NMK-TD-100, a Novel Microtubule Modulating Agent,Blocks Mitosis and Induces Apoptosis in HeLa Cells by Binding to Tubulin

Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC₅₀ value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC₅₀ value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.     

Cellular Effects of Curcumin on Plasmodium falciparum Include Disruption of Microtubules

Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC₅₀ of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.     

Molecular determinants for α-tubulin methylation by SETD2

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2–Rpb1–interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.     

Tubulin isotypes optimize distinct spindle positioning mechanisms during yeast mitosis

Microtubules are dynamic cytoskeleton filaments that are essential for a wide range of cellular processes. They are polymerized from tubulin, a heterodimer of α- and β-subunits. Most eukaryotic organisms express multiple isotypes of α- and β-tubulin, yet their functional relevance in any organism remains largely obscure. The two α-tubulin isotypes in budding yeast, Tub1 and Tub3, are proposed to be functionally interchangeable, yet their individual functions have not been rigorously interrogated. Here, we develop otherwise isogenic yeast strains expressing single tubulin isotypes at levels comparable to total tubulin in WT cells. Using genome-wide screening, we uncover unique interactions between the isotypes and the two major mitotic spindle positioning mechanisms. We further exploit these cells to demonstrate that Tub1 and Tub3 optimize spindle positioning by differentially recruiting key components of the Dyn1- and Kar9-dependent mechanisms, respectively. Our results provide novel mechanistic insights into how tubulin isotypes allow highly conserved microtubules to function in diverse cellular processes.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

Identification of novel non-toxic and anti-angiogenic α-fluorinated chalcones as potent colchicine binding site inhibitors

α-Fluorinated chalcones were prepared and evaluated for their cell growth inhibitory properties against six human cancer cell lines. The most potent chalcone 4c demonstrated excellent selective toxicity against cancer cells versus normal human cells, with IC₅₀ values at nanomolar concentration ranges against 5 cancer cell lines. A further study revealed that 4c could bind to the colchicine site of tubulin, disrupt the cell microtubule networks, and effectively inhibit tubulin polymerisation. Cellular-based mechanism studies elucidated that 4c arrested MGC-803 cell cycle at G2/M phase. In addition, 4c dose-dependently caused Caspase-induced apoptosis of MGC-803 cells through mitochondrial dysfunction. Notably, compound 4c was found to inhibit the HUVECs tube formation, migration, and invasion in vitro. Furthermore, our data suggested that treatment with 4c significantly reduced MGC-803 cells metastasis and proliferation in vitro. Overall, this work showed that chalcone hybrid 4c is a potent inhibitor of tubulin assembly with prominent anti-angiogenesis and anti-cancer properties.     

Rhizoxin binding to tubulin at the maytansine-binding site

The binding of rhizoxin, a potent inhibitor of mitosis and in vitro microtubule assembly, to porcine brain tubulin was studied. Tubulin possesses one binding site for rhizoxin per molecule with a dissociation constant (Kd) of 1.7.10(-7) M. Ansamitocin P-3, a homologue of maytansine, was a competitive inhibitor of rhizoxin binding, with an inhibition constant of 1.3.10(-7) M. Vinblastine also inhibited rhizoxin binding, but was not fully competitive, and the inhibition constant was 2.9.10(-6) M. In contrast, both rhizoxin and ansamitocin P-3 were potent inhibitors of vinblastine binding. Rhizoxin inhibited tau-promoted tubulin assembly, but it, differing from vinblastine, did not induce tubulin aggregation into spirals, even at a concentration as high as 2.10(-5) M. In addition, rhizoxin strongly inhibited vinblastine-induced tau-dependent tubulin aggregation. Rhizoxin binding to tubulin was completely independent from colchicine binding. These effects resemble those of maytansine. The results suggested that rhizoxin binds to the maytansine-binding site and that the binding sites of rhizoxin and vinblastine are not the same.     

Effect of Ansamitocins on Growth of Yeasts

Ansamitocins in combination with amphotericin B produced synergistic inhibition on the growth of several yeasts in liquid cultures, Ansamitocin P–3 at 5 µg/ml completely suppressed the growth of Saccharomyces cerevisiae whereas ansamitocin P–3 alone at 50 µg/ml hardly affected growth. Ansamitocin P–4 and maytansine also showed synergistic activity with amphotericin B against S. cerevisiae. The synergism also occurred in cultures of Candida albicans and Hansenula anomala. Combinations of ansamitocin P–3 with various agents revealed that the synergism depended on the specific property of amphotericin B. Ansamitocins showed no interfering activity against regeneration of protoplasts of S. cerevisiae. These results suggest that the limited activity of ansamitocins against these yeasts is due to the membrane permeability barrier of these cells.     

