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5-Aminolevulinic acid-based photodynamic therapy (ALA-PDT) was first implemented over three decades ago and has since been mainly part of clinical practice for the management of pre-cancerous and cancerous skin lesions. Photodynamic therapy relies on the combination of a photosensitizer, light and oxygen to cause photo-oxidative damage of cellular components. 5-Aminolevulinic acid (ALA) is a natural precursor of the heme biosynthetic pathway, which when exogenously administered leads to the accumulation of the photoactivatable protoporphyrin IX. Although, effective and providing excellent cosmetic outcomes, its use has been restricted by the burning, stinging, and prickling sensation associated with treatment, as well as cutaneous adverse reactions that may be induced. Despite intense research in the realm of drug delivery, pain moderation, and light delivery, a novel protocol design using sunlight has led to some of the best results in terms of treatment response and patient satisfaction. Daylight PDT is the protocol of choice for the management of treatment of multiple or confluent actinic keratoses (AK) skin lesions. This review aims to revisit the photophysical, physicochemical and biological characteristics of ALA-PDT, and the underlying mechanisms resulting in daylight PDT efficiency and limitations. [ABSTRACT FROM AUTHOR]

The administration of 5-aminolevulinic acid (ALA) to generate enhanced intracellular levels of endogenous porphyrins is currently one of the most important approaches for photodynamic therapy (PDT) and photodiagnosis (PDD). Despite the great promise of ALA-based PDT, the physicochemical behaviour and chemical reactivity of ALA are problematic, and a variety of chemical approaches have been brought to bear to improve cellular delivery, enhance porphyrin production, and generate ALA prodrugs that have appropriate stability for convenient clinical use, as well as selectivity for cancerous tissues. While there has been considerable success, there are still a number of challenges to be addressed and opportunities to be exploited through application of chemical insight in this area. [ABSTRACT FROM AUTHOR]

A range of novel BODIPY derivatives with a tripodal aromatic core was synthesized and characterized spectroscopically. These new fluorophores showed promising features as probes for in vitro assays in live cells and offer strategic routes for further functionalization towards hybrid nanomaterials. Incorporation of biotin tags facilitated proof‐of‐concept access to targeted bioconjugates as molecular probes. Computational explorations using DFT and TD‐DFT calculations identified the most stable tripodal linker conformations and predicted their absorption and emission behavior. The uptake and speciation of these molecules in living prostate cancer cells was imaged by single‐ and two‐photon excitation techniques coupled with two‐photon fluorescence lifetime imaging (2P FLIM). [ABSTRACT FROM AUTHOR]

The intestinal paracellular route of absorption is modulated via tight junction (TJ) structures located at the apical neck of polarized intestinal epithelial cells to restrict solute movement through the intercellular space between them. Tight junctions open or close in response to changes in the phosphorylation status of light chain (MLC) at position Ser-19. This phosphorylation event is primarily controlled by MLC kinase (MLCK) and MLC phosphatase (MLCP), the latter being a holoenzyme that involves interaction between protein phosphatase 1 (PP1) and myosin targeting protein 1 (MYPT1). An entirely D-amino acid P ermeant I nhibitor of P hosphatase (PIP) peptide sequence designed to disrupt PP1-MYPT1 interactions at the cytoplasmic surface of TJs, PIP250 (rrfkvktkkrk) localized at intracellular TJ structures, altered expression levels of specific TJ proteins, increased cellular phosphorylated MLC (pMLC) levels, binding to PP1, decreased epithelial barrier function, and significantly increased systemic uptake of the poorly absorbed antibiotic gentamicin in vivo. A series of PIP250 peptide analogues showed that positions phe3 and val5 were critical to its functional properties, with some providing opportunities to tune the dynamic actions of its TJ modulation properties. These data confirm the activity of PIP250 as a rationally designed oral permeation enhancer and validated key amino acids involved in its interaction with PP1 that define its overall actions; the magnitude and duration of these enhancing properties were associated with the MYPT1-mimetic properties of the PIP250 peptide analogues described. [Display omitted] • A membrane permeant peptide (PIP250) reduced intestinal epithelial barrier function and enhanced the in vivo ileal uptake in rats of a poorly absorbed antibiotic, gentamicin. • PIP250 entered epithelial cells to localize near tight junction (TJ) structures, bound protein phosphatase 1 (PP1) and altered expression levels of selected TJ proteins. • The amino acids Phe3 and Val5 within the PIP250 peptide sequences were critical to its mechanism of action and properties to enhance solute uptake. [ABSTRACT FROM AUTHOR]

