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M J Gorbunoff

Publications and source records attributed to M J Gorbunoff.

At least 19 recordsLinked to original sources
PubMed1998

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.

Animals↗
PubMed1998

Linkages in tubulin-colchicine functions: the role of the ring C (C') oxygens and ring B in the controls.

Linkages between structural components of colchicine (COL) and its biphenyl analogues (allocolchicine, ALLO, and its analogues) in the binding to tubulin and its functional consequences were scrutinized. Three ring ALLO analogues with the carbomethoxyl in position 4' of ring C' replaced by a carbomethyl (KAC) and methoxy (MAC) groups were synthesized. The binding properties and consequences of binding (microtubule inhibition, abnormal polymerization, and induction of GTPase activity) were compared within the series of three ring and two ring compounds, as well as between pairs consisting of a two ring and a three ring compound with identical groups in position 4'. Binding measurements showed that the binding of KAC to the COL binding site proceeded with similar chemical characteristics as that of its two ring analogue (TKB), but with the kinetic characteristics of ALLO. The binding constant of KAC was found to be 1.9 x 10(6) M-1 and that of MAC was 4.6 x 10(5) M-1. The binding strength of the three ring analogues in descending order was KAC > ALLO > MAC, with increments similar to the biphenyl compounds, TKB > TCB > TMB. The difference in binding affinities between the pairs of three ring and two ring molecules was invariant (delta delta G degree = -1.3 +/- 0.2 kcal/mol-1), showing that in all cases ring B makes only an entropic contribution by suppressing free rotation about the biaryl bond. In the case of microtubule inhibition, all three ring compounds inhibited strongly with similar potencies, even though the spread in inhibition strength between the corresponding two ring molecules was > 3.3 kcal mol-1 of free energy. This difference was interpreted in terms of the ability of the various molecules to maintain tubulin in the proper conformation for binding in abnormal geometry to the growth end of a microtubule. This ability attains a maximal plateau value for three ring compounds, independently of the oxygen-containing group in ring C' (or C) and is maintained for the methyl ketone whether in a two or three ring compound. The induction of the GTPase activity was found to follow in general the binding affinity, with the exception that molecules that contained a methyl ketone were stronger GTPase inducers than expected from their alignment according to binding affinity. The finding that the binding of tropolone methyl ether (ring C of COL) induced a GTPase activity shows that ring C contains the ability to induce both substoichiometric microtubule inhibition and GTPase activity. Rings A and B act only as anchors in the binding, with ring A making an energetic contribution, while the effect of ring B is only entropic. It was concluded that both microtubule assembly inhibition and induction of GTPase activity were modulated by the same postbinding conformational change in tubulin. The difference between the strengths of these activities induced by ligands reflects the difference between a narrow allosteric effect between two well-defined sites in the case of GTPase activity and a broad effect aimed at the multiple sites involved in the incorporation of a tubulin protomer into the microtubule structure. Thus, there seems to be a loose thermodynamic linkage between binding and GTPase activity, while there is none between binding and microtubule inhibition, the two phenomena being linked only kinetically.

Acetamides↗
PubMed1996

Alterations of rings B and C of colchicine are cumulative in overall binding to tubulin but modify each kinetic step.

The role of the elimination of ring B and/or the modification of ring C of colchicine in tubulin binding kinetics and thermodynamics has been characterized, using four different molecules. These ligands are colchicine (COL); 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC), in which the central ring B has been reduced to one bond; allocolchicine (ALLO), in which ring C has been replaced by a six-membered ring; and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), where the same two modifications are made simultaneously. This paper describes the kinetics of association of ALLO with tubulin. The binding is accompanied by a fluorescence increase with slow biphasic kinetics, indicating binding to fast and slow tubulin isotypes. Binding to each of these isotypes occurs in two steps: a fast initial binding followed by a slower isomerization step. The K1 and k2 values for ALLO at 25 degrees C are 14,000 +/- 2,000 and 25,000 +/- 6,000 M-1 (fast and slow isotypes) and 0.055 +/- 0.003 s-1 and 0.013 +/- 0.001 s-1 (fast and slow isotype), respectively. For ALLO the reaction standard enthalpy change of the initial binding is 68 +/- 5 kJ.mol-1 (fast isotype) and 45 +/- 33 kJ.mol-1 (slow isotype) and the activation energy for the second forward step is 58 +/- 14 kJ.mol-1 (fast isotype) and 81 +/- 17 kJ.mol-1 (slow isotype). Displacement kinetics of bound ALLO by podophyllotoxin was monoexponential. The activation energy for the isomerization in the off direction is 107 +/- 7 kJ.mol-1. Comparison of the thermodynamic parameters for all four compounds shows that the modifications of both rings are cumulative with respect to overall binding. For the intermediate state there is a mutual influence of both modifications, suggesting an alteration of the reaction pathway.

