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Purpose:In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer.Experimental Design: Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay.Results: Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis.Conclusion: TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.

Asparagine deamidation in peptides or in fibronectin fragments containing the asparagine-glycine-arginine sequence generates isoaspartate-glycine-arginine (isoDGR), a new αvβ3 integrin-binding motif. Because αvβ3 is expressed in angiogenic vessels, we hypothesized that isoDGR-containing peptides could be exploited as ligands for targeted delivery of drugs to tumor neovasculature. We found that a cyclic CisoDGRC peptide coupled to fluorescent nanoparticles (quantum dots) could bind αvβ3 integrin and colocalize with anti-CD31, anti-αvβ3, and anti-α5β1 antibodies in human renal cell carcinoma tissue sections, indicating that this peptide could efficiently recognize endothelial cells of angiogenic vessels. Using CisoDGRC fused to tumor necrosis factor α (TNF) we observed that ultralow doses (1–10 pg) of this product (called isoDGR-TNF), but not of TNF or CDGRC-TNF fusion protein, were sufficient to induce antitumor effects when administered alone or in combination with chemotherapy to tumor-bearing mice. The antitumor activity of isoDGR-TNF was efficiently inhibited by coadministration with an excess of free CisoDGRC, as expected for ligand-directed targeting mechanisms. These results suggest that isoDGR is a novel tumor vasculature–targeting motif. Peptides containing isoDGR could be exploited as ligands for targeted delivery of drugs, imaging agents, or other compounds to tumor vasculature. [Cancer Res 2008;68(17):7073–82]

Supplementary Figure 1 Legend from Isoaspartate-Glycine-Arginine: A New Tumor Vasculature–Targeting Motif

Supplementary Figure 2 from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells

Supplementary Figure 3 from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells

Supplementary Figure 6 from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells

A major obstacle for the development of effective immunotherapy is the ability of tumors to escape the immune system. The possibility to kill tumor cells because they are recognized as infected rather than as malignant could help overcome immune escape mechanisms. Here we report a conceptually new approach of cancer immunotherapy based on in vivo infection of tumors and killing of infected tumor cells. Attenuated but still invasive, Salmonella typhimurium can be successfully exploited to invade melanoma cells that can present antigenic determinants of bacterial origin and become targets for anti-Salmonella–specific T cells. However, to fully appreciate the anticancer therapeutic properties of S. typhimurium, tumor-bearing mice need to be vaccinated against S. typhimurium before intratumoral Salmonella injection. Tumor infection when coupled to anti-Salmonella vaccination leads to 50% to 100% tumor-free mice with a better outcome on larger tumors. Invasive Salmonella also exert an indirect toxic effect on tumor cells through the recruitment of inflammatory cells and the cross-presentation of tumor antigens, which allow induction of tumor-specific immune response. This is effective in retarding the growth of untreated established distant tumors and in protecting the mice from subsequent tumor challenges.

Supplementary Figure 1 from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells

Supplementary Figure Legends 2A and 2B from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells

Supplementary Table 4 from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD2 [aGD2-SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced time-dependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD2-SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor- or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor- and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma. (Cancer Res 2006; 66(20): 10073-82)

Supplementary Methods and Figure Legend from Targeting Liposomal Chemotherapy via Both Tumor Cell–Specific and Tumor Vasculature–Specific Ligands Potentiates Therapeutic Efficacy

Supplementary Methods and Legends - PDF file 94K

Supplemental Figures 1 - 7 PDF 2666K, Supplemental Figure 1. Priming with DC-Tag induces a long-lasting antigenspecific immune response. Supplemental Figure 2. Boosting increases the absolute number of rapidly responding Ag-specific CD8+ splenocytes. Supplemental Figure 3. Boosting does not increase the cytolytic activity of Tagspecific CD8+ T cells. Supplemental Figure 4. Repeated DC-Tag boosting helps maintaining the pool of Ag-specific CD8+ TCM cells. Supplemental Figure 5. Different vaccination schedules do not impact on the pool of Ag-specific CD8+ TCM cells.Supplemental Figure 6. A larger pool of Ag-specific CD8+ TCM cells ensures better protection against tumor challenge. Supplemental Figure 7. Donor-derived CD8+ T cells lead the Tag specific immune response in transplanted TRAMP mice

Supplementary Figure S1 from Targeting Liposomal Chemotherapy via Both Tumor Cell–Specific and Tumor Vasculature–Specific Ligands Potentiates Therapeutic Efficacy

Supplementary Figure 1 from Isoaspartate-Glycine-Arginine: A New Tumor Vasculature–Targeting Motif

Supplementary Figure 5 from Cancer Immunotherapy Based on Killing of Salmonella-Infected Tumor Cells

Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.

