Microtubule-targeting agents (MTAs) such as paclitaxel and the vinca alkaloids are among the most important medical weapons available to combat cancer. MTAs interfere with intracellular transport, ...inhibit eukaryotic cell proliferation, and promote cell death by suppressing microtubule dynamics. Recent advances in the structural analysis of MTAs have enabled the extensive characterization of their interactions with microtubules and their building block tubulin. We review here our current knowledge on the molecular mechanisms used by MTAs to hijack the microtubule cytoskeleton, and discuss dual inhibitors that target both kinases and microtubules. We further formulate some outstanding questions related to MTA structural biology and present possible routes for future investigations of this fascinating class of antimitotic agents.
Structural biology has allowed the identification and detailed characterization of six distinct ligand-binding sites on tubulin. Two sites are targeted by microtubule-stabilizing agents (MSAs); four sites are targeted by microtubule-destabilizing agents (MDAs).
MSAs stabilize microtubules by strengthening lateral and/or longitudinal tubulin contacts in microtubules. MDAs destabilize microtubules by either inhibiting the formation of native tubulin contacts or by hindering the curved-to-straight conformational change of tubulin accompanying microtubule formation.
Different types of anticancer agents that were initially developed against kinases were found to bind also to tubulin as an off-target.
The auristatin class of microtubule destabilizers are highly potent cytotoxic agents against several cancer cell types when delivered as antibody drug conjugates. Here we describe the high resolution ...structures of tubulin in complex with both monomethyl auristatin E and F and unambiguously define the trans-configuration of both ligands at the Val-Dil amide bond in their tubulin bound state. Moreover, we illustrate how peptidic vinca-site agents carrying terminal carboxylate residues may exploit an observed extended hydrogen bond network with the M-loop Arg278 to greatly improve the affinity of the corresponding analogs and to maintain the M-loop in an incompatible conformation for productive lateral tubulin-tubulin contacts in microtubules. Our results highlight a potential, previously undescribed molecular mechanism by which peptidic vinca-site agents maintain unparalleled potency as microtubule-destabilizing agents.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their ...molecular mechanisms of action on tubulin and microtubules remain elusive. We determined high-resolution crystal structures of αβ-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane pocket of β-tubulin and used their respective side chains to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. Further, our results offer fundamental structural insights into the control mechanisms of microtubule dynamics.
Tubulin plays essential roles in vital cellular activities and is the target of a wide range of proteins and ligands. Here, using a combined computational and crystallographic fragment screening ...approach, we addressed the question of how many binding sites exist in tubulin. We identified 27 distinct sites, of which 11 have not been described previously, and analyzed their relationship to known tubulin–protein and tubulin–ligand interactions. We further observed an intricate pocket communication network and identified 56 chemically diverse fragments that bound to 10 distinct tubulin sites. Our results offer a unique structural basis for the development of novel small molecules for use as tubulin modulators in basic research applications or as drugs. Furthermore, our method lays down a framework that may help to discover new pockets in other pharmaceutically important targets and characterize them in terms of chemical tractability and allosteric modulation.
How many binding sites exist in the vital anticancer protein target tubulin? A combined computational and crystallographic fragment screening approach now answers this question and reveals 11 novel sites as well as 56 novel small‐molecule ligands in tubulin.
Significance Microtubules are dynamic protein filaments assembled from tubulin subunits, which play a key role for cell division. Ligands that target microtubules and affect their dynamics belong to ...the most successful classes of chemotherapeutic drugs against cancer by inhibiting cell proliferation. Here we have analyzed three structurally unrelated drugs that destabilize microtubules, using X-ray crystallography. The data reveal a new tubulin-binding site for these drugs, which renders their mechanism of action distinct from that of other types of microtubule assembly inhibitors. Similar key interactions with tubulin are observed for all three ligands, thus defining a common pharmacophore. Our results offer an opportunity for the rational design of potent tubulin modulators for the development of more efficient cancer therapies.
The recent success of antibody–drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubule-destabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on β-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubule-destabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.
Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a ...synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.
Microtubule-targeting agents (MTAs) have demonstrated remarkable efficacy as antitumor, antifungal, antiparasitic, and herbicidal agents, finding applications in the clinical, veterinary, and ...agrochemical industry. Recent advances in tubulin and microtubule structural biology have provided powerful tools that pave the way for the rational design of innovative small-molecule MTAs for future basic and applied life science applications. In this mini-review, we present the current status of the tubulin and microtubule structural biology field, the recent impact it had on the discovery and rational design of MTAs, and exciting avenues for future MTA research.
Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new ...antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin-GB1 complex.
Microtubules are highly dynamic polymers of α,β-tubulin dimers which play an essential role in numerous cellular processes such as cell proliferation and intracellular transport, making them an ...attractive target for cancer and neurodegeneration research. To date, a large number of known tubulin binders were derived from natural products, while only one was developed by rational structure-based drug design. Several of these tubulin binders show promising in vitro profiles while presenting unacceptable off-target effects when tested in patients. Therefore, there is a continuing demand for the discovery of safer and more efficient tubulin-targeting agents. Since tubulin structural data is readily available, the employment of computer-aided design techniques can be a key element to focus on the relevant chemical space and guide the design process. Due to the high diversity and quantity of structural data available, we compiled here a guide to the accessible tubulin-ligand structures. Furthermore, we review different ligand and structure-based methods recently used for the successful selection and design of new tubulin-targeting agents.
Laulimalide and peloruside A are microtubule‐stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X‐ray crystallography it is shown that ...laulimalide and peloruside A bind to a unique non‐taxane site on β‐tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane‐site M‐loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M‐loop of β‐tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.
Microtubule‐stabilizing agents: Laulimalide and peloruside A are attractive next‐generation microtubule‐stabilizing agents for use in combination chemotherapy with taxane‐site drugs. X‐ray crystallography shows that both compounds bind to a unique site on β‐tubulin. The data define the mechanism of action of laulimalide and peloruside on tubulin and microtubules.
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