Amino-acid alterations in the β-tubilin gene of Neurospora crassa that confer resistance to carbendazim and diethofencarb

We have previously shown that increased sensitivity to diethofencarb in the carbendazim(MBC)-resistant F914 strain of Neurospora crassa is caused by a single amino-acid change in -tubulin, ¹⁹⁸Glu to Gly. Three diethofencarb-resistant mutants that are also resistant to MBC were isolated from strain F914. They contained single base-pair-substitution mutations in the -tubulin gene. The amino acid changes in -tubulin, Phe from ²⁵⁰Leu, Val from ¹⁶⁵Ala, and Ala from ²³⁷Thr, were responsible for diethofencarb-resistance in the mutant strains FR511, FR513, and FR421, respectively. The amino acid at position 198 of -tubulin in these mutants was Gly, which is the same as in strain F914. -tubulin genes with ¹⁹⁸Glu were constructed by site-directed mutagenesis. The altered -tubulin genes derived from FR511 and FR421 transformed the wild-type strain to resistance to MBC, indicating that ²⁵⁰Phe and ²³⁷Ala in -tubulin are responsible for resistance not only to diethofencarb but also to MBC.     

Electrostatic surface potential calculation on several new halogenated benzimidazole-like dopaminergic ligands

We examined the effects of the electron density distribution (electrostatic surface potential; ESP) of several new benzimidazole-type ligands on their binding affinity for the D(1) and D(2) dopamine receptors (DAR). Receptors were prepared from synaptosomal membranes of bovine caudate nuclei. [(3)H]SCH 23390 and [(3)H]spiperone were used as specific radiolabels for the D(1) and D(2) receptors, respectively. The ESP of these compounds was calculated using Gaussian 98 W software. Calculations performed with known dopaminergic ligands showed that the electron density charge in the aromatic ring of these compounds favors a higher binding affinity for the D(2) DAR. This was confirmed by the synthesis of halogenated analogues of several known dopaminergic ligands. Halogenation resulted in an increase in the positive charge of the aromatic part of the molecule. None of the newly synthesized compounds was efficient in displacing [(3)H]SCH 23390 from the D1 DAR. The introduction of chlorine into the molecule led to a higher binding affinity for the D(2) DAR of the new ligands in comparison to both parent compounds and brominated ligands. This difference probably originates from the difference in the sizes of chlorine and bromine atoms, which could influence the interaction of a ligand with the receptor binding site. However, among the new ligands with bromine as a substituent, two compounds (8b and 10b) expressed a higher binding affinity and two of them (9b and 11b) a lower binding affinity for the D(2) DAR, when compared to unsubstituted parent compounds. These results indicate that the electrostatic surface potential of a ligand is an important factor in its interaction with the D(2) DAR and that this should be taken into account during design and synthesis of dopaminergic compounds.     

Anthelmintic activity of benzimidazole derivatives against Toxocara canis second-stage larvae and Hymenolepis nana adults

The anthelmintic activity of 11 benzimidazole derivatives (A1-A11) and 2 thioureides N,N′-disubstituted (B1-B2) was determined. Each compound and albendazole was tested in vitro against Toxocara canis larvae and in vivo against Hymenolepis nana adult. Compounds A1-A6 and B1-B2 were designed as albendazole prodrugs. Compounds A8-A11 were designed as direct analogues of A7, which had previously proved to be an effective agent against Fasciola hepatica. Results of the in vitro screening showed that A6 was more active than albendazole at 0.18 μM (relative mobility 40% and 80%, respectively). Whereas that the in vivo evaluation against H. nana, compounds A7-A11 demonstrated significant activity in terms of removing cestode adults in the range of 88-97%, displaying better efficacy than albendazole (83%).     

Antimicrobial activity of a new combination system of benzimidazole and various azoles

In the present study a new series of benzimidazole derivatives bearing various (benz)azolylthio moieties were synthesized so as to investigate their antimicrobial activity. Structures of the target compounds (5a-5i) were confirmed by their IR, (1) H-NMR, ES-MS spectral data, and elemental analyses. The synthesized compounds (5a-5i) exhibited poor activity against bacterial strains. On the other hand, antifungal activity of the compounds against Candida species was very significant. Brine-Shrimp lethality assay was performed for determination of toxicity of the compounds. Compounds 5a, 5c, and 5d were evaluated as non-toxic in addition to their attractive antifungal activity. However, the other compounds (5b, e-i) in the series showed toxicity to different extents.     