Peptides seldom retain stable conformations if separated from their native protein structure. In an immunological context, this potentially affects the development of selective peptide-based bioprobes and, from a vaccine perspective, poses inherent limits in the elicitation of cross-reactive antibodies by candidate epitopes. Here, a 1,4-disubstituted-1,2,3-triazole-mediated stapling strategy was used to stabilize the native α-helical fold of the Pal3 peptidic epitope from the protein antigen PalBp (BPSL2765) from Burkholderia pseudomallei, the etiological agent of melioidosis. Whereas Pal3 shows no propensity to fold outside its native protein context, the engineered peptide (Pal3H) forms a stable α-helix, as assessed by MD, NMR, and CD structural analyses. Importantly, Pal3H shows an enhanced ability to discriminate between melioidosis patient subclasses in immune sera reactivity tests, demonstrating the potential of the stapled peptide for diagnostic purposes. With regard to antibody elicitation and related bactericidal activities, the linear peptide is shown to elicit a higher response. On these bases, we critically discuss the implications of epitope structure engineering for diagnostic- and vaccine-oriented applications.

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary praline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-g-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents. (C) 2015 Elsevier B.V. All rights reserved.

Quenching of the triplet state of tryptophan by close contact with cysteine provides a tool for measuring the rate of intramolecular contact formation, one of the most elementary events in the folding process, in peptides and proteins using only natural probes. Here we present a study performed on a stabilized mutant of the second β-hairpin of the GB1 domain, where we combine steady-state fluorescence, laser-induced temperature-jump, and contact formation measurements to unveil the role of elementary structural components on hairpin dynamics and overall stability. In particular, our methodology provides access to the conformational dynamics of both the folded and unfolded state of the hairpin under native conditions, revealing the presence of extremely slow dynamics on the microsecond time scale in the unfolded state and coexistence of structures with partial pairing of the tails in the folded state. Comparing model peptides that mimic the turn sequence, we found that both ion pairing and hydrogen bonding due to the threonine side chain contribute to the propensity of turn formation but not to the much slower dynamics of the hydrophobic core formation. Interestingly, the dynamics of the turn region in isolation are significantly faster than the dynamics measured for the unfolded state of the complete hairpin, suggesting that non-native hydrophobic contacts slow down the reconfiguration dynamics of the unfolded state. Overall, the information extracted from these experiments provides kinetic limits on interconversions among conformational populations, hence enabling a simplified multistate free-energy landscape for the GB1 hairpin to be drawn.

Die Synthese von Disulfidbruckenmimetika ist eine wichtige Strategie zur Optimierung disulfidreicher Peptide beim Wirkstoff-Design. Wir beschreiben Mimetika des Conotoxins MrIA, die durch selektiven Austausch einzelner Disulfidbindungen gegen Brucken aus einem 1,4-disubstituierten 1,2,3-Triazol erhalten wurden. Eine sequenzielle, Kupfer-katalysierte Azid-Alkin-Klick-Reaktion (CuAAC) mit nachfolgender Disulfidbildung fuhrte regioselektiv zu hybriden Triazol-Disulfid-Analoga von MrIA. Mimetika, in denen Triazol die Cys4-Cys13-Disulfidbindung ersetzte, behielten ihre Tertiarstruktur sowie die volle In-vitro- und In-vivo-Aktivitat als Inhibitoren der Noradrenalin-Wiederaufnahme bei. Diese Mimetika sind resistent gegen Reduktion mit Glutathion, was zu einer verbesserten Plasmastabilitat fuhrt und dadurch interessant fur die Wirkstoff-Entwicklung ist.

