carbohydrate-lectins interactions. Such compounds could be used in the treatment of infections by pathogenic bacteria such as Pseudomonas aeruginosa that can be resistant to known antibiotics. Pseudomonas aeruginosa produces two lectins, the fucose binding LecB and the galactose binding LecA. Both lectins have been shown to be virulence factors, involved in cell adhesion and biofilms formation. Screening combinatorial libraries of fucosylated peptide dendrimers led to the glycopeptide dendrimer (C-Fuc-LysProLeu)4(LysPheLysIle)2 LysHisIleNH2. This dendrimer binds the lectin LecB with submicromolar IC50 and shows potent inhibition of P. aeruginosa biofilms for both the laboratory strain PAO1 and for clinical isolates [1]. Appending the peptide dendrimer portion of FD2 with galactosy endgroups gave galactosylpeptide dendrimers as potent ligands for LecA which also act as biofilm inhibitors. Structure-activity relationship studies demonstrated that multivalency was essential for strong binding and biofilm inhibition. [2]The results open the way to develop therapeutic agents based on glycopeptide dendrimers. Peptide dendrimers with antimicrobial properties and good cell penetration are other applications of dendritic peptides we are now investigating.

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

Hepatitis A vaccines and the elderly

Hepatitis A virus (HAV) exposure in unprotected adults may cause severe and serious symptoms, with risk of both morbidity and mortality increasing with age. As seroprevalence of HAV is low in industrialised countries, and an increasing number of people, with an increasing median age, travel from areas of low HAV endemicity to high endemicity, pre-travel vaccination is warranted. Vaccination of the elderly against HAV, however, may be associated with reduced seroprotection, since the immune response decreases with age. Studies with monovalent hepatitis A vaccine or combined hepatitis A and B vaccine show good efficacy in adults in general. Few studies have assessed the immune response in older adults. The only prospective study with monovalent hepatitis A vaccine in the elderly showed a reduced seroprotection of approximately 65% after a single primary dose in subjects over the age of 50 years, while seroprotection was 98% in this age group after receiving a booster dose. The only prospective study with combined hepatitis A and B vaccine in younger subjects or older than 40 years showed similar seroprotection (99-100%) against HAV compared to a monovalent vaccine after receiving three doses. As data on seroprotection for HAV in the elderly are limited, further studies are needed to elucidate how optimal protection in the elderly can be achieved. In the mean time, based on the available data, the suggestion is made to screen elderly travellers to areas endemic for HAV for the presence of naturally acquired immunity, and, if found susceptible, be immunised well in advance of their trip, to allow time for post-vaccination antibody testing and/or administration of a second dose of the vaccine.

Population-based epidemiology of rotavirus hospitalisations in Switzerland

BACKGROUND: Rotaviruses (RV) are the most common cause of dehydrating gastroenteritis requiring hospitalisation in children <5 years of age. A new generation of safe and effective RV vaccines is available. Accurate data describing the current burden of RV disease in the community are needed to devise appropriate strategies for vaccine usage. METHODS: Retrospective, population-based analysis of RV hospitalisations in children <5 years of age during a 5-year period (1999-2003) in a both urban and rural area inhabited by 12% of the Swiss population. RESULTS: Of 406 evaluable cases, 328 were community-acquired RV infections in children <5 years of age. RV accounted for 38% of all hospitalisations for gastroenteritis. The overall hospitalisation incidence in the <5-year-old was 1.5/1000 child-years (peak incidence, 2.6/1000 child-years in children aged 13-24 months). The incidence of community-acquired RV hospitalisations was significantly greater in children of non-Swiss origin (3.0 vs. 1.1/1000 child-years, relative risk 2.7; 95% CI 2.2-3.4), who were younger, but tended to be less severely dehydrated on admission than Swiss children. In comparison with children from urban areas, RV hospitalisation incidence was significantly lower among those residing in the remote mountain area (0.71 vs. 1.71/1000 child years, relative risk 2.2, 95% CI 1.6-3.1). CONCLUSION: Population-based RV hospitalisation incidence was low in comparison with other European countries. Significantly greater hospitalisation rates among children living in urban areas and those from non-Swiss families indicate that factors other than the severity of RV-induced dehydration are important driving forces of hospital admission.

Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.

Macrocyclic chelator-coupled gastrin-based radiopharmaceuticals for targeting of gastrin receptor-expressing tumours

PURPOSE: Diethylenetriamine-pentaacetic acid (DTPA)-coupled minigastrins are unsuitable for therapeutic application with the available beta-emitting radiometals due to low complex stability. Low tumour-to-kidney ratio of the known radiopharmaceuticals is further limiting their potency. We used macrocyclic chelators for coupling to increase complex stability, modified the peptide sequence to enhance radiolytic stability and studied tumour-to-kidney ratio and metabolic stability using (111)In-labelled derivatives. METHODS: Gastrin derivatives with decreasing numbers of glutamic acids were synthesised using (111)In as surrogate for therapeutic radiometals for in vitro and in vivo studies. Gastrin receptor affinities of the (nat)In-metallated compounds were determined by receptor autoradiography using (125)I-CCK as radioligand. Internalisation was evaluated in AR4-2J cells. Enzymatic stability was determined by incubating the (111)In-labelled peptides in human serum. Biodistribution was performed in AR4-2J-bearing Lewis rats. RESULTS: IC(50) values of the (nat)In-metallated gastrin derivatives vary between 1.2 and 4.8 nmol/L for all methionine-containing derivatives. Replacement of methionine by norleucine, isoleucine, methionine-sulfoxide and methionine-sulfone resulted in significant decrease of receptor affinity (IC(50) between 9.9 and 1,195 nmol/L). All cholecystokinin receptor affinities were >100 nmol/L. All (111)In-labelled radiopeptides showed receptor-specific internalisation. Serum mean-life times varied between 2.0 and 72.6 h, positively correlating with the number of Glu residues. All (111)In-labelled macrocyclic chelator conjugates showed higher tumour-to-kidney ratios after 24 h (0.37-0.99) compared to (111)In-DTPA-minigastrin 0 (0.05). Tumour wash out between 4 and 24 h was low. Imaging studies confirmed receptor-specific blocking of the tumour uptake. CONCLUSIONS: Reducing the number of glutamates increased tumour-to-kidney ratio but resulted in lower metabolic stability. The properties of the macrocyclic chelator-bearing derivatives make them potentially suitable for clinical purposes.

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli

The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H(+)-dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.

A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.

DESIGN AND DEVELOPMENT OF BODIPY-BASED FLUORESCENT PROBES FOR SENSING AND IMAGING OF CYANIDE, Zn (II) IONS, LYSOSOMAL pH AND CANCER CELLS

BODIPY (4,4-Difluoro-3a,4a-diaza-s-indacene) dyes have gained lots of attention in application of fluorescence sensing and imaging in recent years because they possess many distinctive and desirable properties such as high extinction coefficient, narrow absorption and emission bands, high quantum yield and low photobleaching effect. However, most of BODIPY-based fluorescent probes have very poor solubilities in aqueous solution, emit less than 650 nm fluorescence that can cause cell and tissue photodamages compared with bio-desirable near infrared (650-900 nm) light. These undesirable properties extremely limit the applications of BODIPY-based fluorescent probes in sensing and imaging applications. In order to overcome these drawbacks, we have developed a very effective strategy to prepare a series of neutral highly water- soluble BODIPY dyes by enhancing the water solubilities of BODIPY dyes via incorporation of tri(ethylene glycol)methyl ether (TEG) and branched oligo(ethylene glycol)methyl ether (BEG) residues onto BODIPY dyes at 1,7-, 2,6-, 3,5-, 4- and meso- positions. We also have effectively tuned absorptions and emissions of BOIDPY dyes to red, deep red and near infrared regions via significant extension of π-conjugation of BODIPY dyes by condensation reactions of aromatic aldehydes with 2,6-diformyl BODIPY dyes at 1,3,5,7-positions. Based on the foundation that we built for enhancing water solubility and tuning wavelength, we have designed and developed a series of water-soluble, BODIPY-based fluorescent probes for sensitive and selective sensing and imaging of cyanide, Zn (II) ions, lysosomal pH and cancer cells. We have developed three BODIPY-based fluorescent probes for sensing of cyanide ions by incorporating indolium moieties onto the 6-position of TEG- or BEG-modified BOIDPY dyes. Two of them are highly water-soluble. These fluorescent probes showed selective and fast ratiometric fluorescent responses to cyanide ions with a dramatic fluorescence color change from red to green accompanying a significant increase in fluorescent intensity. The detection limit was measured as 0.5 mM of cyanide ions. We also have prepared three highly water-soluble fluorescent probes for sensing of Zn (II) ions by introducing dipicoylamine (DPA, Zn ion chelator) onto 2- and/or 6-positions of BEG-modified BODIPY dyes. These probes showed selective and sensitive responses to Zn (II) ion in the range from 0.5 mM to 24 mM in aqueous solution at pH 7.0. Particularly, one of the probes displayed ratiometric responses to Zn (II) ions with fluorescence quenching at 661 nm and fluorescence enhancement at 521 nm. This probe has been successfully applied to the detection of intracellular Zn (II) ions inside the living cells. Then, we have further developed three acidotropic, near infrared emissive BODIPY- based fluorescent probes for detection of lysosomal pH by incorporating piperazine moiety at 3,5-positions of TEG- or BEG-modified BODIPY dyes as parts of conjugation. The probes have low auto-fluorescence at physiological neutral condition while their fluorescence intensities will significant increase at 715 nm when pH shift to acidic condition. These three probes have been successfully applied to the in vitro imaging of lysosomes inside two types of living cells. At the end, we have synthesized one water- soluble, near infrared emissive cancer cell targetable BODIPY-based fluorescent polymer bearing cancer homing peptide (cRGD) residues for cancer cell imaging applications. This polymer exhibited excellent water-solubility, near infrared emission (712 nm), good biocompatibility. It also showed low nonspecific interactions to normal endothelial cells and can effectively detect breast tumor cells.

Virus purification, detection and removal

The biopharmaceutical industry has a growing demand and an increasing need to improve the current virus purification technologies, especially as more and more vaccines are produced from cell-culture derived virus particles. Downstream purification strategies can be expensive and account for 70% of the overall manufacturing costs. The economic pressure and purification processes can be particularly challenging when the virus to be purified is small, as in our model virus, porcine parvovirus (PPV). Our efforts are focused on designing an easy, economical, scalable and efficient system for virus purification, and we focused on aqueous two-phase systems. Industry acceptable standards for virus vaccine recovery can be as low as 30% due to demand of high final titer, virus transduction inhibitors and presence of empty or defective virus capsids as impurities. We have overcome these shortcomings by recovering a high 64% of infectious virus using an aqueous two-phase system. We used high molecular weight polymer and citrate salt to achieve a good yield and eliminated the major contaminant bovine serum albumin. Viruses are also studied for ensuring pure and safe drinking water. Low pressure microfiltrations are continuously being investigated for water filters as they allow high permeate flux and low fouling. Viruses such as PPV are small enough to pass through the microporous membranes. Control of viruses in water is crucial for public health and we have designed an affinity based membrane filter to capture virus. Nanofibers have a high surface to volume ratio providing a highly accessible surface area for virus adsorption. Chitosan an insoluble, biocompatible and biodegradable polymer was used for adsorbing trimer peptide WRW. About 0.2 μmoles of cysteine terminal WRW peptide was conjugated to amine terminal chitosan using maleimide conjugation chemistry. We achieved 90-99% virus removal from water adjusted to a neutral pH. The virus removal from affinity based chitosan was attributed to electrostatic and hydrophobic driven binding effect.

