Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia

Abstract

Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. To investigate the mechanism of action, a photoactive analogue, 1-azidoanthracene, was synthesized. Administration of 1-azidoanthracene to albino stage 40–47 tadpoles was found to immobilize animals upon near-UV irradiation of the forebrain region. The immobilization was often reversible, but it was characterized by a longer duration consistent with covalent attachment of the ligand to functionally important targets. IEF/SDS-PAGE examination of irradiated tadpole brain homogenate revealed labeled protein, identified by mass spectrometry as β-tubulin. In vitro assays with aminoanthracene-cross-linked tubulin indicated inhibition of microtubule polymerization, similar to colchicine. Tandem mass spectrometry confirmed anthracene binding near the colchicine site. Stage 40–47 tadpoles were also incubated 1 h with microtubule stabilizing agents, epothilone D or discodermolide, followed by dosing with 1-aminoanthracene. The effective concentration of 1-aminoanthracene required to immobilize the tadpoles was significantly increased in the presence of either microtubule stabilizing agent. Epothilone D similarly mitigated the effects of a clinical neurosteroid general anesthetic, allopregnanolone, believed to occupy the colchicine site in tubulin. We conclude that neuronal microtubules are “on-pathway” targets for anthracene general anesthetics and may also represent functional targets for some neurosteroid general anesthetics.

Propofol Inhibits SIRT2 Deacetylase through a Conformation-specific, Allosteric Site

“…Because general anesthetics can affect tubulin polymerization (15), and because acetylation of ␣-tubulin is used as a surrogate for microtubule stability, we also confirmed that propofol inhibition of SIRT2 was independent of the polymerization state of tubulin. The microtubule-stabilizing agent epothilone D (15,35) was added to separate assays at a concentration of 2 M, which is sufficient to increase microtubule stability in vivo (15).…”

“…The microtubule-stabilizing agent epothilone D (15,35) was added to separate assays at a concentration of 2 M, which is sufficient to increase microtubule stability in vivo (15). Epothilone D did not affect SIRT2 deacetylation of ␣-tubulin, nor did it affect propofol inhibition of SIRT2 (Fig.…”

“…The applications of [ 3 H]AziPm (11,14) and epothilone D (15) have been described in previous work, and all other chemicals and reagents were commercially available. Recombinant human SIRT2, representing residues 13-319, and recombinant human SIRT1, representing the fulllength protein, were purchased from Sigma-Aldrich, and the identities of the proteins were confirmed with mass spectrometry.…”

Common general anesthetic propofol impairs kinesin processivity

“…This body of work suggests that propofol and other common anesthetics, such as etomidate and ketamine (10)(11)(12)(13)(14), may target additional protein networks of the CNS, including potential interactions with the cytoskeleton to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exist (15)(16)(17)(18). For example, different classes of general anesthetics bind specifically to tubulin and alter its stability (17,18), and entire theories of anesthetic action have been constructed around microtubule (MT) properties (19).…”

Biological Quasicrystals and the Golden Mean: Relevance to Quantum Computation

“…Recent work on anaesthesia, which selectively erases consciousness while sparing nonconscious brain activities, acts via a destabilisation of MTs in brain neurons (Emerson et al, 2013). Microtubules contain a unique arrangement of water molecules in their lumen.…”