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, taltobulin (HTI- 286), 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 β and α subunits. Nitrogen and oxygen atoms from the same backbone formed hydrogen bonds with Asn329 from the α subunit and Asp179 from the β 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 β 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.

The potential for infection by coronaviruses (CoVs) has become a serious concern with the recent emergence of Middle East respiratory syndrome and severe acute respiratory syndrome (SARS) in the human population. CoVs encode two large polyproteins, which are then processed into 15–16 nonstructural proteins (nsps) that make significant contributions to viral replication and transcription by assembling the RNA replicase complex. Among them, nsp9 plays an essential role in viral replication by forming a homodimer that binds single‐stranded RNA. Thus, disrupting nsp9 dimerization is a potential anti‐CoV therapy. However, different nsp9 dimer forms have been reported for alpha‐ and beta‐CoVs, and no structural information is available for gamma‐CoVs. Here we determined the crystal structure of nsp9 from the avian infectious bronchitis virus (IBV), a representative gamma‐CoV that affects the economy of the poultry industry because it can infect domestic fowl. IBV nsp9 forms a homodimer via interactions across a hydrophobic interface, which consists of two parallel alpha helices near the carboxy terminus of the protein. The IBV nsp9 dimer resembles that of SARS‐CoV nsp9, indicating that this type of dimerization is conserved among all CoVs. This makes disruption of the dimeric interface an excellent strategy for developing anti‐CoV therapies. To facilitate this effort, we characterized the roles of six conserved residues on this interface using site‐directed mutagenesis and a multitude of biochemical and biophysical methods. We found that three residues are critical for nsp9 dimerization and its abitlity to bind RNA.

In this study, the sensitivity of the CB2 receptor to methanethiosulfonate (MTS) derivatives was tested, and a native cysteine residue conferring the sensitivity was identified. By incubating human embryonic kidney 293 cells stably transfected with CB2 receptors and MTS derivatives such as MTS ethylammonium (MTSEA), [³H]HU-243 binding was inhibited. Pretreatment of the CB2 receptor with cannabinoid ligands prevented this inhibition, suggesting that MTSEA modification occurred within the binding crevice. To identify the cysteine(s) responsible for the MTSEA sensitivity, 10 CB2 mutants were prepared in which the eight cysteines in transmembrane domains or extracellular loop 2 were mutated to serine or alanine, one at a time or in combination. Five mutants exhibited specific [³H]HU-243 binding, with K_d and B_max values similar to those of wild-type CB2. However, five other mutants had no detectable ligand binding and were not detected on cell membranes by Western blot analysis. Among the five mutants with normal binding, only the sensitivity to MTSEA of the C2.59(89)S mutant was reduced significantly. These data demonstrate that C2.59(89) is the residue that mainly confers the inhibitory effect of MTSEA on ligand binding. Furthermore, the magnitude of the second-order rate constant (1.14 ± 0.28 M^–1s^–1) for the MTSEA reaction with wild-type CB2 suggests that C2.59(89) resides at the margin of the CB2 binding site crevice. The accessibility of C2.59(89) to MTSEA provides experimental evidence for a possible conformational difference between TMH2 of CB2 versus Rho. Modeling studies undertaken to explore the origin of such differences suggest it is possibly caused by the conformational influence of S2.54(84).