Biphenyl Compounds↗
PubMed1996

Different kinetic pathways of the binding of two biphenyl analogues of colchicine to tubulin.

The kinetics of the interaction of tubulin with two biphenyl analogues of colchicine were measured by fluorescence stopped flow. The ligands were 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) and 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB). The binding of both analogues is accompanied by a fluorescence increase with monophasic kinetics, which indicates that these drugs, unlike colchicine, do not discriminate between the isoforms of tubulin. The observed pseudo-first-order rate constant increases in a nonlinear way with the drug concentration, indicating that the binding of the biphenyl analogues to tubulin occurs, like colchicine, in two steps: a fast reversible equilibrium followed by an isomerization of the initial complex. Kinetic analysis shows that TCB and TKB exhibit differences in their K1 values. At 25 degrees C, these are 114,000 +/- 15,000 M(-1) for TCB and 8,300 +/- 900 M(-1) for TKB. Both molecules show a much higher affinity than colchicine for the initial binding site. Also at 25 degrees C, the k2 value is 0.66 +/- 0.04 s(-1) for TCB and 3.0 +/- 0.2 s(-1) for TKB. From the temperature dependence, a reaction enthalpy change for the initial binding (deltaH(zero)1) of 44 +/- 9 kJ x mol(-1) (TCB) and -40 +/- 14 kJ x mol(-1) (TKB) and an activation energy for the second forward step of 64 +/- 2 kJ x mol(-1) (TCB) and 101 +/- 10 kJ x mol(-1) (TKB) were calculated. The dissociation kinetics were studied by displacement experiments, in which podophyllotoxin was used as a displacing ligand. The rate constant for the second step in the off direction (k(-2)) is 0.25 +/- 0.05 s(-1) for TCB and 0.093 +/- 0.009 s(-1) for TKB at 25 degrees C. The activation energies for the backward isomerization of the complexes were found to be 86 +/- 20 kJ x mol(-1) (TCB) and 79 +/- 5 kJ x mol(-1) (TKB). Combination of these results with the kinetic parameters for association gives a full characterization of the enthalpy pathway for the binding of TCB and TKB. The pathway of TCB binding is shown to differ considerably from that of TKB binding. Since their structural difference is located in ring C', this result points to their use of the ring C' in the first binding step. The competitiveness of the binding of TCB and TKB with those of podophyllotoxin, MTC, and MDL 27048 indicates that the two biphenyls interact as well with the trimethoxyphenyl-specific subsite.

Animals↗
PubMed1996

Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues.