Imported bancroftian filariasis: Diethylcarbamazine response and benzimidazole susceptibility of Wuchereria bancrofti in dynamic cross-border migrant population targeted by the National Program to Eliminate Lymphatic Filariasis in South Thailand

The implementation on the Thailand-Myanmar border of annual mass drug administration (MDA) of a single 6 mg/kg dose of diethylcarbamazine (DEC) plus 400 mg albendazole, part of the National Program to Eliminate Lymphatic Filariasis (PELF), has been challenging. In particular, chain migration of cross-border Myanmar workers at risk for nocturnally periodic Wuchereria bancrofti infection can lead to imported bancroftian filariasis (IBF) in Thailand. IBF is targeted for multiple-dose MDA with 300 mg DEC, in addition to what is recommended by the World Health Organization (WHO). The dynamic Myanmar migrants in Phang-nga, southern Thailand were sampled to test whether the responsible W. bancrofti has a genetic predisposition of benzimidazole exposure, and IBF exhibits DEC susceptibility. The long-term migrants had more access to DEC. IBF in W. bancrofti antigenemic (microfilaremic vs. amicrofilaremic) short-term migrants exhibited susceptibility to a 300-mg single-dose DEC treatment. During the course of a 3-month follow-up, antigenemia was significantly reduced, but microfilaremia was fluctuated. Surprisingly, a newly recognized Mansonella infection co-existing among W. bancrofti-affected Myanmar migrants elicited microfilaremia clearance within a month after treatment. As a result of the presence of genetically stable W. bancrofti β-tubulin (Wbtubb) gene responsible for benzimidazole susceptibility, IBF did not possess a genetic predisposition for benzimidazole exposure. Point mutations at positions Phe167Tyr and Phe200Tyr were not detected by Wbtubb locus-specific nested PCR and sequencing. This study has the potential to help guide not only the Thai/Myanmar PELF surveillance and monitoring of mass treatment impacts on W. bancrofti, but also the other endemic countries allied with the Global Program to Eliminate Lymphatic Filariasis (GPELF).     

Synthesis, physicochemical properties and antimicrobial activity of some new benzimidazole derivatives

Some derivatives of benzimidazole were synthesized by nucleophilic substitution of 2-substituted-1H-benzimidazole. The resulting ethyl (2-substituted-1H-benzimidazol-1-yl) acetate on treatment with hydrazine hydrate yielded 2-(2-substituted-1H-benzimidazol-1-yl) acetohydrazide, which on further reaction with one equivalent of different aliphatic or aromatic carboxylic acids in the presence of phosphoryl chloride afforded the corresponding target compounds, 2-substituted-1-[{(5-substituted alkyl/aryl)-1,3,4-oxadiazol-2-yl} methyl]-1H-benzimidazole. The structures of the synthesized compounds were evaluated by spectral and elemental methods of analyses. All the synthesized compounds were screened for their antimicrobial activities. All of the derivatives showed good activity towards Gram-positive bacteria and negligible activity towards Gram-negative bacteria. Some of the synthesized compounds showed moderate activity against tested fungi.     

Action Mechanisms of Cell-Division-Arresting Benzimidazoles and of Sterol Biosynthesis-Inhibiting Imidazoles, 1, 2, 4-Triazoles, and Pyrimidines

Summary: In spite of a formal similarity between benzimidazoles and azoles, two completely different action mechanisms are in operation, namely inhibition of tubuline association by the benzimidazoles, and inhibition of ergosterol biosynthesis at the stage of C₁₄ demethylation by imidazoles, 1, 2, 4-triazoles, and pyrimidines.
Zusammenfassung: Trotz formaler Ähnlichkeit zwischen Benzimidazolen und Azolen werden zwei vöüig verschiedene Wirkungsmechanismen verwirklicht, nämlich Hemmung der Tubulinassoziation durch Benzimidazole und Hemmung der Ergosterolbiosynthese auf der Stufe der C₁₄-Desmethylierung durch Imidazole, 1, 2, 4-Triazole und Pyrimidine.     

Benzimidazole -Resistance in Haemonchus contortus: New PCR-RFLP Method for the Detection of Point Mutation at Codon 167 of Isotype 1 β-Tubulin Gene

Background

Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT-HC167 MF-primer), to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubulin gene of Haemonchus contortus.

Methods

There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleotide polymorphism (SNP) at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer), in which the nucleotide T at the position 443 was substituted through a nucleotide A creating a restriction site for restriction endonuclease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp) was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achieving a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, respectively.

Results

All worms had two alleles encoding for phenylalanine (BZ^ss homozygote) for both codons.

Conclusion

Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP.