Chitinase Inhibitor Allosamidin Research Articles

Chitinase-Induced Airway Hyperreactivity and Inflammation in a Mouse Model of Nonallergic Asthma

Introduction: Environmental exposure to mites and fungi has been proposed to critically contribute to the development of IgE-mediated asthma. A common denominator of such organisms is chitin. Human chitinases have been reported to be upregulated by interleukin-13 secreted in the context of Th2-type immune responses and to induce asthma. We assessed whether chitin-containing components induced chitinases in an innate immune-dependent way and whether this results in bronchial hyperresponsiveness. Materials and Methods: Monocyte/macrophage cell lines were stimulated with chitin-containing or bacterial components in vitro. Chitinase activity in the supernatant and the expression of the chitotriosidase gene were measured by enzyme assay and quantitative PCR, respectively. Non-sensitized mice were stimulated with chitin-containing components intranasally, and a chitinase inhibitor was administered intraperitoneally. As markers for inflammation leukocytes were counted in the bronchoalveolar lavage (BAL) fluid, and airway hyperresponsiveness was assessed via methacholine challenge. Results: We found both whole chitin-containing dust mites as well as the fungal cell wall component zymosan A but not endotoxin-induced chitinase activity and chitotriosidase gene expression in vitro. The intranasal application of zymosan A into mice led to the induction of chitinase activity in the BAL fluid and to bronchial hyperresponsiveness, which could be reduced by applying the chitinase inhibitor allosamidin. Discussion: We propose that environmental exposure to mites and fungi leads to the induction of chitinase, which in turn favors the development of bronchial hyperreactivity in an IgE-independent manner.

Glycal Metallanitrenes for 2-Amino Sugar Synthesis: Amidoglycosylation of Gulal-, Allal-, Glucal-, and Galactal 3-Carbamates.

The rhodium(II)-catalyzed oxidative cyclization of glycal 3-carbamates with in situ incorporation of an alcohol nucleophile at the anomeric position provides access to a range of 2-amino sugars having 1,2-trans-2,3-cis stereochemistry, a structural motif present in compounds of medicinal and biological significance such as the streptothricin group of antibiotics and the Chitinase inhibitor allosamidin. All of the diastereomeric d-glycal 3-carbamates have been investigated, revealing significant differences in anomeric stereoselectivity depending on substrate stereochemistry and protecting groups. In addition, some substrates were prone to forming C3-oxidized dihydropyranone byproducts under the reaction conditions. Allal- and gulal 3-carbamates provided uniformly high stereo- and chemoselectivity, while for glucal substrates, acyclic, electron-withdrawing protecting groups at the 4 O and 6 O positions were required. Galactal 3-carbamates have been the most challenging substrates; formation of their amidoglycosylation products is most effective with an electron-withdrawing 6 O-Ts substituent and a sterically demanding 4 O-TBS group. These results suggest a mechanism whereby conformational and electronic factors determine the partitioning of an intermediate acyl nitrenoid between alkene addition, leading to amidoglycosylation, and C3-H insertion, providing the dihydropyranone byproduct. Along the amidoglycosylation pathway, high anomeric selectivity results when a glycosyl aziridine intermediate is favored over an aziridine-opened oxocarbenium donor.

Thermodynamic analysis of allosamidin binding to the human chitotriosidase

Human chitotriosidase (HCHT) is one of two active family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the innate human defense mechanism against fungal parasites. Recently, it has been shown that levels of HCHT bioactivity and protein are significantly increased in the circulation and lungs of systemic sclerosis patients and for this reason is a suggested therapeutic target. For this reason, we have undertaken a detailed thermodynamic investigation using isothermal titration calorimetry of the binding interaction of HCHT with the well-known family 18 chitinase inhibitor allosamidin. The binding is shown to be strong (Kd=0.20±0.03μM and ΔGr°=−38.9±0.4kJ/mol) and driven by favorable changes in enthalpy (ΔHr°=−50.2±1.2kJ/mol) and solvation entropy (−TΔSsolv°=−41.8±4.4kJ/mol). It is accompanied with a large penalty in conformational entropy change (−TΔSconf°=43.1±4.2kJ/mol).