The mechanism of the stoichiometric and substoichiometric inhibitions of tubulin self-assembly by several structural analogues of colchicine (COL) was investigated. The inhibition data were analyzed in terms of a simple model that takes into consideration Kg, the normal microtubule growth constant, equal to Cr-1 (Cr is the critical concentration for microtubule formation), and Kb, the binding constant of the drug to tubulin. In this manner, the value of the microtubule inhibition constant (Ki), which is the binding constant of the tubulin-drug complex to the end of a growing microtubule (which stops the microtubule growth), was determined. The results of the analysis of microtubule inhibition by the various colchicine analogues show that all the inhibitions can be expressed reasonably by this model. The strongest inhibitors found were colchicine (COL), allocolchicine (ALLO), and the biphenyl keto analogue 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB), which had essentially identical values of Ki = (2.1 +/- 0.3) x 10(6) M(-1). MTC, the two-ring analogue of colchicine, was weaker (Ki = 5.6 x 10(5) M(-1). A most striking result was that tropolone methyl ether (TME), which is ring C of COL, and which binds very weakly to tubulin (Kb = 3.5 x 10(2) M(-1)), is a substoichiometric inhibitor. Its Ki value of 8.7 x 10(5) M(-1) makes it identical in strength to MTC, suggesting that ring A makes little or no contribution to the induction of assembly inhibition. The three biphenyls, which bind to tubulin with similar affinity, spanned the spectrum from strong substoichiometric inhibition (TKB) to stoichiometric inhibition for 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) and an intermediate mode for the methoxy derivative 2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB). The extent of tubulin bound to drugs at 50% inhibition (r) was ca. 2% for TKB, ALLO, and COL, i.e. one liganded tubulin for every 40-50 molecules of free protein (substoichiometric). This ratio was 1:1.5 for TCB (stoichiometric) and 1:6 for TMB (intermediate). For TME, which is a single ring compound, it was 1:25. The progression of the stoichiometries varied directly with Ki and was totally unrelated to the values of Kb, which indicated the control of the stoichiometry by Ki and the close thermodynamic linkage between r and Ki. Comparison of the inhibitory capabilities of the various drugs identified the need for strong substoichiometric inhibition of a carbonyl group on ring C or C'. Furthermore, this group must be properly oriented by interaction with the protein or by the structural rigidity imparted by ring B, as in ALLO. The simple linked equilibrium model developed in this paper permits the alignment of drugs along a continuum that ranges from stoichiometric to strong substoichiometric modes of microtubule inhibition. Furthermore, it shows that the previously identified two classes are the two ends of a monotonously progressing spectrum described by a single mechanism of action.

Animals↗
PubMed1996

Structural analysis of the substoichiometric and stoichiometric microtuble-inhibiting biphenyl analogues of colchicine.

The structures of the colchicine (COL) analogues, 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB)and 2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB), were solved by X-ray diffraction. Their comparison with the structure of colchicine indicated the ability of both compounds to enter into a colchicine binding pocket. Comparison of TKB with 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) showed that the methyl group of the carbomethoxy group in position 4' of TCB protrudes beyond the (C=O)-CH3 group in the same position in TKB. Superposition of both structures on the van der Waals surface of COL clearly demonstrates that TKB can fully fit within that domain, while the CH3 group of TCB protrudes beyond the COL contour. This is proposed to be the source of the inability of TCB to inhibit microtubule assembly substoichiometrically, while TKB is a very strong inhibitor. While the same steric hindrance to entering into the COL site on tubulin must exist in allocolchicine (ALLO), in its case, this is overcome by the rigidity of the three-ring structure which abolishes the loss on binding of the entropy of free rotation between the two rings of the biphenyl TCB.

Biphenyl Compounds↗
PubMed1991

Roles of ring C oxygens in the binding of colchicine to tubulin.

The roles of the oxygens in ring C of colchicine in its binding to tubulin were probed by a study of the interactions of two allocolchicine biphenyl analogues, 2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB) and 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB), the first one containing a methoxy group in position 4', the second a keto group. Both analogues were found to bind specifically to the colchicine-binding site on tubulin in a rapidly reversible equilibrium. The standard free energies of binding at 25 degrees C were delta G zero (TKB) = 7.19 +/- 0.11 kcal mol-1 and delta G zero (TMB) = -6.76 +/- 0.22 kcal mol-1. The binding of TKB induced the same perturbation in protein circular dichroism at 220 nm as colchicine and allocolchicine, as well as quenching of protein tryptophan fluorescence. Binding of TMB did not affect the protein CD spectrum within experimental error and induced only a marginal quenching of protein fluorescence. Comparison with the binding properties of allocolchicine and its des(ring B) analogue 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) [Medrano et al. (1989) Biochemistry 28, 5589-5599] has shown that the binding properties of the 4'-keto analogue (TKB) were closer to those of allocolchicine, even though the substituent in the 4'-position of TCB is identical with that of allocolchicine. It has been proposed that binding in the ring C subsite on tubulin, which is stabilized thermodynamically by stacking interactions, can be modulated in a nonidentical fashion by the carbonyl and the ether oxygens in the para position of ring C.

Binding, Competitive↗
PubMed1991

Mechanism of colchicine binding to tubulin. Tolerance of substituents in ring C' of biphenyl analogues.

The limits of structural variation of the substituent in position 4' of ring C' of biphenyl colchicine analogues (ring C in colchicine) were probed by the synthesis of a number of analogues and the examination of their binding to tubulin and its consequences. Binding was found to require the location in three-dimensional space of the oxygen in the 4'-substituent at a locus not far distant from those of the colchicine ring C oxygens. All those analogues that bind to the colchicine site of tubulin induced the GTPase activity and inhibited microtubule assembly, those containing a carbonyl group substoichiometrically and the others stoichiometrically. A similar relation was found for the induction of the abnormal polymerization of the colchicine analogue-tubulin complex, with methoxy-containing compounds requiring a higher temperature to induce the polymerization. A concerted analysis of the binding thermodynamics of colchicine and its various analogues has shown full consistency with the previously proposed two-step binding pathway that involves two nonidentical binding moieties in the ligand [Andreu, J. M., & Timasheff, S. N. (1982) Biochemistry 21, 534-543]. Comparison of the binding parameters of colchicine, its des(ring B) analogue (MTC), and ring A and C compounds individually with the thermodynamic parameters deduced for the first steps of the bindings of colchicine and MTC [Engelborghs, Y., & Fitzgerald, T. J. (1987) J. Biol. Chem. 262, 5204-5209] have led to the conclusion that binding can occur by two pathways leading to the identical product. In the first pathway, ring A binds first; this is followed by a rate-determining thermodynamically indifferent reaction (protein conformation change), and finally a rapid binding of ring C. In the second pathway, the events are the same except that the order of binding of the rings is reversed. Colchicine, due to the steric hindrance of ring B, can follow only the second pathway. For MTC, both kinetic pathways are open and binding may be initiated by random first contact of either ring A or ring C.

Animals↗
PubMed1989

Roles of colchicine rings B and C in the binding process to tubulin.

The interactions of tubulin with colchicine analogues in which the tropolone methyl ether ring had been transformed into a p-carbomethoxybenzene have been characterized. The analogues were allocolchicine (ALLO) and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), the first being transformed colchicine and the second transformed colchicine with ring B eliminated. The binding of both analogues has been shown to be specific for the colchicine binding site on tubulin by competition with colchicine and podophyllotoxin. Both analogues bind reversibly to tubulin with the generation of ligand fluorescence. The binding of ALLO is slow, the fluorescence reaching a steady state in the same time span as colchicine; that of TCB is rapid. The displacement of ALLO by podophyllotoxin proceeds with a half-life of ca. 40 min. Binding isotherms generated from gel filtration and fluorescence measurements have shown that both analogues bind to tubulin with a stoichiometry of 1 mol of analogue/mol of alpha-beta tubulin. The equilibrium binding constants at 25 degrees C have been found to be (9.2 +/- 2.5) x 10(5) M-1 for ALLO and (1.0 +/- 0.2) X 10(5) M-1 for TCB. Binding of both analogues was accompanied by quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Both inhibited microtubule assembly in vitro, ALLO substoichiometrically, and both induced the abnormal cooperative polymerization of tubulin, which is characteristic of the tubulin-colchicine complex. Analysis in terms of the simple bifunctional ligand binding mechanism developed for colchicine [Andreu, J.M., & Timasheff, S.N. (1982) Biochemistry 21, 534-543] and comparison with the binding of the colchicine two-ring analogue, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one [Andreu, J. M., Gorbunoff, M. J., Lee, J. C., & Timasheff, S. N. (1984) Biochemistry 23, 1742-1752], have shown that transformation of the tropolone methyl ether part of colchicine into p-carbomethoxybenzene weakens the standard free energy of binding to tubulin by 1.4 +/- 0.1 kcal/mol, while elimination of ring B weakens it by 1.0 +/- 0.1 kcal/mol. The roles of rings C and B of colchicine in the thermodynamic and kinetic mechanisms of binding to tubulin were analyzed in terms of these findings.

Animals↗
PubMed1989

Colchicine analogues that bind reversibly to tubulin define microtubular requirements for newly synthesized protein secretion in rat lacrimal gland.