Tight junction (TJ) structures restrict the movement of solutes between adjacent epithelial cells to maintain homeostatic conditions. A peptide, termed PIP 640, with the capacity to regulate the transient opening of intestinal TJ structures through an endogenous mechanism involving the induction of myosin light chain (MLC) phosphorylation at serine 19 (MLC-pS 19 ) has provided a promising new method to enhance the in vivo oral bioavailability of peptide therapeutics. PIP 640 is a decapeptide composed of all D-amino acids (rrdykvevrr-NH 2 ) that contains a central sequence designed to emulates a specific domain of C-kinase potentiated protein phosphatase-1 inhibitor-17 kDa (CPI-17) surrounded by positively-charged amino acids that provide a cell penetrating peptide (CPP)-like character. Here, we examine compositional requirements of PIP 640 with regard to its actions on MLC phosphorylation, its intracellular localization to TJ structures, and its interactions with MLC phosphatase (MLCP) elements that correlate with enhanced solute uptake. These studies showed that a glutamic acid and tyrosine within this peptide are critical for PIP 640 to retain its ability to increase MLC-pS 19 levels and enhance the permeability of macromolecular solutes of the size range of therapeutic peptides without detectable cytotoxicity. On the other hand, exchange of the aspartic acid for alanine and then arginine resulted in an increasingly greater bias toward protein phosphatase-1 (PP1) relative to MLCP inhibition, an outcome that resulted in increased paracellular permeability for solutes in the size range of therapeutic peptides, but with a significant increase in cytotoxicity. Together, these data further our understanding of the composition requirements of PIP 640 with respect to the desired goal of transiently altering the intestinal epithelial cell paracellular barrier properties through an endogenous mechanism, providing a novel approach to enhance the oral bioavailability of poorly absorbed therapeutic agents of < ~ 5 kDa. [ABSTRACT FROM AUTHOR]

Abstract: Photochemical internalisation (PCI) is a site-specific technique for improving cellular delivery of macromolecular drugs. In this study, a cell penetrating peptide, containing the core HIV-1 Tat 48–57 sequence, conjugated with a porphyrin photosensitiser has been shown to be effective for PCI. Herein we report an investigation of the photophysical and photobiological properties of a water soluble bioconjugate of the cationic Tat peptide with a hydrophobic tetraphenylporphyrin derivative. The cellular uptake and localisation of the amphiphilic bioconjugate was examined in the HN5 human head and neck squamous cell carcinoma cell line. Efficient cellular uptake and localisation in endo/lysosomal vesicles was found using fluorescence detection, and light-induced, rupture of the vesicles resulting in a more diffuse intracellular fluorescence distribution was observed. Conjugation of the Tat sequence with a hydrophobic porphyrin thus enables cellular delivery of an amphiphilic photosensitiser which can then localise in endo/lysosomal membranes, as required for effective PCI treatment. PCI efficacy was tested in combination with a protein toxin, saporin, and a significant reduction in cell viability was measured versus saporin or photosensitiser treatment alone. This study demonstrates that the cell penetrating peptide-photosensitiser bioconjugation strategy is a promising and versatile approach for enhancing the therapeutic potential of bioactive agents through photochemical internalisation. [Copyright &y& Elsevier]

Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate–cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 μM CETP eliciting a remarkable ∼10-fold induction of NQO1. Furthermore, 50 μM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1−/− mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1−/− MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2+/+ MEFs, but not Nrf2−/− MEFs, with 15 μM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the α,β-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics. [ABSTRACT FROM PUBLISHER]

