Fluorescence spectroscopy of solid-state tubulin and 3D-QSAR of colchicine and analogs using comparative molecular field analysis (CoMFA)

A method was formulated and validated which protected the protein tubulin during lyophilization. Tubulin was freeze-dried in two separate buffer systems using different cryoprotectant/stabilizer systems. The reconstituted protein was tested for biological activity and fluorescent spectral properties relative to conventionally prepared solution samples. To examine the native properties of the protein in the solid state, a steady state fluorescent technique was developed. The new technique supported results achieved with Fourier-transform infrared (FTIR) spectroscopy, known to be useful for determining protein structure in the solid state. In addition lyophilized tubulin was analyzed by frequency domain fluorescent lifetime spectroscopy in solution and solid form. The results indicated that steady state and frequency domain fluorescent spectroscopy could be used to evaluate native properties of tubulin in solid form and that Iyophilization of tubulin is a viable and more convenient method of protein preparation.;In Part II, forty-five C ring variants of colchicine and allocolchicine were evaluated for inhibition of microtubule assembly in vitro. The structural variation in the series was confined to the C-9 and C-10 regions of colchicine, and the C-9 region of allocolchicine. This was done to probe the C ring region of the colchicine-binding site on tubulin. It was found that replacing the oxygen atoms of the C ring ester of allocolchicine with methylene groups had a minor affect on analog potency, indicating that hydrogen bonding between the ligand and the C ring region of the binding is not required for high antimicrotubule activity. The structure-activity relationship for the C ring region of colchicinoid, ligands was quantitatively assessed through the use of comparative molecular field analysis (CoMFA). The resulting model is a robust predictor of activity for drugs in this series. Molecular analysis and graphics are presented and discussed relative to the CoMFA results.