The role of microtubules in 3H-labeled protein secretion in rat lacrimal glands was probed by the use of colchicine and two of its analogues that reversibly bind to tubulin. These analogues were 2-methoxy-5-(2,3,4,4'-trimethoxyphenyl)-2,4,6-cycloheptatriene-1-o ne and 2,3,4,4'-tetramethoxy-1,1'-biphenyl, the latter having been synthesized for these studies. Immunofluorescence revealed that untreated exocrine acinar cells contained an intact microtubule network, which was totally abolished by drug addition. Subsequent drug removal restored the network for the two reversibly binding drugs--more rapidly so for the biphenyl, but this was not the case with colchicine. The protein-secretory process was examined by adding the three drugs at various stages--prepulse incubation, pulse, maturation, apical storage of granules, and discharge under cholinergic stimulation. Comparison with the kinetics of microtubular network restoration, which differed for the two reversibly binding drugs, led to the conclusion that the microtubular system is critical to the maturation phase of secretion.

Animals↗
PubMed1984

Interaction of tubulin with bifunctional colchicine analogues: an equilibrium study.

The interaction of tubulin with simple analogues of colchicine that contain both its tropolone and trimethoxyphenyl rings has been characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine [ Andreu , J. M., & Timasheff , S. N. (1982) Biochemistry 21, 534-543] on the basis of interactions of tubulin with single-ring analogues. The compound 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6- cycloheptatrien -1-one has been found to bind reversibly to 0.86 +/- 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 +/- 0.3) X 10(5) M-1 (25 degrees C), delta H degrees app = -1.6 +/- 0.7 kcal mol-1, and delta S degrees app = 20.5 +/- 2.5 eu. The binding appears specific for the colchicine site. The closely related compound 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino] -2,4,6- cycloheptatrien -1-one interacts weakly with tubulin. Binding of the first analogue is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analogue inhibit microtubule assembly in vitro. Excess analogue concentration under microtubule-promoting conditions induces an abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.

Animals↗
PubMed1984

The interaction of proteins with hydroxyapatite. I. Role of protein charge and structure.

The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11.0); (2) ionic nature of eluant (Na salts of PO4, F-, Cl-, SCN-, ClO-4, and CaCl2); and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PO4, F-, Cl-, SCN-, and ClO-4, and low (less than 0.003 M) Ca2+; (2) acidic proteins which elute at about equal moderate molarities of PO4 and F-, but do not elute with Ca2+ and usually not with Cl-; (3) neutral proteins, which elute with PO4, F-, and Cl-, but show a strong anion specificity, and do not elute with Ca2+ or SCN-. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction.

Chemical Phenomena↗
PubMed1984

The interaction of proteins with hydroxyapatite. II. Role of acidic and basic groups.

The elution behavior from hydroxyapatite columns of the modification products of seven basic and three acidic proteins has been investigated. Three classes of NH2 derivatives were prepared. These consisted of (1) replacement by a guanidyl group with no change in charge; (2) blocking with loss of charge; and (3) replacement of positive charges by negative ones. Two types of COOH derivatives were prepared: (1) blocking with loss of charge; and (2) replacement of COOH by SO3H with no change in charge. The elution behavior of the derivatives in PO4, F-, Cl-, ClO-4, and Ca2+ ion eluants showed that (1) the elution patterns are determined by the isoelectric points of the proteins, there being no symmetry between the binding or elution behavior of acidic and basic proteins; (2) the binding of basic proteins requires the presence of a high density of positively charged groups; (3) the binding of all proteins to hydroxyapatite equilibrated with phosphate buffer is enhanced by a decrease in the number of their negative charges; and (4) calcium ions affect the binding of proteins to hydroxyapatite at the level of carboxyls, since clusters of carboxyls strengthen both the interaction with Ca2+ and the binding to hydroxyapatite.

Chemical Phenomena↗
PubMed1984

The interaction of proteins with hydroxyapatite. III. Mechanism.

The mechanism of protein binding to and elution from hydroxyapatite (HA) has been established. Binding occurs both by nonspecific attraction between protein positive charges and HA and by specific complexing of protein carboxyls with calcium loci on the mineral. Elution can take place either as the result of the nonspecific ion screening of charges or by the specific displacement of protein groups from sites on the column with which they had complexed.

Adsorption↗