Although α6-contaning (α6*) nicotinic acetylcholine receptors (nAChRs) are densely expressed in the visual system, their role is not well known. We have characterized a family of toxins that are antagonists for α6β2* receptors and used one of these [RDP-MII(E11R)] to localize α6* nAChRs and investigate their impact on retinal function in adult Long-Evans rats. The α6*nAChRs in retinal tissue were localized using either a fluorescently tagged [RDP-MII(E11R)] or anti-α6-specific antibodies and found to be predominantly at the level of the ganglion cell layer. After intraocular injection of RDP-MII(E11R) in one eye and vehicle or inactive MII in contralateral eyes as controls, we recorded flash electroretinograms (F-ERGs), pattern ERGs (P-ERGs), and cortical visual-evoked potential (VEPs). There was no significant difference in F-ERG between the RDP-MII(E11R)-treated and control eyes. In contrast, P-ERG response amplitude was significantly reduced in the RDP-MII(E11R)-injected eye. Blocking α6* nAChRs at retinal level also decreased the VEP amplitude recorded in the visual cortex contralateral to the injected eye. Because both the cortical and inner retina output were affected by RDP-MII(E11R), whereas photoreceptor output was preserved, we conclude that the reduced visual response was due to an alteration in the function of α6* nAChRs present in the ganglion cell layer.-Barloscio, D., Cerri, E., Domenici, L., Longhi, R., Dallanoce, C., Moretti, M., Vilella, A., Zoli, M., Gotti, C., and Origlia, N. In vivo study of the role of α6-containing nicotinic acetylcholine receptor in retinal function using subtype-specific RDP-MII(E11R) toxin.

Although cancer vaccines are in the clinic, several issues remain to be addressed to increase vaccine efficacy. In particular, whether how and how frequently a patient should be boosted remains to be defined. Here, we have assessed the ability of dendritic cell (DC)-based vaccines to induce a long-lasting tumor-specific CTL response in either prophylactic or therapeutic settings by taking advantage of transplantable and spontaneous mouse tumor models. Implementing a 24-hour ex vivo intracellular cytokine production assay, we have found that priming with a DC-based vaccine induced a long-lasting CTL response in wild-type mice, and homologous boosting better sustained the pool of central memory T cells, which associated with potent protection against B16F1 melanoma challenge. Appropriate timing of booster vaccination was also critical, as a tight boosting schedule hindered persistence of IFN-γ–competent memory CD8+ T cells and mice survival in prophylactic settings. Conversely, prime/boost vaccination proved to be of no advantage or even detrimental in therapeutic settings in B16F1 and transgenic adenocarcinoma of the mouse prostate (TRAMP) models, respectively. Although DC priming was indeed needed for tumor shrinkage, restoration of immune competence, and prolonged survival of TRAMP mice, repeated boosting did not sustain the pool of central memory CTLs and was detrimental for mice overall survival. Thus, our results indicate that booster vaccinations impact antitumor immunity to different extents, depending on their prophylactic or therapeutic administration, and suggest evaluating the need for boosting in any given patient with cancer depending on the state of the disease. Cancer Res; 73(12); 3545–54. ©2013 AACR.

The development of new vaccines remains an attractive goal for disease prevention and therapy, in combination or alternative to drug-based treatment. In parallel, a growing awareness of the importance of early diagnosis in successful disease management is driving the demand for new reliable diagnostic tools. As a consequence, over the last decades an impressive amount of work has been directed toward the search for new solutions to address vaccine design and biomarker discovery. In this context, peptides have generated considerable interest thanks to their general accessibility and ease of manipulation. The aim of this review is to provide the reader a general picture of the traditional peptide-based strategies adopted in immunology and to report on recent advances made in this field, highlighting advantages and limitations of classic versus innovative approaches. Case studies are described to provide illustrative examples, and cross references to more topic-focused and exhaustive reviews are proposed throughout the text.

Summary We present an approach integrating structural and computational biology with immunological tests to identify epitopes in the OppA antigen from the Gram-negative pathogen Burkholderia pseudomallei , the etiological agent of melioidosis. The crystal structure of OppA Bp , reported here at 2.1 A resolution, was the basis for a computational analysis that identified three potential epitopes. In parallel, antigen proteolysis and immunocapturing allowed us to identify three additional peptides. All six potential epitopes were synthesized as free peptides and tested for their immunoreactivity against sera from healthy seronegative, healthy seropositive, and recovered melioidosis patients. Three synthetic peptides allowed the different patient groups to be distinguished, underlining the potential of this approach. Extension of the computational analysis, including energy-based decomposition methods, allowed rationalizing results of the predictive analyses and the immunocapture epitope mapping. Our results illustrate a structure-based epitope discovery process, whose application may expand our perspectives in the diagnostic and vaccine design fields.

A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15 min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid. (C) 2012 Elsevier B.V. All rights reserved.

We present a new multidisciplinary strategy integrating computational biology with high-throughput microarray analysis aimed to translate molecular understanding of protein-antibody recognition into the design of efficient and selective protein-based analytical and diagnostic tools. The structures of two proteins with different folds and secondary structure contents, namely, the beta-barrel FABP and the α-helical S100B, were used as the basis for the prediction and design of potential antibody-binding epitopes using the recently developed MLCE computational method. Starting from the idea that the structure, dynamics, and stability of a protein-antigen play a key role in the interaction with antibodies, MLCE integrates the analysis of the dynamical and energetic properties of proteins to identify nonoptimized, low-intensity energetic interaction-networks on the surface of the isolated antigens, which correspond to substructures that can aptly be recognized by a binding partner. The identified epitopes were next synthesized as free peptides and used to elicit specific antibodies in rabbits. Importantly, the resulting antibodies were proven to specifically and selectively recognize the original, full-length proteins in microarray-based tests. Competition experiments further demonstrated the specificity of the molecular recognition between the target immobilized proteins and the generated antibodies. Our integrated computational and microarray-based results demonstrate the possibility to rationally discover and design synthetic epitopes able to elicit antibodies specific for full-length proteins starting only from three-dimensional structural information on the target. We discuss implications for diagnosis and vaccine development purposes.

A new cyclic peptide containing the isoDGR motif that, after coupling to albumin, selectively binds αvβ3, an integrin overexpressed in the tumor vasculature. IsoDGR-tagged albumin binds tumor vessels and can be exploited as a carrier for the preparation of tumor vasculature-selective nanomedicines, such as gold nanoparticles (Au) carrying tumor necrosis factor α (TNF), a potent vascular damaging agent.

Ordered and reproducible bioprobe immobilization onto sensor surfaces is a critical step in the development of reliable analytical devices. A growing awareness of the impact of the immobilization scheme on the consistency of the generated data is driving the demand for chemoselective approaches to immobilize biofunctional ligands, such as peptides, in a predetermined and uniform fashion. Herein, the most intriguing strategies to selective and oriented peptide immobilization are described and discussed. The aim of the current work is to provide the reader a general picture on recent advances made in this field, highlighting the potential associated with each chemoselective strategy. Case studies are described to provide illustrative examples, and cross-references to more topic-focused and exhaustive reviews are proposed throughout the text.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

No abstract is available for this article.

Chromogranin A (CgA), a secretory protein expressed by many neuroendocrine cells, neurons, cardiomyocytes, and keratinocytes, is the precursor of various peptides that regulate the carbohydrate/lipid metabolism and the cardiovascular system. We have found that CgA, locally administered to injured mice, can accelerate keratinocyte proliferation and wound healing. This biological activity was abolished by the Asp45Glu mutation. CgA and its N-terminal fragments, but not the corresponding Asp45Glu mutants, could selectively recognize the αvβ6-integrin on keratinocytes (a cell-adhesion receptor that is up-regulated during wound healing) and regulate keratinocyte adhesion, proliferation, and migration. No binding was observed to other integrins such as αvβ3, αvβ5, αvβ8, α5β1, α1β1, α3β1, α6β4, α6β7 and α9β1. Structure–activity studies showed that the entire CgA39–63 region is crucial for αvβ6 recognition (K i = 7 nM). This region contains an RGD site (residues CgA43–45) followed by an amphipathic α-helix (residues CgA47–63), both crucial for binding affinity and selectivity. These results suggest that the interaction of the RGD/α-helix motif of CgA with αvβ6 regulates keratinocyte physiology in wound healing.

SummaryCCR5, the major HIV-1 coreceptor, is a primary target for HIV-1 entry inhibition strategies. CCL5/RANTES, a natural CCR5 ligand, is one of the most potent HIV-1 entry inhibitors and, therefore, an ideal candidate to derive HIV-1 blockers. Peptides spanning the RANTES N-loop/β1-strand region act as specific CCR5 antagonists, with their hydrophobic N- and C termini playing a crucial role in virus blockade. Here, hydrophobic surfaces were enhanced by tryptophan substitution of aromatic residues, highlighting position 27 as a critical hot spot for HIV-1 blockade. In a further molecular evolution step, C-terminal engraftment of RANTES 40′ loop produced a peptide with the highest solubility and anti-HIV-1 activity. These modified peptides represent leads for the development of effective HIV-1 inhibitors and microbicides.

