Design of Bright Chemogenetic Reporters Based on the Combined Engineering of Fluorogenic Molecular Rotors and of the HaloTag Protein.

The combination of fluorogenic probes (fluorogens) and self-labeling protein tags represent a promising tool for imaging biological processes with high specificity but it requires the adequation between the fluorogen and its target to ensure a good activation of its fluorescence. In this work, we report a strategy to develop molecular rotors that specifically target HaloTag with a strong enhancement of their fluorescence. The divergent design facilitates the diversification of the structures to tune the photophysical and cellular properties. Four bright fluorogens with emissions ranging from green to red were identified and applied in wash-free live cell imaging experiments with good contrast and selectivity. A HaloTag mutant adapted from previous literature reports was also tested and shown to further improve the brightness and reaction rate of the most promising fluorogen of the series both in vitro and in cells. This work opens new possibilities to develop bright chemogenetic reporters with diverse photophysical and biological properties by exploring a potentially large chemical space of simple dipolar fluorophores in combination with protein engineering.

FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Förster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

Charge-driven arrested phase-separation of polyelectrolyte-gold nanoparticle assemblies leading to plasmonic oligomers.

Aggregates of charged metal particles obtained by electrostatic coupling with a compound of opposite charge in the vicinity of the net zero charge ratio are of interest in the field of plasmonics because the inter-particle distance is minimal, which favours plasmonic coupling. However, these structures present a low colloidal stability limiting the development of applications. In this article we show that globally neutral aggregates formed by electrostatic complexation of citrate-stabilized gold particles and a quaternized chitosan (i.e., polycation) around the net zero charge ratio could be stabilized at a nanometric size by the subsequent addition of polyelectrolyte chains. Furthermore, the sign of the charge carried by the stabilizing chains determines the sign of the global charge carried by the stabilized complexes. The stabilization is demonstrated in saline environment on a broad pH range as well as in a cell culture media over periods of several days. Contrarily to stabilization by charged particles, our stabilized complexes are found to retain their initial characteristics (i.e. shape, size, internal structure and optical properties) after stabilization. Hence, the plasmonic coupling allows to maximize the optical absorption around the 800 nm wavelength at which the lasers used for thermoplasmonic and surface enhanced Raman scattering analysis operate.

A Conjugate between Lqh-8/6, a Natural Peptide Analogue of Chlorotoxin, and Doxorubicin Efficiently Induces Glioma Cell Death.

Natural peptides isolated from animal venoms generally target cell surface receptors with high affinity and selectivity. On many occasions, some of these receptors are over-expressed in cancer cells. Herein, we identified Lqh-8/6 as a natural peptide analog of chlorotoxin, a proven and useful compound for the diagnosis and treatment of glioma. Lqh-8/6 and two other natural analogues were chemically synthesized for the first time and evaluated for their ability to label, detect and prevent glioma growth in vitro. We demonstrate that a biotinylated version of Lqh-8/6 allows both the labeling of glioma cell lines and the detection of glioma in brain sections of glioma allograft Fisher rats. Lqh-8/6 has intrinsic anti-invasive properties but is non-toxic to glioma cells. To confer anti-tumor properties to Lqh-8/6, we chemically coupled doxorubicin to the glioma-targeting peptide using click chemistry. To this end, we successfully chemically synthesized Lqh-8/6-azide and doxorubicin-alkyne without impairing the toxic nature of doxorubicin. The toxin-drug conjugate efficiently promotes the apoptosis of glioma cells in vitro. This example contributes to the concept that animal venom peptides constitute exquisite warheads for delivering toxic chemical conjugates, a parallel to the popular concept of antibody-drug conjugates for the treatment of cancer.

Fluorogenic and genetic targeting of a red-emitting molecular calcium indicator.

We introduce a strategy for the fluorogenic and genetic targeting of a calcium indicator by combining a protein fluorogen with the BAPTA sensing group. The resulting dual-input probe acts like a fluorescent AND logic gate with a Ca2+-sensitive red emission that is activated only upon reaction with HaloTag with a 25-fold intensity enhancement and can be used for wash-free calcium imaging in HeLa cells. The modular all-molecular design relying on a well-established self-labeling protein tag opens future possibilities for tuning the photophysical properties or targeting different analytes.

An expanded palette of fluorogenic HaloTag probes with enhanced contrast for targeted cellular imaging.

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths to design a palette of fluorogens with emissions spanning the green to red range, opening new possibilities for multicolor imaging. The probes were studied in glycerol and in the presence of HaloTag and exhibited good fluorogenic properties thanks to a viscosity-sensitive emission. In live-cell confocal imaging, the fluorogens yielded only a very low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast. Combining experimental studies and theoretical investigation of the protein/fluorogen complexes by molecular dynamics, these results offer new insight into the design of molecular rotor-based fluorogenic HaloTag probes in order to improve reaction rates and the imaging contrast.

