Thesis advisor: Jianmin Gao / Covalent drugs present a unique situation in the clinical world. Formation of a covalent bond between a drug molecule and its target protein can lead to significant increases in a number of desirable traits such as residence time, potency, and efficacy of a drug. From a kinetic perspective, the formation of a covalent bond between a drug and its target functionally eliminates the dissociation rate (koff) of the compound, ensuring that the compound will stay engaged with its target. However, development of covalent drugs has been met with caution and concern, as an irreversible covalent bond forming on the wrong target can have disastrous results, so specificity is of the utmost importance. One option for increasing specificity is by linking a covalent binding electrophile, or warhead, to a peptide. Peptide-based therapeutics have already been shown to serve as effective protein-targeting modalities with high specificity, a specificity that would greatly benefit covalent drugs. Phage display is a powerful technique for the discovery of selective peptides which utilizes the screening of vast libraries of randomized peptides to identify strong binders. This technology has been used to discover a large number of protein-targeting peptides, but also a smaller number of cyclic, covalent binding peptides that function as enzymatic inhibitors. Herein, this study aimed to explore the idea of adding covalent-binding functionality to phage libraries in novel ways and expand upon the scope of proteins that can be targeted with phage libraries containing covalent libraries. We sought to develop a mechanistic and chemical understanding of the interactions between bacteriophage and chemical warheads to best understand both the limits and the potential of this technology. In order to best understand the relationship between chemical warhead and phage particle, a model system was developed based on the M13KE pIII protein. It was found that the extracellular N-terminal domains of this protein could be expressed and purified in low yields in bacterial cells and that these domains would behave similarly in solution as in the membrane of the M13KE bacteriophage. With this protein in hand, experiments previously performed using small, cysteine containing peptides, could be performed on a full protein to mimic the phage labeling environment. This protein was used to identify efficient cysteine crosslinkers, most notably dichloroacetone (DCA) and bis-chlorooxime (BCO). The pIII protein system was then used to study the viability of bifunctional warhead molecules containing a covalent warhead and a cysteine crosslinker. Based on preliminary analyses with the pIII protein, aryl sulfonyl fluoride was chosen as a novel warhead candidate that warranted further pursuit. Kinetic NMR studies verified that aryl sulfonyl fluoride was capable of forming covalent bonds with phenols under phage labeling conditions. Labeling experiments analyzed with LC/MS seemed to indicate a degradation of the warhead. However, as the source of the degradation was not able to be determined, it was decided that various affinity assays would be used to identify if phage could be labeled with an aryl sulfonyl fluoride-DCA conjugate. Both streptavidin-bead pulldown assays and ELISA assays were used, however both assays yielded results that could not conclusively verify the integrity of the warhead. During phage labeling experiments, a phenomenon was noted that phage titers after modification showed a 2-3 order of magnitude drop in phage count. Covalent modification of phage beyond what is intended could have troubling consequences for all covalent phage libraries, and so a more in-depth approach was taken to identify and better understand phage toxicity as it relates to covalent warheads. As a model, a well-studied diazaborine-mediated warhead with a slow dissociation rate was selected and used in a range of phage toxicity screenings. Despite statistical fluctuations between trials, toxicity screenings using this warhead served to highlight a unique concern for bifunctional covalent warheads. A concentration-dependent toxicity can be seen in phage incubated with bifunctional small molecules that is not present when incubated with the monofunctional equivalents. The presence of this toxicity even towards a phage with no free thiols highlights a unique challenge of off-target labeling within phage particles that, if solved, could provide the next significant step towards developing novel covalent phage libraries. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Clostridium difficile est un pathogène entérique qui cause d'importantes infections nosocomiales dont le traitement est parfois problématique. Il n'existe, à l'heure actuelle, que deux antibiotiques approuvés pour traiter les infections à C. difficile et le taux de rechute est assez important. Ce projet a initialement visé à isoler et caractériser des phages virulents contre C. difficile en vue de les utiliser en phagothérapies comme outils thérapeutiques alternatifs. Les eaux usées et les selles de patients infectés par C. difficile ont été utilisées pour isoler et détecter les phages virulents. Or, quatre phages différents (9MMPOI-O4) ont été isolés mais aucun de ces phages ne s'est révélé être virulent. Les quatre phages tempérés ont donc été caractérisés et leur impact a été évalué sur quelques phénotypes bactériens chez C. difficile dont la motilité et la production des toxines A et B. La caractérisation morphologique des phages (pMMPOl-04 a permis de déterminer que ceux-ci appartiennent à la familles des Myoviridae alors que la caractérisation génomique a permis de démontrer que certains de ces phages sont assez différents entre eux mais également par rapport aux autres phages tempérés, isolés et caractérisés dans la littérature. De façon générale, les phages (pMMPOl-04 ne semblent pas s'induire spontanément de manière plus importante mais suggère que la présence de certains antibiotiques pourrait augmenter l'induction de certains de ces phages. L'impact des phages (pMMPOl-04 sur la motilité chez C. difficile n'a pas démontré que ceux-ci avaient un rôle à jouer sur ce phénotype. Par contre, certains des phages (pMMP semblent augmenter ou diminuer la production en toxines A et B. Les résultats de nos travaux indiquent donc que certains des phages caractérisés présentent des différences importantes qui suggèrent une grande diversité parmi les phages tempérés chez C. difficile. De plus, certains des phages cpMMP auraient la capacité de participer aux transferts horizontaux de matériels génétiques et d'affecter la régulation de certains facteurs de virulence chez C. difficile tel que la production en toxines A et B. Évidemment, des travaux supplémentaires seront nécessaires pour confirmer la modification du phénotype de production en toxines par ces phages mais également sur d'autres phénotypes associés aux autres facteurs de virulence de cette bactérie. [Symboles non conformes]

Interferon regulatory factor 1 (IRF-1) is a tumour suppressor protein and transcription factor. It has been shown to modulate target gene expression in response to stimuli, which include viral infection and DNA damage, and to be down-regulated in several forms of cancer. This thesis details the development of an intrabody, an intracellular antibody, that binds specifically to endogenous IRF-1. The binding of the intrabody to IRF-1 enhanced transcription from IRF-1-responsive reporter gene constructs and endogenous promoters, thus it was shown to activate IRF-1. Intrabody binding also increased the rate at which IRF-1 was degraded, suggesting that the intrabody epitope may be regulating both IRF-1 activity and turnover. These results were supported point mutation within the intrabody epitope (P325 to A) as the resultant mutant also displayed both a higher transcriptional activity and increased rate of degradation. In an effort to understand the mechanisms which regulate IRF-1 activity a search for novel IRF-1-interacting proteins was carried out using phage peptide display. This in vitro technique enables the identification of peptides able to bind a specific target protein. The sequence of these peptides can then be used to search protein databases for homologous, full-length proteins that could also bind the target protein. This led to the identification of an IRF-1-binding peptide that held sequence similar to a region of Zinc Finger 350 (ZNF350), a transcription factor involved in regulating the DNA damage response. Subsequently, endogenous ZNF350 and IRF-1 were co-immunoprecipitated from a human cancer cell line. The extreme C-terminus of IRF-1 was shown to be sufficient for an interaction with ZNF350, although a second, more N-terminal site was also shown to be essential for a stable intracellular interaction. This data sheds new light on the role of the extreme C-terminus of IRF-1 in modulating the protein‟s activity. This study also provides new and IRF-1-specific molecular tools, in the form of intrabodies and IRF-1-binding peptides, which could be used in the future to further characterise the activity and regulation of this tumour suppressor protein.

