A fluorescence-based high-throughput assay for antimicrotubule drugs

Barron, Donna M; Chatterjee, Sabarni K; Ravindra, Rudravajhala; Roof, Rebecca A.; Baloglu, Erkan; Kingston, David G. I.; Bane, Susan

doi:10.1016/s0003-2697(02)00691-7

Cited by 72 publications

(38 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Microtubule assembly was monitored by DAPI fluorescence according to the protocol described previously (Barron et al, 2003). Polymerization was performed using 10 M microtubule-associated protein (MAP)-rich tubulin (Cytoskeleton Inc) in BRB80 buffer, 1 mM GTP, 10 M DAPI, and recombinant HSPB1 at 5 M when stated.…”

Section: Methodsmentioning

confidence: 99%

Small Heat-Shock Protein HSPB1 Mutants Stabilize Microtubules in Charcot-Marie-Tooth Neuropathy

Almeida-Souza¹,

Asselbergh²,

d’Ydewalle³

et al. 2011

J. Neurosci.

View full text Add to dashboard Cite

Mutations in the small heat shock protein HSPB1 (HSP27) are causative for Charcot-Marie-Tooth (CMT) neuropathy. We previously showed that a subset of these mutations displays higher chaperone activity and enhanced affinity to client proteins. We hypothesized that this excessive binding property might cause the HSPB1 mutant proteins to disturb the function of proteins essential for the maintenance or survival of peripheral neurons. In the present work, we explored this hypothesis further and compared the protein complexes formed by wild-type and mutant HSPB1. Tubulin came out as the most striking differential interacting protein, with hyperactive mutants binding more strongly to both tubulin and microtubules. This anomalous binding leads to a stabilization of the microtubule network in a microtubule-associated protein-like manner as reflected by resistance to cold depolymerization, faster network recovery after nocodazole treatment, and decreased rescue and catastrophe rates of individual microtubules. In a transgenic mouse model for mutant HSPB1 that recapitulates all features of CMT, we could confirm the enhanced interaction of mutant HSPB1 with tubulin. Increased stability of the microtubule network was also clear in neurons isolated from these mice. Since neuronal cells are particularly vulnerable to disturbances in microtubule dynamics, this mechanism might explain the neuron-specific CMT phenotype caused by HSPB1 mutations.

show abstract

Section: Methodsmentioning

confidence: 99%

Small Heat-Shock Protein HSPB1 Mutants Stabilize Microtubules in Charcot-Marie-Tooth Neuropathy

Almeida-Souza¹,

Asselbergh²,

d’Ydewalle³

et al. 2011

J. Neurosci.

View full text Add to dashboard Cite

show abstract

“…The potential antivascular effects of the synthesized compounds were assessed by their ability to inhibit tubulin polymerization (fluorimetric assay) [29] and to rapidly induce a rounding up of endothelial cells. [30] This morphological test is considered to be predictive of potential in vivo antivascular activity, and it allows determination of the lowest active concentration that causes rounding of immortalized HUVEC (EAhy 926 cells) within 2 h. Compounds were also evaluated for their growth inhibitory effects against two murine cancer cell lines: B16 melanoma cells and Lewis lung carcinoma (3LL) cells.…”

Section: Biological Evaluationmentioning

confidence: 99%

Synthesis and Structure–Activity Relationships of Constrained Heterocyclic Analogues of Combretastatin A4

et al. 2011

View full text Add to dashboard Cite

A series of combretastatin A4 (CA4) analogues with a lactam or lactone ring fused to the trimethoxyphenyl or the B-phenyl moiety were synthesized in an efficient and stereoselective manner by using a domino Heck-Suzuki-Miyaura coupling reaction. The vascular-disrupting potential of these conformationally restricted CA4 analogues was assessed by various in vitro assays: inhibition of tubulin polymerization, modification of endothelial cell morphology, and disruption of endothelial cell cords. Compounds were also evaluated for their growth inhibitory effects against murine and human tumor cells. B-ring-constrained derivatives that contain an oxindole ring (in contrast to compounds with a benzofuranone ring) as well as analogues bearing a six-membered lactone core fused to the trimethoxyphenyl ring are endowed with significant biological activity. The most potent compound of this series (oxindole 9 b) is of particular interest, as it combines chemical stability and a biological activity profile characteristic of a vascular-disrupting agent.

show abstract

“…The combination of high throughput turbidity measurements with accelerated stability tests, by means of mechanical stress, was shown to be useful in protein formulation (13). However, high throughput turbidity screening of protein solutions often is hindered by the requirement of high protein concentrations and the high variation in absorbance signal (14). This variation can be caused due to the relatively small diameter (Ø~0.75 mm) of the light beam, which corresponds to less than 1% of the surface of a 96 well (Ø~9.0 mm), and the irregular growth of protein aggregates.…”

Section: Discussionmentioning

confidence: 99%

A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

2008

View full text Add to dashboard Cite

Purpose. The feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied. Materials and Methods. A hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.5 and 10.5. The stability of the sCT formulations was analyzed over 1 week by the following assays: (1) protein concentration, (2) volume control by measuring pathlength, (3) turbidity (absorbance at 350 nm), (4) intrinsic tyrosine fluorescence, (5) 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence, (6) Nile Red fluorescence. Addition of the dyes (Nile Red and ANS) was used to study protein conformational changes. Results. After 1 day, 27 out of the 100 formulations of salmon calcitonin were stable. After 7 days, 12 stable sCT formulations remained. The best salmon calcitonin formulation was in 10 mM sodium acetate buffer with pH values between 3.5 and 5.5. Conclusions. The findings are in accordance with the sCT formulations that were patented and used commercially. This can be considered as a proof of concept for the high throughput protein formulation platform.

show abstract

A fluorescence-based high-throughput assay for antimicrotubule drugs

Cited by 72 publications

References 21 publications

Small Heat-Shock Protein HSPB1 Mutants Stabilize Microtubules in Charcot-Marie-Tooth Neuropathy

Small Heat-Shock Protein HSPB1 Mutants Stabilize Microtubules in Charcot-Marie-Tooth Neuropathy

Synthesis and Structure–Activity Relationships of Constrained Heterocyclic Analogues of Combretastatin A4

A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

Contact Info

Product

Resources

About