Selective Capture of Anti-N-glucosylated NTHi Adhesin Peptide Antibodies by a Multivalent Dextran Conjugate.

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N3 )-NH2 to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular.

Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of ß-1,2 mannotriose protective epitopes.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and ß anomery complemented with a synthetic ß-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or ß linkages, excepted for MAb B6.1 (protective epitope) reacting with ß-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients' sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing ß- and α-Man epitopes and detection of antibodies against ß-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and ß-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/ß ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Anti-Inflammatory Effects of Analogues of N-Acyl Homoserine Lactones on Eukaryotic Cells.

BACKGROUND: Since acyl-homoserine lactone (AHL) profiling has been described in the gut of healthy subjects and patients with inflammatory bowel disease (IBD), the potential effects of these molecules on host cells have raised interest in the medical community. In particular, natural AHLs such as the 3-oxo-C12-HSL exhibit anti-inflammatory properties. Our study aimed at finding stable 3-oxo-C12-HSL-derived analogues with improved anti-inflammatory effects on epithelial and immune cells. METHODS: We first studied the stability and biological properties of the natural 3-oxo-C12-HSL on eukaryotic cells and a bacterial reporter strain. We then constructed and screened a library of 22 AHL-derived molecules. Anti-inflammatory effects were assessed by cytokine release in an epithelial cell model, Caco-2, and a murine macrophage cell line, RAW264.7, (respectively, IL-8 and IL-6) upon exposure to the molecule and after appropriate stimulation (respectively, TNF-α 50 ng/mL and IFN-γ 50 ng/mL, and LPS 10 ng/mL and IFN-γ 20 U/mL). RESULTS: We found two molecules of interest with amplified anti-inflammatory effects on mammalian cells without bacterial-activating properties in the reporter strain. The molecules furthermore showed improved stability in biological medium compared to the native 3-oxo-C12-HSL. CONCLUSIONS: We provide new bio-inspired AHL analogues with strong anti-inflammatory properties that will need further study from a therapeutic perspective.

Distinct Postprandial Bile Acids Responses to a High-Calorie Diet in Men Volunteers Underscore Metabolically Healthy and Unhealthy Phenotypes.

Bile acids (BAs) regulate dietary lipid hydrolysis and absorption in the proximal intestine. Several studies have highlighted a determinant role of circulating levels and/or metabolism of BAs in the pathogenesis of major cardiometabolic diseases. Whether changes in BA profiles are causative or are consequence of these diseases remains to be determined. Healthy male volunteers (n = 71) underwent a postprandial exploration following consumption of a hypercaloric high fat typical Western meal providing 1200 kcal. We investigated variations of circulating levels of 28 BA species, together with BA synthesis marker 7α-hydroxy-4-cholesten-3-one (C4) over an approximately diurnal 12 h period. Analysis of BA variations during the postprandial time course revealed two major phenotypes with opposite fluctuations, i.e., circulating levels of each individual species of unconjugated BAs were reduced after meal consumption whereas those of tauro- and glyco-conjugated BAs were increased. By an unbiased classification strategy based on absolute postprandial changes in BA species levels, we classified subjects into three distinct clusters; the two extreme clusters being characterized by the smallest absolute changes in either unconjugated-BAs or conjugated-BAs. Finally, we demonstrated that our clustering based on postprandial changes in BA profiles was associated with specific clinical and biochemical features, including postprandial triglyceride levels, BMI or waist circumference. Altogether, our study reveals that postprandial profiles/patterns of BAs in response to a hypercaloric high fat challenge is associated with healthy or unhealthy metabolic phenotypes that may help in the early identification of subjects at risk of developing metabolic disorders.

Hyperglucosylated adhesin-derived peptides as antigenic probes in multiple sclerosis: Structure optimization and immunological evaluation.

Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide-antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that the N-linked ß-d-glucopyranosyl moieties (N-Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin C-terminal portion HMW1(1205-1526) were essential for high-affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347-1354) fragment bearing one or two N-Glc respectively on Asn-1349 and/or Asn-1352. The N-glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC50 in the range 10-8 -10-10 M by competitive indirect enzyme-linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di-N-glucosylated adhesin peptide Ac-KAN (Glc)VTLN (Glc)TT-NH2 as the shortest sequence able to inhibit high-avidity interaction with N-Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)- and circular dichroism (CD)-based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to two N-glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.

Fluorogenic Protein Probes with Red and Near-Infrared Emission for Genetically Targeted Imaging*.

