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R H Himes

Publications and source records attributed to R H Himes.

At least 19 recordsLinked to original sources
PubMed1997

Mechanism of action of the unusually potent microtubule inhibitor cryptophycin 1.

Cryptophycin 1 is a remarkably potent antiproliferative compound that shows excellent antitumor activity against mammary, colon, and pancreatic adenocarcinomas in mouse xenographs. At picomolar concentrations, cryptophycin 1 blocks cells in the G2/M phase of the cell cycle by an apparent action on microtubules. The compound binds to tubulin, inhibits microtubule polymerization, and depolymerizes preformed microtubules in vitro. Its exceptionally powerful antitumor activity (many-fold greater than paclitaxel or the vinca alkaloids) raises important questions about its mechanism of action. By quantitative video microscopy, we examined the effects of cryptophycin 1 on the dynamics of individual microtubules assembled to steady state from bovine brain tubulin. At low nanomolar concentrations, in the absence of net microtubule depolymerization, cryptophycin 1 potently stabilized microtubule dynamics. It reduced the rate and extent of microtubule shortening and growing and increased the frequency of rescue. The results suggest that cryptophycin 1 exerts its antiproliferative and antimitotic activity by binding reversibly and with high affinity to the ends of microtubules, perhaps in the form of a tubulin-cryptophycin 1 complex, resulting in the most potent suppression of microtubule dynamics yet described.

Animals↗
PubMed1997

Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues.

To determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent analogues in which a (dimethylamino)benzoyl group has been introduced into the 7- and 10-positions, and the benzoyl groups at the 2- and N- as well as the 3'-phenyl ring have been modified with dimethylamino functions. In a tubulin assembly assay, the N-m- and N-p-(dimethylamino)benzoyl derivatives had activities comparable to the activity of paclitaxel. The 2-, 3'-, and 10-analogues had slightly reduced activity, and the 7-derivative was about 5% as active as paclitaxel. On the basis of the results of studies of the effect of solvents on the fluorescence emission spectra, it is proposed that the unbound analogues form hydrogen bonds with protic solvents. But the 7- and 10-substituted analogues appear to be more affected by protic solvents than the other analogues. Previously, we studied the binding of the N-meta derivative to tubulin and microtubules [Sengupta, S., et al. (1995) Biochemistry 34, 11889-11894]. In this study, we extended the studies to include the 2-, 7-, and 10-derivatives. Similar to the N-substituted analogue, binding of the 2-derivative to tubulin was accompanied by a large blue shift, whereas a very small shift occurred when the 7- and 10-substituted derivatives bound. The 2- and N-substituted analogues bind to microtubules with an increase in fluorescence intensity over that which was observed with tubulin, whereas binding of the 7- and 10-substituted analogues was accompanied by a large quenching in fluorescence. This quenching may be due to the presence of charged residues in the protein near the 7- and 10-(dimethylamino)benzoyl groups or to pi stacking of the groups with an aromatic side chain. The presence of paclitaxel with microtubules prevented the fluorescence increase of the 2- and N-derivatives and quenching of the 7- and 10-derivatives. The difference in behavior of the fluorescent analogues upon binding to polymerized tubulin, coupled with the solvent studies on the free drugs, suggests that the 2- and N-benzoyl groups of paclitaxel bind in a hydrophobic pocket of tubulin but could participate in hydrogen bonding, and the 7- and 10-positions are in a more hydrophilic environment.

Animals↗
PubMed1996

Studies on the nocodazole-induced GTPase activity of tubulin.

The tubulin dimer contains two guanine nucleotide binding sites, a nonexchangeable site occupied by GTP and an exchangeable site (E-site) occupied by GTP or GDP. Under the conditions used in this study the E-site GTP was hydrolyzed at a rate of 8 x 10(-5) s(-1) at 37 degrees C. This rate is stimulated four- to fivefold by nocodazole, an antimitotic drug. We studied the characteristics of this drug-stimulated reaction to learn more about the hydrolytic center of tubulin. The reaction, studied using single turnover kinetics, i.e., in the absence of added GTP, has a pH optimum of 6.6 to 7.2 and an activation energy of 88 kJ x mol(-1). It is first-order with respect to tubulin-GTP, indicating that hydrolysis is not dependent on an aggregation process. Divalent cations stimulate the hydrolysis three- to six-fold over the rate in the presence of EDTA. The reaction has a requirement for Na+ that is not satisfied effectively by other monovalent cations. In contrast, Na+ and K+ are almost equally effective in the tubulin assembly reaction. Different purine nucleoside triphosphates can bind to the E-site and are hydrolyzed. GTP and ITP are hydrolyzed at equivalent rates and XTP and ATP are hydrolyzed at a rate about half as fast. Hydrolysis is inhibited by the reagents diethylpyrocarbonate and N-ethylmaleimide, although the GTPase activity is less sensitive than the assembly reaction. The reaction rate in D2O is twice the rate in H2O. This inverse isotope effect suggests the involvement of a sulfhydryl group in the rate-limiting step in the reaction.

