Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed. Received: 25 May 1998 / Accepted: 29 June 1998     

Computer-aided discovery of two novel chalcone-like compounds active and selective against Leishmania infantum

Leishmaniasis are infectious diseases caused by parasites of genus Leishmania that affect affects 12 million people in 98 countries mainly in Africa, Asia, and Latin America. Effective treatments for this disease are urgently needed. In this study, we present a computer-aided approach to investigate a set of 32 recently synthesized chalcone and chalcone-like compounds to act as antileishmanial agents. As a result, nine most promising compounds and three potentially inactive compounds were experimentally evaluated against Leishmania infantum amastigotes and mammalian cells. Four compounds exhibited EC₅₀ in the range of 6.2–10.98 μM. In addition, two compounds, LabMol-65 and LabMol-73, exhibited cytotoxicity in macrophages >50 μM that resulted in better selectivity compared to standard drug amphotericin B. These two compounds also demonstrated low cytotoxicity and high selectivity towards Vero cells. The results of target fishing followed by homology modeling and docking studies suggest that these chalcone compounds could act in Leishmania because of their interaction with cysteine proteases, such as procathepsin L. Finally, we have provided structural recommendations for designing new antileishmanial chalcones.     

Identification, characterization and functional analysis of a GH-18 chitinase from Streptomyces roseolus

A 40 kDa chitinase from Streptomyces roseolus DH was purified to homogeneity from culture medium. The N-terminal sequence was TPPPAKAVKLGYFTNWGVYG, which was highly homologous to the glycoside hydrolase (GH) 18 conserved domain of Streptomyces chitinases and included the two crucial Trp and Tyr sites. The purified enzyme showed maximal activity at 60 °C, pH 6.0 and exhibited good thermal and pH stabilities. The enzyme displayed strict substrate specificity on colloidal or glycol chitin, but not on chitosan derivatives. It was activated by Mg²⁺, Ba²⁺ and Ca²⁺, and inhibited by Cu²⁺, Co²⁺, Mn²⁺, whereas Zn²⁺ and ethylenediamine tetraacetic acid showed little inhibitory effects. Morphological changes observed by scanning electron microscopy revealed the occurrence of regular pores on the surface with the progress of enzymatic chitinolysis. Additionally, this GH-18 chitinase had a marked inhibitory effect on fungal hyphal extensions. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.     

Isolation and characterization of chitinase from a marine bacterium Vibrio sp.

A chitinase was separated from the culture broth of Vibrio sp. 11211 isolated from sediment from the South China Sea. The chitinase was purified 18.3-fold with 33% recovery by ammonium sulphate precipitation and chromatography. The subunit molecular weight of the enzyme was estimated by SDS-PAGE to be about 30kDa. The enzyme showed optimum pH at 6.5 and optimum temperature at 50^°C, and was stable in the pH range of 4 to 9 and at the temperature below 40^°C.     

Design,synthesis, in silico studies and in vitro evaluation of isatin-pyridine oximes hybrids as novel acetylcholinesterase reactivators

Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the availability of medical countermeasures, the clinically available compounds lack a broad spectrum, are not effective towards all organophosphorus toxins, and have poor pharmacokinetics properties to allow them crossing the blood-brain barrier, hampering cholinesterase reactivation at the central nervous system. In this work, we designed and synthesised novel isatin derivatives, linked to a pyridinium 4-oxime moiety by an alkyl chain with improved calculated properties, and tested their reactivation potency against paraoxon- and NEMP-inhibited acetylcholinesterase in comparison to the standard antidote pralidoxime. Our results showed that these compounds displayed comparable in vitro reactivation also pointed by the in silico studies, suggesting that they are promising compounds to tackle organophosphorus poisoning.     

Design and synthesis of a novel BODIPY-labeled PSMA inhibitor

Prostate-specific membrane antigen (PSMA) is a zinc-bound metalloprotease which is highly expressed in metastatic prostate cancer. It has been considered an excellent target protein for prostate cancer imaging and targeted therapy because it is a membrane protein and its active site is located in the extracellular region. We successfully synthesized and evaluated a novel PSMA ligand conjugated with BODIPY_650/665. Compound 1 showed strong PSMA-inhibitory activity and selective uptake into PSMA-expressing tumors. Compound 1 has the potential to be utilized as a near infrared (NIR) optical imaging probe targeting PSMA-expressing cancers.     

Enhancement of nematicidal potential through cloning and expression of chitinase gene from Bacillus subtilis subsp. Subtilis BTN7A strain

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.     