Fluorogenic probes are important tools to image proteins with high contrast and no wash protocols. In this work, we rationally designed and synthesized a small set of four protein fluorogens with red or near-infrared emission. The fluorophores were characterized in the presence of albumin as a model protein environment and exhibited good fluorogenicity and brightness (fluorescence quantum yield up to 36 %). Once conjugated to a haloalkane ligand, the probes reacted with the protein self-labeling tag HaloTag with a high fluorescence enhancement (up to 156-fold). The spectroscopic properties of the fluorogens and their reaction with HaloTag were investigated experimentally in vitro and with the help of molecular dynamics. The two most promising probes, one in the red and one in the near-infrared range, were finally applied to image the nucleus or actin in live-cell and in wash-free conditions using fluorogenic and chemogenetic targeting of HaloTag fusion proteins.

Glycoreplica peptides to investigate molecular mechanisms of immune-mediated physiological versus pathological conditions.

Investigation of the role of saccharides and glycoconjugates in mechanisms of immune-mediated physiological and pathological conditions is a hot topic. In fact, in many autoimmune diseases cross-reactivity between sugar moieties exposed on exogenous pathogens and self-molecules has long been hinted. Several peptides have been reported as mimetics of glycans specifically interacting with sugar-binding antibodies. The seek for these glycoreplica peptides is instrumental in characterizing antigen mimicry pathways and their involvement in triggering autoimmunity. Therefore, peptides mimicking glycan-protein interactions are valuable molecular tools to overcome the difficulties of oligosaccharide preparations. The clinical impact of peptide-based probes for autoimmune diseases diagnosis and follow-up is emerging only recently as just the tip of the iceberg of an overlooked potential. Here we provide a brief overview of the relevance of the structural and functional aspects of peptide probes and their mimicry effect in autoimmunity mechanisms for promising applications in diagnostics and therapeutics.

Mannose-Coated Fluorescent Lipid Microparticles for Specific Cellular Targeting and Internalization via Glycoreceptor-Induced Phagocytosis.

In this work, we report on the development of mannose-coated fluorescent lipid microparticles to study the role of C-type lectin membrane receptors in phagocytosis. The micrometric droplets of soybean oil-in-water emulsion were functionalized with a tailor-made fluorescent mannolipid. The amphiphilic ligand was built from a mannose unit, a lipid C11 spacer, and a naphthalimide fluorophore. The functionalization of the droplets was monitored by fluorescence microscopy as well as their interaction with concanavalin A, which was used as a model lectin in vitro. The use of a monovalent ligand on the surface of emulsion droplets yielded particles with an affinity approximately 40 times higher than that of free mannose. In cellulo, the coated droplets were shown to be specifically internalized by macrophages in a receptor-dependent phagocytic pathway. The naked droplets, on the other hand, displayed very little internalization because of their low immunogenicity. This work thus brings evidence that C-type lectin membrane receptors may act as phagocytic receptors. The functionalization of the droplets with the tailored amphiphilic fluorescent ligand also provides insights into the development of organic fluorescent particles that may prove useful for developing targeted imaging and delivery tools.

Inter-kingdom effect on epithelial cells of the N-Acyl homoserine lactone 3-oxo-C12:2, a major quorum-sensing molecule from gut microbiota.

BACKGROUND AND AIMS: N-acyl homoserine lactones (AHLs), which are autoinducer quorum-sensing molecules involved in the bacterial communication network, also interact with eukaryotic cells. Searching for these molecules in the context of inflammatory bowel disease (IBD) is appealing. The aims of our study were to look for AHL molecules in faecal samples from healthy subjects (HS) and IBD patients to correlate AHL profiles with the microbiome and investigate the effect of AHLs of interest on epithelial cells. METHODS: Using mass spectrometry, we characterised AHL profiles in faecal samples from HS (n = 26) and IBD patients in remission (n = 24) and in flare (n = 25) and correlated the presence of AHLs of interest with gut microbiota composition obtained by real-time qPCR and 16S sequencing. We synthesised AHLs of interest to test the inflammatory response after IL1ß stimulation and paracellular permeability on Caco-2 cells. RESULTS: We observed 14 different AHLs, among which one was prominent. This AHL corresponded to 3-oxo-C12:2 and was found significantly less frequently in IBD patients in flare (16%) and in remission (37.5%) versus HS (65.4%) (p = 0.001). The presence of 3-oxo-C12:2 was associated with significantly higher counts of Firmicutes, especially Faecalbacterium prausnitzii, and lower counts of Escherichia coli. In vitro, 3-oxo-C12:2 exerted an anti-inflammatory effect on Caco-2 cells. Interestingly, although 3-oxo-C12, the well-known AHL from Pseudomonas aeruginosa, increased paracellular permeability, 3-oxo-C12:2 did not. CONCLUSIONS: We identified AHLs in the human gut microbiota and discovered a new and prominent AHL, 3-oxo-C12:2, which correlates with normobiosis and exerts a protective effect on gut epithelial cells.