<p>In affinity protein biotechnology the selective bindingbetween a chosen protein and an interacting biomolecule isutilized for a variety of applications including bioseparation,detection and therapy. Traditionally, affinity proteinsrecruited for such applications have been derived from naturalproteins or immunoglobulins generated via immunization routes.More recently, advances in the construction and handling oflarge collections of proteins(denoted libraries) generated invitro have opened up for new routes for the development ofaffinity proteins with desired properties.</p><p>In this study, phage display selection technology was usedfor the isolation of novel human CD28 (hCD28)-specific affinityproteins from a protein library constructed by combinatorialprotein engineering of a 58 aa protein domain (Z) derived fromstaphylococcal protein A (SPA). From selections using hCD28 asa target molecule, several hCD28-specific affinity proteins(denoted affibodies) could be identified and analysis of theisolated affibody variants revealed a high degree of sequencehomology between the different clones. The biosensor analysisshowed that all variants bound to hCD28 with micromolardissociation constants (KD) and no significant cross-reactivitytowards the structurally related T-cell receptor hCTLA-4 couldbe observed. The apparent binding affinity for hCD28 of one ofthe isolated affibodies was further improved through fusion toa human Fc fragment fusion partner, resulting in a homodimericversion of the affibody ligand showing avidity effects uponhCD28 binding. Further, a co-culture experiment involvingJurkat T-cells and CHO cell lines tranfected to express eitherhuman CD80 or LFA-3 on the cell surface showed that apreincubation of Jurkat cells with one of the affibody variantsresulted in a specific concentration-dependent inhibition ofthe CD80 induced IL-2 production. This indicates that thisaffibody binds to hCD28 and specifically interferes with theco-stimulation signal mediated via hCD28 and hCD80. ACD28-specific binding protein could have potential as an agentfor various immunotherapy applications. In a second study, anaffinity protein-based strategy was investigated forsite-specific anchoring of proteins onto cellulose for woodfiber engineering purposes. Here, affinity proteins derivedfrom different sources were used for the assembly of acellulosome-like complex for specific and reversible anchoringof affinity domain-tagged reporter proteins to acellulose-anchored fusion protein. A fusion protein between acellulose binding module (Cel6A CBM1) derived from the fungalTrichoderma reesei and a five-domain staphylococcal protein A(SPA) moiety was constructed to serve as a platform for thedocking of reporter proteins produced as fusion to two copiesof a SPA-binding affibody affinity protein (denoted ZSPA-1),selected by phage display technology from a Z domain basedprotein library. In a series of experiments, involving repeatedwashing and low pH elutions, affinity tagged Enhanced GreenFluorescent Protein (EGFP) and Fusarium solani pisi lipasecutinase reporter proteins were both found to be specificallydirected from solution to a region of a cellulose-based filterpaper where the SPA-CBM fusion protein previously had beenpositioned. This showed that the cellulose-anchored SPA-Cel6ACBM1 fusion protein had been stably anchored to the surfacewith retained binding activity and that the interaction betweenSPA and the ZSPA-1 affibody domain was selective.</p><p>phage display, combinatorial, selection, CD28, cellulosome,cellulose, affibody</p>

Protein kinases function as key regulators in a variety of signaling pathways by executing the phosphorylation of a variety of protein substrates. Perturbation in the activity of numerous proteins kinases has been implicated in a large number of diseases including cancer, diabetes, inflammation and neurological disorders. Therefore, selective modulation of kinase activity is highly desirable for the dissection of complex signaling pathways and substantiating therapeutic targets. To develop potent and selective inhibitors for an array of kinases, our group has developed a fragment based bivalent methodology utilizing phage display. The strategy involves an ATP active site targeted small molecule which directs the selection of cyclic peptides, from a phage displayed library, on the target kinase surface through coiled coil interactions. The selected cyclic peptides can be conjugated to the ATP mimetic to generate bivalent inhibitors. In this thesis, I have expanded the scope of the bivalent phage-display selection approach. To interrogate the generality of this approach, we targeted several kinases from different groups within the human kinome using the staurosporine warhead. Fyn and PDGFRβ represented the tyrosine kinase group and CLK2 and Pim-1 kinases represented the CMGC and CaMK groups respectively. The selections against these four kinases did not result in potent inhibitors though they provided an avenue for the refinement of the bivalent phage-display approach as well as method development. Application of this methodology to AKT2 in the AGC family resulted in bivalent inhibitors which were interrogated for their selectivity and mode of action. The bivalent strategy was further explored for its utility to target inactive kinases, and success was achieved against AKT1. Finally, we demonstrated the modularity of ATP site targeted ligand by carrying out a selection against STK33 kinase using a new small molecule warhead, sunitinib. This resulted in potent and selective bivalent inhibitors for STK33. The use of different ATP site targeting molecules potentially increases the number of targetable kinases with our strategy. In all the selections, the identified cyclic peptides inhibited the kinase and showed a non-competitive mode of inhibition with respect to the kinase substrate. This suggests that the selected peptides do not target the substrate site and possibly bind to unidentified pockets on the kinase surface, which potentially provides new methods to target kinases outside the traditional ATP binding cleft. The strategy may prove to be a robust method to discover new allosteric sites on kinases as well as other proteins. The potent and selective bivalent inhibitors obtained by our strategy have the potential to provide insight towards the design of new non-ATP targeted approaches for inhibiting protein kinases and elucidating their specific functions.

No description available.

