Oligodendroglioma cells lack glutamine synthetase and are auxotrophic for glutamine, but do not depend on glutamine anaplerosis for growth

In cells derived from several types of cancer, a transcriptional program drives high consumption of glutamine (Gln), which is used for anaplerosis, leading to a metabolic addiction for the amino acid. Low or absent expression of Glutamine Synthetase (GS), the only enzyme that catalyzes de novo Gln synthesis, has been considered a marker of Gln-addicted cancers. In this study, two human cell lines derived from brain tumors with oligodendroglioma features, HOG and Hs683, have been shown to be GS-negative. Viability of both lines depends from extracellular Gln with EC of 0.175 ± 0.056 mM (Hs683) and 0.086 ± 0.043 mM (HOG), thus suggesting that small amounts of extracellular Gln are sufficient for OD cell growth. Gln starvation does not significantly affect the cell content of anaplerotic substrates, which, consistently, are not able to rescue cell growth, but causes hindrance of the Wnt/β-catenin pathway and protein synthesis attenuation, which is mitigated by transient GS expression. Gln transport inhibitors cause partial depletion of intracellular Gln and cell growth inhibition, but do not lower cell viability. Therefore, GS-negative human oligodendroglioma cells are Gln-auxotrophic but do not use the amino acid for anaplerosis and, hence, are not Gln addicted, exhibiting only limited Gln requirements for survival and growth

In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules

Antibody Complementarity-Determining Regions (CDRs): A Bridge between Adaptive and Innate Immunity

Background: It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities. Methodology/Principal Findings: In this study we demonstrate that a synthetic peptide with sequence identical to VHCDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, V HCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. Conclusions/Significance: These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscen

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents

In Vitro and In Vivo Anti-Candida Activity and Structural Analysis of Killer Peptide (KP)-Derivatives

The previously described decapeptide AKVTMTCSAS (killer peptide, KP), derived from the variable region of a recombinant yeast killer toxin-like anti-idiotypic antibody, proved to exert a variety of antimicrobial, antiviral, and immunomodulatory activities. It also showed a peculiar self-assembly ability, likely responsible for the therapeutic effect in animal models of systemic and mucosal candidiasis. The present study analyzed the biological and structural properties of peptides derived from KP by substitution or deletion of the first residue, leaving unchanged the remaining amino acids. The investigated peptides proved to exert differential in vitro and/or in vivo anti-Candida activity without showing toxic effects on mammalian cells. The change of the first residue in KP amino acidic sequence affected the conformation of the resulting peptides in solution, as assessed by circular dichroism spectroscopy. KP-derivatives, except one, were able to induce apoptosis in yeast cells, like KP itself. ROS production and changes in mitochondrial transmembrane potential were also observed. Confocal and transmission electron microscopy studies allowed to establish that selected peptides could penetrate within C. albicans cells and cause gross morphological alterations. Overall, the physical and chemical properties of the first residue were found to be important for peptide conformation, candidacidal activity and possible mechanism of action. Small antimicrobial peptides could be exploited for the development of a new generation of antifungal drugs, given their relative low cost and ease of production as well as the possibility of devising novel delivery systems

Antimicrobial, antiviral and immunomodulatory activity of antibody-derived peptides