Chitinase Inhibitor Allosamidin and Its Analogues: An Update

The pseudotrisaccharide allosamidin 1 is a potent family-18 chitinase inhibitor, and it demonstrates biological activities against insects, fungi, and the Plasmodium falciparum life cycle. Compound 1 contains two N-acetylhexosamine residues with the unusual D-allo-configuration and a novel aminocyclitol (i.e. allosamizoline 2), joined by two β-(1→4) glycosidic bonds. Many traditional syntheses of compounds 1-2 and their analogues have been reported. Herein, recent development for the synthesis of compounds 1-2 and their analogues was reviewed. Donohoe and Rosas approach to allosamizoline 2 involves a key-step ring-closing metathesis (RCM) to form the cyclopentene core followed by halocyclization to form the oxazoline unit. Rojas et al. reported that rhodium-catalyzed oxidative cyclization of glucal 3-carbamates led to oxazolidinone-protected mannosamine derivatives. Huang et al. described the solid-phase synthesis of allosamidin 1 and its analogues. The compound 1 and its analogues were obtained by iterative glycosylation reactions, catalytic hydrogenation, acetylation, and deacetylation, respectively. Withers and his co-workers research shows that chitobiose and chitotriose thiazolines exhibit chitinase inhibition activity. In a word, the goal is to investigate the novel methods for the synthesis of allosamidin and its analogues, which is convenient for the discovery of allosamidin analogues with high activity. Keywords: Allosamidin, allosamizoline, analogues, synthesis, recent development, Plasmodium falciparum, ring-closing metathesis (RCM), iterative glycosylation reactions, acetylation, catalytic hydrogenation, deacetylation, chitinase inhibition activity, allosamidin analogues, pseudotrisaccharide, aminocyclopentitol moiety

Chitinase Gene Expression in Response to Environmental Stresses inArabidopsis thaliana: Chitinase Inhibitor Allosamidin Enhances Stress Tolerance

The expression levels of three chitinase genes in Arabidopsis thaliana, AtChiA (class III), AtChiB (class I), and AtChiV (class IV), were examined under various stress conditions by semi-quantitative RT-PCR. Under normal growth conditions, the AtChiB and AtChiV genes were expressed in most organs of Arabidopsis plants at all growth stages, whereas the AtChiA gene was not expressed at all. The class III AtChiA gene was expressed exclusively when the plants were exposed to environmental stresses, especially to salt and wound stresses. Treatment of Arabidopsis plants with allosamidin, which inhibits class III chitinases, did not affect the growth rate. Surprisingly, however, the plants treated with allosamidin were more tolerant of abiotic stresses (cold, freezing, heat, and strong light) than the control plants. It also appeared that allosamidin enhances AtChiA and AtChiB expression under heat and strong light stresses. Allosamidin is likely to enhance abiotic stress tolerance, probably through crosstalk between the two signaling pathways for biotic and abiotic stress responses.

Chitinase inhibitor allosamidin promotes chitinase production of Streptomyces generally

Allosamidin is a family 18 chitinase inhibitor produced by Streptomyces. In its producing strain, Streptomyces sp. AJ9463, allosamidin promotes production of the family 18 chitinase originated from chi65 in a chitin medium through the two-component regulatory system encoded by chi65R and chi65S, which were present at the 5′-upstream region of chi65. In this study, we showed generality of the allosamidin's effect. Allosamidin enhanced production of the family 18 chitinases originated from chi65h of Streptomyces halstedii MF425, another allosamidin producer, chiC of Streptomyces coelicolor A3(2) and chiIII of Streptomyces griseus. All the three chitinase genes had high homology to chi65 and two genes homologous to chi65S and chi65R were present at their 5′-upstream regions. When allosamidin's effect was tested with six Streptomyces strains randomly isolated from soil, allosamidin enhanced chitinase production of all strains. All six strains possessed a set of three genes homologous to chi65, chi65S and chi65R. Analysis of 16S rDNA indicated that allosamidin-sensitive strains are distributed widely in Streptomyces. These observations suggested that allosamidin can affect the common regulatory system for production of a chitinase with a two-component regulatory system in Streptomyces.

