A Photoaffinity Analogue of Discodermolide Specifically Labels a Peptide in β-Tubulin

Abstract

Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.

Hallmarks of Molecular Action of Microtubule Stabilizing Agents

“…Materials-Tubulin was isolated from the marginal bands of chicken erythrocytes and from bovine brain as previously described (24,25). BBT was stored in 0.1 M MES, 1 mM EGTA, 0.5 mM MgCl 2 , and 3 M glycerol, pH 6.6, in liquid nitrogen.…”

“…Drug Displacement Studies-Three separate experiments were done as described previously (8,25) with the following modifications; tubulin and [ 3 H]Taxol concentrations were increased to 3 M, 0.3 mM GTP was used, and the total reaction volume was reduced to 160 l.…”

Structural and chemical basis for anticancer activity of a series of²-tubulin ligands: molecular modeling and 3D QSAR studies

“…The importance of the C 19 -carbamate, (R)-C 17 -hydroxyl, C 14 -methyl and (S)-C 7 -hydroxyl substituents, and the lactone ring corroborates with the β-tubulin interaction sites Asp26, Gly360, Phe270, Arg276 and His227, as previously reported. 25,37,[46][47][48] As can be seen in the docking position obtained for discodermolide (Figure 10), the lactone ring fits in the Asp224/His227 pocket and leads to the formation of a hydrogen bond between the carbonyl oxygen and the side chain of Arg276, while the C 19 -carbamate NH 2 group is oriented toward the side chain of Asp26 to form an additional hydrogen bond. The (S)-C 7 -hydroxyl substituent is also hydrogen-bonded to Arg276 and the C 14 -methyl group interacts with Phe270 through hydrophobic contacts.…”

“…These observations are in agreement with prior experimental evidence that suggests the importance of the substituents at C 19 -carbamate, (R)-C 17 -hydroxyl, and (S)-C 7 -hydroxyl, and the carbonyl oxygen of the lactone ring to the observed discodermolide cytotoxicity. 25,37,46,47 The corresponding steric fields for the two generated molecular alignments are depicted in Figure 9. The green contours surrounding the lactone ring of the discodermolide analog (compound 32, Table 1) suggest that bulky substituents would be favorable in that region according to both rigid-body fit and receptor-based steric contour maps.…”

Developmental Biology of Neoplastic Growth

“…Discodermolide binds competitively with paclitaxel to tubulin dimers (Hung et al 1996). Recently, it was shown, using a photoaffinity analogue, that it binds to amino acid residues 355-359 of β-tubulin in close proximity to the taxol binding site (Xia et al 2006).…”