Rudravajhala Ravindra

A fluorescence-based high-throughput assay for antimicrotubule drugs.

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.

Site-Specific Orthogonal Labeling of the Carboxy Terminus of α-Tubulin

A fluorescent probe has been attached to the carboxy terminus of the alpha-subunit of alpha,beta-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of alpha-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophores absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.

High-throughput screening of microtubule-interacting drugs.

Drugs that affect microtubule dynamics are among the most effective anticancer agents in routine clinical use. The standard assay for antimicrotubule agents observes the ability of a particular substance to affect in vitro microtubule assembly. We have modified these procedures so that they can be performed in 96-well plates using a standard fluorescence plate reader. Two different protocols are provided in this chapter. One of these protocols is for ligands that inhibit microtubule polymerization, such as colchicine and related molecules. The second is for ligands that promote in vitro microtubule assembly, such as Taxol.

Corrigendum to “Synthesis and biological activity of 2,5-diaryl-3-methylpyrimido[4,5-c]quinolin-1(2H)-one derivatives” [Bioorg. Med. Chem. 15 (2007) 2434–2440]

Please cite this article in press as: Metwally K., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.025 Kamel Metwally a,⇑, Omar Aly , Enayat Aly , Abhijit Banerjee , Rudravajhala Ravindra , Susan Bane d Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt Department of Medicinal Chemistry, Faculty of Pharmacy, Elminia University, Elminia, Egypt Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt Department of Chemistry, Binghamton University, Binghamton, NY, USA