Chitinase Inhibitor Allosamidin Is a Signal Molecule for Chitinase Production in Its Producing Streptomyces

In Streptomyces sp. AJ9463, a producer of chitinase inhibitor allosamidin, allosamidin strongly enhances production of the chitinase mainly secreted to the culture broth in a chitin medium. To clarify the mechanism for regulation of the chitinase production by allosamidin, a disruption experiment of genes encoding proteins constructing a two-component regulatory system present at 5'-upstream region of the chitinase gene was performed. In the disruptant obtained, allosamidin could not promote the chitinase production, but N, N'-diacetylchitobiose, which also enhances production of the same chitinase more weakly than allosamidin, promoted the chitinase production similarly to the case observed in the wild-type strain. Furthermore, by the experiment in an inorganic salt solution, it was shown that allosamidin could not induce the chitinase production without addition of N, N'-diacetylchitobiose. These results show that allosamidin can activate transcription of the chitinase gene through the two-component regulatory system in the presence of N, N'-diacetylchitobiose.

Chitinase Inhibitor Allosamidin Is a Signal Molecule for Chitinase Production in Its Producing Streptomyces

Allosamidin, a typical secondary metabolite of Streptomyces, has been known as a chitinase inhibitor. We found that allosamidin can dramatically promote chitinase production and growth of its producer, Streptomyces sp. AJ9463, in a chitin medium at a few hundred nM. Allosamidin promoted production of the main chitinase detected in the culture filtrate and the chitin-hydrolytic activity of the chitinase was not inhibited by allosamidin at the concentration. The gene encoding the chitinase showed that it is a family 18 chitinase and it was revealed that two genes encoding proteins constructing two-component regulatory system were present at 5'-upstream region of the chitinase gene. Allosamidin is located in the microbial mycelia cultured in a medium without chitin, but it was released from the mycelia by responding to chitin. These results show that allosamidin acts as a key signal molecule for chitinase production in its producing strain, which may be useful for its growth in chitin-rich environment.

Molecular cloning and functional characterization of mouse chitotriosidase

Mammalian chitinase and chitinase-like proteins are members of a recently discovered gene family. Thus far, neither chitin nor chitin synthase has been found in mammals. The existence of chitinase genes in mammals is intriguing and the physiologic functions of chitinases are not clear. Human chitotriosidase, also called chitinase 1 (chit1), has been cloned. It has been found that high levels of serum chitotriosidase are associated with several diseases, but the physiologic functions of this enzyme are still unclear. To facilitate the studies in animal models we cloned and characterized a cDNA that encodes the mouse chitotriosidase. The open reading frame of this cDNA predicts a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytic domain and a chitin-binding domain. The predicted amino acid sequence is highly homologous to that of human chitotriosidase and to that of mouse acidic mammalian chitinase. Sequence analysis indicates that the mouse chitotriosidase gene has 12 exons, spanning a 40-kb region in mouse chromosome 1. The constitutive expression of mouse chitotriosidase is restricted to brain, skin, bone marrow, kidney, tongue, stomach and testis. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted and has chitinolytic activity that is sensitive to the specific chitinase inhibitor allosamidin and has the ability to bind to chitin particles. Substitution mutations at the conserved catalytic site completely abolished the enzymatic activity of the recombinant protein. These studies illustrate that mouse chitotriosidase is a typical chitinase that belongs to the mammalian chitinase gene family.

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Structure of Human Chitotriosidase: IMPLICATIONS FOR SPECIFIC INHIBITOR DESIGN AND FUNCTION OF MAMMALIAN CHITINASE-LIKE LECTINS

Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.

Growth of chitinolytic dune soil beta-subclass Proteobacteria in response to invading fungal hyphae.