Ordered and reproducible bioprobe immobilization onto sensor surfaces is a critical step in the development of reliable analytical devices. A growing awareness of the impact of the immobilization scheme on the consistency of the generated data is driving the demand for chemoselective approaches to immobilize biofunctional ligands, such as peptides, in a predetermined and uniform fashion. Herein, the most intriguing strategies to selective and oriented peptide immobilization are described and discussed. The aim of the current work is to provide the reader a general picture on recent advances made in this field, highlighting the potential associated with each chemoselective strategy. Case studies are described to provide illustrative examples, and cross-references to more topic-focused and exhaustive reviews are proposed throughout the text.

The new tumor-specific antigens Bcr/Abl-OOF, identified in Philadelphia chromosome (Ph)-positive leukemia cells, are derived from an alternative splicing event involving BCR exons 1, 13, or 14 and ABL exons 4 and 5. The COOH-terminus of these transcription products contain an amino acid portion derived from an out-of-frame (OOF) reading of the ABL gene; these variants are expressed in Ph-positive chronic myelogenous leukemia (CML) and acute lymphocytic leukemia patients. Previously, we confirmed the presence of out-of-frame peptide-specific T cells in the peripheral blood of CML patients with the ability to lyse primary autologous CML cells. We also demonstrated that the out-of-frame Abl portion was immunogenic in HLA-A2.1 transgenic mice. Here we describe the production and characterization of monoclonal antibody 1D8G8, a new tool for localization and functional studies of the tumor antigen Bcr/Abl-OOF. This antibody recognizes the out-of-frame protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence, and binds to a specific epitope of this antigen presented in association with HLA-A2.1 molecules at the surface of these cells, as demonstrated by flow cytometry. Thus this MAb could be useful to better understand how this new protein presents in Ph-positive cells beside the canonical Bcr/Abl fusion proteins.

The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV-1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure-guided approach to design peptide mimetics of the N-loop and β1-strand regions of regulated on activation normal T-cell-expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV-1 blockade. Rational modifications were sequentially introduced into the N-loop/β1-strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5-specific HIV-1 isolates (IC50 range: 104–640 nM) but lacking activity against CXCR4-specific HIV-1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the β-extended conformation adopted in full-length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in silico-optimized linker segment designed using de novo structure-prediction algorithms to stabilize the C-terminal α-helix and experimentally validated by NMR. Our mimetics exerted CCR5-antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV-1 entry inhibitors.—Lusso, P., Vangelista, L., Cimbro, R., Secchi, M., Sironi, F., Longhi, R., Faiella, M., Maglio, O., Pavone, V. Molecular engineering of RANTES peptide mimetics with potent anti-HIV-1 activity.

The therapeutic index of anti-cancer drugs is increased when encapsulating them in tumor-targeted liposomes. Liposome-entrapped doxorubicin (DXR), targeting the tumor vasculature marker, aminopeptidase N (APN), displayed enhanced anti-tumor effects and prolonged survival in human neuroblastoma (NB)-bearing mice. Here we exploited a peptide ligand of aminopeptidase A (APA), discovered by phage display technology for delivery of liposomal DXR to perivascular tumor cells. Immunohistochemistry, performed in NB-bearing mice, showed APA expression in the vascular wall of NB primary and metastatic lesions. APA-targeted peptides displayed specific binding to APA-transfected cells in vitro, and also accumulation in the tumor of NB-bearing mice. Consequently, novel, APA-targeted, DXR-liposomes were developed and in vivo proof-of-principle was established, alone and in combination with APN-targeted DXR-loaded liposomes, in NB-bearing mice. Mice receiving APA-targeted liposomal DXR exhibited an increased life span in comparison to control mice, but to a lesser extent relative to that in mice treated with APN-targeted formulation, moreover the greatest increase in TUNEL-positive tumor cells was observed in animals treated with APN-targeted formulations. Mice treated with a combination of APA- and APN-targeted, liposomal DXR had a significant increase in life span compared to each treatment administered separately. There was a significant increase in the level of apoptosis in the tumors of mice on the combination therapy, and a pronounced destruction of the tumor vasculature with nearly total ablation of endothelial cells and pericytes. The availability of novel ligands binding to additional tumor vasculature-associated antigens will allow the design of sophisticated combinations of ligand-targeted liposomal anti-cancer drugs. (C) 2010 Elsevier B.V. All rights reserved.

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycinearginine (isoDGR), a ligand of alpha v beta 3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alpha v beta 3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alpha v beta 3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (alpha v beta 5, alpha v beta 6, alpha v beta 8, and alpha 5 beta 1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I(f) current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.