Characterisation of benzimidazole binding with recombinant tubulin from Giardia duodenalis, Encephalitozoon intestinalis, and Cryptosporidium parvum

MacDonald, L.M.; Armson, A.; Thompson, R.C.A.; Reynoldson, J.A.

doi:10.1016/j.molbiopara.2004.08.001

Cited by 73 publications

(47 citation statements)

References 36 publications

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“…That study concluded that furazolidone resistance is due to the reduced expression of ferredoxin and increased NADH oxidase activity, which differs from metronidazole resistance. In contrast, albendazole-resistant G. intestinalis strains revealed changes in the cytoskeleton, especially for ␤-tubulin (309), suggesting that qualitative changes in ␤-tubulin lead to decreased sensitivity to albendazole (191). In that study, benzimidazole analogues showed higher affinity for monomeric ␤-and heterodimeric ␣␤-tubulin derived from benzimidazole-sensitive parasites than for those from benzimidazole-insensitive organisms (191).…”

Section: Drug Resistance In G Intestinalismentioning

confidence: 80%

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Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

2007

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SUMMARY The “amitochondriate” protozoan parasites of humans Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis share many biochemical features, e.g., energy and amino acid metabolism, a spectrum of drugs for their treatment, and the occurrence of drug resistance. These parasites possess metabolic pathways that are divergent from those of their mammalian hosts and are often considered to be good targets for drug development. Sulfur-containing-amino-acid metabolism represents one such divergent metabolic pathway, namely, the cysteine biosynthetic pathway and methionine γ-lyase-mediated catabolism of sulfur-containing amino acids, which are present in T. vaginalis and E. histolytica but absent in G. intestinalis. These pathways are potentially exploitable for development of drugs against amoebiasis and trichomoniasis. For instance, l-trifluoromethionine, which is catalyzed by methionine γ-lyase and produces a toxic product, is effective against T. vaginalis and E. histolytica parasites in vitro and in vivo and may represent a good lead compound. In this review, we summarize the biology of these microaerophilic parasites, their clinical manifestation and epidemiology of disease, chemotherapeutics, the modes of action of representative drugs, and problems related to these drugs, including drug resistance. We further discuss our approach to exploit unique sulfur-containing-amino-acid metabolism, focusing on development of drugs against E. histolytica.

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Section: Drug Resistance In G Intestinalismentioning

confidence: 80%

“…Resistant organisms have been isolated from patients and characterized in various laboratories (1,6,7,95,150,191,310,312,314). Cross-resistance to tinidazole has also been demonstrated in metronidazole-resistant strains (310,312,314).…”

Section: Drug Resistance In G Intestinalismentioning

confidence: 99%

Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

2007

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“…Initially, these oryzalin analogs were evaluated in vitro for their ability to inhibit the polymerization of tubulin purified from L. amazonensis (Werbovetz et al, 1999), and ongoing drug discovery efforts against this promising target will continue to require in vitro evaluation of novel compounds against purified leishmanial tubulin. Reports detailing the expression of tubulin from parasites have appeared (MacDonald et al, 2003;Fennell et al, 2006), some of which have demonstrated the assembly of recombinant αβ tubulin (Oxberry et al, 2001;MacDonald et al, 2004). No such expression system currently exists for the leishmanial protein, however, and antileishmanial drug discovery work will continue to rely on the purification of tubulin directly from the parasite in the near future.…”

Section: Introductionmentioning

confidence: 99%

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Yakovich

Ragone

Alfonzo

et al. 2006

Experimental Parasitology

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Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, Leishmania amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of α-and β-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly-disassembly cycle in 1% overall recovery based on total cellular protein. L. tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae α-and β-tubulin genes were 99.6% and 99.4% identical to the corresponding amino acid sequences from the L. major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.

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“…and other protists (e.g., Giardia lamblia and Cryptococcus neoformans) are very sensitive to several derivatives of a group of drugs called the benzimidazoles, which are widely used to treat helminth infections in humans and animals and as systemic fungicides in agriculture (13,17). Benzimidazoles have been shown to disrupt mitosis in sensitive organisms through binding to the ␤-tubulin subunit of microtubules.…”

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confidence: 99%

Analysis of the β-Tubulin Gene from Vittaforma corneae Suggests Benzimidazole Resistance

Franzen

Salzberger

2008

Antimicrob Agents Chemother

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We amplified, cloned, and sequenced the ␤-tubulin gene of Vittaforma corneae, a microsporidium causing human infections. The ␤-tubulin gene sequence has a substitution at Glu 198 (with glutamine), which is one of six amino acids reported to be associated with benzimidazole sensitivity. Benzimidazoles were assayed for antimicrosporidial activity and showed poor parasite inhibition.Microsporidia are obligate intracellular protozoan parasites that have emerged as major opportunistic human pathogens since the onset of the AIDS pandemic (6, 11). Vittaforma corneae is responsible for keratitis in otherwise healthy persons (2, 9, 10, 18), but disseminated infections have occurred in human immunodeficiency virus-infected patients as well (3). V. corneae was first described as Nosema corneum in a stromal biopsy from a non-human immunodeficiency virus-infected man who suffered from central disciform keratitis for 18 months (2). The parasite was cultivated in cell culture and identified as a new species, N. corneum (19). Infection with this organism was subsequently established in athymic mice (20), and new taxonomically significant features found warranted placing this organism within a new genus, Vittaforma, as V. corneae (21). In the second reported case of V. corneae infection, dual microsporidial infection was detected in a patient with AIDS-Encephalitozoon hellem in the sinonasal aspirate and V. corneae in the urine (3), indicating that V. corneae is capable of dissemination and survival in deep tissues, at least in an immunocompromised host. Three further cases involving V. corneae infection of the corneal stroma of immunocompetent patients were published (9,10,18). In addition to these confirmed cases, there is molecular evidence (obtained by PCR) for Vittaforma infections as a cause of keratitis in India (15 cases) (12) and of enteritis in Portugal (25 cases) (23).Benzimidazoles are widely used as anthelmintic drugs in veterinary and human medicine and have been used as antifungal agents in agriculture. Albendazole is one of the most commonly used drugs for treating microsporidiosis in humans (5). Infections with Encephalitozoon spp. respond especially well to therapy with albendazole, whereas infections with Enterocytozoon bieneusi do not respond to albendazole, and the sequence of the ␤-tubulin gene of E. bieneusi suggests that this species is resistant to albendazole (1,5). No clinical data about the response of Vittaforma infections to albendazole are available, and molecular data on the ␤-tubulin gene are not available for this species either. We therefore tested the susceptibility of V. corneae to different benzimidazoles in vitro and amplified, cloned, and sequenced the ␤-tubulin gene of V. corneae.MRC-5 cells were seeded on 24-well culture plates at a concentration of 5 ϫ 10 5 /ml in minimal essential medium and incubated overnight to confluence. Confluent monolayers of cells were inoculated with 2 ϫ 10 6 microsporidian spores (V. corneae and E. cuniculi as a control). Three hours later, noninternalized sp...

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Characterisation of benzimidazole binding with recombinant tubulin from Giardia duodenalis, Encephalitozoon intestinalis, and Cryptosporidium parvum

Cited by 73 publications

References 36 publications

Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Analysis of the β-Tubulin Gene from Vittaforma corneae Suggests Benzimidazole Resistance

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