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International audience; ISOTOPICS (H2020 - Marie Sklodowska-Curie Actions - Innovative Training Network; grant number 675071) combines researchers and industry in an innovative effort to de-risk drug development, create new tools for medicine and train first-class radiochemists with dual academic-industrial expertise.

International audience; Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/$^{129}$Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized $^{129}$Xe NMR. This promising system should find important applications for theranostic use.

Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/

International audience; Gaining an understanding of the nature of host–guest interactions in supramolecular complexes involving heavy atoms is a difficult task. Described herein is a robust simulation method applied to complexes between xenon and members of a cryptophane family. The calculated chemical shift of xenon caged in a H2O2 probe, as modeled by quantum chemistry with complementary-orbital, topological, and energy-decomposition analyses, is in excellent agreement with that observed in hyperpolarized 129Xe NMR spectra. This approach can be extended to other van der Waals complexes involving heavy atoms.

The parallel oxorhenium-mediated assembly of 288 noncyclic RGD analogues is reported. All complexes contain a NS(2) +S chelating motif that enables the unambiguous coordination of the oxorhenium and oxotechnetium cores. In this study, "modules S" contain a variety of pending guanidinium groups whereas the "NS(2) modules" are made of a series of N-acylated amino acids. Combination of sets of "NS(2) " and "S modules" together with tetrabutylammonium tetrachlorooxorhenate gave the corresponding oxorhenium complexes in good yields and satisfactory purities. Evaluation of these metalloconstructs towards integrins α(V) β(3) , α(IIb) β(3) , and α(V) β(5) led to the identification of micromolar and submicromolar antagonists of theses integrins. These compounds exhibit interesting selectivities and promise attractive applications for the molecular imaging of integrin-dependent pathologies.

We report the design of a new ligand of integrins that might be used for the molecular imaging of tumor neoangiogenesis. For this purpose, we designed a modified RGD tripeptide bearing a N-terminal N -bis(thioethyl)glycinate (NS 2 ) motif and a thioethyl moiety at the C-terminus. Simultaneous coordination of an oxorhenium core by the NS 2 and thioethyl moieties led to peptide cyclization and gave the corresponding monomers 13a and b (major isomer) resulting from the syn / anti -isomerism, along with dimers' species 16a and b . Cyclometallated peptide 13b showed the most promising activity with an IC 50 of 86 nM for integrin α V β 3 whereas it binds integrin α IIb β 3 with an affinity lower by an order of magnitude. Labeling with [ 99m Tc]oxotechnetium gluconate led exclusively to complex 17 , the equivalent of compound 13b , which displayed satisfactory stabilities in mice plasma and towards glutathione.

We report the design of a new ligand of integrins that might be used for the molecular imaging of tumor neoangiogenesis. For this purpose, we designed a modified RGD tripeptide bearing a N-terminal N -bis(thioethyl)glycinate (NS 2 ) motif and a thioethyl moiety at the C-terminus. Simultaneous coordination of an oxorhenium core by the NS 2 and thioethyl moieties led to peptide cyclization and gave the corresponding monomers 13a and b (major isomer) resulting from the syn / anti -isomerism, along with dimers' species 16a and b . Cyclometallated peptide 13b showed the most promising activity with an IC 50 of 86 nM for integrin α V β 3 whereas it binds integrin α IIb β 3 with an affinity lower by an order of magnitude. Labeling with [ 99m Tc]oxotechnetium gluconate led exclusively to complex 17 , the equivalent of compound 13b , which displayed satisfactory stabilities in mice plasma and towards glutathione.