Animals↗
PubMed1996

Site-directed mutagenesis of putative catalytic and nucleotide binding sites in N10-formyltetrahydrofolate synthetase.

To determine the importance of specific amino-acid residues in catalysis and substrate binding by N10-formylH4 folate synthetase, one lysine and three histidine residues in the enzyme from Clostridium cylindrosporum were mutated to glutamine and serine residues, respectively. These residues, Lys-71, His-125, His-131, and His-268, are conserved in four bacterial and five eukaryotic proteins for which the amino-acid sequences are known. Previous evidence indicated that a histidine residue may play a role in catalysis and it has been proposed that Lys-71 could be a member of a putative nucleotide binding consenus sequence. The histidine mutations, H125S, H131S, and H268S, produced proteins that were unstable and were proteolytically degraded to different extents. No activity of purified H268S could be detected and the 240 kDa native tetramer was also absent. Activities of the H125S and H131S mutants could be measured and the Km values of the substrates were similar to those for the wild-type enzyme. It is concluded that the mutations resulted in monomers that do not fold properly and/or do not associate to the active tetramer and, as a consequence, are susceptible to intracellular proteolytic digestion. On the other hand, the K71Q mutation did not produce proteolyzed material. The resulting protein had a kcat value which was reduced by a factor of 3.3 x 10(-4). Km values of the substrates were not affected, nor were the affinty constants for MgATP and H4PteG3. CD and fluorescence spectra demonstrated that little change in the tertiary structure of the protein had occurred as a result of the mutation. The monomer form of K71Q was less stable than the monomer of the wild-type enzyme and reassociated less efficiently than the wild-type. From these results it is suggested that Lys-71 plays a critical role in catalysis by N10-formylH4 folate synthetase and that this residue may reside at an intersubunit interface.

Amino Acid Sequence↗
PubMed1996

Preparation of phenolic paclitaxel metabolites.

The synthesis and biological evaluation of the two known phenolic metabolites of paclitaxel are described. The C3'-phenolic metabolite 2 of paclitaxel was prepared from 7-(triethylsilyl)-baccatin III (8) and enantioenriched N-benzoyl-2-azetidinone 7. The C2-phenolic metabolite 3 was synthesized from paclitaxel (1a) via selective C2 debenzoylation and reacylation.

Antineoplastic Agents, Phytogenic↗
PubMed1995

Interaction of cryptophycin 1 with tubulin and microtubules.

The cryptophycins are newly discovered antimitotic agents isolated from the cyanobacterium Nostoc. Previous studies using cultured cells demonstrated that microtubules are the target of these compounds. We have studied the interaction of cryptophycin 1 with tubulin and microtubules in vitro. Cryptophycin 1 is an effective inhibitor of tubulin polymerization, causes tubulin to aggregate, and depolymerizes microtubules to linear polymers somewhat similar to the spiral-like structures produced by the Vinca alkaloids. Cryptophycin 1 also inhibits vinblastine binding to tubulin but not colchicine binding. Thus, it appears that the cryptophycins may bind to the Vinca site in tubulin or to a site that overlaps with the Vinca site.

Animals↗
PubMed1995

Interaction of a fluorescent paclitaxel analogue with tubulin.

To study the mechanism of binding of the antitumor agent paclitaxel to microtubules and tubulin, we have synthesized a fluorescent analogue of the drug. A dimethylamino group was introduced onto the 3'-N-benzoyl group of paclitaxel. This compound was synthesized from N-debenzoylpaclitaxel and 3-(dimethylamino)benzoyl chloride in 67% yield. N-Debenzoyl-N-[3-(dimethylamino)benzoyl]-paclitaxel has activity similar to paclitaxel in inducing microtubule assembly and binds to tubulin at the paclitaxel-binding site. Under assembly conditions, binding of this paclitaxel analogue to tubulin occurs in a time-dependent manner and is accompanied by a large increase in fluorescence intensity, as well as a large blue shift in the emission maximum. In addition, evidence is presented to show that this compound also binds to tubulin in the dimeric state, but the binding affinity is much lower (Kd = 49 +/- 8 microM at 25 degrees C) than that reported for polymeric tubulin. The fluorescent paclitaxel analogue, with a high quantum yield, will be a useful tool in studying the mechanism of paclitaxel binding to tubulin and the environment of the paclitaxel-binding site on tubulin.

