Antiproliferative mechanism of action of cryptophycin-52: kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>/=10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC50 = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC50 = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [3H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (Kd, 47 nM, maximum of approximately 19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that approximately 5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.     

Mechanism of action of E7010, an orally active sulfonamide antitumor agent: inhibition of mitosis by binding to the colchicine site of tubulin

E7010 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonami de), an orally active sulfonamide antitumor agent that is currently in a Phase I clinical trial, showed rather consistent growth-inhibitory activities against a panel of 26 human tumor cell lines (IC50 = 0.06-0.8 microg/ml), in contrast to vincristine (VCR; IC50 = 0.0002-0.04 microg/ml), 5-fluorouracil (IC50 = 0.2-30 microg/ml), Adriamycin (IC50 = 0.002-0.7 microg/ml), mitomycin C (IC50 = 0.007-3 microg/ml), 1-beta-D-arabinofuranoxylcytosine (IC50 = 0.005 to >30 microg/ml), camptothecin (IC50 = 0.002-0.4 microg/ml), and cisplatin (IC50 = 0.5-20 microg/ml). It caused a dose-dependent increase in the percentage of mitotic cells in parallel with a decrease in cell proliferation, like VCR. It also showed a dose-dependent inhibition of tubulin polymerization, which correlated well with the cell growth-inhibitory activity. 14C-labeled E7010 bound to purified tubulin, and this binding was inhibited by colchicine but not by VCR. However, its binding properties were different from those of colchicine, as well as those of VCR. E7010 was active against two kinds of VCR-resistant P388 cell lines, one of which showed multidrug resistance due to the overexpression of P-glycoprotein (resistant to Taxol), and the other did not show multidrug resistance (sensitive to Taxol). Furthermore, four E7010-resistant P388 cell lines showed no cross-resistance to VCR, a different pattern of resistance to podophyllotoxin, and collateral sensitivity to Taxol. Therefore, E7010 is a novel tubulin-binding agent that has a wider antitumor spectrum than VCR and has different properties from those of VCR or Taxol.     

Inhibition of [3H]mebendazole binding to tubulin by structurally diverse microtubule inhibitors which interact at the colchicine binding site

A rapid and convenient radioligand assay was used to characterise the interaction of several structurally diverse microtubule inhibitors with the colchicine binding domain of tubulin. Values determined for the inhibition of [3H]mebendazole binding to tubulin by colchicine, combretastatin A4, NSC 181928, NSC 321567, podophyllotoxin and tubulozole-C provided an independent measure of the relative potency of these compounds. This methodology has several advantages over the inhibition of [3H]colchicine binding as a technique for investigating the molecular mechanisms involved in determining tubulin-ligand interactions.     

Role of the colchicine ring A and its methoxy groups in the binding to tubulin and microtubule inhibition

The roles of the methoxy substituents on ring A of two ring colchicine (COL) analogues were probed by the synthesis of a number of drugs and the examination of their effect on binding to tubulin, inhibition of microtubule assembly, and induction of GTPase activity. Selective elimination of ring A methoxy groups at positions 2, 3, and 4 weakened all three processes. The effects on binding and inhibition were independent of the nature of ring C (or C'). Specifically, excision of the 2- or 3-methoxy groups weakened binding by ca. 0.4 kcal mol-1, while that of the 4-methoxy group of ring A was weakened by 1.36 +/- 0.15 kcal mol-1. The effect on the inhibition of microtubule assembly, expressed as the equilibrium constant for the binding of the tubulin-drug complex to the end of a microtubule, was more complex and strongly dependent on the nature of ring C (or C'). This was attributed to the abilities of various groups on ring C' to overcome the wobbling in the tubulin-drug complex introduced by the weakening of the anchoring provided by ring A. It is concluded that ring A of COL is not germane to the mechanism of the inhibition of tubulin self-assembly. It serves only as a complex-stabilizing anchor. The control of this process resides in the interactions that key oxygen atoms of ring C of COL or C' of structural analogues establish with the protein. It is proposed that the 4-methoxy group of ring A serves as a key attachment point for immobilization of the drugs on the protein.     

Structural basis for specificity of Grb2-SH2 revealed by a novel ligand binding mode

The procedures of the arbitration committee of north Germany for medical liability claims are discussed. This procedure is set into relation to that at court. Due to the continuously maintained communication between lawyers and physicians, which does not occur in a comparable manner in court, the choice to proceed at a arbitration committee and an expert board is seen as more useful and pertinent than at court. This is specifically explained.     

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.     

Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin

Melatonin has been suggested as a physiological antagonist of calmodulin. In this work, we have characterized melatonin binding sites in Xenopus laevis oocyte membranes. Binding of [125I]melatonin by X. laevis oocyte membranes fulfills all criteria for binding to a receptor site. Binding was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. The binding site was also pharmacologically characterized. Stoichiometric studies showed a high-affinity binding site with a Kd of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (Kd=0.12 nM). In competition studies, we observed a low-affinity binding site (Kd=63.41 microM). Moreover, the binding site was characterized as calmodulin. Thus, binding was dependent on calcium and blocked by anti-CaM antibodies in a concentration-dependent manner. Calmodulin inhibitor chlorpromazine also inhibited binding of the tracer. From these results, it is suggested that membrane-bound calmodulin acts as a melatonin binding site in Xenopus laevis oocytes, where it might couple cellular activities to rhythmic circulating levels of melatonin. This hypothesis correlates with the previous findings describing melatonin as a physiological antagonist of calmodulin.     

Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography

The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331-338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant.     

Inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action

We propose here a new and detailed model for the antimalarial action of chloroquine (CQ), based on the its ability to inhibit degradation of heme by glutathione. Heme, which is toxic to the malaria parasite, is formed when the intraerythrocytic malaria parasite ingests and digests inside its food vacuole its host cell cytosol, which consists mainly of hemoglobin. The parasite protects itself against the toxicity of heme by polymerizing some of it to insoluble hemozoin (HZ). We show here that in Plasmodium falciparum at the trophozoite stage only ca. 30% of the heme is converted into hemozoin. We suggest that nonpolymerized heme exits the food vacuole and is subsequently degraded by glutathione, as has been shown before for uninfected erythrocytes. Marginal amounts of free heme could be detected in the membrane fraction of infected cells but nowhere else. It is well established that CQ and amodiaquine (AQ) accumulate in the parasite's food vacuole and inhibit heme polymerization, thereby increasing its efflux out of the food vacuole. We found that these drugs competitively inhibit the degradation of heme by glutathione, thus allowing heme to accumulate in membranes. Incubation of intact infected cells with CQ and AQ results in a marked increase in membrane-associated heme in a dose- and time-dependent manner, and a relationship exists between membrane heme levels and the extent of parasite killing. Heme has been shown to disrupt the barrier properties of membranes and to upset ion homeostasis in CQ-treated malaria-infected cells. In agreement with the predictions of our model, increasing the cellular levels of glutathione leads to increased resistance to CQ, whereas decreasing them results in enhanced sensitivity to the drug. These results insinuate a novel mechanism of drug resistance.     

Supramolecular self-assembly of glutamine synthetase: mutagenesis of a novel intermolecular metal binding site required for dodecamer stacking

Dodecameric glutamine synthetase (GS) from Escherichia coli assembles into highly ordered supramolecular protein tubes in the presence of several divalent metal ions. The molecular mechanism for this metal-induced self-assembly of the E. coli GS has been studied by molecular modeling and site-directed mutagenesis. The X-ray crystal structure of the nearly identical Salmonella typhimurium GS has been used to construct a model of the "stacked" complex between two dodecamers. A complementary fit, based on steric constraints, reveals a possible interaction between the N-terminal helices from adjacent dodecamers. The amino acid side chains of His and Met residues within the helices from each of the subunits of one face of a dodecamer lie within approximately 3.5 A of the analogous side chains in the subunits from the adjacent dodecamer in the stacked complex. His-4, Met-8, and His-12 from adjacent helices provide potential ligands for a binuclear metal binding site. Replacement of each of these surface residues with aliphatic amino acids has negligible effects on the enzymatic activity, the regulation of activity via adenylylation, and gross dodecameric structure. However, the rate and extent of metal ion-mediated self-assembly of GS tubules are reduced to < 2% of the wild-type protein in the single mutants H4A, H12L, and H12D. The M8L mutant demonstrates a 3-fold decrease in the bimolecular rate constant for stacking, but electron microscopy indicates that this mutant does form stacked tubes. The cysteine-containing mutants H4C, M8C, and H12C were also constructed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.     

A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule

We describe a mutation in the outer vestibule region of the adult rat skeletal muscle voltage-gated Na+ channel (microliter) that dramatically alters binding of mu-conotoxin GIIIA (mu-CTX). Mutating the glutamate at position 758 to glutamine (E758Q) decreased mu-CTX binding affinity by 48-fold. Because the mutant channel showed both low tetrodotoxin (TTX) and mu-CTX affinities, these results suggested that mu-CTX bound to the outer vestibule and implied that the TTX- and mu-CTX-binding sites partially overlapped in this region. The mutation decreased the association rate of the toxin with little effect on the dissociation rate, suggesting that Glu-758 could be involved in electrostatic guidance of mu-CTX to its binding site. We propose a mechanism for mu-CTX block of the Na+ channel based on the analogy with saxitoxin (STX) and TTX, on the requirement of mu-CTX to have an arginine in position 13 to occlude the channel, and on this experimental result suggesting that mu-CTX binds in the outer vestibule. In this model, the guanidinium group of Arg-13 of the toxin interacts with two carboxyls known to be important for selectivity (Asp-400 and Glu-755), with the association rate of the toxin increased by interaction with Glu-758 of the channel.     

