Structural and Biochemical Characterization of the Interaction of Tubulin with Potent Natural Analogues of Podophyllotoxin

Abstract

Four natural analogues of podophyllotoxin obtained from the Mexican medicinal plant Bursera fagaroides, namely, acetyl podophyllotoxin (2), 5'-desmethoxy-β-peltatin A methyl ether (3), 7',8'-dehydro acetyl podophyllotoxin (4), and burseranin (5), have been characterized, and their interactions with tubulin have been investigated. Cytotoxic activity measurements, followed by immunofluorescence microscopy and flow cytometry studies, demonstrated that these compounds disrupt microtubule networks in cells and cause cell cycle arrest in the G2/M phase in the A549 cell line. A tubulin binding assay showed that compounds 1-4 were potent assembly inhibitors, displaying binding to the colchicine site with Kb values ranging from 11.75 to 185.0 × 10(5) M(-1). In contrast, burseranin (5) was not able to inhibit tubulin assembly. From the structural perspective, the ligand-binding epitopes of compounds 1-3 have been mapped using STD-NMR, showing that B and E rings are the major points for interaction with the protein. The obtained results indicate that the inhibition of tubulin assembly of this family of compounds is more effective when there are at least two methoxyl groups at the E ring, along with a trans configuration of the lactone ring in the aryltetralin lignan core.

Identification and molecular characterization of the tubulin‐podophyllotoxin‐type lignans binding site on Giardia lamblia

“…According to our docking results, on Giardia , the mode of interactions of 5‐DES, APOD, and POD to residues of tubulin was distinct since these lignans recognize a single binding pocket in a hydrophobic cavity to the tubulin heterodimer interface; the binding was highly biased toward β‐tubulin, D327, L331, Q334, M349, K350, V351, partially interactions with α‐tubulin were also observed. These results agree with the experimental data reported by Mayra Antúnez and coworkers (2016) since they demonstrated that in mammalian cells these same lignans bind to the tubulin heterodimer interface inhibiting its polymerization (Antúnez‐Mojica et al, ).…”

“…These results could explain the higher selectivity of APOD toward tubulin from Giardia reported in our previous experimental findings, whose growth kinetics revealed a dose‐dependent inhibition, with an IC 50 value of 2.12 μM for APOD and 42.22 μM for BUR (Gutiérrez‐Gutiérrez, Puebla‐Pérez, et al, ). BUR was not able to bind tubulin, any effect on polymerized tubulin was observed, as previously reported (Antúnez‐Mojica et al, ). On the other hand, some works suggest that podophyllotoxin derivatives are colchicine‐site ligands that inhibit microtubule polymerization (Antúnez‐Mojica et al, ; Escudero‐Martínez et al, ; Kamal et al, ); the zones of the interaction of colchicine have been highly biased toward β‐tubulin.…”

“…BUR was not able to bind tubulin, any effect on polymerized tubulin was observed, as previously reported (Antúnez‐Mojica et al, ). On the other hand, some works suggest that podophyllotoxin derivatives are colchicine‐site ligands that inhibit microtubule polymerization (Antúnez‐Mojica et al, ; Escudero‐Martínez et al, ; Kamal et al, ); the zones of the interaction of colchicine have been highly biased toward β‐tubulin. Recently, Naresh Kumar Manchukonda and coworkers (2013) reported that the amino acids that are involved in the interaction of colchicine with tubulin from mammal cells are V236, L246, N247, A248, L253, N255, M256, A314, A315, V316, K350, and A352 (Manchukonda et al, ).…”

“…In mammalian cells, it has been reported that BUR, 5‐DES, and APOD affect microtubule dynamics by targeting the interface of the tubulin dimer, binding on specific residues (Antúnez‐Mojica et al, ). To evaluate whether BUR, 5‐DES, APOD, and POD exhibit affinity to tubulin of Giardia , molecular docking simulations were carried out.…”

Antigiardial Activity of Podophyllotoxin-Type Lignans from Bursera fagaroides var. fagaroides

“…It is largely known that POD is an anti-tubulin agent; it binds at the interface between α- and β-tubulin, which inhibits the assembly of tubulin into microtubules. In addition, it was recently described that 5-DES and APOD also disrupt microtubule networks in mammalian cells [ 31 ]. Considering that microtubules are an essential part of the Giardia cytoskeleton, our data suggest that POD, 5-DES, and APOD may inhibit Giardia proliferation by perturbing microtubule assembly, and we are currently conducting studies to identify the molecular targets involved.…”