Animals↗
PubMed1995

18Oxygen incorporation into inorganic phosphate in the reaction catalyzed by N5,10-methenyltetrahydrofolate synthetase.

The mechanism of the N5,10-methenyltetrahydrofolate synthetase reaction was probed by determining the source of the oxygen atom introduced between the beta- and gamma-phosphates as ATP is converted to ADP and Pi. The reaction was performed using a mixture of [18O]- and [16O]N5-formyltetrahydrofolate in the presence of [16O]H2O and using [16O]N5-formyltetrahydrofolate in the presence of a 1:1 mixture of [18O]H2O and [16O]H2O. 31P NMR spectroscopy was used to examine the products. It was found that 18O from the formyl group was incorporated into Pi, and that 18O was not incorporated from the solvent. The results are consistent with a mechanism involving phosphorylation of the formyl group at the N5-position, followed by displacement of the phosphate by the 10-nitrogen.

Adenosine Diphosphate↗
PubMed1994

Polymerization of Antarctic fish tubulins at low temperatures: role of carboxy-terminal domains.

We have proposed previously that the efficient polymerization of tubulins from Antarctic fishes at low, physiological temperatures (-1.8 to +2 degrees C) may result in part from adaptations (e.g., reductions in acidic residues) located in their carboxy-terminal (C-terminal) tails [Detrich & Overton (1986) J. Biol. Chem. 261, 10922-10930]. To test this hypothesis, we have examined the polymerization of Antarctic fish neural tubulins modified at their C termini by proteolysis or by neutralization of carboxyl groups. Addition of subtilisin to low concentrations of Notothenia coriiceps tubulin induced a biphasic assembly reaction: stage I corresponded to the C-terminal cleavage of beta chains to produce alpha beta s dimers, and stage II coincided with the slower, C-terminal cleavage of alpha chains to yield alpha s beta s. Both stage I and stage II polymers consisted of protofilament sheets and microtubules with attached sheets. The critical concentration for assembly of the stage II polymer was at least 10-fold lower than that of untreated tubulin. Neutralization of Glu and Asp carboxyls in Gobionotothen gibberifrons microtubules by the carbodiimide-catalyzed incorporation of glycine ethyl ester (GEE) moieties produced a tubulin, modified largely in its C termini, that assembled more readily than did control tubulin. When 12 GEE groups were incorporated per dimer, the critical concentrations for assembly of modified tubulin at 5-10 degrees C were 2-3-fold smaller than those for the unmodified protein. Comparably modified bovine tubulin (10 GEE/dimer) assembled at 37 degrees C with a critical concentration 2.6-fold lower than that for the unmodified tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1994

The effect of the aromatic rings of taxol on biological activity and solution conformation: synthesis and evaluation of saturated taxol and taxotere analogues.

The synthesis and biological evaluation of novel cyclohexyl analogues of taxol and taxotere are detailed. 2-(Cyclohexylcarbonyl)-2-debenzoylbaccatin III (6) was prepared from baccatin III by hydrogenation. Subsequent coupling of 6 with N-t-BOC-3-[(tert-butyldimethylsilyl)oxy]-4-phenyl-2-azetidinone (7), followed by removal of the protecting groups, afforded 2-(cyclohexylcarbonyl)-2-debenzoyltaxotere (9). In a similar synthetic sequence, 3'-cyclohexyl-3'-dephenyltaxol (14) was prepared from N-benzoyl-3-[(tert-butyldimethylsilyl)oxy]-4-cyclohexyl-2-azetidinone (12) and (triethylsilyl)baccatin III. The taxol analogue 15, in which all three taxol phenyl groups are substituted by a cyclohexyl moiety, was synthesized in one step from taxol via hydrogenation. All three analogues (9, 14, and 15) exhibited strong activity in the microtubule assembly assay and cytotoxicity comparable to taxol against B16 melanoma cells. It was also shown that 9, like taxol and taxotere, has an extended side chain in chloroform, but in DMSO/water mixtures preferentially adopts a different conformation in which the 2-(cyclohexylcarbonyl), 3'-phenyl, and 4-acetyl groups cluster. However, this behavior does not appear to occur for 3'-cyclohexyl analogues 14 and 15, in which the side chain conformation remains extended independent of solvent. These results suggest the aromaticity of the 3'-phenyl ring significantly stabilizes the clustered conformation.

