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Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αβ-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryoelectron microscopy structure of cryptophycin-52 bound to the maytansine site of β-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of β-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "βT5-loop site” that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple β-tubulin binding sites. [ABSTRACT FROM AUTHOR]

Microtubules (MTs) are dynamic filaments of the cytoskeleton, which are formed by the polymerization of their building block tubulin. Perturbation of MT dynamics by MT-targeting agents (MTAs) leads to cell cycle arrest or cell death, a strategy that is pursued in chemotherapy. We recently performed a combined computational and crystallographic fragment screening approach and identified several tubulin-binding fragments. Here, we sought to capitalize on this study with the aim to demonstrate that low affinity tubulin-binding fragments can indeed be used as valuable starting points for the development of active, lead-like antitubulin small molecules. To this end, we report on a new, rationally designed series of 2-aminobenzimidazole derivatives that destabilize MTs by binding tubulin at the colchicine-binding site (CBS). We applied a fragment growing strategy by combining X-ray crystallography and computer-aided drug design. Preliminary structure-activity-relationship studies afforded compound 18 that inhibits HeLa cell viability with a submicromolar activity (IC 50 of 0.9 μM). X-ray crystallography confirmed the compound pose in the CBS, while immunostaining experiments suggested a molecular mechanism of action alike classical CBS ligands with antimitotic and antitumor activity associated with MTs destabilization. This promising outcome underpins that our previously performed combined computational and crystallographic fragment screening approach provides promising starting points for developing new MTAs binding to the CBS of tubulin and, eventually, to further tubulin pockets. [Display omitted] • A fragment growing strategy afforded the new tubulin colchicine-site binder 18. • The representative compound 18 showed low micromolar activity in HeLa cell viability assays. • X-ray crystallography confirmed compound 18 binding to tubulin at the colchicine site. • 18 interferes with microtubule formation in HMF3a cells alike classical colchicine-site ligands. [ABSTRACT FROM AUTHOR]

The selectin family consisting of E−, P - and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P - and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E -selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E−, as well as P -selectin. [Display omitted] • P -selectin ligands require sulfate groups for high binding affinity. • E -selectin can functionally bind sLex-modified glycolipids and glycoproteins. • Optimized glycomimetics of rivipansel improve affinity for E− and P -selectin. • The superior affinity of PSGL-1 for P -selectin results from sulfate interactions. [ABSTRACT FROM AUTHOR]