Modular synthesis of functional libraries by accelerated SuFEx click chemistry.

Accelerated SuFEx Click Chemistry (ASCC) is a powerful method for coupling aryl and alkyl alcohols with SuFEx-compatible functional groups. With its hallmark favorable kinetics and exceptional product yields, ASCC streamlines the synthetic workflow, simplifies the purification process, and is ideally suited for discovering functional molecules. We showcase the versatility and practicality of the ASCC reaction as a tool for the late-stage derivatization of bioactive molecules and in the array synthesis of sulfonate-linked, high-potency, microtubule targeting agents (MTAs) that exhibit nanomolar anticancer activity against multidrug-resistant cancer cell lines. These findings underscore ASCC's promise as a robust platform for drug discovery.

Microtubule specialization by +TIP networks: from mechanisms to functional implications.

To fulfill their actual cellular role, individual microtubules become functionally specialized through a broad range of mechanisms. The 'search and capture' model posits that microtubule dynamics and functions are specified by cellular targets that they capture (i.e., a posteriori), independently of the microtubule-organizing center (MTOC) they emerge from. However, work in budding yeast indicates that MTOCs may impart a functional identity to the microtubules they nucleate, a priori. Key effectors in this process are microtubule plus-end tracking proteins (+TIPs), which track microtubule tips to regulate their dynamics and facilitate their targeted interactions. In this review, we discuss potential mechanisms of a priori microtubule specialization, focusing on recent findings indicating that +TIP networks may undergo liquid biomolecular condensation in different cell types.

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers.

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips.

Kinesins are microtubule-dependent motor proteins, some of which moonlight as microtubule polymerases, such as the yeast protein Kip2. Here, we show that the CLIP-170 ortholog Bik1 stabilizes Kip2 at microtubule ends where the motor domain of Kip2 promotes microtubule polymerization. Live-cell imaging and mathematical estimation of Kip2 dynamics reveal that disrupting the Kip2-Bik1 interaction aborts Kip2 dwelling at microtubule ends and abrogates its microtubule polymerization activity. Structural modeling and biochemical experiments identify a patch of positively charged residues that enables the motor domain to bind free tubulin dimers alternatively to the microtubule shaft. Neutralizing this patch abolished the ability of Kip2 to promote microtubule growth both in vivo and in vitro without affecting its ability to walk along microtubules. Our studies suggest that Kip2 utilizes Bik1 as a cofactor to track microtubule tips, where its motor domain then recruits free tubulin and catalyzes microtubule assembly.

Structural insight into the stabilization of microtubules by taxanes.

Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules' diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a ßM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.

Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography.

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

Multivalency ensures persistence of a +TIP body at specialized microtubule ends.

Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential for cell division and morphogenesis. Here we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIP proteins Kar9 (functional homologue of APC, MACF and SLAIN), Bim1 (orthologous to EB1) and Bik1 (orthologous to CLIP-170). A multivalent web of redundant interactions links the three +TIPs together to form a '+TIP body' at the end of chosen microtubules. This body behaves as a liquid condensate that allows it to persist on both growing and shrinking microtubule ends, and to function as a mechanical coupling device between microtubules and actin cables. Our study identifies nanometre-scale condensates as effective cellular structures and underlines the power of dissecting the web of low-affinity interactions driving liquid-liquid phase separation in order to establish how condensation processes support cell function.

Chemical modulation of microtubule structure through the laulimalide/peloruside site.

Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics. Here, we use a combination of the parental natural compounds and derived analogs to unravel the stabilization mechanism through this site. These drugs settle lateral interactions without engaging the M loop, which is part of the key and lock involved in the inter-protofilament contacts. Importantly, these drugs can modulate the angle between protofilaments, producing microtubules of different diameters. Among the compounds studied, we have found some showing low cytotoxicity and able to induce stabilization without compromising microtubule native structure. This opens the window of new applications for microtubule-stabilizing agents beyond cancer treatment.

VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules.