New branched amino acids for high affinity dendrimeric DC-SIGN ligands.

A branched amino acid was synthesized from methyl glucopyranoside; this amino acid presents three amino groups protected by Fmoc and one acid group and can be used in classic peptide synthesis. In parallel, similar azido terminated blocks were synthesized. Successive coupling reaction and deprotection afforded dendrimers with up to 27 azido functional groups. As an example of application, d-mannose and l-fucose residues were linked through CuAAC coupling and resulting glycodendrimers were evaluated in their interaction with DC-SIGN using SPR competition assay.

A Secondary Structural Element in a Wide Range of Fucosylated Glycoepitopes.

The increasing understanding of the essential role of carbohydrates in development, and in a wide range of diseases fuels a rapidly growing interest in the basic principles governing carbohydrate-protein interactions. A still heavily debated issue regarding the recognition process is the degree of flexibility or rigidity of oligosaccharides. Combining NMR structure determination based on extensive experimental data with DFT and database searches, we have identified a set of trisaccharide motifs with a similar conformation that is characterized by a non-conventional C-H⋅⋅⋅O hydrogen bond. These motifs are present in numerous classes of oligosaccharides, found in everything from bacteria to mammals, including Lewis blood group antigens but also unusual motifs from amphibians and marine invertebrates. The set of trisaccharide motifs can be summarized with the consensus motifs X-ß1,4-[Fucα1,3]-Y and X-ß1,3-[Fucα1,4]-Y-a secondary structure we name [3,4]F-branch. The wide spectrum of possible modifications of this scaffold points toward a large variety of glycoepitopes, which nature generated using the same underlying architecture.

Supramolecular Chitosan Micro-Platelets Synergistically Enhance Anti-Candida albicans Activity of Amphotericin B Using an Immunocompetent Murine Model.

PURPOSE: The aim of this work is to design new chitosan conjugates able to self-organize in aqueous solution in the form of micrometer-size platelets. When mixed with amphotericin B deoxycholate (AmB-DOC), micro-platelets act as a drug booster allowing further improvement in AmB-DOC anti-Candida albicans activity. METHODS: Micro-platelets were obtained by mixing oleoyl chitosan and α-cyclodextrin in water. The formulation is specifically-engineered for mucosal application by dispersing chitosan micro-platelets into thermosensitive pluronic® F127 20 wt% hydrogel. RESULTS: The formulation completely cured C. albicans vaginal infection in mice and had a superior activity in comparison with AmB-DOC without addition of chitosan micro-platelets. In vitro studies showed that the platelets significantly enhance AmB-DOC antifungal activity since the IC50 and the MIC90 decrease 4.5 and 4.8-times. Calculation of fractional inhibitory concentration index (FICI = 0.198) showed that chitosan micro-platelets act in a synergistic way with AmB-DOC against C. albicans. No synergy is found between spherical nanoparticles composed poly(isobutylcyanoacrylate)/chitosan and AmB-DOC. CONCLUSION: These results demonstrate for the first time the ability of flattened chitosan micro-platelets to have synergistic activity with AmB-DOC against C. albicans candidiasis and highlight the importance of rheological and mucoadhesive behaviors of hydrogels in the efficacy of the treatment.

Dimerization of the fungal defense lectin CCL2 is essential for its toxicity against nematodes.

Lectins are used as defense effector proteins against predators, parasites and pathogens by animal, plant and fungal innate defense systems. These proteins bind to specific glycoepitopes on the cell surfaces and thereby interfere with the proper cellular functions of the various antagonists. The exact cellular toxicity mechanism is in many cases unclear. Lectin CCL2 of the mushroom Coprinopsis cinerea was previously shown to be toxic for Caenorhabditis elegans and Drosophila melanogaster. This toxicity is dependent on a single, high-affinity binding site for the trisaccharide GlcNAc(Fucα1,3)ß1,4GlcNAc, which is a hallmark of nematode and insect N-glycan cores. The carbohydrate-binding site is located at an unusual position on the protein surface when compared to other ß-trefoil lectins. Here, we show that CCL2 forms a compact dimer in solution and in crystals. Substitution of two amino acid residues at the dimer interface, R18A and F133A, interfered with dimerization of CCL2 and reduced toxicity but left carbohydrate-binding unaffected. These results, together with the positioning of the two carbohydrate-binding sites on the surface of the protein dimer, suggest that crosslinking of N-glycoproteins on the surface of intestinal cells of invertebrates is a crucial step in the mechanism of CCL2-mediated toxicity. Comparisons of the number and positioning of carbohydrate-binding sites among different dimerizing fungal ß-trefoil lectins revealed a considerable variability in the carbohydrate-binding patterns of these proteins, which are likely to correlate with their respective functions.

A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets.