Abstract
Clinical microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymerization with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small molecules with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degradation by covalent binding to Cys-239 of β-tubulin. The small molecules highlighted in this study include T0070907 (a peroxisome proliferator-activated receptor γ inhibitor), T007-1 (a T0070907 derivative), T138067, N,N'-ethylene-bis(iodoacetamide) (EBI), and allyl isothiocyanate (AITC). Label-free quantitative proteomic analysis revealed that T007-1 promotes tubulin degradation with high selectivity. Mass spectrometry findings showed covalent binding of both T0070907 and T007-01 to Cys-239 of β-tubulin. Furthermore, T007-1 exerted a degradative effect on tubulin isoforms possessing Cys-239 (β2, β4, and β5(β)) but not those containing Ser-239 (β3, β6) or mutant β-tubulin with a C239S substitution. Three small molecules (T138067, EBI, and AITC) also reported to bind covalently to Cys-239 of β-tubulin similarly induced tubulin degradation. Our results strongly suggest that covalent modification of Cys-239 of β-tubulin by small molecules could serve as a novel strategy to promote tubulin heterodimer degradation. We propose that these small molecules represent a third novel class of tubulin inhibitor agents that exert their effects through degradation activity.
Highlights
Clinical microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents
Our results strongly suggest that covalent modification of Cys-239 of -tubulin by small molecules could serve as a novel strategy to promote tubulin heterodimer degradation
Labelfree quantitative proteomic analysis of T007-1-treated HeLa cells showed that T007-1 induced suppression of the protein levels of ␣- and -tubulin isoforms with high specificity
Summary
T0070907, a peroxisome proliferator-activated receptor ␥ (PPAR␥) inhibitor (Fig. 1A), promotes proteasome-dependent tubulin degradation and exerts anticancer effects on colorectal carcinoma cells [13, 19]. Promotes ␣-tubulin and -tubulin degradation in HeLa and Hct116 cells in a dose-dependent manner (Fig. 1E). A and B, T007-1 induced time-dependent down-regulation of ␣-tubulin and -tubulin proteins in both HeLa and Hct116 cells but exerted no inhibitory effects on the corresponding mRNA levels. Immunoblot analysis consistently revealed that T007-1 promotes degradation of 2-, 4-, and 5-tubulin isoforms containing cysteine at position 239, whereas exerting no effect on 3- and 6-tubulin isoforms, which possess serine at this position (Fig. 5B) These findings gave rise to the speculation that covalent modification of Cys-239 by T007-1 contributes to its tubulin degradation activity. B, in vitro tubulin polymerization assay: optical density at 340 nm of purified tubulin incubated with the indicated compounds was detected once every minute for 30 min at 37 °C This result is representative of two independent experiments. Our results clearly demonstrate that small molecules that covalently bind Cys-239 of -tubulin promote tubulin degradation
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