It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse living fungal hyphae, thereby releasing potential growth substrate. However, the conditions used in such assays (high bacterial density, rich media, fragmented hyphae) make it difficult to determine whether mycolytic activity is actually of importance for the growth and survival of chitinolytic bacteria in soils. An unidentified group of beta-subclass Proteobacteria (CbetaPs) was most dominant among the culturable nonfilamentous chitinolytic bacteria isolated from Dutch sand dune soils. Here we demonstrate that the CbetaPs grew at the expense of extending fungal mycelium of three dune soil fungi (Chaetomium globosum, Fusarium culmorum, and Mucor hiemalis) under nutrient-limiting, soil-like conditions. Aggregates of CbetaPs were also often found attached to fungal hyphae. The growth of a control group of dominant nonchitinolytic dune soil bacteria (beta- and gamma-subclass Proteobacteria) was not stimulated in the mycelial zone, indicating that growth-supporting materials were not independently released in appreciable amounts by the extending hyphae. Therefore, mycolytic activities of CbetaPs have apparently been involved in allowing them to grow after exposure to living hyphae. The chitinase inhibitor allosamidin did not, in the case of Mucor, or only partially, in the cases of Chaetomium and Fusarium, repress mycolytic growth of the CbetaPs, indicating that chitinase activity alone could not explain the extent of bacterial proliferation. Chitinolytic Stenotrophomonas-like and Cytophaga-like bacteria, isolated from the same dune soils, were only slightly stimulated by exposure to fungal hyphae.

Knockout of the rodent malaria parasite chitinase pbCHT1 reduces infectivity to mosquitoes.

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite, Plasmodium berghei. We show that the gene, named PbCHT1, is a structural ortholog of PgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity in Anopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds-an artificial situation where midgut invasion occurs before PM formation-suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of Plasmodium-mosquito interactions.

Chitinase Gene Expression during Mycoparasitic Interaction ofTrichoderma harzianumwith Its Host

Zeilinger, S., Galhaup, C., Payer, K., Woo, S. L., Mach, R. L., Fekete, C., Lorito, M., and Kubicek, C. P. 1999. Chitinase gene expression during mycoparasitic interaction ofTrichoderma harzianumwith its host.Fungal Genetics and Biology26,131–140. For monitoring chitinase expression during mycoparasitism ofTrichoderma harzianum in situ,we constructed strains containing fusions of green fluorescent protein (GFP) to the 5′-regulatory sequences of theT. harzianum nag1(N-acetyl-β-d-glucosaminidase-encoding) andech42(42-kDa endochitinase-encoding) genes. Confronting these strains withRhizoctonia solaniled to induction of gene expression before (ech42) or after (nag1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolishedech42expression, indicating that macromolecules are involved in its precontact activation. Noech42expression was triggered by culture filtrates ofR. solanior by placingT. harzianumonto plates previously colonized byR. solani.Instead, high expression occurred upon incubation ofT. harzianumwith the supernatant ofR. solanicell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded byech42) fromT. harzianum.The chitinase inhibitor allosamidin blockedech42expression and reduced inhibition ofR. solanigrowth during confrontation. The results indicate thatech42is expressed before contact ofT. harzianumwithR. solaniand its induction is triggered by soluble chitooligosaccharides produced by constitutive activity of CHIT42 and/or other chitinolytic enzymes.

Substrate assistance in the mechanism of family 18 chitinases: theoretical studies of potential intermediates and inhibitors

Based on first principles and molecular mechanics calculations, we conclude that the mechanism of hevamine (a family 18 chitinase) involves an oxazoline ion intermediate stabilized by the neighboring C2′ acetamido group. In this intermediate, the acetamido carbonyl oxygen atom forms a covalent bond to C1′ of N-acetyl-glucosamine and has a transferred positive charge from the pyranose ring onto the acetamido nitrogen atom, leading to an anchimeric stabilization of 38.1 kcal/mol when docked with hevamine.This double displacement mechanism involving an oxazoline intermediate distinguishes the family 18 chitinase (which have one acidic residue near the active site) from family 19 chitinase and from hen egg-white lysozyme, which have two acidic residues near the active site.The structural and electronic properties of the oxazoline intermediate are similar to the known chitinase inhibitor allosamidin, suggesting that allosamidins act as transition state analogs of an oxazoline intermediate. Structural and electronic features of the oxazoline ion likely to be important in the design of new chitinase inhibitors are discussed.