Antineoplastic Agents↗
PubMed1994

Synthesis of a photoaffinity taxol analogue and its use in labeling tubulin.

A photoaffinity analogue of taxol, N-([3,5-3H]-4-azidobenzoyl)-N-debenzoyltaxol (7), was synthesized and used to photolabel microtubules. Approximately 20% of the noncovalently bound analogue becomes covalently bound upon irradiation at 300 nm. Incorporated label was stable to a 50% ethanol solution and sodium dodecyl sulfate. About 80% of the incorporated label was found in the beta-subunit and 20% in the alpha-subunit. Incorporation did not occur into unpolymerized tubulin, consistent with the fact that taxol binds only to polymerized tubulin, and was decreased by the presence of taxol. Little or no nonspecific labeling occurs. This analogue is currently being used to identify taxol binding site(s) on tubulin.

Affinity Labels↗
PubMed1994

The binding of a ciliary microtubule plus-end binding protein complex to microtubules is regulated by ciliary protein kinase and phosphatase activities.

Using a human autoimmune CREST antiserum, we identified a 97-kDa polypeptide at the plus ends of Tetrahymena ciliary microtubules and an antigen associated with mammalian kinetochores (Miller, J.M., Wang, W., Balczon, R., and Dentler, W.L. (1990) J. Cell. Biol. 110, 703-714). The ciliary protein is part of a 1,500-2,000-kDa complex that can be released from ciliary microtubules and from in vitro assembled brain microtubules with ATP and ATP gamma S (Wang, W., Suprenant, K.A., and Dentler, W. L. (1993) J. Biol. Chem. 268, 24796-24807). Here we show that the ATP-dependent release of the 97-kDa protein from microtubules is inhibited, in a concentration-dependent manner, by calf intestine phosphatase and by the protein kinase inhibitor 6-dimethylaminopurine. Sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, stimulated the ATP-dependent release of the 97-kDa protein from microtubules. Therefore, the ciliary fraction contains both protein kinase and phosphatase activities, and selective inhibition of these activities is necessary for the ATP-dependent binding and release of the 97-kDa protein from microtubules. When incubated with [gamma-32P]ATP, a portion of the 97-kDa protein is phosphorylated as are several other polypeptides associated with it. ATP sensitivity requires a low molecular weight heat-stable factor associated with axonemes. These results suggest that the association of the 97-kDa protein with microtubules is regulated by protein phosphorylation by axoneme-associated kinases and phosphatases.

Adenine↗
PubMed1994

Conformation of ATP and ADP bound to N10-formyltetrahydrofolate synthetase determined by TRNOE NMR spectroscopy.

ATP and ADP bind to N10-CHO-H4folate synthetase from Clostridium cylindrosporum at four identical sites. Although both ADP and ATP bind to the enzyme with essentially the same Ka values as the Mg2+.nucleotide complexes, only the Mg2+.nucleotides are kinetically active. Using transferred nuclear Overhauser effect (TRNOE) NMR spectroscopy, we have measured the time-dependent NOE buildup rates of selected protons in ADP and ATP bound to N10-CHO-H4folate synthetase after preirradiating protons H1', H2', H3', and H4'. The results were used to calculate interproton distances. In order to define the conformations of ADP and ATP bound to the enzyme, we used the TRNOE distance constraints in a distance geometry algorithm. The results of the distance geometry calculations suggest that, within experimental error, the conformations of both ADP and ATP (with or without Mg2+) have an average glycosidic torsion angle X (O4'-C1'-N9-C8) of 100 degrees +/- 20 degrees and a sugar pucker angle psi' (C5'-C4'-C3'-O3') of 85 degrees +/- 5 degrees. These values are consistent with a nucleotide structure generated by computer modeling after energy minimization, which has X = 90 degrees +/- 6 degrees and psi' = 81 degrees, indicating a high-anti and C3'-endo conformation.

Adenosine Diphosphate↗
PubMed1993

Solvent oxygen is not incorporated into N10-formyltetrahydrofolate in the reaction catalyzed by N10-formyltetrahydrofolate synthetase.