*INSERM U-575 and Department of Biochemistry, Faculty of Medicine, Strasbourg, France CEA/Saclay, Department d’Inge´nierie et d’Etudes des prote´ines, Gif Sur Yvette, FranceThe kynurenine pathway is the principal metabolic route forcatabolism of tryptophan. Several intermediates of the path-way are active at central neurotransmitter sites, for example,the NMDA receptor agonist quinolinic acid and the gluta-mate receptor antagonist kynurenic acid. The latter is mainlysynthesized in rat brain astrocytes by kynurenine aminotrans-ferase and is released into the immediate vicinity of gluta-matergic synapses to possibly reduce the neurotoxic effectsof excitatory amino acid (Guidetti et al. 1995, 2007; Ceresoliet al. 1997). These findings raised the possibility that thekynurenine pathway and the metabolites of tryptophan couldbe involved in various neuroregulatory and neuropatholo-gical phenomenon in the CNS (Stone and Darlington 2002).Xanthurenic acid (Xa) is one metabolite of this kynureninepathway and could be considered as the parent compound ofkynurenic acid. Indoleamine 2,3 dioxygenase which ispresent in the brain transforms tryptophan into kynureninethat is either transaminated into kynurenic acid by kynure-nine aminotransferase or hydroxylated into 3-hydroxykynur-enine by kynurenine 3-hydroxylase. Transamination of3-hydroxykynurenine leads to xanthurenic acid (Fig. 1).The latter pathway is generally considered to be part of adetoxification process that reduces the concentration of 3-hydroxykynurenine because spontaneous oxidation of thissubstance induces free radical generation and apoptosis, bothin vitro and in vivo (Okuda et al. 1996; Wei et al. 2000).However, a transaminase specific for 3-hydroxykynureninehas not been identified in man. In mammals, it is generallythought that 3-hydroxykynurenine is transformed to Xa bythe same kynurenine transaminase isoenzymes that catalyzethe transamination of kynurenine to kynurenic acid (Urenjakand Obrenovitch 2000).

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA–gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA–gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal inflammation. Our findings strongly implicate CD71 in the pathogenesis of CD.

Background & Aims: Intestinal epithelial cells release antigen-presenting vesicles (exosomes) bearing major histocompatibility complex class II/peptide complexes stimulating specific immune responses in vivo. To characterize further the role of human epithelial exosomes in antigen presentation, their capacity to load antigenic peptides, bind immune target cells, and induce T-cell activation was analyzed in vitro. Methods: The capacity of exosomes derived from the HLA-DR4-expressing, intestinal epithelial cell line T84 to load the HLA-DR4-specific peptide 3H-HSA 64–76 and to activate a HLA-DR4-restricted T-cell hybridoma was tested in the presence or absence of human monocyte-derived dendritic cells (DCs). Interaction of fluorescein isothiocyanate-labeled exosomes with T cells and DCs was analyzed by flow cytometry and confocal microscopy. Results: T84-derived exosomes, enriched in CD9, CD81, CD82, and A33 antigen, were capable of binding specifically human serum albumin (HSA) 64–76 peptide on HLA-DR4 molecules and of interacting preferentially with DCs. HSA-loaded exosomes were unable to activate the T-cell hybridoma directly but induced a productive T-cell activation through DCs. When HSA peptide was bound to exosomal HLA-DR4 molecules instead of in a soluble form, the threshold of peptide presentation by DCs was markedly decreased (×10−3). Conclusions: Exosomes released by intestinal epithelial cells bear exogenous peptides complexed to major histocompatibility complex class II molecules and interact preferentially with DCs, strongly potentiating peptide presentation to T cells. Epithelial exosomes constitute a powerful link between luminal antigens and local immune cells by mediating the transfer of tiny amounts of luminal antigenic information and facilitating immune surveillance at mucosal surfaces.

International audience; The activation of C-H bonds has revolutionized modern synthetic chemistry. However, no general strategy for enantiospecific C-H activation has been developed to date. We herein report an enantiospecific C-H activation reaction followed by deuterium incorporation at stereogenic centers. Mechanistic studies suggest that the selectivity for the α-position of the directing heteroatom results from a four-membered dimetallacycle as the key intermediate. This work paves the way to novel molecular chemistry on nanoparticles.

Peptide metalloconstructs display interesting conformations, activities, and resistance to proteolysis. However, introduction of a metal core close to the residues that interact with the protein might strongly affect the binding. We investigated the effects of a coordinated oxorhenium core on the binding of model peptides to cyclophilin hCyp-18, a protein implicated in important biological processes and several diseases. For this purpose, we synthesized a series of linear metalloconstructs bearing an oxorhenium(V) core (ReO3+), as well as a peptide cyclized through oxorhenium(V) coordination. All these peptides contain an Ala-Pro-Xaa-pNA moiety (Xaa = Cys derivative) and are anticipated to bind simultaneously to the S1-S1' and S2'-S3' subsites of hCyp-18. Therefore, the metal core is coordinated to both the cysteine residue and exogenous or endogenous NS2 tridentate systems. Cyclization of the peptide through metal coordination did not affect the affinity whereas bimolecular oxorhenium metalloconstructs bind hCyp-18 with a slightly better affinity than the corresponding nonmetalated peptide. Peptide labeling with a 99mTcO3+ core was also carried out successfully.