The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of action is little known. Here, we show in reconstituted systems and cells that VASH1-SVBP and VASH2-SVBP drive the global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH region is critical for microtubule detachment and diffusion of VASH-SVBP enzymes on lattices. Our results suggest a mechanism by which VASH1-SVBP and VASH2-SVBP could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.

Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts.

Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions, including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent αß-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the αß-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in Xenopus egg cytoplasmic extracts. We find that in almost all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.

Development of [1,2]oxazoloisoindoles tubulin polymerization inhibitors: Further chemical modifications and potential therapeutic effects against lymphomas.

Lymphomas are among the ten most common cancers, and, although progress has been achieved in increasing survival, there is still an unmet need for more effective therapeutic approaches, including better options for patients with refractory tumors that initially respond but then relapse. The lack of effective alternative treatment options highlights the need to develop new therapeutic strategies capable of improving survival prospects for lymphoma patients. Herein, we describe the identification and exploration of the SAR of a series of [1,2]oxazolo[5,4-e]isoindoles as potent small molecules that bind to the colchicine site of tubulin and that have promise for the treatment of refractory lymphomas. Exploration of the chemical space of this class of compounds at the pyrrole moiety and at the [1,2]oxazole ring highlighted two compounds bearing a 3,5-dimethoxybenzyl and a 3,4,5-trimethoxybenzyl group as potent candidates and showed that structural modifications at the isoxazole moiety are generally not favorable for activity. The two best candidates showed efficacy against different lymphoma histotypes and displayed 88 and 80% inhibition of colchicine binding fitting well into the colchicine pocket, as demonstrated by X-ray crystallography T2R-TTL-complexes, docking and thermodynamic analysis of the tubulin-colchicine complex structure. These results were confirmed by transcriptome data, thus indicating [1,2]oxazolo[5,4-e]isoindoles are promising candidates as antitubulin agents for the treatment of refractory lymphomas.

Novel fragment-derived colchicine-site binders as microtubule-destabilizing agents.

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 (IC50 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.

Crystallization Systems for the High-Resolution Structural Analysis of Tubulin-Ligand Complexes.

Since the first moderate resolution, structural description of Taxol bound to tubulin by electron crystallography in 1998, several tubulin crystal systems have been developed and optimized for the high-resolution analysis of tubulin-ligand complexes by X-ray crystallography. Here we describe three tubulin crystal systems that have allowed investigating the molecular mechanisms of action of a large number of diverse anti-tubulin agents.

Rational Design of a Novel Tubulin Inhibitor with a Unique Mechanism of Action.

In this study, we capitalized on our previously performed crystallographic fragment screen and developed the antitubulin small molecule Todalam with only two rounds of straightforward chemical synthesis. Todalam binds to a novel tubulin site, disrupts microtubule networks in cells, arrests cells in G2/M, induces cell death, and synergizes with vinblastine. The compound destabilizes microtubules by acting as a molecular plug that sterically inhibits the curved-to-straight conformational switch in the α-tubulin subunit, and by sequestering tubulin dimers into assembly incompetent oligomers. Our results describe for the first time the generation of a fully rationally designed small molecule tubulin inhibitor from a fragment, which displays a unique molecular mechanism of action. They thus demonstrate the usefulness of tubulin-binding fragments as valuable starting points for innovative antitubulin drug and chemical probe discovery campaigns.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Inhibiting parasite proliferation using a rationally designed anti-tubulin agent.

Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand-binding sites in tubulin makes this protein an attractive target for anti-parasite drug discovery. However, despite remarkable successes as anti-cancer agents, the rational development of protozoan parasite-specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug-binding sites. Based on this information, we rationally designed the parasite-specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species-specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much-needed, novel parasite inhibitors.

Preclinical and Early Clinical Development of PTC596, a Novel Small-Molecule Tubulin-Binding Agent.