Evaluation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated bombesin-based radioantagonist for the labeling with single-photon emission computed tomography, positron emission tomography, and therapeutic radionuclides

PURPOSE: G protein-coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new [111In/68Ga]-labeled bombesin-based antagonist RM1 with the agonist [111In]-AMBA for targeting the gastrin-releasing peptide receptor (GRPR). EXPERIMENTAL DESIGN: IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy. RESULTS: The IC50 value of [(nat)In]-RM1 was 14 +/- 3.4 nmol/L. [(nat/111)In]-RM1 was found to bind to the GRPR with a Kd of 8.5 +/- 2.7 nmol/L compared with a Kd of 0.6 +/- 0.3 nmol/L of [111In]-AMBA. A higher maximum number of binding site value was observed for [111In]-RM1 (2.4 +/- 0.2 nmol/L) compared with [111In]-AMBA (0.7 +/- 0.1 nmol/L). [(nat)Lu]-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas [(nat)In]-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 +/- 0.8% IA/g versus 3.69 +/- 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios. CONCLUSION: Despite their relatively low GRPR affinity, the antagonists [111In/68Ga]-RM1 showed superior targeting properties compared with [111In]-AMBA. As found for somatostatin receptor-targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors.

Toward global prevention of sexually transmitted infections (STIs): The need for STI vaccines.

An estimated 499 million curable sexually transmitted infections (STIs; gonorrhea, chlamydia, syphilis, and trichomoniasis) occurred globally in 2008. In addition, well over 500 million people are estimated to have a viral STI such as herpes simplex virus type 2 (HSV-2) or human papillomavirus (HPV) at any point in time. STIs result in a large global burden of sexual, reproductive, and maternal-child health consequences, including genital symptoms, pregnancy complications, cancer, infertility, and enhanced HIV transmission, as well as important psychosocial consequences and financial costs. STI control strategies based primarily on behavioral primary prevention and STI case management have had clear successes, but gains have not been universal. Current STI control is hampered or threatened by several behavioral, biological, and implementation challenges, including a large proportion of asymptomatic infections, lack of feasible diagnostic tests globally, antimicrobial resistance, repeat infections, and barriers to intervention access, availability, and scale-up. Vaccines against HPV and hepatitis B virus offer a new paradigm for STI control. Challenges to existing STI prevention efforts provide important reasons for working toward additional STI vaccines. We summarize the global epidemiology of STIs and STI-associated complications, examine challenges to existing STI prevention efforts, and discuss the need for new STI vaccines for future prevention efforts.

Immune responses against major histocompatibility complex and interleukin-2 gene transfected K1735 murine melanoma: Potential vaccines for the immunotherapy of cancer