Structural basis for the binding of a globular antifreeze protein to ice

Antifreeze proteins (AFPs) have the unique ability to adsorb to ice and inhibit its growth. Many organisms ranging from fish to bacteria use AFPs to retard freezing or lessen the damage incurred upon freezing and thawing. The ice-binding mechanism of the long linear alpha-helical type I AFPs has been attributed to their regularly spaced polar residues matching the ice lattice along a pyramidal plane. In contrast, it is not known how globular antifreeze proteins such as type III AFP that lack repeating ice-binding residues bind to ice. Here we report the 1.25 A crystal structure of recombinant type III AFP (QAE isoform) from eel pout (Macrozoarces americanus), which reveals a remarkably flat amphipathic ice-binding site where five hydrogen-bonding atoms match two ranks of oxygens on the [1010] ice prism plane in the <0001> direction, giving high ice-binding affinity and specificity. This binding site, substantiated by the structures and properties of several ice-binding site mutants, suggests that the AFP occupies a niche in the ice surface in which it covers the basal plane while binding to the prism face.     

Oxytocin and renal function in the rat; an investigation of a possible proximal site of action

Direct measurements of proximal tubular fluid reabsorption have been employed to examine the possible renal site of action of oxytocin. In whole kidney studies the natriuresis and chloriuresis, which occurred during the period of oxytocin infusion, did not coincide with the associated diuresis. The latter reached a peak 10-20 min after hormone administration has ceased. The separation in the saliuretic and diuretic responses underlines the apparent independence of these actions of oxytocin on the renal handling of water and electrolytes. The disturbances in renal function were not related to any change in glomerular filtration rate (gfr) and an examination of single nephron function failed to detect any significant effect of oxytocin on proximal tubular reabsorption. The renal actions of oxytocin would therefore appear to emanate from altered tubular rather than glomerular function, though the present study provides no support for a proximal site of action.     

Electron transfer dynamics of Rhodopseudomonas viridis reaction centers with a modified binding site for the accessory bacteriochlorophyll

Femtosecond spectroscopy in combination with site-directed mutagenesis was used to study the influence of histidine L153 in primary electron transfer in the reaction center of Rhodopseudomonas viridis. Histidine was replaced by cysteine, glutamate, or leucine. The exchange to cysteine did not lead to significant changes in the primary reaction dynamics. In the case of the glutamate mutation, the decay of the excited electronic level of the special pair P* is slowed by a factor of 3. The exchange to leucine caused the incorporation of a bacteriopheophytin b instead of a bacteriochlorophyll b molecule at the BA site. As a consequence of this chromophore exchange, the energy level of the electron transfer state P+BA- is lowered to such an extent that repopulation from the next electron transfer intermediate state P+HA- takes place, resulting in a long-lasting P+BA- population. The observed differences in time constants are discussed in the scope of nonadiabatic electron transfer theory considering the influence of the amino acids at position L153 and the chromophore exchange on the energy level of the intermediate state P+BA-. The results show that the high efficiency of primary electron transfer is reduced substantially, if the energy level of P+BA- is lowered or raised by several hundred wave numbers.     

RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations

Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.     

Characterization of a specific binding site for angiotensin II in chicken liver

We have characterized a specific binding site for angiotensin II (AngII) in chicken liver membranes. Pseudo-equilibrium studies at 22 degrees C for 30 min have shown that this binding site recognizes AngII with a high affinity (pKD of 8.13 +/- 0.21). The binding sites are saturable and relatively abundant (maximal binding capacity varies from 0.318 to 0.88 pmol/mg of protein). Nonequilibrium kinetic analyses at 22 degrees C revealed a calculated kinetic pKD of 8.77 +/- 0.20. The binding site is pharmacologically distinct from the classic AngII receptors AT1 and AT2. Competitive binding studies with chicken liver membranes demonstrated the following rank order of effectiveness: AngII (human; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > AngI(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) > AngIII(Arg-Val-Tyr-Ile-His-Pro-Phe) > AngIV (Val-Tyr-Ile-His-Pro-Phe) > Ang(1-7) (Asp-Arg-Val-Tyr-Ile-His-Pro) > PD123319 (1-[4(dimethylamino)3-methylphenyl] methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-6-carboxylic acid) > DuP753 (2-n-butyl-4-chloro-5 hydroxymethyl-1-[(2'-1H-tetrazol-5-yl)biphenyl-4-yl)methyl] imidazole. This atypical AngII binding site (chicken AT) was sensitive to increasing concentrations of DTT and Mn2+. The structure-activity relationship on position 1 of AngII showed that the primary N-terminal amine was essential for binding affinity ([Asp1]AngII > [Suc1]AngII > or = [Sar1]AngII), but modifications of the side chain in position 1 had less influence on the affinity ([Gly1]AngII > [Cys1]AngII approximately [aminoisobutyryl1]AngII approximately [Ser1]AngII > > > [Sar1]AngII). The presence of substantial quantities of this binding site in chicken liver membranes suggests the possibility that the chicken AT may play an important, yet unrecognized, role in the renin-angiotensin system.