Abstract: Sulforaphane can stimulate cellular adaptation to redox stressors through transcription factor Nrf2. Using mouse embryonic fibroblasts (MEFs) as a model, we show herein that the normal homeostatic level of glutathione in Nrf2−/− MEFs was only 20% of that in their wild-type counterparts. Furthermore, the rate of glutathione synthesis following its acute depletion upon treatment with 3 µmol/l sulforaphane was very substantially lower in Nrf2−/− MEFs than in wild-type cells, and the rebound leading to a ∼1.9-fold increase in glutathione that occurred 12–24 h after Nrf2 +/+ MEFs were treated with sulforaphane was not observed in Nrf2−/− fibroblasts. Wild-type MEFs that had been pre-treated for 24 h with 3 µmol/l sulforaphane exhibited between 1.4- and 3.2-fold resistance against thiol-reactive electrophiles, including isothiocyanates, α,β-unsaturated carbonyl compounds (e.g. acrolein), aryl halides and alkene epoxides. Pre-treatment of Nrf2 +/+ MEFs with sulforaphane also protected against hydroperoxides (e.g. cumene hydroperoxide, CuOOH), free radical-generating compounds (e.g. menadione), and genotoxic electrophiles (e.g. chlorambucil). By contrast, Nrf2−/− MEFs were typically ∼50% less tolerant of these agents than wild-type fibroblasts, and sulforaphane pre-treatment did not protect the mutant cells against xenobiotics. To test whether Nrf2-mediated up-regulation of glutathione represents the major cytoprotective mechanism stimulated by sulforaphane, 5 µmol/l buthionine sulfoximine (BSO) was used to inhibit glutathione synthesis. In Nrf2 +/+ MEFs pre-treated with sulforaphane, BSO diminished intrinsic resistance and abolished inducible resistance to acrolein, CuOOH and chlorambucil, but not menadione. Thus Nrf2-dependent up-regulation of GSH is the principal mechanism by which sulforaphane pre-treatment induced resistance to acrolein, CuOOH and chlorambucil, but not menadione. [Copyright &y& Elsevier]

Abstract Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). While TNF-α/IFN-γ treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes. Graphical Abstract Unlabelled Image Highlights • PIP 640 increases the paracellular permeability of macromolecules in vitro and in vivo. • PIP 640 increases claudin-2 at tight junction structures in vitro and in vivo. • PIP 640 induces a permeability increase that is selective for cationic macromolecules. [ABSTRACT FROM AUTHOR]

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4 kDa fluorescent dextran but not 70 kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70 kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier. [ABSTRACT FROM AUTHOR]

We have developed a fluorescent peptide conjugate (TrpNDIRGDfK) based on the coupling of cyclo(RGDfK) to a new tryptophan-tagged amino acid naphthalenediimide (TrpNDI). Confocal fluorescence microscopy coupled with fluorescence lifetime imaging (FLIM) mapping, single and two-photon fluorescence excitation, lifetime components and corresponding decay profiles were used as parameters able to investigate qualitatively the cellular behavior regarding the molecular environment and biolocalisation of TrpNDI and TrpNDI–RGDfK in cancer cells. [ABSTRACT FROM AUTHOR]

The chitinase-like proteins YKL-39 (chitinase 3-like-2) and YKL- 40 (chitinase 3-like-1) are highly expressed in a number of human cells independent of their origin (mesenchymal, epithelial or haemapoietic). Elevated serum levels of YKL-40 have been associated with a negative outcome in a number of diseases ranging from cancer to inflammation and asthma. YKL-39 expression has been associated with osteoarthritis. However, despite the reported association with disease, the physiological or pathological role of these proteins is still very poorly understood. Although YKL-39 is homologous to the two family 18 chitinases in the human genome, it has been reported to lack any chitinase activity. In the present study, we show that human YKL-39 possesses a chitinase-like fold, but lacks key active-site residues required for catalysis. A glycan screen identified oligomers of N-acetylglucosamine as preferred binding partners. YKL-39 binds chitooligosaccharides and a newly synthesized derivative of the bisdionin chitinase-inhibitor class with micromolar affinity, through a number of conserved tryptophan residues. Strikingly, the chitinase activity of YKL-39 was recovered by reverting two non-conservative substitutions in the active site to those found in the active enzymes, suggesting that YKL-39 is a pseudo-chitinase with retention of chitinase-like ligand-binding properties. [ABSTRACT FROM AUTHOR]

Summary: Acidic mammalian chitinase (AMCase) is produced in the lung during allergic inflammation and asthma, and inhibition of enzymatic activity has been considered as a therapeutic strategy. However, most chitinase inhibitors are nonselective, additionally inhibiting chitotriosidase activity. Here, we describe bisdionin F, a competitive AMCase inhibitor with 20-fold selectivity for AMCase over chitotriosidase, designed by utilizing the AMCase crystal structure and dicaffeine scaffold. In a murine model of allergic inflammation, bisdionin F-treatment attenuated chitinase activity and alleviated the primary features of allergic inflammation including eosinophilia. However, selective AMCase inhibition by bisdionin F also caused dramatic and unexpected neutrophilia in the lungs. This class of inhibitor will be a powerful tool to dissect the functions of mammalian chitinases in disease and represents a synthetically accessible scaffold to optimize inhibitory properties in terms of airway inflammation. [ABSTRACT FROM AUTHOR]

Summary: Natural products are often large, synthetically intractable molecules, yet frequently offer surprising inroads into previously unexplored chemical space for enzyme inhibitors. Argifin is a cyclic pentapeptide that was originally isolated as a fungal natural product. It competitively inhibits family 18 chitinases by mimicking the chitooligosaccharide substrate of these enzymes. Interestingly, argifin is a nanomolar inhibitor of the bacterial-type subfamily of fungal chitinases that possess an extensive chitin-binding groove, but does not inhibit the much smaller, plant-type enzymes from the same family that are involved in fungal cell division and are thought to be potential drug targets. Here we show that a small, highly efficient, argifin-derived, nine-atom fragment is a micromolar inhibitor of the plant-type chitinase ChiA1 from the opportunistic pathogen Aspergillus fumigatus. Evaluation of the binding mode with the first crystal structure of an A. fumigatus plant-type chitinase reveals that the compound binds the catalytic machinery in the same manner as observed for argifin with the bacterial-type chitinases. The structure of the complex was used to guide synthesis of derivatives to explore a pocket near the catalytic machinery. This work provides synthetically tractable plant-type family 18 chitinase inhibitors from the repurposing of a natural product. [Copyright &y& Elsevier]

Abstract: The development of a reliable sensitive method for the HPLC determination of 5-aminolaevulinic acid (ALA) and ALA esters in cell lysates is described. The method relies on the quantification of a fluorescent derivative of ALA following its derivatisation with acetylacetone and formaldehyde. Following this procedure it is possible to quantify ALA in cell lysates with no need for pre-purification of the sample. The method has been validated in two ranges of concentration (0.6–65μM, 0.1–10μg/mL, and 30–600μM, 5–100μg/mL), and has also been extended and validated for the determination of ALA released from ALA prodrugs after acidic hydrolysis. [Copyright &y& Elsevier]

Summary: Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides, and antiasthmatics. Argifin, a natural product cyclopentapeptide, competitively inhibits family 18 chitinases in the nanomolar to micromolar range and shows extensive substrate mimicry. In an attempt to map the active fragments of this large natural product, the cyclopentapeptide was progressively dissected down to four linear peptides and dimethylguanylurea, synthesized using a combination of solution and solid phase peptide synthesis. The peptide fragments inhibit chitinase B1 from Aspergillus fumigatus (AfChiB1), the human chitotriosidase, and chitinase activity in lung homogenates from a murine model of chronic asthma, with potencies ranging from high nanomolar to high micromolar inhibition. X-ray crystallographic analysis of the chitinase-inhibitor complexes revealed that the conformations of the linear peptides were remarkably similar to that of the natural product. Strikingly, the dimethylguanylurea fragment, representing only a quarter of the natural product mass, was found to harbor all significant interactions with the protein and binds with unusually high efficiency. The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors. [Copyright &y& Elsevier]

Abstract: An efficient synthesis of selectively N-substituted xanthine derivatives is described. Cyclocondensation of a suitably protected aminoimidazole with methyl-2-phenylthioethyl carbamate, followed by oxidation of sulfur to the sulfone, provides access to an orthogonally 1,7-protected xanthine, which may then be regioselectively alkylated and deprotected under mild conditions. [Copyright &y& Elsevier]

Abstract: The first example of the synthesis of a peptide incorporating 5-aminolaevulinic acid (5-ALA) using standard Fmoc solid-phase chemistry is reported. The synthesised peptide contains residues 52–58 of the cell-permeable peptide Penetratin and represents a prototype for the enhanced topical delivery of 5-ALA using such oligopeptide vectors. Effective intracellular conversion of the peptide to the endogenous photosensitiser, protoporphyrin IX, is observed in PAM212 cells, thus demonstrating the potential of this approach for the development of novel peptide prodrugs for use in photodynamic therapy. [Copyright &y& Elsevier]

Family 18 chitinases play key roles in the life cycles of a variety of organisms ranging from bacteria to man. Very recently it has been shown that one of the mammalian chitinases is highly over-expressed in the asthmatic lung and contributes to the pathogenic process through recruitment of inflammatory cells. Although several potent natural product chitinase inhibitors have been identified, their chemotherapeutic potential or their use as cell biological tools is limited due to their size, complex chemistry, and limited availability. We describe a virtual screening-based approach to identification of a novel, purine-based, chitinase inhibitor. This inhibitor acts in the low micromolar (Ki = 2.8 ± 0.2 µM) range in a competitive mode. Dissection of the binding mode by x-ray crystallography reveals that the compound, which consists of two linked caffeine moieties, binds in the active site through extensive and not previously observed stacking interactions with conserved, solvent exposed tryptophans. Such exposed aromatics are also present in the structures of many other carbohydrate processing enzymes. The compound exhibits favorable chemical properties and is likely to be useful as a general scaffold for development of pan-family 18 chitinase inhibitors. [ABSTRACT FROM AUTHOR]

O-linked N-acetylglucosamine (O-GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O-GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O-GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O-GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate-assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (Ki=5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala-Cys(-S-GlcNAc)-Ala, and has OGA activity against O-GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA. [ABSTRACT FROM AUTHOR]

Abstract: The macrocyclic spermidine alkaloid lunarine 1 from Lunaria biennis is a competitive, time-dependent inhibitor of the protozoan oxidoreductase trypanothione reductase (TryR), a promising target in drug design against tropical parasitic diseases. Various molecules related to 1 and the alkaloid itself have been synthesized in racemic form and evaluated against TryR in order to determine the key features of 1 that are associated with time-dependent inhibition. Kinetic data are consistent with an inactivation mechanism involving a conjugate addition of an active site cysteine residue onto the C-24–C-25 double bond of the tricyclic nucleus of 1. Comparison of data for synthetic (±)-1, the natural product, and other derivatives 7–10 from L. biennis confirms the importance of the unique structure of the tricyclic core as a motif for inhibitor design and reveals that the non-natural enantiomer may be a more suitable scaffold upon which thiophilic groups may be presented. [Copyright &y& Elsevier]

Trypanothione replaces glutathione in defence against cellular damage caused by oxidants, xenobiotics and methylglyoxal in the trypanosomatid parasites, which cause trypanosomiasis and leishmaniasis. In Leishmania major, the first step in methylglyoxal detoxification is performed by a trypanothione-dependent glyoxalase I (GLO1) containing a nickel cofactor; all other characterized eukaryotic glyoxalases use zinc. In kinetic studies L. major and human enzymes were active with methylglyoxal derivatives of several thiols, but showed opposite substrate selectivities: N 1-glutathionylspermidine hemithioacetal is 40-fold better with L. major GLO1, whereas glutathione hemithioacetal is 300-fold better with human GLO1. Similarly, S-4-bromobenzylglutathionylspermidine is a 24-fold more potent linear competitive inhibitor of L. major than human GLO1 ( Kis of 0.54 µM and 12.6 µM, respectively), whereas S-4-bromobenzylglutathione is > 4000-fold more active against human than L. major GLO1 ( Kis of 0.13 µM and > 500 µM respectively). The crystal structure of L. major GLO1 reveals differences in active site architecture to both human GLO1 and the nickel-dependent Escherichia coli GLO1, including increased negative charge and hydrophobic character and truncation of a loop that may regulate catalysis in the human enzyme. These differences correlate with the differential binding of glutathione and trypanothione-based substrates, and thus offer a route to the rational design of L. major-specific GLO1 inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: The first synthesis of the cyclopentapeptide family 18 chitinase inhibitor argifin has been achieved by a combination of solid phase and solution chemistry. Synthetic argifin is a nanomolar inhibitor of chitinase B1 from Aspergillus fumigatus and the high-resolution X-ray structure of the synthesized material in complex with the same enzyme is identical to that previously obtained for the natural product. [Copyright &y& Elsevier]

Abstract: An efficient and cost-effective procedure has been devised for the preparation of urethane-protected 5-aminolaevulinic acid (5-ALA) dipeptide ester derivatives which avoids problems associated with the instability of 5-ALA under basic conditions. The procedure is also applicable to the direct synthesis of N-(α)-acetyl amino acid-ALA dipeptides in high enantiomeric purity as potential novel prodrugs for photodynamic therapy (PDT). [Copyright &y& Elsevier]

The synthesis and evaluation of 3,5-disubstituted benzofuran derivatives as time-dependent inhibitors of the protozoan oxidoreductase trypanothione reductase are reported. These molecules were designed as simplified mimetics of the naturally occurring spermidine-bridged macrocyclic alkaloid lunarine 1, a known time-dependent inhibitor of trypanothione reductase. In this series of compounds the bis-polyaminoacrylamide derivatives 2–4 were all shown to be competitive inhibitors, but only the bis-4-methyl-piperazin-1-yl-propylacrylamide derivative 4 displayed time-dependent activity. The kinetics of time dependent inactivation of trypanothione reductase by 1 and 4 have been determined and are compared and discussed herein. [Copyright &y& Elsevier]