Tumor specific immunity is mediated by cytotoxic T lymphocytes (CTL) that recognize peptide antigen (Ag) in the context of major histocompatibility complex (MHC) class I molecules and by helper T (Th) lymphocytes that recognize peptide Ag in the context of MHC class II molecules. The purpose of this study is (1) to induce or augment the immunogenicity of nonimmunogenic or weakly immunogenic tumors by genetic modification of tumor cells, and (2) to use these genetically altered cells in cancer immunotherapy. To study this, I transfected a highly tumorigenic murine melanoma cell line (K1735) that did not express constitutively either MHC class I or II molecules with syngeneic cloned MHC class I and/or class II genes, and then determined the tumorigenicity of transfected cells in normal C3H mice. K1735 transfectants expressing either $\rm K\sp{k}$ or $\rm A\sp{k}$ molecules alone produced tumors in normal C3H mice, whereas most transfectants that expressed both molecules were rejected in normal C3H mice but produced tumors in nude mice. The rejection of K1735 transfectants expressing $\rm K\sp{k}$ and $\rm A\sp{k}$ Ag in normal C3H mice required both $\rm CD4\sp+$ and $\rm CD8\sp+$ T cells. Interestingly, the $\rm A\sp{k}$ requirement can be substituted by IL-2 because transfection of $\rm K\sp{k}$-positive/A$\sp{\rm k}$-negative K1735 cells with the IL-2 gene also resulted in abrogation of tumorigenicity in normal C3H mice but not in nude mice. In addition, 1735 $(\rm I\sp+II\sp+)$ transfected cells can function as antigen presenting cells (APC) since they could process and present native hen egg lysozyme (HEL) to HEL specific T cell hybridomas. Furthermore, the transplantation immunity induced by K1735 transfectants expressing both $\rm K\sp{k}$ and $\rm A\sp{k}$ molecules completely cross-protected mice against challenge with $\rm K\sp{k}$-positive transfectants but weakly protected them against challenge with parental K1735 cells or $\rm A\sp{k}$-positive transfectants. Finally, I demonstrated that MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells can function as anti-cancer vaccines since they can abrogate the growth of established tumors and metastasis.^ In summary, my results indicate that expression of either MHC class I or II molecule alone is insufficient to cause the rejection of K1735 melanoma in syngeneic hosts and that both molecules are necessary. In addition, my data suggest that the failure of $\rm K\sp{k}$-positive K1735 cells to induce a primary tumor-rejection response in normal C3H mice may be due to their inability to induce the helper arm of the anti-tumor immune response. Finally, the ability of MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells to prevent growth of established tumors or metastasis suggests that these cell lines can serve as potential vaccines for the immunotherapy of cancer. (Abstract shortened by UMI.) ^

Peptide nucleic acids: Mechanism of tight binding and application in artificial nuclease

Peptide nucleic acids (PNA) are mimics of nucleic acids with a peptidic backbone. Duplexes and triplexes formed between PNA and DNA or RNA possess remarkable thermal stability, they are resistant to nuclease cleavage and can better discriminate mismatches. Understanding the mechanism for the tight binding between PNA and oligonucleotides is important for the design and development of better PNA-based drugs.^ We have performed molecular dynamics (MD) simulations of 8-mer PNA/DNA duplex and two analogous duplexes with chiral modification of PNA strand (D- or L-Alanine modification). MD simulations were performed with explicit water and Na$\sp{+}$ counter ions. The 1.5-ns simulations were carried out with AMBER using periodic boundary and particle mesh Ewald summation. The point charges for PNA monomers were derived from fitting electrostatic potentials, obtained from ab initio calculation, to atomic centers using RESP. Derived charges reveal significantly altered charge distribution on the PNA bases and predict the Watson-Crick H-bonds involving PNA to be stronger. Results from NMR studies investigating H-bond interactions between DNA-DNA and DNA-PNA base pairs in non-polar environment are consistent with this prediction. MD simulations demonstrated that the PNA strand is more flexible than the DNA strand in the same duplex. That this flexibility might be important for the duplex stability is tested by introducing modification into the PNA backbones. Results from MD simulation revealed dramatically altered structures for the modified PNA-DNA duplexes. Consistent with previous NMR results, we also found no intrachain hydrogen bonds between O7$\sp\prime$ and N1$\sp\prime$ of the neighboring residues in our MD study. Our study reveals that in addition to the lack of charge repulsion, stronger Watson-Crick hydrogen bonds together with flexible backbone are important factors for the enhanced stability of the PNA-DNA duplex.^ In a related study, we have developed an application of Gly-Gly-His-(Gly)$\sb3$-PNA conjugate as an artificial nuclease. We were able to demonstrate cleavage of single stranded DNA at a single site upon Ni(II) binding to Gly-Gly-His tripeptide and activation of nuclease with monoperoxyphthalic acid. ^

N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging

UNLABELLED Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.