Background & Aims: The resistance of prolamines to digestive enzymes is thought to be a key contributor to the pathogenesis of celiac disease by promoting the intestinal entrance of peptides able to trigger inflammation in at-risk individuals. Oral administration of a bacterial prolyl-endopeptidase (PEP) therefore was proposed as a treatment for celiac disease. To delineate the feasibility of this treatment, the effect of PEP on gliadin peptides was assessed in vitro, and ex vivo during their transport across intestinal biopsy specimens of active celiac disease patients. Methods: In vitro degradation by PEP of 3H-labeled gliadin peptides 56–88 (33-mer) and 31–49, was analyzed by radio–reverse-phase high-performance liquid chromatography and mass spectrometry. For ex vivo studies, PEP and 3H-peptides were added together onto the mucosal side of duodenal biopsy specimens mounted in Ussing chambers, and peptide transport and digestion were assessed by radio–reverse-phase high-performance liquid chromatography. Results: Gliadin peptides were degraded partly by 20 mU/mL PEP both in vitro and ex vivo. This concentration of PEP decreased the amount of intact peptides 31–49 and 56–88 crossing the intestinal biopsy specimens of celiac disease patients, but could not prevent the intestinal passage of toxic or immunostimulatory metabolites. PEP concentrations of at least 500 mU/mL for 3 hours were required to achieve full detoxification of peptides and to prevent intestinal transport of active fragments. Conclusions: After prolonged exposure to high concentrations of PEP, the amount of immunostimulatory gliadin peptides reaching the local immune system in celiac patients is decreased. These results provide a basis to establish whether such conditions are achievable in vivo.

[(3)H]BBL454, a new CCK(2) selective tritiated agonist was prepared via the reductive tritiation of a 5-aminopentyn-1-yl moiety introduced on the N-terminal end of a pentapeptide derivative of cholecystokinin. The binding properties of this labelled compound were determined on CHO cells transfected with the rat CCK(2) receptor. [(3)H]BBL454 is able to discriminate two affinity states of the CCK(2) receptor a supplementary indication of its validity for further exploring the heterogeneity of this receptor.

Tripeptides bearing aza-phenylalanine derivatives Aphe-X-(4-nitrophenyl), where X is CH2, O or NH, were synthesized starting from benzylhydrazine via a 4-step strategy. The pseudopeptides were evaluated as ligands of cyclophilin hCyp-18, an important human peptidyl-prolyl isomerase (PPlase), All pseudopeptides bind to hCyp-18, although only Suc-Ala-Pro-Phe-pNA11 and Suc-Ala-Pro-Aphe-pNB (X=CH2)4 are able to inhibit the PPIase activity, suggesting that they can bind to the S1-S1′ and S2′-S3′ subsites of hCyp-18 simultaneously. A circular dichroism study showed that only compounds4 and11 have β-turns conformations in 0.47 M LiCl/TFE (which favors acis-Ala-Pro conformation). In addition, the hydrazide (X=CH2)4 as well as the aza-urea (X=NH)6 are resistant to both trypsin and alpha-chymotrypsin. The corresponding carbazate (X=O)10 readily reacts with alpha-chymotrypsin and is also hydrolyzed by trypsin.

The human cyclophilin hCyp-18, an abundant peptidyl-prolyl cis-trans isomerase (PPIase) implicated in protein folding, controls the infection of CD4(+) T-cells by HIV-1, the pathologic agent of AIDS. Therefore, hCyp-18 is an interesting target for the development of novel anti-HIV-1 therapeutics. We focused on the design of transition-state analogue inhibitors of the PPIase activity of cyclophilin. Most experimental results reported in the literature suggest that hCyp-18 catalyzes the pyramidalization of the nitrogen of pyrrolidine via an H-bond network which results in the deconjugation of the amino acyl-prolyl peptide bond. We proposed the Glypsi(PO(2)R(1)-N)Pro motif (R = alkyl or H) as a selective transition-state analogue inhibitor of cyclophilin. This motif was inserted in Suc-Ala-Ala-Pro-Phe-pNA, a peptide substrate of hCyp-18. The pseudopeptide Suc-Ala-Glypsi(PO(2)Et-N)Pro-Phe-pNA 1b bound to hCyp-18 (K(d) = 20 +/- 5 microM) and was able to selectively inhibit its PPIase activity (IC(50) = 15 +/- 1 microM) but not hFKBP-12, another important PPIase. Deprotection of the phosphonamidate moiety resulted in a complete lack of inhibition. We previously demonstrated that reduction of the Phe-pNA moiety caused a quantitative reduction of the affinity; however, Suc-Ala-Glypsi(PO(2)Et-N)Pro-Phepsi(CH(2)-NH)pNA 7b still bound and inhibited hCyp-18, suggesting that the Glypsi(PO(2)Et-N)Pro motif plays the major role in the binding to cyclophilin. Consequently, we propose compound 1b as being a novel transition-state mimic inhibitor of hCyp-18.