PTC596 is an investigational small-molecule tubulin-binding agent. Unlike other tubulin-binding agents, PTC596 is orally bioavailable and is not a P-glycoprotein substrate. So as to characterize PTC596 to position the molecule for optimal clinical development, the interactions of PTC596 with tubulin using crystallography, its spectrum of preclinical in vitro anticancer activity, and its pharmacokinetic-pharmacodynamic relationship were investigated for efficacy in multiple preclinical mouse models of leiomyosarcomas and glioblastoma. Using X-ray crystallography, it was determined that PTC596 binds to the colchicine site of tubulin with unique key interactions. PTC596 exhibited broad-spectrum anticancer activity. PTC596 showed efficacy as monotherapy and additive or synergistic efficacy in combinations in mouse models of leiomyosarcomas and glioblastoma. PTC596 demonstrated efficacy in an orthotopic model of glioblastoma under conditions where temozolomide was inactive. In a first-in-human phase I clinical trial in patients with cancer, PTC596 monotherapy drug exposures were compared with those predicted to be efficacious based on mouse models. PTC596 is currently being tested in combination with dacarbazine in a clinical trial in adults with leiomyosarcoma and in combination with radiation in a clinical trial in children with diffuse intrinsic pontine glioma.

Structure and regulation of the microtubule plus-end tracking protein Kar9.

In many eukaryotes, coordination of chromosome segregation with cell cleavage relies on the patterned interaction of specific microtubules with actin filaments through dedicated microtubule plus-end tracking proteins (+TIPs). However, how these +TIPs are spatially controlled is unclear. The yeast +TIP Kar9 drives one of the spindle aster microtubules along actin cables to align the mitotic spindle with the axis of cell division. Here, we report the crystal structure of Kar9's folded domain, revealing spectrin repeats reminiscent of the +TIPs MACF/ACF7/Shot and PRC1/Ase1. Point mutations abrogating spectrin-repeat-mediated dimerization of Kar9 reduced and randomized Kar9 distribution to microtubule tips, and impaired spindle positioning. Six Cdk1 sites surround the Kar9 dimerization interface. Their phosphomimetic substitution inhibited Kar9 dimerization, displaced Kar9 from microtubules, and affected its interaction with the myosin motor Myo2. Our results provide molecular-level understanding on how diverse cell types may regulate and pattern microtubule-actin interactions to orchestrate their divisions.

1,3-Benzodioxole-Modified Noscapine Analogues: Synthesis, Antiproliferative Activity, and Tubulin-Bound Structure.

Since the revelation of noscapine's weak anti-mitotic activity, extensive research has been conducted over the past two decades, with the goal of discovering noscapine derivatives with improved potency. To date, noscapine has been explored at the 1, 7, 6', and 9'-positions, though the 1,3-benzodioxole motif in the noscapine scaffold that remains unexplored. The present investigation describes the design, synthesis and pharmacological evaluation of noscapine analogues consisting of modifications to the 1,3-benzodioxole moiety. This includes expansion of the dioxolane ring and inclusion of metabolically robust deuterium and fluorine atoms. Favourable structural modifications were subsequently incorporated into multi-functionalised noscapine derivatives that also possessed modifications previously shown to promote anti-proliferative activity in the 1-, 6'- and 9'-positions. Our research efforts afforded the deuterated noscapine derivative 14 e and the dioxino-containing analogue 20 as potent cytotoxic agents with EC50 values of 1.50 and 0.73 µM, respectively, against breast cancer (MCF-7) cells. Compound 20 also exhibited EC50 values of <2 µM against melanoma, non-small cell lung carcinoma, and cancers of the brain, kidney and breast in an NCI screen. Furthermore, compounds 14 e and 20 inhibit tubulin polymerisation and are not vulnerable to the overexpression of resistance conferring P-gp efflux pumps in drug-resistant breast cancer cells (NCIADR/RES ). We also conducted X-ray crystallography studies that yielded the high-resolution structure of 14 e bound to tubulin. Our structural analysis revealed the key interactions between this noscapinoid and tubulin and will assist with the future design of noscapine derivatives with improved properties.

Comprehensive Analysis of Binding Sites in Tubulin.

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.