Enterohaemorrhagic Escherichia coli (EHEC) strains are associated with gastrointestinal and severe systemic disease in humans. EHEC O157:H7 is the most common serotype causing human infections in North America and the UK. Human infections mainly originate from cattle, through either direct contact with infected animals or indirectly through contamination of food or water with animal faeces. From the sequencing of EHEC O157 strains, it is clear that the genomes contain multiple prophages, many of them cryptic, which define this E. coli pathotype. These regions include the locus of enterocyte effacement (LEE) which is a critical horizontally acquired pathogenicity island and encodes a type III secretion system (T3SS). The T3SS translocates effector proteins into epithelial cells that enable tight attachment to these host cells and also modify innate responses and other cellular functions to promote persistence in the animal host. The T3SS is essential for the colonisation of cattle by EHEC O157 where it is localised to the terminal rectum. The regulation of T3S is complex with many regulators and environmental factors already identified. Previous work has demonstrated marked variation in the levels of T3S among EHEC O157 strains. The aim of this research was to further investigate the regulation of T3S towards two objectives: (1) to understand the localisation of EHEC O157 at the terminal rectum of cattle; (2) to understand the strain variation in T3S. (1) In relation to rectal and mucosal colonisation, established aerobic/anaerobic regulators were investigated including arcA, fnr, narX, narQ. Briefly, arcA, fnr, narX, narQ were deleted in an E. coli O157 strain ZAP198 by lambda red recombination. Apart from the fnr mutant which showed lower levels of T3S, the remaining mutants displayed similar T3S protein levels compared to the wild type strain. In addition, no significant changes in adherence and A/E lesion formation capacity were measured for the mutants following interaction with bovine epithelial cells. (2) Strain secretion variation was approached in two ways; the first was to control expression from the LEE1 operon, required for T3S expression, in order to both induce expression and examine the importance of downstream regulation. The second was to investigate variation in T3S between different phages types of EHEC O157. While attempts to construct an inducible T3SS were not successful, intermediate strains made in the process have been useful to dissect how regulators being studied in the laboratory control T3S. The main novel insights from the research have come from examining T3S in different EHEC O157 phage types. We found that the average level of T3S in PT 21/28 strains was lower than in PT 32 strains. Interestingly, most (90%) of PT 21/28 strains contained both Stx2 and Stx2c phages. In contrast, only 28% of PT 32 strains had both phages. Taken together, this raised the possibility that Stx phage integration might have a repressive impact on T3SS regulation in E.coli O157:H7. This hypothesis was addressed using a number of different approaches. Deletions of Stx phages were constructed and these had increased levels of T3S when compared to the parental strains. This phage regulation of T3SS was confirmed in an E. coli K12 background by examining an induced LEE1 reporter in the presence and absence of a transduced Stx2 phage. In addition, it was shown that deletion of the CII phage regulator led to increased T3S and may contribute to the Stx phage repression reported above. This work demonstrates for the first time that Stx phage integration represses T3S expression. It is proposed that this control may limit immune exposure of this critical colonisation factor and that the repression actually allows activation by prophage encoded regulators, including PchA/B, that co-ordinate T3S and non LEE-encoded effector expression to promote epithelial cell colonisation.

Bacteria are under constant threat by their viral parasites, the bacteriophages (phages) and have evolved a range of anti-phage systems to defend themselves. One of these systems is termed abortive infection (Abi) where, upon phage infection, an Abi system may be activated which initiate a bacteriostatic or bactericidal response. While the infected bacteria do not obviously benefit from the activation of these systems, the cessation of bacterial growth or premature cellular suicide prevents the release of phage progeny. Thus Abi can be viewed as an altruistic process as only the remaining clonal bacterial population benefits. The Type III toxin-antitoxin systems have previously been shown to be involved in Abi, however the mechanisms through which these systems are activated are still poorly understood. A common approach to reveal the phage product involved in triggering these systems is to first determine the mutations that a previously sensitive phage evolves to escape after exposure to an Abi system. Analysis of viral "escape" mutants has been used in this study to try to elucidate the activation mechanism(s) of two Type III systems (ToxIN$_P$$_a$ and TenpIN$_P$$_l$) of several environmental phages. Several new phage products were identified in escape mutants as candidate factors involved in circumventing Abi - and possible roles in phage metabolism predicted. Furthermore, the genomes of several phages that could not evolve escapes, or were insensitive to Abi, are sequenced and these data exposed interesting curiosities regarding Abi (as well as the discovery of several novel and rare phages). Previously, no coliphage was identified that was capable of escape of the ToxIN$_P$$_a$ or TenpIN$_P$$_l$ systems. However, this study defined and characterised the first ToxIN$_P$$_a$ and TenpIN$_P$$_l$ coliphage escapes as well as a new method for isolating host-dependent coliphage escapes. Finally, multiple phages that infect the insect pathogen $\textit{Photorhabdus luminescens}$ TT01 (the bacterial strain from which the TenpIN$_P$$_l$ system originated) were isolated, genomically sequenced and characterised in terms of host range. The results revealed a large superfamily of flagellum-dependent phages that exhibit remarkable host promiscuity, possibly defining the most promiscuous phages thus far identified.