Design,synthesis and antifungal activity of novel fenfuram-diarylamine hybrids

Ten novel fenfuram-diarylamine hybrids were designed and synthesized. And their antifungal activities against four phytopathogenic fungi have been evaluated in vitro and most of the compounds demonstrated a significant antifungal activities against Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 5e exhibited the most potent antifungal activity against R. solani with an EC₅₀ value of 0.037 mg/L, far superior to the commercially available fungicide boscalid (EC₅₀ = 1.71 mg/L) and lead fungicide fenfuram (EC₅₀ = 6.18 mg/L). Furthermore, scanning electron microscopy images showed that the mycelia on treated media grew abnormally with tenuous, wizened and overlapping colonies compared to the negative control. Molecular docking studies revealed that compound 5e featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 3-chlorophenyl group in compound 5e formed a CH-π interaction with B/Trp-206 and a Cl-π interaction with D/Tyr-128, rendering compound 5e more active than fenfuram against SDH.     

Design and synthesis of a novel tyrosine kinase inhibitor template

We report the design and synthesis of an insulin receptor kinase family-targeted inhibitor template using the inhibitor conformation observed in an IGF1R/inhibitor co-crystal complex by application of a novel molecular design approach that we have recently published. The synthesis of the template involves a one pot Opatz cyclization reaction that provides a versatile indole ester in good yields. We also developed the required chemistry to elaborate this template with additional substituents and have used this chemistry to prepare some initial compounds that show selective inhibition of anaplastic lymphoma kinase (ALK).     

Design,synthesis and biological evaluation of amide-pyridine derivatives as novel dual-target (SE,CYP51) antifungal inhibitors

Based on the analysis of the squalene cyclooxygenase (SE) and 14α-demethylase (CYP51) inhibitors pharmacophore feature and the dual-target active sites, a series of compounds with amide-pyridine scaffolds have been designed and synthesized to treat the increasing incidence of drug-resistant fungal infections. In vitro evaluation showed that these compounds have a certain degree of antifungal activity. The most potent compounds 11a, 11b with MIC values in the range of 0.125–2 μg/ml had a broad-spectrum antifungal activity and exhibited excellent inhibitory activity against drug-resistant pathogenic fungi. Preliminary mechanism studies revealed that the compound 11b might play an antifungal role by inhibiting the activity of SE and CYP51. Notably compounds did not show the genotoxicity through plasmid binding assay. Finally, this study of molecular docking, ADME/T prediction and the construction of 3D QSAR model were performed. These results can point out the direction for further optimization of the lead compound.     

Design,synthesis, and in vitro evaluation of novel antifungal triazoles

Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and synthesized. Most of the compounds exhibited potent in vitro antifungal activities against 8 fungal isolates. Especially, compounds a10, a13, and a14 exhibited superior or comparable antifungal activity to ravuconazole against all the tested fungi. Structure-activity relationship study indicated that replacing 4-cyanophenylthioazole moiety of ravuconazole with fluorophenylisoxazole resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.     

Design and synthesis of spiro[indole-thiazolidine]spiro[indole-pyrans] as antimicrobial agents

A series of novel spiro[indole-thiazolidine]spiro[indole-pyran] derivatives were synthesized from N-(bromoalkyl)indol-2,3-diones via monospiro-bisindole intermediates; the two indole nuclei being connected via N-(CH(2))(n)-N linker. Synthesized compounds were evaluated for their antimicrobial activities in vitro against three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermis), four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia) as well as four fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Candida albicans) using Cup plate method. Bis spiro-indoles exhibited stronger antibacterial and antifungal efficiency than their corresponding mono spiro-indoles. Compound 10e, the most active derivative was shown to inhibit the growth of all bacterial strains and two fungal strains (A. niger and C. albicans).     

Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene.

Summary The class I chitinases are vacuolar proteins implicated in the defense of plants against pathogens. Leaves of transgenic Nicotiana sylvestris plants homozygous for a chimeric tobacco (Nicotiana tabacum) chitinase gene with Cauliflower Mosaic Virus (CaMV) 35S RNA expression signals usually accumulate high levels of chitinase relative to comparable leaves of non-transformed plants. Unexpectedly, some transgenic plants accumulated lower levels of chitinase than nontransformed plants. We call this phenomenon silencing. The incidence of silencing depends on the early rearing conditions of the plants. When grown to maturity in a greenhouse, 25% of plants raised as seedlings in closed culture vessels were of the silent type; none of the plants raised from seed in a greenhouse showed this phenotype. Silencing is also developmentally regulated. Plants showed three patterns of chitinase expression: uniformly high levels of expression in different leaves, uniformly low levels of expression in different leaves, and position-dependent silencing in which expression was uniform within individual leaves but varied in different leaves on the same plant. Heritability of the silent phenotype was examined in plants homozygous for the transgene. Some direct descendants exhibited a high-silent-high sequence of activity phenotypes in successive sexual generations, which cannot be explained by simple Mendelian inheritance. Taken together, the results indicate that silencing results from stable but potentially reversible states of gene expression that are not meiotically transmitted. Gene-specific measurements of chitinase and chitinase mRNA showed that silencing results from co-suppression, i.e. the inactivation of both host and transgene expression in trans. The silent state was not correlated with cytosine methylation of the transgene at the five restriction sites investigated.These authors have both made an equal contribution to this work     