Gaining an understanding of the nature of host-guest interactions in supramolecular complexes involving heavy atoms is a difficult task. Described herein is a robust simulation method applied to complexes between xenon and members of a cryptophane family. The calculated chemical shift of xenon caged in a H2O2 probe, as modeled by quantum chemistry with complementary-orbital, topological, and energy-decomposition analyses, is in excellent agreement with that observed in hyperpolarized (129)Xe NMR spectra. This approach can be extended to other van der Waals complexes involving heavy atoms.

1521-3773; An efficient H/D exchange method allowing the deuteration of pyridines, quinolines, indoles, and alkyl amines with D2 in the presence of Ru@PVP nanoparticles is described. By a general and simple procedure involving mild reaction conditions and simple filtration to recover the labeled product, the isotopic labeling of 22 compounds proceeded in good yield with high chemo- and regioselectivity. The viability of this procedure was demonstrated by the labeling of eight biologically active compounds. Remarkably, enantiomeric purity was conserved in the labeled compounds, even though labeling took place in the vicinity of the stereogenic center. The level of isotopic enrichment observed is suitable for metabolomic studies in most cases. This approach is also perfectly adapted to tritium labeling because it uses a gas as an isotopic source. Besides these applications to molecules of biological interest, this study reveals a rich and underestimated chemistry on the surface of ruthenium nanoparticles.

(2S,4RS)-Boc-4-cyanoglutamate γ-methyl ester (ee = 95%) and (2S)-Boc-2-amino-4-bis(cyano)butyrate (ee = 96%) were synthesised in respectively 43% and 40% overall yields by addition of sodium malonate derivatives onto Boc-dehydroalanine methyl ester followed by regio- and stereoselective hydrolysis of α-methyl ester by α-chymotrypsin. These regio-and stereoselectivities were strongly dependent on the nature of the γ-substituents.

Boc- cis -4-fluoro-L-proline and 4-difluoro-L-proline, usable in classical peptide synthesis, were obtained in respectively 71% (3 steps) and 65% (4 steps) overall yields from the readily available trans -4-hydroxy-L-proline methyl ester. The corresponding fluorinated trans -isomer was isolated in 24% yield (5 steps). Transformation of Boc-protected compounds to their Fmoc-equivalents was performed in high yields.

International audience; : In celiac disease, enhanced permeability to gliadin peptides can result from their apico-basal transport by secretory immunoglobulin A1 (SIgA1) binding to the CD71 receptor ectopically expressed at the gut epithelial surface. Herein, we have established a mouse model in which there is apico-basal transport of the model antigen ovalbumin (OVA) by specific SIgA1 and have analyzed local T-cell activation. Transgenic DO11.10 mice were grafted with a hybridoma-secreting OVA-specific humanized IgA1, which could bind mouse CD71 and which were released in the intestinal lumen as SIgA. CD71 expression was induced at the gut apical surface by treating the mice with tyrphostin A8. Following gavage of the mice with OVA, OVA-specific CD4(+) T cells isolated from the mesenteric lymph nodes displayed higher expression of the activation marker CD69 and produced more interferon gamma in mice bearing the hybridoma-secreting OVA-specific IgA1, than in ungrafted mice or in mice grafted with an irrelevant hybridoma. These results indicate that the protective role of SIgA1 might be jeopardized in human pathological conditions associated with ectopic expression of CD71 at the gut surface.Mucosal Immunology advance online publication 10 July 2013; doi:10.1038/mi.2013.49.

The development of optimized xenon host systems is of crucial importance for the success of molecular imaging using hyperpolarized (129)Xe MRI. Cryptophane-111 is a promising candidate because of its encapsulation properties. The synthesis of cryptophane-111-based biosensors requires both water-solubilizing and chemically activatable groups. An expeditious synthesis of a water-soluble and functionalizable cryptophane-111 is described.

A new triazole oxotechnetium chelating agent was synthesized via a 'Click-to-Chelate' strategy. In vivo evaluation of the corresponding (99m)Tc complex shows that the tracer exhibits very interesting properties for molecular imaging.

The dynamic combinatorial assembly of libraries of modular cyclophilin hCyp18 oxorhenium inhibitors of general formula [A˙ReO˙B] was accelerated by addition of increasing concentrations of hCyp18 (‘Cyclophilin Enhancing Effect’, CEE). This result suggested that modules assembly might proceed through an in situ coordination chemistry process. However, we observed that the CEE was not strictly related to the affinity of the complexes for hCyp18. The CEE was not altered by cyclosporine A, a potent competitive inhibitor of hCyp18. The use of a non-degassed buffer caused a fall in complexation yields that could be reversed upon addition of hCyp18. All these data suggested that the CEE results from a partial protection of exogenous thiols against reoxidation. As anticipated, carbamido-methylation of cyclophilin Cys52 and Cys62 residues with iodoacetamide annihilated the CEE. All these results highlight the role of hCyp18 in maintaining chemical modules in a reduced state.

New activated disulfides bearing a 4-diazocyclohexa-2,5-dienone precursor were synthesized in order to build up photoactivatable and cleavable peptides via cysteine modification.

Fmoc-(L)-serine ethers bearing either 2-(2′-methoxyethoxymethyl ether)-5-terbutyloxycarbonylaminobenzyl (ortho) or 5-(2′-methoxyethoxymethyl ether)-2-terbutyloxycarbonylaminobenzyl (meta) moieties were synthesized. Spectral characteristics of the corresponding photoactivatable 4-diazocyclohexa-2,5-dienones were determined.

International audience; Assembly of independent chemical modules through oxorhenium coordination by a NS$_2$ + S chelation motif was applied to the synthesis of RGD (Arg-Gly-Asp) analogs. Modules were assembled through oxorhenium chelation to give a series of 18 metal complexes in good yields and satisfactory purities. Screening of these oxorhenium coordinates as antagonists of integrins αVβ3, αIIbβ3 and αVβ5 led to the identification of 3 bioactive compounds that exhibit submicromolar affinities for the 3 integrins. Preliminary studies showed that the corresponding oxotechnetium complexes are stable in mice plasma and therefore could be proposed for the molecular imaging of pathologies that overexpress integrins αVβ3 and αVβ5.

Pseudopeptides containing the phosphinic analogue of the alanyl-proline motif Alaψ(PO 2 R-CH)Pro, were synthesized via three- (R=H) and four-step (R=CH 3 ) procedures. The mixtures of diastereomers were evaluated as inhibitors of the human cyclophilin hCyp-18, an important peptidyl-prolyl isomerase.

Non-protein amino acids (2R,3R)- and (2R,3S)-3-fluoroprolines were synthesized as novel probes for studying the cis/trans isomerization of the amino acylproline peptide bond. The N-Boc-protected target compounds were obtained as optically pure material starting from (2S,3S)-3-hydroxyproline.

The intestinal permeability of undegraded α9-gliadin peptide 31-49 (p31-49) and 33-mer gliadin peptides is increased in active celiac disease. Two distinct transport pathways have been proposed: paracellular leakage through epithelial tight junctions and protected transcellular transport. To analyze the relative contribution of these pathways, we compared mucosa-to-serosa permeability of small and large permeability markers [ionic conductance (G), mannitol, 182 Da; horseradish peroxidase, 40 kDa] and gliadin peptides [33-mer (p56-88, 3900 Da), 19-mer (p31-49, 2245 Da; and p202-220, 2127 Da), and 12-mer (p57-68, 1453 Da)] in duodenal biopsy specimens mounted in Ussing chambers. The permeability of intact peptides was much higher for p31-49 or 33-mer than for horseradish peroxidase, p202-220, and p57-68. A positive correlation was observed between G, an index of paracellular diffusion of ions, and mannitol permeability. The absence of correlation between G and permeability to intact 33-mer or p31-49 did not favor paracellular diffusion of the peptides. Immunofluorescence studies indicated that 33-mer enters the early endosome antigen 1–positive compartment but escapes the lysosomal-associated protein 2–positive compartment. The results underline that mannitol and ionic conductance G cannot be considered markers of permeability to gliadin peptides. In active celiac disease, increases in transcellular permeability to intact gliadin peptides might be considered in treatment strategies aimed at controlling epithelial permeability to gluten.