Il progetto complessivo si è proposto di studiare le potenzialità immunoterapeutiche ed immunomodulanti di peptidi sintetici di derivazione anticorpale. I peptidi correlati ai CDR e molti decapeptidi, rappresentanti la regione variabile di un Ab anti-idiotipico ricombinante immagine interna di una tossina killer di lievito, hanno esibito attività fungicida in vitro nei confronti di Candida albicans. Un derivato alaninico di un decapeptide candidacida ha mostrato una incrementata efficacia terapeutica in vivo nei confronti di candidosi vaginale e sistemica (Polonelli L. et al. Infect. & Immun. 2003). Peptidi sintetici, rappresentanti i CDR di Ab monoclonali murini ed umani specifici per diversi epitopi irrilevanti, hanno dimostrato di esplicare in modo differenziale attività antifungina (C. albicans), antivirale (HIV-1) e/o antitumorale (cellule di melanoma) in vitro, ex vivo e/o in vivo. I bersagli molecolari dei CDR candidacidi sono supposti essere i β-glucani, che neutralizzano la loro attività, e una sequenza N-terminale della famiglia ALS3. Derivati alaninici sintetici dei CDR candidacidi, usati come surrogati di mutazioni puntiformi naturali, hanno mostrato attività antimicrobica, antivirale e/o antitumorale aumentata, inalterata o diminuita (Polonelli L. et al. PLoS ONE, 2008). Alcuni peptidi candidacidi correlati ai CDR hanno dimostrato di essere caratterizzati in soluzione da proprietà di auto-aggregazione e rilascio, catalizzati da β-1,3-glucani. Mentre la auto-aggregazione assicura la protezione nei confronti di proteasi, la lenta cinetica di dissociazione assicura il rilascio della forma attiva nel tempo, e l’affinità per il recettore è responsabile del rilascio mirato (Pertinhez T.A. et al. Mol. Pharmaceut. 2009). In questo studio, è stato dimostrato che peptidi sintetici selezionati, con omologia di sequenza con frammenti derivanti dal taglio proteolitico della regione costante (Fc) di tutte le classi di Ab, esibiscono una attività antimicrobica, antivirale (HIV-1) e/o antitumorale (cellule di melanoma) differenziale in vitro e/o in vivo. Un peptide Fc sintetico ha dimostrato di avere proprietà immunomodulanti con la stimolazione di: i) produzione di citochine pro infiammatorie e ii) pathway Dectina-1. Alcuni peptidi candidacidi correlati a Fc hanno dimostrato di essere caratterizzati in soluzione da proprietà di auto-aggregazione, catalizzata da β-1,3-glucani. Infine, un peptide sintetico con sequenza identica a VHCDR3 di un mAb murino, specifico per l’antigene difucosilico del gruppo sanguigno A umano, ha dimostrato di legarsi ai macrofagi stimolando: i) produzione di citochine proinfiammatorie, ii) pathway PI3K-Akt e iii) espressione di TLR-4. VHCDR3 ha esercitato un effetto terapeutico nei confronti di candidosi sistemica senza possedere proprietà candidacida (Gabrielli E. et al. PLoS ONE, 2009). L’elevata frequenza di frammenti anticorpali bioattivi, che non mostrano alcuna omologia di sequenza con altri peptidi antimicrobici naturali, e i recenti progressi nello sviluppo e nella somministrazione di peptidi bioattivi stabili, sembrerebbero suggerire che le Ig possano rappresentare una fonte pressoché illimitata di peptidi potenzialmente attivi nei confronti di agenti infettivi e cellule tumorali ed utilizzabili per la messa a punto di nuovi agenti antinfettivi ed antitumorali.The overall project has been proposed to study the immunotherapeutic and immunomodulatory potential of antibody (Ab)-derived synthetic peptides. CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab exerted fungicidal activity in vitro against Candida albicans. An alanine-substitute of a candidacidal decapeptide displayed increased therapeutic effect in vitro and in vivo against vaginal and systemic candidiasis (Polonelli L. et al. Infect. & Immun. 2003). CDR-based synthetic peptides of murine and human monoclonal Abs directed to irrelevant epitopes, showed differential antifungal (C. albicans), antiviral (HIV-1) and/or anti-cancer (melanoma cells) in vitro, ex vivo and/or in vivo activity. Molecular targets to candidacidal CDRs were supposed to be β-glucans, that neutralize their activity, and N-terminal sequence in ALS3 family. Alanine substitutes of synthetic candidacidal CDRs, used as surrogates of natural point mutations, showed differential differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities (Polonelli L. et al. PLoS ONE, 2008). Some candidacidal CDR-related peptides showed to be characterized in solution by self-aggregation-releasing property, catalyzed by b-1,3-glucan. While the self-assembled state provides protection against proteases and the slow kinetic of dissociation assures a release of the active form over time, the receptor affinity is responsible for targeted delivery (Pertinhez T.A. et al. Mol. Pharmaceut. 2009). In this study, it was demonstrated that selected synthetic peptides possessing sequence homology with fragments deriving from the proteolytic cleavage of the constant region (Fc) of all classes of Abs, proved to display differential antimicrobial, antiviral (HIV-1) and/or anti-cancer (melanoma cells) in vitro and/or in vivo activities. A synthetic Fc peptide has been shown to have immunomodulatory properties with the stimulation of: i) production of proinflammatory cytokines and ii) Dectin-1 pathway. Some candidacidal peptides related to Fc have been shown to be characterized in solution to self-aggregation, catalyzed by β-1,3-glucans. Finally, a synthetic peptide with sequence identical to VHCDR3 of a mouse mAb specific for difucosyl human blood group A showed to be taken up by macrophages with stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. VHCDR3 exerted a therapeutic effect against systemic candidiasis without possessing candidacidal properties (Gabrielli E. et al. PLoS ONE, 2009). The high frequency of bioactive Ab fragments, based on Ab sequences that show no sequence homology with other antimicrobial natural peptides, and recent advances in peptide delivery, stability and design, would seem to suggest that the Igs may represent a virtually unlimited source of peptides potentially active against infectious agents and cancer cells and usable for the development of new anti-infective and anticancer agents