Angiogenesis and anti-leukaemia activity of novel indole derivatives as potent colchicine binding site inhibitors

The screened compound DYT-1 from our in-house library was taken as a lead (inhibiting tubulin polymerisation: IC₅₀=25.6 µM, anti-angiogenesis in Zebrafish: IC₅₀=38.4 µM, anti-proliferation against K562 and Jurkat: IC₅₀=6.2 and 7.9 µM, respectively). Further investigation of medicinal chemistry conditions yielded compound 29e (inhibiting tubulin polymerisation: IC₅₀=4.8 µM and anti-angiogenesis in Zebrafish: IC₅₀=3.6 µM) based on tubulin and zebrafish assays, which displayed noteworthily nanomolar potency against a variety of leukaemia cell lines (IC₅₀= 0.09–1.22 µM), especially K562 cells where apoptosis was induced. Molecular docking, molecular dynamics (MD) simulation, radioligand binding assay and cellular microtubule networks disruption results showed that 29e stably binds to the tubulin colchicine site. 29e significantly inhibited HUVEC tube formation, migration and invasion in vitro. Anti-angiogenesis in vivo was confirmed by zebrafish xenograft. 29e also prominently blocked K562 cell proliferation and metastasis in blood vessels and surrounding tissues of the zebrafish xenograft model. Together with promising physicochemical property and metabolic stability, 29e could be considered an effective anti-angiogenesis and -leukaemia drug candidate that binds to the tubulin colchicine site.     

Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

The trypanosomatid cytoskeleton is responsible for the parasite''s shape and it is modulated throughout the different stages of the parasite''s life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite''s hypotonic stress-induced shape change, as well as its recovery.     

The Amino Acid Exchange R28E in Ciliary Neurotrophic Factor (CNTF) Abrogates Interleukin-6 Receptor-dependent but Retains CNTF Receptor-dependent Signaling via Glycoprotein 130 (gp130)/Leukemia Inhibitory Factor Receptor (LIFR)

Ciliary neurotrophic factor (CNTF) is a neurotrophic factor with therapeutic potential for neurodegenerative diseases. Moreover, therapeutic application of CNTF reduced body weight in mice and humans. CNTF binds to high or low affinity receptor complexes consisting of CNTFR·gp130·LIFR or IL-6R·gp130·LIFR, respectively. Clinical studies of the CNTF derivative Axokine revealed intolerance at higher concentrations, which may rely on the low-affinity binding of CNTF to the IL-6R. Here, we aimed to generate a CNTFR-selective CNTF variant (CV). CV-1 contained the single amino acid exchange R28E. Arg²⁸ is in close proximity to the CNTFR binding site. Using molecular modeling, we hypothesized that Arg²⁸ might contribute to IL-6R/CNTFR plasticity of CNTF. CV-2 to CV-5 were generated by transferring parts of the CNTFR-binding site from cardiotrophin-like cytokine to CNTF. Cardiotrophin-like cytokine selectively signals via the CNTFR·gp130·LIFR complex, albeit with a much lower affinity compared with CNTF. As shown by immunoprecipitation, all CNTF variants retained the ability to bind to CNTFR. CV-1, CV-2, and CV-5, however, lost the ability to bind to IL-6R. Although all variants induced cytokine-dependent cellular proliferation and STAT3 phosphorylation via CNTFR·gp130·LIFR, only CV-3 induced STAT3 phosphorylation via IL-6R·gp130·LIFR. Quantification of CNTF-dependent proliferation of CNTFR·gp130·LIFR expressing cells indicated that only CV-1 was as biologically active as CNTF. Thus, the CNTFR-selective CV-1 will allow discriminating between CNTFR- and IL-6R-mediated effects in vivo.     

