Marilena Manea

Therapeutically effective antibodies against amyloid-β peptide target amyloid-β residues 4−10 and inhibit cytotoxicity and fibrillogenesis

Immunization of transgenic mouse models of Alzheimer disease using amyloid-β peptide (Aβ) reduces both the Alzheimer disease–like neuropathology and the spatial memory impairments of these mice. However, a therapeutic trial of immunization with Aβ42 in humans was discontinued because a few patients developed significant meningo-encephalitic cellular inflammatory reactions. Here we show that beneficial effects in mice arise from antibodies selectively directed against residues 4–10 of Aβ42, and that these antibodies inhibit both Aβ fibrillogenesis and cytotoxicity without eliciting an inflammatory response. These findings provide the basis for improved immunization antigens as well as attempts to design small-molecule mimics as alternative therapies.

Receptor-mediated tumor targeting based on peptide hormones

Importance of the field: Tumor targeting with peptides is based on the discovery that receptors for many regulatory peptides are overexpressed in tumor cells, compared with their expression in normal tissues. Consequently, these peptides and their analogues can be used as carriers/targeting moieties for the preparation of diagnostic and therapeutic agents that have increased selectivity and decreased peripheral toxicity. Areas covered in this review: Here an overview is given of the most relevant gonadotropin-releasing hormone (GnRH) and somatostatin derivatives, as well as of their applications in cancer diagnosis and therapy. For this purpose, recently published data in these areas (mostly articles published from 2000 to 2009) were reviewed. What the reader will gain: In contrast to other regulatory peptides that stimulate the tumor growth, GnRH and somatostatin derivatives have inhibitory effect; therefore, they were used primarily for the preparation of various conjugates to be used in targeted chemotherapy, targeted radiotherapy, photodynamic therapy, boron neutron capture therapy and cancer diagnosis. Some of these conjugates have already found clinical applications, whereas others are now in preclinical and clinical trials. Take home message: Tumor targeting with hormone peptides provides a basis for the development of new diagnostic and therapeutic approaches for cancer.

Development of an Oxime Bond Containing Daunorubicin-Gonadotropin-Releasing Hormone-III Conjugate as a Potential Anticancer Drug

Here, we report on the synthesis and biological properties of a conjugate in which daunorubicin (Dau) as chemotherapeutic agent was attached through an oxime bond to gonadotropin-releasing hormone-III (GnRH-III) as targeting moiety. In vitro toxicity and the cytostatic effect of the conjugate on MCF-7 human breast and C26 murine colon cancer cell lines were determined, and the results were compared with those obtained for the free daunorubicin, as well as with the doxorubicin containing derivative. In vivo antitumor effect of daunorubicin-GnRH-III was studied on Balb/c female mice transplanted with C26 tumor. Our data indicate that the daunorubicin-GnRH-III conjugate had a lower toxic effect than the free daunorubicin and it was essentially nontoxic up to 15 mg (Dau content)/kg body weight. The treatment of the C26 tumor bearing mice with the conjugate led to tumor growth inhibition and longer survival time in comparison with the controls and with the administration of the free drug. When mice were treated twice with the conjugate (on days 4 and 7 after tumor transplantation), 46% tumor growth inhibition was obtained. In this case, the increase of the median survival time was 38% compared to the controls.

Gonadotropin-releasing hormone receptors as molecular therapeutic targets in prostate cancer : Current options and emerging strategies

