Structural Insights into the Pharmacophore of Vinca Domain Inhibitors of Microtubules

Abstract

Antibody-drug conjugates (ADCs) have achieved great success in cancer therapy in recent years. Some peptidyl microtubule inhibitors consisting of natural and unnatural amino acids, such as monomethyl auristatin E (MMAE) and F (MMAF), are extremely cytotoxic and have been used as a payload in ADCs.However, their precise molecular interaction with tubulin and microtubules remains unclear. We determined the crystal structures of tubulin in complex with three ultra-potent peptidyl microtubule inhibitors [MMAE,, and tubulysin M] at 2.5 Å. Our data showed that the three peptides bound to the vinca domain and shared a common and key pharmacophore containing two consecutive hydrophobic groups (Val, Ile-like side chain). These groups protruded in opposite directions into hydrophobic pockets on the tubulin b and a subunits. Nitrogen and oxygen atoms from the same backbone formed hydrogen bonds with Asn329 from the a subunit and Asp179 from the b subunit in a direction normal to the surface formed by the aforementioned hydrophobic groups. In addition, our crystal structure data indicated that tubulysin M bound to the b subunit alone, providing a structural explanation for its higher affinity. We also compared the conformations of two representative structurally different vinca domain compounds, ustiloxin D and vinblastine, with those of the aforementioned peptidyl ligands, and found that they shared a similar pharmacophore. Our findings lay a foundation for the rational design of novel vinca domain ligands and may facilitate the development of microtubule inhibitors with high specificity, affinity, and efficiency as payloads for ADCs in cancer therapy.

Key analogs of a uniquely potent synthetic vinblastine that contain modifications of the C20′ ethyl substituent

“…X-ray crystal structure of tubulin-bound vinblastine (pdb 1Z2B) 16a highlighting the C20′ ethyl binding site at the dimer–dimer interface where vinblastine binds (left) and site of binding with top of proteins removed to visualize bound vinblastine (right).…”

“…The upper catharanthine-derived (velbanamine) subunit is deeply imbedded in the tubulin binding site located at the α/β-tubulin dimer-dimer interface. 16 In contrast to the C20′ ureas that reside at a site that permits their extension along and further disruption of the continuing protein–protein interaction, the indole group and ethyl substituent each are anchored tightly in small hydrophobic pockets found on the α- or β-tubulin subunits, respectively. Each occupies the opposite top corners of a T-shaped tubulin-bound conformation of vinblastine with the core of the velbanamine subunit filling the intervening space and serving as a rigid scaffold that fixes the placement of these two anchoring groups.…”

“…The results are presented below and displayed remarkable trends. Little free space is present in the hydrophobic pocket surrounding the ethyl group in the X-ray crystal structures 16 of vinblastine bound to tubulin (Figure 2). The earlier and remarkable improvements in potency and tubulin binding affinity for 6 , which incorporates the ethyl group constrained in a six-membered ring adding only two sp 3 methylenes and the C15′ stereocenter, may be attributed to its ability to more favorably occupy and fill the ethyl binding site without changing the intrinsic conformational or structural features of the natural product.…”

(−)-Rhazinilam and the diphenylpyridazinone NSC 613241: Two compounds inducing the formation of morphologically similar tubulin spirals but binding apparently to two distinct sites on tubulin

“…The (−)-rhazinilam- and DPP-induced spirals are very different morphologically from those induced by the vinca alkaloids, as shown above. Nevertheless, the crystal structures of tubulin with bound vinblastine [40] or a bound dolastatin 10 analogue [41,42] strongly suggest that the binding sites of compounds that induce aberrant assembly reactions are generated by the interaction of two tubulin dimers. In these crystals, the binding sites are formed by the interaction of α-tubulin on one dimer with β-tubulin on a second dimer.…”

Tubulin as a sensitive target of nanosecond-scale intense electric field: quantitative insights from molecular dynamics simulations

“…Residues of drug binding sites 425 The paclitaxel, colchicine/nocodazole and vinca binding sites were identified based on 426 the RSCB PDB structures 1JFF (paclitaxel is named as residue TA1, site identifier 427 AC5) [65], 1SA0 (colchicine is named as residue CN2, site identifier AC8) [66] and 428 5BMV (vinblastine is named as residue VLB, site identifier AE1) [67], respectively. The 429 residues belonging to the respective binding sites are identified there using an algorithm 430 from [68] and are available in a respective .pdb file.…”