A library of RGD tripeptide analogs cyclized through oxorhenium coordination by an NS2/S chelation motif was synthesized. Screening towards integrins αVβ3, αIIbβ3 and αVβ5 led to the identification of 6 oxorhenium complexes that bind to integrin αVβ3 in the submicromolar range. In vivo evaluation of five of the corresponding oxotechnetium complexes using nude mice bearing a U87MG human tumor xenograft showed a significant and specific accumulation of radioactivity inside the tumor. The best results in vivo were obtained with complexes Tc-16 and Tc-50 that displayed a higher tumor accumulation and a lower distribution in other tissues relative to a reference cyclopentapeptide tracer.

Intramolecular Michael addition of β-ketoesters on enones 1, 2 and 3 with two conjugated unsaturations along the connecting chain led to cyclic monomers and dimers. These reactions work best with a cis alkene; they give poor yield of monomer with a trans alkene and no monomer with an alkyne.

The reactivity of electrophiles derived from N -tritylserine, threonine and allo -threonine esters toward a selection of nucleophiles was investigated. Best yields from substitution products were obtained with N -trityliodoalanine and soft nucleophiles such as thiols. This strategy was applied to the synthesis of lanthionines, the monosulfide analogs of cystine. Orthogonally protected sulfide adducts from L- and D-cysteines, threo - β -methyl-L-cysteine and D-penicillamine were isolated in 81–88% yield (ee>98%). This strategy was applied to the prepration of lanthionine and cyclolanthionine suitably protected for peptide synthesis.

Boc- cis -4-fluoro-L-proline and 4-difluoro-L-proline, usable in classical peptide synthesis, were obtained in respectively 71% (3 steps) and 65% (4 steps) overall yields from the readily available trans -4-hydroxy-L-proline methyl ester. The corresponding fluorinated trans -isomer was isolated in 24% yield (5 steps). Transformation of Boc-protected compounds to their Fmoc-equivalents was performed in high yields.

The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis . However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 Å), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 Å apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.

The dynamic combinatorial assembly of independent modules A and B through oxorhenium(V) coordination by a NS2+S motif in the presence of cyclophilin hCyp-18-an important peptidyl-prolyl isomerase-was investigated. Increasing glutathione (GSH) concentrations were used to dissociate [ARe(V)OB] complexes that displayed low affinity for hCyp-18. Conversely, coordinates that displayed submicromolar affinities for hCyp-18 were protected against thiol exchange and could be detected by LC-MS. Determination of the GSH concentration that decreased the extracted ionic current of the complex by 50 % (CC(50)) enabled the selection of three oxorhenium coordinates that were shown to bind to the active site of hCyp-18 and to inhibit its peptidyl-prolyl isomerase activity in the micromolar to submicromolar range.

Purpose: This study aimed to characterize the transepithelial transport of miltefosine (HePC), the first orally effective drug against visceral leishmaniasis, across the intestinal barrier to further understand its oral absorption mechanism.Materials and Methods: Caco-2 cell monolayers were used as an in vitro model of the human intestinal barrier. The roles of active and passive mechanisms in HePC intestinal transport were investigated and the relative contributions of the transcellular and paracellular routes were estimated.Results: HePC transport was observed to be pH-independent, partially temperature-dependent, linear as a function of time and non-saturable as a function of concentration. The magnitude of HePC transport was quite similar to that of the paracellular marker mannitol, and EDTA treatment led to an increase in HePC transport. Furthermore, HePC transport was found to be similar in the apical-to-basolateral and basolateral-to-apical directions, strongly suggesting that HePC exhibits non-polarized transport and that no MDR-mediated efflux was involved.Conclusions: These results demonstrate that HePC crosses the intestinal epithelium by a non-specific passive pathway and provide evidence supporting a concentration-dependent paracellular transport mechanism, although some transcellular diffusion cannot be ruled out. Considering that HePC opens epithelial tight junctions, this study shows that HePC may promote its own permeation across the intestinal barrier.