Prostate cancer is androgen-dependent in its early stages and androgen deprivation therapy represents the most effective first-line therapeutic approach. However, after an initial remission, prostate cancer progresses towards the castration resistant prostate cancer (CRPC) stage, with increased malignancy and resistance to conventional chemotherapy. Pituitary gonadotropin-releasing hormone receptors (GnRH-Rs) represent the most effective molecular target for the treatment of steroid-dependent prostate cancer. GnRH agonists (through GnRH-Rs desensitization) suppress the pituitary-testicular axis and, therefore, represent the treatment of choice for prostate cancer patients. GnRH-Rs are also expressed in prostate cancer, even when the tumor has reached the CRPC stage, and are endowed with antitumor activity, supporting the notion that they might represent a molecular target for GnRH analog-based therapeutic strategies. In addition to GnRH agonists and antagonists, GnRH-based bioconjugates (cytotoxic GnRH bioconjugates, GnRH-conjugated lytic peptides and GnRH-toxin bioconjugates) have been developed and are now undergoing intensive investigations; some of them (i.e., AN-152, Dox-[d-Lys(6)]-GnRH) have entered clinical trials. The advantage of these treatments is the specific delivery of cytotoxic agents to cancer cells. Interestingly, other isoforms of the peptide have been identified. One of them is GnRH-III, which was isolated from sea lamprey. GnRH-III specifically binds to GnRH-Rs in cancer cells and exerts antiproliferative effects; on the other hand, its endocrine effects at pituitary level are insignificant, supporting its selective antitumor activity. Based on these observations, different cytotoxic GnRH-III bioconjugates have recently been synthesized; preliminary in vitro studies suggest that these compounds might represent a new promising treatment strategy for prostate cancer.

New derivatives of GnRH as potential anticancer therapeutic agents

GnRH (gonadotropin-releasing hormone), a decapeptide produced by the hypothalamus, plays an important role in the reproduction by regulating the pituitary-gonadal axis. Continuous high doses of GnRH or its superactive agonists result in desensitization of the pituitary gonadotropes and a suppression of sex steroid production by the gonads (chemical castration). Based on these effects, the treatment with GnRH agonists has become a widely used hormonal therapy of the sex-steroid dependent tumors. It was also demonstrated that most tumor cells contain GnRH receptors, and the direct antiproliferative effect of GnRH analogs on cancer cells might be mediated by these receptors. Development of new GnRH derivatives is focused on the decrease of their hormonal potency resulting in higher selectivity of the antitumor activity. One of the most promising natural GnRH analogs, lamprey (l) lGnRH-III, was isolated from see lamprey. This variant of GnRH binds to GnRH receptors and inhibits proliferation of various cancer cells. However, its endocrine effect is insignificant in mammals. lGnRH-III dimers and conjugates were prepared and were shown to have increased antiproliferative effects on various cancer cells, while their hormonal activity was lower than that of the native hormone. lGnRH-III was applied as targeting moiety to deliver anticancer agents to tumor cells. Research data concerning lGnRH-III and its analogs represent a new outlook for research trends of the application of GnRH compounds in cancer chemotherapy. Studies on the effects of lGnRH-III derivatives including antiproliferative effects, cytotoxicity, hormonal actions, and enzymatic stability are reviewed in this article.

Mass spectrometric approaches for elucidation of antigen–antibody recognition structures in molecular immunology

Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, “affinity proteomics” using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a ß-amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimers disease. Using an immobilized antigen and antibody-proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; “parex-prot”). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of “molecular antibody-recognition signatures” offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.

Interaction of β-amyloid(1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study

The stoichiometries and the affinity toward simple and paired metal ions of synthetic amyloid-beta(1-40) peptide (Abeta1-40) were investigated by electrospray ion trap mass spectrometry (ESI-MS), circular dichroism (CD), and atomic force microscopy (AFM). The results lead to the working hypothesis that pH-dependent metal binding to Abeta1-40 may induce conformational changes, which affect the affinity toward other metals. A significant copper and zinc binding to Abeta1-40 peptide at pH 5.5 was found, whereas nickel ions commonly bind to each molecule of beta-amyloid peptide. Some complexes of Abeta1-40 with more than one nickel ion were identified by ESI-MS. In addition, nickel ions proved to enhance Abeta oligomerization. On increasing pH, up to 12 ions of zinc may bind to a single Abeta molecule. Under the same pH and concentration conditions, the binding pattern of the independent copper and silver ions to Abeta1-40 was different from that of the equimolecular mixture of the two metal ions. One might assume that some conformational changes due to water loss altered the capacity of Abeta peptide to bind certain heavy metal ions. As a consequence, copper-silver interaction with the binding process to Abeta1-40 became highly complex. A competition between silver and nickel ions for Abeta1-40 binding sites at high pH was also observed. New strategies were proposed to identify the characteristic signals for some important metal ion-peptide complexes in the spectra recorded at high pH or high concentrations of metal ions. To explain the formation of such a large number of high metal ion-Abeta complexes, we took into consideration the participation of both histidine residues and free amino groups as well as carboxylate ones in the binding process. Finally, CD and AFM studies supported the mass spectrometric data.

Anthracycline-GnRH derivative bioconjugates with different linkages: Synthesis, in vitro drug release and cytostatic effect

To increase the selectivity and consequently to minimize the side effects of chemotherapeutic agents, receptor mediated tumor targeting approaches have been developed. In the present work, various anthracycline-GnRH derivative bioconjugates were synthesized with the aim of investigating the influence of (i) different anthracycline anticancer drugs, (ii) different linkages between the targeting moiety and the anticancer drug, and (iii) different targeting moieties (e.g., GnRH-III and [D-Lys⁶]-GnRH-I) on their in vitro drug release and cytostatic effect. The anthracyclines, daunorubicin or doxorubicin, were attached to the ε-amino group of Lys of GnRH-III or [D-Lys⁶]-GnRH-I through oxime, hydrazone or ester bonds. In another bioconjugate, a self-immolative p-aminobenzyloxycarbonyl spacer was used to link daunorubicin to GnRH-III. The in vitro degradation of the bioconjugates was investigated in the presence of rat liver lysosomal homogenate and cathepsin B. The cellular uptake of the compounds was evaluated by flow cytometry and their in vitro cytostatic effect was determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. The results indicate that on the tested cancer cell lines there is no significant difference in the cellular uptake and in vitro cytostatic effect of bioconjugates containing GnRH-III or [D-Lys⁶]-GnRH-I as a targeting moiety. The bioconjugates containing ester bond, hydrazone bond and the self-immolative spacer exert the highest cytostatic effect, followed by oxime bond-linked compounds.

Increased Levels of Antigen-Bound β-Amyloid Autoantibodies in Serum and Cerebrospinal Fluid of Alzheimer’s Disease Patients

Recent studies have suggested a protective role of physiological β-amyloid autoantibodies (Aβ-autoantibodies) in Alzheimer’s disease (AD). However, the determination of both free and dissociated Aβ-autoantibodies in serum hitherto has yielded inconsistent results regarding their function and possible biomarker value. Here we report the application of a new sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of antigen-bound Aβ-autoantibodies (intact Aβ-IgG immune complexes) in serum and cerebrospinal fluid (CSF) of a total number of 112 AD patients and age- and gender-matched control subjects. Both serum and CSF levels of Aβ-IgG immune complexes were found to be significantly higher in AD patients compared to control subjects. Moreover, the levels of Aβ-IgG complexes were negatively correlated with the cognitive status across the groups, increasing with declining cognitive test performance of the subjects. Our results suggest a contribution of IgG-type autoantibodies to Aβ clearance in vivo and an increased immune response in AD, which may be associated with deficient Aβ-IgG removal. These findings may contribute to elucidating the role of Aβ-autoantibodies in AD pathophysiology and their potential application in AD diagnosis.

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β‐amyloid peptide revealed by affinity mass spectrometry and molecular modeling

Humanin (HN) is a linear 24‐aa peptide recently detected in human Alzheimers disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß‐amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1–40) and characterization of the interaction structure through a molecular modeling study. Wild‐type HN and HN‐sequence mutations were synthesized by SPPS and the HPLC‐purified peptides characterized by MALDI‐MS. The interaction epitopes between HN and Aß(1–40) were identified by affinity‐MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity‐bound peptides. The affinity‐MS analyses revealed HN(5–15) as the epitope sequence of HN, whereas Aß(17–28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1–40) and by ELISA with Aß(1–40) and Aß‐partial sequences as ligands to immobilized HN. The specificity and affinity of the HN‐Aß interaction were characterized by direct ESI‐MS of the HN‐Aß(1–40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a KD of the complex of 610 n m. A molecular dynamics simulation of the HN‐Aß(1–40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1–40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN. Copyright