Structural and Thermodynamic Characterization of Cadherin·β-Catenin·α-Catenin Complex Formation

The classical cadherin·β-catenin·α-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although α-catenin binds to β-catenin and to F-actin, β-catenin significantly weakens the affinity of α-catenin for F-actin. Moreover, α-catenin self-associates into homodimers that block β-catenin binding. We investigated quantitatively and structurally αE- and αN-catenin dimer formation, their interaction with β-catenin and the cadherin·β-catenin complex, and the effect of the α-catenin actin-binding domain on β-catenin association. The two α-catenin variants differ in their self-association properties: at physiological temperatures, αE-catenin homodimerizes 10× more weakly than does αN-catenin but is kinetically trapped in its oligomeric state. Both αE- and αN-catenin bind to β-catenin with a K_d of 20 nm, and this affinity is increased by an order of magnitude when cadherin is bound to β-catenin. We describe the crystal structure of a complex representing the full β-catenin·αN-catenin interface. A three-dimensional model of the cadherin·β-catenin·α-catenin complex based on these new structural data suggests mechanisms for the enhanced stability of the ternary complex. The C-terminal actin-binding domain of α-catenin has no influence on the interactions with β-catenin, arguing against models in which β-catenin weakens actin binding by stabilizing inhibitory intramolecular interactions between the actin-binding domain and the rest of α-catenin.     

Conformational changes in tubulin upon binding cryptophycin-52 reveal its mechanism of action

Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC₅₀ values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we have determined the binding site of Cp-52, and its parent compound, cryptophycin-1, on HeLa tubulin, to a resolution of 3.3 Å and 3.4 Å, respectively, by cryo-EM and characterized this binding further by molecular dynamics simulations. The binding site was determined to be located at the tubulin interdimer interface and partially overlap that of maytansine, another cytotoxic tubulin inhibitor. Binding induces curvature both within and between tubulin dimers that is incompatible with the microtubule lattice. Conformational changes occur in both α-tubulin and β-tubulin, particularly in helices H8 and H10, with distinct differences between α and β monomers and between Cp-52-bound and cryptophycin-1-bound tubulin. From these results, we have determined: (i) the mechanism of action of inhibition of both microtubule polymerization and depolymerization, (ii) how the affinity of Cp-52 for tubulin may be enhanced, and (iii) where linkers for targeted delivery can be optimally attached to this molecule.     

A radioligand binding assay for antitubulin activity in tumor cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.     

Design,synthesis and biological evaluation of novel thiazole-naphthalene derivatives as potential anticancer agents and tubulin polymerisation inhibitors

A novel series of thiazole-naphthalene derivatives as tubulin polymerisation inhibitors were designed, synthesised, and evaluated for the anti-proliferative activities. The majority of the tested compounds exhibited moderate to potent antiproliferative activity on the MCF-7 and A549 cancer cell lines. Among them, compound 5b was found to be the most active compound with IC₅₀ values of 0.48 ± 0.03 and 0.97 ± 0.13 μM. Moreover, mechanistic studies revealed that 5b significantly inhibited tubulin polymerisation with an IC₅₀ value of 3.3 µM, as compared to the standard drug colchicine (IC₅₀ = 9.1 μM). Further cellular mechanism studies elucidated that 5b arrested the cell cycle at G2/M phase and induced apoptosis in MCF-7 cancer cells. Molecular modelling study indicated that 5b binds well to the colchicine binding site of tubulin. In summary, these results suggest that 5b represents a promising tubulin polymerisation inhibitor worthy of further investigation as potential anticancer agents.     

Rational Design,Synthesis, and Biological Evaluation of Third Generation α-Noscapine Analogues as Potent Tubulin Binding Anti-Cancer Agents

Systematic screening based on structural similarity of drugs such as colchicine and podophyllotoxin led to identification of noscapine, a microtubule-targeted agent that attenuates the dynamic instability of microtubules without affecting the total polymer mass of microtubules. We report a new generation of noscapine derivatives as potential tubulin binding anti-cancer agents. Molecular modeling experiments of these derivatives 5a, 6a-j yielded better docking score (-7.252 to -5.402 kCal/mol) than the parent compound, noscapine (-5.505 kCal/mol) and its existing derivatives (-5.563 to -6.412 kCal/mol). Free energy (ΔG _bind) calculations based on the linear interaction energy (LIE) empirical equation utilizing Surface Generalized Born (SGB) continuum solvent model predicted the tubulin-binding affinities for the derivatives 5a, 6a-j (ranging from -4.923 to -6.189 kCal/mol). Compound 6f showed highest binding affinity to tubulin (-6.189 kCal/mol). The experimental evaluation of these compounds corroborated with theoretical studies. N-(3-brormobenzyl) noscapine (6f) binds tubulin with highest binding affinity (K_D, 38 ± 4.0 µM), which is ~ 4.0 times higher than that of the parent compound, noscapine (K_D, 144 ± 1.0 µM) and is also more potent than that of the first generation clinical candidate EM011, 9-bromonoscapine (K_D, 54 ± 9.1 µM). All these compounds exhibited substantial cytotoxicity toward cancer cells, with IC₅₀ values ranging from 6.7 µM to 72.9 µM; compound 6f showed prominent anti-cancer efficacy with IC₅₀ values ranging from 6.7 µM to 26.9 µM in cancer cells of different tissues of origin. These compounds perturbed DNA synthesis, delayed the cell cycle progression at G2/M phase, and induced apoptotic cell death in cancer cells. Collectively, the study reported here identified potent, third generation noscapinoids as new anti-cancer agents.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

Plasma Testosterone and Testosterone Binding Affinities in Men with Impotence,Oligospermia, Azoospermia,and Hypogonadism

Mean plasma testosterone levels (± S.D.), using Sephadex LH-20 and competitive protein binding, were 629 ± 160 ng/100 ml for a group of 27 normal adult men, 650 ± 205 ng/100 ml for 27 impotent men with normal secondary sex characteristics, 644 ± 178 ng/100 ml for 20 men with oligospermia, and 563 ± 125 ng/100 ml for 16 azoospermic men. None of these values differ significantly. For 21 men with clinical evidence of hypogonadism the mean plasma testosterone (± S.D.), at 177 ± 122 ng/100 ml, differed significantly (P < 0·001) from that of the normal men.The mean testosterone binding affinities (as measured by the reciprocal of the quantity of plasma needed to bind 50% of ³H-testosterone tracer) were similar for normal, impotent, and oligospermic men. Though lower for azoospermic men the difference was not significant (P >0·1). For 12 of the 16 hypogonadal males the testosterone binding affinity was normal, but raised binding affinities, similar to those found in normal adult females or prepubertal boys (about twice normal adult male levels), were found in four cases of delayed puberty. These findings help to explain why androgen therapy is usually useless in the treatment of impotence.     

Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

Microtubule-associated proteins regulate microtubule (MT) dynamics spatially and temporally, which is essential for proper formation of the bipolar mitotic spindle. The XMAP215 family is comprised of conserved microtubule-associated proteins that use an array of tubulin-binding tumor overexpressed gene (TOG) domains, consisting of six (A–F) Huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin (HEAT) repeats, to robustly increase MT plus-end polymerization rates. Recent work showed that TOG domains have differentially conserved architectures across the array, with implications for position-dependent TOG domain tubulin binding activities and function within the XMAP215 MT polymerization mechanism. Although TOG domains 1, 2, and 4 are well described, structural and mechanistic information characterizing TOG domains 3 and 5 is outstanding. Here, we present the structure and characterization of Drosophila melanogaster Mini spindles (Msps) TOG3. Msps TOG3 has two unique features as follows: the first is a C-terminal tail that stabilizes the ultimate four HEAT repeats (HRs), and the second is a unique architecture in HR B. Structural alignments of TOG3 with other TOG domain structures show that the architecture of TOG3 is most similar to TOG domains 1 and 2 and diverges from TOG4. Docking TOG3 onto recently solved Stu2 TOG1· and TOG2·tubulin complex structures suggests that TOG3 uses similarly conserved tubulin-binding intra-HEAT loop residues to engage α- and β-tubulin. This indicates that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence. The similarity of TOG domains 1–3 and the divergence of TOG4 suggest that a TOG domain array with polarized structural diversity may play a key mechanistic role in XMAP215-dependent MT polymerization activity.     

Binding selectivity of rhizoxin, phomopsin A, vinblastine, and ansamitocin P-3 to fungal tubulins: differential interactions of these antimitotic agents with brain and fungal tubulins.

The binding of four potent antimitotic agents, rhizoxin (RZX), phomopsin A (PMS-A), ansamitocin P-3 (ASMP-3), and vinblastine (VLB), to tubulins from RZX-sensitive and -resistant strains of Aspergillus nidulans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae was investigated. Mycelial extracts to which RZX could bind contained beta-tubulin with Asn as the 100th amino acid residue (Asn-100) in all cases, and those without affinity for RZX contained beta-tubulins with either Ile-100 or Val-100. Though PMS-A shares the same binding site as RZX and ASMP-3 on porcine brain tubulin (Asn-100), only ASMP-3 bound Asn-100 fungal tubulins in a competitive manner with respect to RZX. PMS-A and VLB, which strongly bind to porcine brain tubulin, did not bind to any of the fungal mycelial extracts examined. The results indicate differential interactions of these antimitotic agents with brain and fungal tubulins.