Recent Approaches to the Identification of Novel Microtubule-Targeting Agents

Abstract

Microtubules are key components of the eukaryotic cytoskeleton with essential roles in cell division, intercellular transport, cell morphology, motility, and signal transduction. They are composed of protofilaments of heterodimers of α-tubulin and β-tubulin organized as rigid hollow cylinders that can assemble into large and dynamic intracellular structures. Consistent with their involvement in core cellular processes, affecting microtubule assembly results in cytotoxicity and cell death. For these reasons, microtubules are among the most important targets for the therapeutic treatment of several diseases, including cancer. The vast literature related to microtubule stabilizers and destabilizers has been reviewed extensively in recent years. Here we summarize recent experimental and computational approaches for the identification of novel tubulin modulators and delivery strategies. These include orphan small molecules, PROTACs as well as light-sensitive compounds that can be activated with high spatio-temporal accuracy and that represent promising tools for precision-targeted chemotherapy.

Keywords: PROTAC; artificial intelligence; chemotherapeutic agents; microtubule drugs; photocaged; photopharmacology; photoswitch; tubulin drugs.

FIGURE 1

Functional roles of microtubules (MTs), and chemical molecules affecting their dynamic instability. (A) In mitosis, MTs contribute to the formation of the mitotic spindle, ensuring equal chromosomes segregation into two daughter cells. The mitotic spindle consists of kinetochore, interpolar and astral MTs that exert mechanical forces first to align the metaphase plate and then to divide sister chromatids equally. (B) In neural cells, the cytoskeleton dynamic generated by MTs and actin ensures the proper regeneration and polarization of axons. Bundled MTs are responsible for the elongation of axons, whereas in the growth cone cooperation of filopodial actin and polymerizing MTs leads to elongation. (C) MT protofilament architecture. α- and β-tubulin subunits assemble in a heterodimer (surface representation in light and dark green respectively, (PDB-ID 1JFF)), that form polarized protofilaments. MTs are hollow structures (PDB-ID 6O2T) formed by 13 protofilaments of α/β-tubulin dimers. (D) To elongate the protofilaments, during polymerization GTP-loaded tubulin dimers are added to the growing plus end of the MT, where the dynamics is more rapid compared to the minus end. During MT shrinking, GDP-loaded tubulin dimers dissociate from the lattice.

FIGURE 2

MTAs binding sites, mechanism of action and the photo-pharmacological approach. (A) Binding sites of the major classes of MT stabilizers and destabilizers. Cartoon and surface representation of the α/β-tubulin dimer with the six major sites targeted by MT binding molecules. Most molecules associate with pockets on the β-tubulin subunit, including maytansine (PDB-ID 4TV8, light blue), vinblastine (PDB-ID 5J2T, pink), the Taxane vincadermolide (PDB-ID 5LXT, gold), laulimalide (PDB-ID 4O4H, brown). Colchicine fits at the interface between α -tubulin and β -tubulin subunits of the hetero-dimer (PDB-ID 4O2B, orange). The only characterized MT destabilizer binding to α-tubulin is pironetin (PDB-ID 5LA6, purple). (B) Mechanism of action of the microtubule-binding agents. Diagram of the mechanisms of action of the MSAs Taxane (gold) and laulimalide (brown): these ligands bind between two adjacent β-tubulin subunits stabilizing the lateral interaction between protofilaments in the MT. In the MDAs category, colchicine (orange) inhibits MT growth by preventing the conformational change of the α/β-tubulin dimer required for MT-lattice formation. Vinca-site agents (pink) intercalate between two longitudinally-aligned α/β-tubulin dimers along the MT lattice leading to MT destabilization. Also maytansine agents (light blue) fit on top of the β-tubulin subunit, constraining longitudinal interaction between two α/β-tubulin dimers. Pironetin agents (purple) bind to the α-tubulin subunit and prevent its binding to the α/β-tubulin dimer in the same protofilament. (C) Activation of photocaged compounds. Prior to illumination, the pharmacologically active moiety of the drug is shielded by a photolabile element, which acts as a hindering functionality. Upon illumination, cleavage of the photolabile moiety occurs and the drug becomes irreversibly activated and primed for binding to the target. (D) A photoswitchable element in the scaffold of the drug allows a reversible conformational change upon illumination with a specific wavelength of light, thus causing drug activation. The back reaction can either occur spontaneously upon light cessation (thermal activation), or by illumination with a different wavelength of light. (E) Existing photoswitchable versions of known MTAs. The name of the original MTA drug is indicated first, followed by the type of scaffold used for the synthesis of the photoswitchable analog and by the name of the corresponding photoswitchable MTA thus obtained. (F) Selected examples of microtubule-targeting agents. i) Lead structures of selected microtubule-destabilizing agents used for the development of photoswitchable analogues. ii) Trans-cis photoisomerization reaction of photostatin (PST-1) iii) Trans-cis photoisomerization reaction of styrylbenzothiazole (SBTub3) iv) Chemical structure of a hemithioindigo-based indanone-like tubulin inhibitor (HITub-4) v) Chemical structure of a hemithioindigo colchicinoid tubulin inhibitor (HOTub-31) and a pyrrole hemithioindigo tubulin inhibitor (PHTub-7).