Anti-Infective Antibody-Derived Peptides Active against Endogenous and Exogenous Fungi

Mycoses still represent relevant opportunistic infections worldwide, although overshadowed in recent years by other severe and more widespread infections. Moreover, deep-seated mycoses are often accompanied by unacceptably high mortality rates. Etiologic agents include endogenous components of the mycobiota, Candida and Malassezia species above all, and exogenous species, both yeasts and filamentous fungi. Old and new fungal pathogens are increasingly characterized by resistance to the existing antifungal agents, making imperative the search for effective and safe new therapeutics. Among the candidate molecules proposed in recent decades, synthetic peptides derived from the complementarity determining and constant regions of diverse antibodies (Abs), as well as the translated products of Ab-encoding genes, have proved of considerable interest. Their anti-infective activities, regardless of the specificity and isotype of the originating Ab, will be briefly presented and discussed in the light of their different mechanisms of action. Intriguing suggestions on the possible function of Abs after their half-life will be presented, following the recent detection, in human serum, of an antimicrobial Ab-derived peptide. Overall, Abs could represent a source of biologically active, highly flexible peptides, devoid of detectable toxicity, which can be easily synthesized and manipulated to be used, alone or in association with already available drugs, for new anti-infective strategies

Anti-Infective Antibody-Derived Peptides Active against Endogenous and Exogenous Fungi

Mycoses still represent relevant opportunistic infections worldwide, although overshadowed in recent years by other severe and more widespread infections. Moreover, deep-seated mycoses are often accompanied by unacceptably high mortality rates. Etiologic agents include endogenous components of the mycobiota, Candida and Malassezia species above all, and exogenous species, both yeasts and filamentous fungi. Old and new fungal pathogens are increasingly characterized by resistance to the existing antifungal agents, making imperative the search for effective and safe new therapeutics. Among the candidate molecules proposed in recent decades, synthetic peptides derived from the complementarity determining and constant regions of diverse antibodies (Abs), as well as the translated products of Ab-encoding genes, have proved of considerable interest. Their anti-infective activities, regardless of the specificity and isotype of the originating Ab, will be briefly presented and discussed in the light of their different mechanisms of action. Intriguing suggestions on the possible function of Abs after their half-life will be presented, following the recent detection, in human serum, of an antimicrobial Ab-derived peptide. Overall, Abs could represent a source of biologically active, highly flexible peptides, devoid of detectable toxicity, which can be easily synthesized and manipulated to be used, alone or in association with already available drugs, for new anti-infective strategies