Despite intense research efforts, a safe and effective HIV-1/AIDS vaccine still remains far away. HIV-1 escapes the humoral immune response through various mechanisms and until now, only a few nAbs have been identified. A promising strategy to identify new epitopes that may elicit such nAbs is to dissect and analyze the humoral immune response of sera with broadly reactive nAbs. The identified epitopes recognized by these antibodies might then be incorporated into a vaccine to elicit similar nAbs and thus provide protection from HIV-1 infection. Using random peptide phage display libraries, the Ruprecht laboratory has identified the epitopes recognized by polyclonal antibodies of a rhesus monkey with high-titer, broadly reactive nAbs that had been induced after infection with a SHIV encoding env of a recently transmitted HIV-1 clade C. The laboratory analyzed phage peptide inserts for conformational and linear homology with computational assistance. Several of the identified peptides mimicked domains of the original HIV-1 clade Env, such as conformational V3 loop epitopes and the conserved linear region of the gp120 C-terminus. As part of this work, these mimotopes were analyzed for cross-reactivity with other sera obtained from rhesus monkeys with nAbs and antibody recognition was shown for several mimotopes, particularly those representing the V3 loop. In addition, these mimotopes were incorporated into a novel DNA prime/phage boost strategy to analyze the immunogenicity of such phage-displayed peptides. Mice were primed only once with HIV-1 clade C gp160 DNA and subsequently boosted with mixtures of recombinant phages. This strategy was designed to focus the humoral immune response on a few, selected Env epitopes (immunofocusing) and induced HIV-1 clade C gp160 binding antibodies and cross-clade nAbs. Furthermore, the C-terminus of gp120, a conserved HIV Env region, was linked to the induction of nAbs for the first time. The identification of such conserved antigens may lead to the development of a vaccine that is capable of inducing broadly reactive nAbs that might confer protection form HIV-1 infection. / Trotz enormer Forschungsleistungen liegt ein sicherer und effektiver Impfstoff gegen HIV-1/AIDS immer noch in weiter Ferne. HIV-1 entkommt der humoralen Immunantwort aufgrund mehrerer Mechanismen und daher wurden bis zu diesem Zeitpunkt nur wenige neutralisierende Antikörper identifiziert. Eine vielversprechende Strategie zur Identifizierung neuer Epitope, die neutralisierende Antikörper induzieren könnten, ist die Analyse von Seren mit solchen Antikörper. Die dabei identifizierten Epitope könnten dann zur Herstellung eines Impfstoffes verwendet werden, der ähnliche neutralisierende Antikörper induziert und damit vor einer Infektion mit HIV-1 schützt. Mittels Phage-Display hat das Labor von Ruth Ruprecht mehrere solcher Epitope von polyklonalen Antikörpern aus Rhesus Affen mit hochtitrigen, breit-neutralisierenden Antikörpern identifiziert. Diese Antikörper wurden nach einer Infektion mit einem SHIV induziert, das das virale Hüllprotein eines kürzlich übertragenen HIV-1 clade C Virus enthielt. Die Phagenpeptide wurden auf konformelle und lineare Homologie mittels einer Computer Software untersucht. Mehrere dieser Peptide entsprachen Domänen des viralen Hüllproteins, wie z.B. konformelle V3 loop Epitope and Epitope des linearen C-Terminus von gp120. Im Rahmen dieser Arbeit wurden diese Mimotope auf Kreuzreaktivität mit anderen Seren von Rhesus Affen mit neutralisierenden Antikörpern untersucht. Dabei wurden insbesondere die Mimotope des V3 loops von anderen Seren erkannt. Des Weiteren wurden diese Phagen-Mimotope im Rahmen einer neuen DNA prime/phage boost Strategie zur Immunisierung verwendet. Mäuse wurden einmalig mit HIV-1 clade C gp160 DNA immunisiert und anschließend mehrfach mit rekombinanten Phagen geboostet. Mittels dieser Strategie sollte das Immunsystem auf einige, spezielle Epitope des viralen Hüllproteins fokusiert werden (Immunofocusing). Hierbei wurden HIV-1 clade gp160-bindende Antikörper und breit-neutralisierende Antikörper induziert. Des Weiteren konnten zum ersten Mal neutralisierende Antikörper gegen den C-terminus von gp120, einer konservierten Region des viralen Hüllproteins, induziert werden. Die Identifikation solcher konservierter Mimotope kann zur Entwicklung von einem HIV-1 Impfstoff beitragen, der breit neutralisierende Antikörper induziert, die vor einer Infektion schützen können.

La détection de l'apoptose revêt un intérêt considérable en raison de son implication dans de nombreuses pathologies d'incidence élevée, comme le cancer ou la maladie d'Alzheimer pour n'en citer que deux. Des méthodes de mise en évidence de ce phénomène se multiplient et se développent sans cesse visant à détecter in vitro voire même in vivo, cette forme de mort cellulaire programmée. Grâce à son pouvoir de résolution élevé et à des agents de contraste spécifiques, l'Imagerie par Résonance Magnétique (IRM) offre la possibilité de détecter, de manière non invasive, les cellules apoptotiques. Cette approche trouvera son utilité pour la mise au point de traitements anti-tumoraux et pour leur suivi en clinique. Aujourd'hui, il existe des agents de contraste magnétiques capables de reconnaître les cellules apoptotiques, mais leurs molécules vectrices sont constituées de protéines, d'anticorps ou de leurs domaines dont on ne peut ignorer l'important pouvoir immunogène. Des molécules mimétiques ou des oligomères de petite taille comme des peptides seraient plus adaptés et plus simples à synthétiser chimiquement. Notre travail porte sur la sélection, de peptides capables de reconnaître spécifiquement les cellules apoptotiques, en se fixant à la phosphatidylsérine (molécule marqueur des cellules apoptotiques). Ces peptides ont été sélectionnés par la méthode du phage display. Le phage display est une technique de biologie moléculaire permettant de sélectionner, entre autre, des peptides de longueur déterminée, en fonction de leur affinité pour une cible donnée. Plusieurs milliards de séquences peptidiques exposées par les phages constituant la bibliothèque, peuvent être testés simultanément. Les meilleures séquences sont ensuite synthétisées chimiquement et testées indépendamment du phage. Dans la première partie de ce travail, plusieurs protocoles de phage display basés sur différentes méthodes d'immobilisation de la cible, et différents types de bibliothèques de phages ont été testés et comparés. A l'issue de ces sélections, plusieurs phages présentant une affinité élevée pour la phosphatidylsérine ont été sélectionnés. Un des peptides exposés par un de ces phages, nommé E3, a été synthétisé et étudié dans la seconde partie de ce travail. Le peptide E3 a été d'abord greffé à un contrastophore magnétique de type superparamagnétique (Ultra Small Particle Iron Oxide) pour être testé in vitro sur des systèmes biologiques (apoptose induite chez des cellules Hep G2 par la camptothécine). Après avoir été testé avec succès sur culture cellulaire, ce nouvel agent de contraste magnétique a été utilisé, in vivo dans le modèle murin d'apoptose hépatique induite par injection i.v. d'anticorps anti-Fas. Comme tout agent de contraste de type particulaire, le GG-E3-USPIO (peptide E3 greffé aux USPIOs par un pont diglycyle) serait capté de manière non spécifique par le foie. Il doit donc être rendu "furtif" pour permettre l'exploitation de sa spécificité. Afin de contourner le problème de capture non spécifique par le foie, le GG-E3-Gd-DTPA (peptide E3 greffé au Gd-DTPA par un pont diglycyle), un autre agent de contraste, cette fois de type paramagnétique, a été synthétisé. Les agents de contraste GG-E3-USPIO-PEG (peptide E3 greffé aux USPIO recouvertes de polyéthylèneglycol par un pont diglycyle) et GG-E3-Gd-DTPA se sont avérés efficaces, provoquant respectivement une diminution et un rehaussement du signal des foies apoptotiques de souris en IRM.