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It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to βHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by βIII-tubulin (βIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with βIII-tubulin and βIII tubulin-mediated drug resistance. This supports the idea that overexpression of βIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands.

In this research article, we report on the strengthening of a non‐classical hydrogen bond (C−H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E‐selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre‐organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre‐organization, the stabilization by a non‐classical hydrogen bond between the H−C5 of l‐fucose and the ring oxygen O5 of the neighboring d‐galactose moiety is essential and yields 7.4 kJ mol−1. This effect could be further strengthened by replacing l‐fucose by 6,6,6‐trifluoro‐l‐fucose leading to an improved non‐classical H‐bond of 14.9 kJ mol−1, i.e. an improved pre‐organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR‐shifts of the H−C5Fuc, by X‐ray diffraction analysis of glycomimetics co‐crystallized with E‐selectin as well as by isothermal titration calorimetry. Furthermore, the electron‐withdrawing character of the CF3‐group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid‐stability, a prerequisite for gastrointestinal stability, could be substantially improved. [ABSTRACT FROM AUTHOR]

Carbohydrate‐binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent‐exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec‐8, a monomeric I‐type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra‐ and hexavalent and, to an even larger extent, multivalent ligands to Siglec‐8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect. [ABSTRACT FROM AUTHOR]

The d-GlcNAc moiety in sialyl Lewisx (sLex, 1) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of GlcNAc pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLex (1). To test this hypothesis, GlcNAc mimetics consisting of (R,R)-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols (3b-n) was applied. These cyclohexanediol derivatives were incorporated into the sLex mimetics 2b-n. For analyzing the relationship of affinity and core conformation, a 1H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLex mimetics and therefore could be used to quantify the contribution of the R-groups. [ABSTRACT FROM AUTHOR]

To investigate the class‐dependent properties of anti‐viral IgM antibodies, we use membrane antigen capture activated cell sorting to isolate spike‐protein‐specific B cells from donors recently infected with SARS‐CoV‐2, allowing production of recombinant antibodies. We isolate 20, spike‐protein‐specific antibodies of classes IgM, IgG, and IgA, none of which shows any antigen‐independent binding to human cells. Two antibodies of class IgM mediate virus neutralization at picomolar concentrations, but this potency is lost following artificial switch to IgG. Although, as expected, the IgG versions of the antibodies appear to have lower avidity than their IgM parents, this is not sufficient to explain the loss of potency. Synopsis: Virus‐specific B cells of all classes can be isolated from human donors by membrane antigen capture activated cell sorting (MACACS). IgM antibodies include potent neutralizers, but potency is impaired by artificial class switch to IgG. MACACS is a powerful technique for isolating B cells specific for membrane antigens.Serum IgM from recently infected donors is self‐reactive, but monoclonal IgM from MACACS B cells are antigen specific.Isolated IgM include potent virus‐neutralizing antibodies, but this potency is dependent on the IgM structure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Affinity data, such as dissociation constants (KD) or inhibitory concentrations (IC50), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein–ligand complexes and their half‐life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure–kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re‐analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off‐rate constant koff) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on‐rate constant kon). Based on congeneric ligand modifications and structural input from co‐crystal structures, a strong relationship between the formed hydrogen‐bond network and koff could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on kon. [ABSTRACT FROM AUTHOR]

Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established. [ABSTRACT FROM AUTHOR]

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1--dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubulebinding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin- 1 activators, with which the motor transiently interacts as it moves along microtubules. [ABSTRACT FROM AUTHOR]

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (©2021 The Authors; Published by the American Association for Cancer Research.)

The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC 50 ; 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to β -tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

There is an unmet need for new antimitotic drug combinations that target cancer-specific vulnerabilities. Based on our finding of elevated biomolecule oxidation in mitotically arrested cancer cells, we combined Plk1 inhibitors with TH588, an MTH1 inhibitor that prevents detoxification of oxidized nucleotide triphosphates. This combination showed robust synergistic killing of cancer, but not normal, cells that, surprisingly, was MTH1-independent. To dissect the underlying synergistic mechanism, we developed VISAGE, a strategy integrating experimental synergy quantification with computational-pathway-based gene expression analysis. VISAGE predicted, and we experimentally confirmed, that this synergistic combination treatment targeted the mitotic spindle. Specifically, TH588 binding to β-tubulin impaired microtubule assembly, which when combined with Plk1 blockade, synergistically disrupted mitotic chromosome positioning to the spindle midzone. These findings identify a cancer-specific mitotic vulnerability that is targetable using Plk1 inhibitors with microtubule-destabilizing agents and highlight the general utility of the VISAGE approach to elucidate molecular mechanisms of drug synergy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Affinity data, such as dissociation constants (K D ) or inhibitory concentrations (IC 50 ), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein-ligand complexes and their half-life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure-kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re-analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off-rate constant k off ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant k on ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and k off could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on k on ., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)