Identification of novel small molecule non-peptidomimetic inhibitor for prolyl oligopeptidase through in silico and in vitro approaches

Abstract

Prolyl oligopeptidase (POP) enzyme has been studied for various disorders, viz. Schizophrenia, Alzheimer’s, Parkinson’s, Depression, Inflammation, etc., for three decades, but no drug has passed through the clinical trials, possibly because of indigent pharmacokinetics. This might have been a result of similar structures of drug candidates. This study aimed at identifying novel small non-peptidomimetic inhibitors for POP enzyme that could serve as a lead for developing newer drugs. Structure-based virtual screening of molecules of MolMall database was conducted on the POP enzyme (PDB ID 3DDU) to identify potential hits. The hits identified were subjected to computational pharmacokinetic screening followed by molecular mechanics/generalized Born and surface area studies to estimate the binding free energy of the docked complexes. After that, nine hits were selected and tested for POP inhibitory activity, among which one compound MM 4 was found to be most potent with EC₅₀ of 100 µM. Compound MM 4 was further subjected to molecular dynamics simulations to study the overall stability of the ligand–protein complex. The compound interacted strongly with catalytic amino acid Arg643 by forming salt and water bridges; it also interacted well with amino acids Phe173, Arg252 and Met235. This study provides a lead molecule for further development of POP inhibitors.

Communicated by Ramaswamy H. Sarma     

Design,synthesis, in vivo and in silico evaluation of phenacyl triazole hydrazones as new anticonvulsant agents

A series of phenacyl triazole hydrazones 3 have been designed based on the hybridization of (arylalkly)triazole and aroyl hydrazone scaffolds as new anticonvulsant agents. The target compounds 3 were easily synthesized from appropriate phenacyl triazoles and aryl acid hydrazides and characterized by IR, NMR and Mass spectroscopy. The in vivo anticonvulsant evaluation of synthesized compounds by using MES and PTZ tests revealed that they are more effective in MES model respect to PTZ test. All compounds showed 33–100% protection against MES-induced seizures at the dose of 100 mg/kg. However, the isonicotinic acid hydrazide derivative 3h showed the best profile of activity in both models. Molecular docking studies of compound 3h with different targets (NMDA, AMPA, GABA_A and sodium channel), postulated that the compound acts mainly via GABA_A receptors. In silico molecular properties predictions indicated that all compounds have favourable oral bioavailability and BBB permeability.     

Design and synthesis of novel substituted quinazoline derivatives as antileishmanial agents

4-(Substituted-benzylidine)-2-substituted-5,6-dihydrobenzo[h]quinazoline (5a–p) and 4-(substituted-benzylidine)-2-substituted-3, 4, 5, 6-tetrahydrobenzo[h]quinazoline (6a–p) have been synthesized from 2-(substituted-benzylidine)tetralone-1(3a–d) and several substituted guanidine sulfates(4a–d).These compounds were tested for their in vitro antileishmanial activity. The compounds 6i, 6f, 6g show promising antileishmanial activity against Leishmania donovani.     

Isolation and characterization of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities

A novel protease inhibitor, designated mungoin, with both antifungal and antibacterial activities, and exhibiting a molecular mass of 10 kDa in SDS-polyacrylamide gel electrophoresis, was isolated from mung bean (Phaseolus mungo) seeds. The isolation procedure involved a combination of extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography (HPLC) on SP-Toyopearl. Its isoelectric point was estimated to be 9.8 by isoelectric focusing. Its N-terminal amino acid sequence was determined to be EMPGKPACLDTDDFCYKP, demonstrating some resemblance to the C-terminal sequences of other protease inhibitors and inhibitor precursors from leguminous plants. It exerted a potent inhibitory action toward a variety of fungal species including Physalospora piricola, Mycosphaerella arachidicola, Botrytis cinerea, Pythium aphanidermatum, Sclerotium rolfsii and Fusarium oxysporum, as well as an antibacterial action against Staphylococcus aureus. In addition, this novel plant protease inhibitor displayed anti-proliferative activity toward tumor cells.