Purification, characterization, and drug susceptibility of tubulin from Leishmania

Werbovetz, Karl A.; Brendle, JamesJ.; Sackett, DanL.

doi:10.1016/s0166-6851(98)00146-7

Cited by 90 publications

(90 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A modification of the earlier protocol for purification of tubulin from L. amazonensis was employed for the isolation of L. tarentolae tubulin (Werbovetz et al, 1999). PBS washed parasites (7.3 × 10 11 cells) were thawed and resuspended in three 40 mL portions in PME + P buffer (PME plus protease inhibitors, consisting of 0.1M PIPES (pH 6.9), 1 mM MgCl 2 , 1 mM EGTA, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin) on ice.…”

Section: Purification Of Tubulinmentioning

confidence: 99%

“…tarentolae tubulin was purified based on a previous protocol for the isolation of L. amazonensis tubulin (Werbovetz et al, 1999). The initial step in the earlier protocol involved lysis of cells by sonication, followed by centrifugation to generate a cytoplasmic fraction and a pellet rich in tubulin (most likely from subpellicular microtubules and flagella).…”

Section: Purification Of L Tarentolae Tubulinmentioning

confidence: 99%

“…N 1 -phenyl-3,5-dinitro-N 4 ,N 4 -di-n-propylsulfanilamide (GB-II-5) (Werbovetz et al, 2003) and N 1 -phenyl-3,5-dinitro-N 4 ,N 4 -di-n-butylsulfanilamide (GB-II-150) (Bhattacharya et al, 2004) are derivatives of the dinitroaniline herbicide oryzalin that have displayed potent antimitotic effects on kinetoplastid parasites (see Figure 1 for structures). 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).…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, L. tarentolae's natural host is the Gecko lizard (Tarentola mauritanica) (Wenyon, 1921), and this parasite species does not infect humans. Furthermore, L. tarentolae can be cultured to higher cell densities than L. amazonensis; typical achievable cell densities are 1.8 × 10 8 parasites/ml (Sbicego et al, 2003) and 5 × 10 7 parasites/ml (Werbovetz et al, 1999), respectively. In addition, L. amazonensis requires supplemented Schneider's Drosophila medium for high density growth (Werbovetz et al, 1999), whereas L. tarentolae can be grown in Brain Heart Infusion supplemented with hemin (Sbicego et al, 2003), with the latter medium being considerably less expensive.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, L. tarentolae can be cultured to higher cell densities than L. amazonensis; typical achievable cell densities are 1.8 × 10 8 parasites/ml (Sbicego et al, 2003) and 5 × 10 7 parasites/ml (Werbovetz et al, 1999), respectively. In addition, L. amazonensis requires supplemented Schneider's Drosophila medium for high density growth (Werbovetz et al, 1999), whereas L. tarentolae can be grown in Brain Heart Infusion supplemented with hemin (Sbicego et al, 2003), with the latter medium being considerably less expensive. Herein we report the purification and amino acid sequence of tubulin from L. tarentolae, along with a comparison of the effects of selected antimicrotubule agents on tubulin from L. tarentolae and L. amazonensis.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Yakovich

Ragone

Alfonzo

et al. 2006

Experimental Parasitology

View full text Add to dashboard Cite

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.

show abstract

Section: Purification Of Tubulinmentioning

confidence: 99%

Section: Purification Of L Tarentolae Tubulinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Yakovich

Ragone

Alfonzo

et al. 2006

Experimental Parasitology

View full text Add to dashboard Cite

show abstract

Pharmacological and biological screening of ascorbigen: protection against glucose‐induced endothelial cell toxicity

Joshi

Bauer

Werbovetz³

et al. 2008

Phytotherapy Research

View full text Add to dashboard Cite

Cruciferous vegetables contain significant amounts of ascorbigen and related substances with known molecular structures. This study tested the hypothesis that ascorbigen demonstrates antioxidant properties and protects human umbilical cord endothelial cells against hyperglycemic toxicity in vitro. It was observed that ascorbigen, in micromolar concentrations, protected against endothelial cell death from glucose toxicity. Additionally, ascorbigen at 3.0 mm shifted the concentration response curve of l-phenylephrine to the right, with a reduction in the maximal contractile effects of the agonist. This action was not related to alpha-adrenoceptor blockade. Ascorbigen also relaxed the vascular tone induced by l-phenylephrine, which is not mediated by an endothelial cell nitric oxide-dependent mechanism. On the guinea-pig ileum, the spasmogenic effects of carbachol, histamine and serotonin were reduced in the presence of 3 mM ascorbigen. Spasm of the gut induced by the acetylcholinesterase inhibitor, physostigmine, was antagonized by ascorbigen with an IC50 of 286 microM. This natural product also has a weak antiparasitic activity. The cytoprotective effects of ascorbigen may be highly relevant in the optimum physiological regulation of the function and the therapeutic value of this substance in disease settings needs to be further investigated.

show abstract

Synthesis, Structural Determination, and Pharmacology of Putative Dinitroaniline Antimalarials

et al. 2018

View full text Add to dashboard Cite

A series of novel, homologous compounds possessing the general formula N1‐Nn‐bis(2,6‐dinitro‐4‐trifluormethylphenyl)‐1,n‐diamino alkanes (where n=4, 6, 10 or 12), were designed to probe inter‐ and intra‐ binding site dimensions in malarial parasite (Plasmodium) tubulin. Various crystal structures, including chloralin and trifluralin, an isopropyl dimer, and 2,6‐dinitro‐4‐trifluoromethyl‐phenylamine, were determined. Dinitroanilines, when soluble, were evaluated both in culture and in vivo. Trifluralin was up to 2‐fold more active than chloralin against cultured parasites. The isopropyl dimer was water soluble (>5 mM) and revealed activity superior to that of chloralin in culture. The effects of selected dinitroanilines upon the mitotic microtubular structures of Plasmodium, the putative target of these dinitroanilines, were also determined. Electronic properties of the molecules were calculated using DFT (B3LYP/6‐31+G* level) to ascertain whether incorporation of such a pharmacophore could allow both QSAR and rational development of more selectively toxic antiparasitic agents.

show abstract

Purification, characterization, and drug susceptibility of tubulin from Leishmania

Cited by 90 publications

References 38 publications

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

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

Pharmacological and biological screening of ascorbigen: protection against glucose‐induced endothelial cell toxicity

Synthesis, Structural Determination, and Pharmacology of Putative Dinitroaniline Antimalarials

Contact Info

Product

Resources

About