Cloning and expression of chitinases of Entamoebae

Entamoeba histolytica ( Eh) and Entamoeba dispar ( Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens ( Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cyst formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene ( Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respectively. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats ( Eh and Ed) or repeats varying in length ( Ei). The aa compositions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Manduca sexta (29%). Recombinant Entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h.

In vitro degradation rates of partially N-acetylated chitosans in human serum

The initial degradation rates ( r) in human serum of three chitosans with F A = 0.42, 0.51, and 0.60 were determined by measuring the decrease in viscosity as a function of time. A strong increase in r with increasing F A of the chitosans was observed, with r increasing proportionally to F A 4.5. With increasing concentrations of lysozyme added to the reaction mixtures of chitosan and serum, the relative increase in degradation rate of chitosans with increasing F A was almost the same as that without lysozyme added. Addition of the chitinase inhibitor allosamidin (50 μM) did not inhibit the degradation rate of chitosan (F A = 0.60) by human serum. The results suggest that chitosans are actually mainly depolymerized by lysozyme in human serum, and not by other enzymes or other depolymerization mechanisms.

Mechanism of the cyclopentane ring formation of allosamizoline, an aminocyclitol derivative of the chitinase inhibitor allosamidin

Feeding experiments to study the mechanism of cyclopentane ring formation of allosamizoline with [3- 2H], [4- 2H]-, [5- 2H]-, and [6- 2H 2]- d-glucosamine indicated that the cyclization to form the cyclopentanoid moiety of allosamizoline proceeds via an intermediate 6-aldehyde glucosamine derivative.

Chitinase activity in human serum and leukocytes.

Using colloidal [3H] chitin as a substrate, we provide the first demonstration of a chitinase in human leukocytes; chitinolytic activity in whole and disrupted leukocyte preparations (approximately 0.6 and 5.5 nmol of N-acetylglucosamine [GlcNAc] released min-1 mg of protein-1, respectively) was partially inhibited by the specific chitinase inhibitor allosamidin (9 microM). Following fractionation of the leukocytes, much higher levels of chitinase activity were detected in granulocyte-rich homogenates (approximately 7.2 nmol of GlcNAc released min-1 mg of protein-1) than in lymphocyte- and monocyte-rich homogenates (approximately 0.22 and 0.26 nmol of GlcNAc released min-1 mg of protein-1, respectively). Low levels of chitinase activity were detected in human serum (approximately 4 pmol of GlcNAc released min-1 mg of protein-1). Chitinolytic activity in granulocyte-rich homogenates and serum was partially inhibited by allosamidin (9 microM). Proteins with chitinolytic activities (approximate molecular masses, 48 and 56 kDa) distinct from lysozyme (14.3 kDa) were detected on polyacrylamide gels following the electrophoresis of human granulocyte-rich preparations. Chitinase activity, detected consistently in serum and leukocytes from all human volunteers investigated, may contribute to the protection of the host by cleaving chitin in the cell walls of fungal pathogens.

Preparation of a Highly Functionalized Cyclopentane Derivative Suitable for the Synthesis of Allosamidin Analogs

Abstract Methyl 3,6-di-O-benzyl-4-O-benzoyl-2-deoxy-2-methoxycarbonylamino-α-D-glucopyranoside (8) was prepared form D-glucosamine via its 4,6-O-benzylidene derivative. The methyl glycoside moiety of 8 was hydrolyzed in the presence of d-camphorsulfonic acid in acetic acid to give hemiacetal 12. The oxime 14 derived from the latter was subjected to the radical cyclization mediated by tributyltin hydride, providing three types of cyclopentane derivatives. One isomer, 15, having an allosamizoline (2)-like configuration was converted into the N,N′-isopropylidene derivative 3, which is a potential intermediate for the syntheses of analogs of chitinase inhibitor allosamidin (1).