The mechanism of the reaction catalyzed by N10-formyltetrahydrofolate synthetase involves the formation of formyl phosphate as an intermediate which then formylates tetrahydrofolate at the N-10 position. Previous studies demonstrated that the non-enzymic formylation of tetrahydrofolate by formyl phosphate occurs exclusively at the more nucleophilic 5-nitrogen in the reduced pyrazine ring. The experiments described in this report were designed to determine whether N5-formyltetrahydrofolate might be the first product to be formed on the enzyme, followed by formyl transfer to the 10-nitrogen via the cyclic intermediate N5,10-methenyltetrahydrofolate. If this were the case, oxygen from solvent H2O would be incorporated into the formyl group of the N10-derivative. By conducting the reaction in a 1:1 mixture of [16O]H2O and [18O]H2O and using 13C NMR spectroscopy we show that no 18O is incorporated into the product and conclude that the reaction proceeds via a direct formylation of the N-10 position by formyl phosphate.

Catalysis↗
PubMed1993

Exchangeable GTP binding site of beta-tubulin. Identification of cysteine 12 as the major site of cross-linking by direct photoaffinity labeling.

After direct photoaffinity cross-linking of [3H]GTP to the beta-subunit of tubulin, followed by tryptic digestion and alkaline phosphatase treatment, we employed cis-diol-specific boronate gel chromatography and reversed-phase high-pressure liquid chromatography to purify a peptide containing most of the covalently bound radioactivity. The sequence of this peptide corresponded to that of residues 3-19 of beta-tubulin. Residue 10 of the peptide, which is Cys-12 in beta-tubulin, could not be identified. The fast atom bombardment mass spectrum of this peptide showed the presence of a predominant species with a molecular mass of 2022 kDa (2021 kDa for the 12C variant), which is 255 Da greater than the molecular mass of the peptide. Fast atom bombardment collision-activated decomposition mass spectrometry analysis produced fragments which are consistent with the beta(3-19) peptide but having a unit of mass of 358 at position 12. Thermolysin digestion of the tryptic peptide restricted the cross-linking site to the 9-amino acid sequence, I(L)QAGQXGNQ. The molecular mass of this peptide was 1174 kDa, which is equal to the mass of the beta(7-15) peptide containing an extra group of mass 255. To explain the molecular masses of the two labeled peptides, which are 26 atomic mass units less than expected, a mechanism of photolabeling is proposed that involves opening of the guanine ring and loss of the C-6 carbonyl function as CO2.

Affinity Labels↗
PubMed1993

Sequence and expression of the gene for N10-formyltetrahydrofolate synthetase from Clostridium cylindrosporum.

Sau3 A and Hind III restriction fragments of Clostridium cylindrosporum genomic DNA were used to isolate clones containing 80% of the N10-H4folate synthetase gene in a 5' fragment and the remaining 20% of the gene in the 3' fragment. These fragments were joined at a common SnaB I restriction site and expressed in Escherichia coli at a level equivalent to what is normally found in C. cylindrosporum. Sequence comparisons show a large degree of homology with genes from two other clostridial species, including a thermophile. Certain conserved sequences found in the three clostridial proteins and in the N10-H4folate synthetase portion of eukaryotic C1-H4folate synthases may represent consensus sequences for nucleotide and H4folate binding.

Amino Acid Sequence↗
PubMed1993

A comparison of microtubule assembly in brain extracts from young and old rats.

Microtubule assembly was examined in the high-speed supernatant from homogenates of young (2-4 months old) and old (more than 24 months old) rat brains and significant age-related differences in microtubule assembly were found in the absence of exogenous GTP. In extracts from young brains, the increase in absorbance at 350 nm, which reflects the assembly reaction, was characterized by three phases (lag, elongation, and steady state) superimposed on a slow continuous increase due to non-specific aggregation. However, assembly in extracts from old brains, was very sluggish, in some cases barely more rapid than the non-specific aggregation reaction. Two to three times as much protein was assembled into cold-labile microtubules in extracts from young brains than from old brains. When 1 mM GTP was included in the assembly solutions the patterns of assembly in extracts from young and old brains became similar, with about the same amount of protein assembled into cold-labile microtubules. The assembly of tubulin purified from rat brains showed no differences between young and old. Results showed that extracts from old brains contained a higher GTPase activity than extracts from young brains. The sluggish assembly in extracts from old brains could be due to a deficiency in GTP or an inefficient regeneration of GTP.

Age Factors↗