Oxalone and lactone moieties of podophyllotoxin exhibit properties of both the B and C rings of colchicine in its binding with tubulin

Abstract

Thermodynamics of podophyllotoxin binding to tubulin and its multiple points of attachment with tubulin has been studied in detail using isothermal titration calorimetry. The calorimetric enthalpy of the association of podophyllotoxin with tubulin is negative and occurs with a negative heat capacity change (ΔC_p = -2.47 kJ mol_-1 K_-1). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen-bonding forces with unfavorable negative entropic contribution at higher temperature, favored with an enthalpy-entropy compensation. Interestingly, the binding of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (AC, a colchicine analogue without the B ring) with tubulin is enthalpy-favored. However, the podophyllotoxin-tubulin association depending upon the temperature of the reaction has a favorable entropic and enthalpic component, which resembles both B- and C-ring properties of colchicine. On the basis of the crystal structure of the podophyllotoxin-tubulin complex, distance calculations have indicated a possible interaction between threonine 179 of α-tubulin and the hydroxy group on the D ring of podophyllotoxin. To confirm the involvement of the oxalone moiety as well as the lactone ring of podophyllotoxin in tubulin binding, analogues of podophyllotoxin are synthesized with methoxy substitution at the 4' position of ring D along with its isomer and another analogue epimerized at ring E. From these results, involvement of oxalone as well as the lactone ring of the drug in a specific orientation inclusive of ring A is indicated for podophyllotoxin-tubulin binding. Therefore, podophyllotoxin, like colchicine, behaves as a bifunctional ligand having properties of both the B and C rings of colchicine by making more than one point of attachment with the protein tubulin.