Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The epothilones (Epos) are a group of natural products isolated from the myxobacterium, Sorangium cellulosum. They have a mechanism of action similar to that of Taxol, i.e., they stabilize microtubules and induce the formation of microtubule bundles in cells. Because they are simpler in structure than Taxol and preserve their activity in P-glycoprotein-expressing cells, they are being studied as potential antitumor drugs. In this work, a series of Epo-resistant A549 and HeLa cell lines have been selected and analyzed. Class I beta-tubulin, the major isotype of beta-tubulin in these Epo-resistant cell lines, has been sequenced in a search for mutations. In the Epo B-resistant A549 cells, there is a mutation at beta 292 from Gln to Glu, in the Epo A-resistant HeLa cell line there is a mutation at beta 173 from Pro to Ala, and in the Epo B-resistant HeLa cell line there is a heterozygous mutation at beta 422 from Tyr to a mixture of Tyr and Cys. These mutations are close to the M-loop, the nucleotide-binding site, and the microtubule-associated protein binding sites, respectively. It is likely that these mutations in beta-tubulin provide cells with a mechanism of resistance to the Epos and taxanes. Among these resistant cell lines, A549.EpoB40 is hypersensitive to microtubule-destabilizing drugs, such as vinblastine and colchicine, and HeLa.EpoB1.8 is dependent on the Epos or taxanes for growth. Our studies provide evidence that the M-loop, the GTP binding site, and the microtubule-associated protein binding sites at the COOH terminus in beta-tubulin are critical for the regulation of microtubule stability.
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PMID:Mutations in beta-tubulin map to domains involved in regulation of microtubule stability in epothilone-resistant cell lines. 1246 33

During the course of a mechanism-based screening program designed to identify new microtubule-disrupting agents from natural products, we identified a crude extract from Tacca chantrieri that initiated Taxol-like microtubule bundling. Bioassay-directed purification of the extract yielded the highly oxygenated steroids taccalonolides E and A. The taccalonolides caused an increased density of cellular microtubules in interphase cells and the formation of thick bundles of microtubules similar to the effects of Taxol. Mitotic cells exhibited abnormal mitotic spindles containing three or more spindle poles. The taccalonolides were evaluated for antiproliferative effects in drug-sensitive and multidrug-resistant cell lines. The data indicate that taccalonolide E is slightly more potent than taccalonolide A in drug-sensitive cell lines and that both taccalonolides are effective inhibitors of cell proliferation. Both taccalonolides are poorer substrates for transport by P-glycoprotein than Taxol. The ability of the taccalonolides to circumvent mutations in the Taxol-binding region of beta-tubulin was examined using the PTX 10, PTX 22, and 1A9/A8 cell lines. The data suggest little cross-resistance of taccalonolide A as compared with Taxol, however, the data from the PTX 22 cell line indicate a 12-fold resistance to taccalonolide E, suggesting a potential overlap of binding sites. Characteristic of agents that disrupt microtubules, the taccalonolides caused G(2)-M accumulation, Bcl-2 phosphorylation, and initiation of apoptosis. The taccalonolides represent a novel class of plant-derived microtubule-stabilizers that differ structurally and biologically from other classes of microtubule-stabilizers.
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PMID:Taccalonolides E and A: Plant-derived steroids with microtubule-stabilizing activity. 1281 Jun 50

Single-step selections were used to obtain Chinese hamster ovary cell lines resistant to Colcemid and vinblastine. Verapamil was included in the selections to circumvent the isolation of cells with P-glycoprotein-mediated multidrug resistance and thereby enrich for cells with tubulin alterations. The isolated cell lines were 2-fold resistant to the selecting drug, exhibited cross-resistance to other drugs that inhibit microtubule assembly, and had enhanced sensitivity to the microtubule-stabilizing drug paclitaxel. The concomitant resistance to microtubule-destabilizing drugs and enhanced sensitivity to paclitaxel suggested that these cell lines have changes in microtubule assembly. Consistent with this interpretation, drug-resistant cell lines exhibited altered alpha- or beta-tubulin mobility on two-dimensional gels and higher levels (47-54%) of assembled tubulin compared with wild-type (39%) or paclitaxel-resistant cells (25%). Some drug-resistant cells also had bundled microtubules as judged by immunofluorescence. Genomic sequencing of 11 drug-resistant cell lines predicted five different alterations (D45Y, C211F, D224N, S234N, and K350N) in beta-tubulin and four different alterations (H283Y, E55K, A383V, and R390C) in alpha-tubulin. The amino acid substitutions are dispersed on the primary and tertiary structures of tubulin and, together with the other mutant properties, argue against a mechanism involving changes in drug binding. Rather, we propose that the alterations in alpha- and beta-tubulin increase microtubule stability by promoting longitudinal interdimer and intradimer interactions and/or lateral interactions between protofilaments. This enhanced stability of microtubules increases their resistance to drugs that inhibit assembly.
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PMID:Mutations in alpha- and beta-tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine. 1288 31

The taxanes, paclitaxel (PTX) and docetaxel (DTX), belong to a novel class of anticancer drugs that stabilize microtubules and lead to tumor cell death. While both agents are widely used for the treatment of lung, breast, and ovarian cancer, many tumor types are refractory or develop resistance to these drugs. We describe here a novel analogue of DTX, designated MAC-321 [Microtubule/Apoptosis/Cytotoxic: 5beta, 20-epoxy-1, 2alpha-, 4-, 7beta-, 10beta-, 13alpha-hexahydroxytax-11-en-9-one 4 acetate 2 benzoate 7-propionate 13-ester with (2R,3S)-N-tertbutoxycarbonyl-3-(2-furyl)isoserine], that overcomes P-glycoprotein-mediated resistance to PTX and DTX in preclinical model systems. Similar to PTX or DTX, MAC-321 enhanced the rate of tubulin polymerization in vitro and caused the bundling of microtubules in cells. MAC-321 inhibited proliferation of a panel of 14 tumor cell lines with minimal variation in potency (IC(50) = 2.2 +/- 1.4 nM; range = 0.6-5.3 nM). Unlike PTX or DTX, the IC(50) of MAC-321 did not vary in cells that expressed low to moderate levels of P-glycoprotein. Even under extraordinary conditions in KB-V1 cells, which highly overexpress P-glycoprotein, resistance to MAC-321 was 80-fold compared with that of PTX (1400-fold) and DTX (670-fold). In addition, equivalent or less resistance to MAC-321 compared with PTX or DTX was observed in four cell lines that contain distinct point mutations within the taxane-binding site of beta-tubulin. Most importantly, MAC-321 displayed superior in vivo efficacy because: (a) MAC-321 either partially or completely inhibited tumor growth in three tumor models that overexpressed P-glycoprotein and were resistant to PTX; and (b) unlike PTX or DTX, MAC-321 was highly effective when given orally. MAC-321 was also highly effective when given as single i.v. dose. Our findings suggest that MAC-321, which is currently under clinical evaluation, may have broad therapeutic value.
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PMID:MAC-321, a novel taxane with greater efficacy than paclitaxel and docetaxel in vitro and in vivo. 1455 6

Paclitaxel (PTX) is a potent anti-neoplastic agent that is highly effective in treating ovarian cancer. Nevertheless, the emergence of PTX resistance has limited the control of this disease. To gain insight into the molecular alterations accompanying drug resistance in ovarian cancer, we generated a new stable PTX-resistant ovarian carcinoma cell line. CABA I cells, which display an intrinsic PTX resistance (IC50 = 800 ng/ml), were subjected to continuous exposure to PTX. From the residual surviving cells, the highly PTX-resistant line CABA-PTX (IC50 = 256000 ng/ml) was generated and stably maintained in vitro. Analysis of beta-tubulin expression indicated that only the HM40 and Hbeta9 isotypes were expressed in both parental and resistant cells. No specific point mutations in the HM40 were detected in either cell line, but expression levels of this isotype were significantly reduced (40%) in CABA-PTX cells. Hbeta9 levels were unchanged. In those cells, PTX resistance was associated with cross-resistance to vinblastine but not to methotrexate or 5-fluorouracil. Verapamil treatment did not reverse the intrinsic drug resistance of parental cells, but partially modulated the sensitivity of CABA-PTX cells to PTX and induced total sensitivity to vinblastine. No changes in the cell surface expression of the drug efflux pumps MRP1, MRP2 and P-glycoprotein were observed. PTX influx, monitored using a fluorescent drug derivative, was significantly reduced and delayed in CABA-PTX cells as compared to the parental cells. Together, these findings suggest that more than one mechanism is involved in PTX resistance, making CABA-PTX cell line a potentially valuable in vitro tool to study multifactorial acquired drug resistance in ovarian cancer.
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PMID:Induction of a multifactorial resistance phenotype by high paclitaxel selective pressure in a human ovarian carcinoma cell line. 1514 55

Peloruside A (peloruside), a microtubule-stabilizing agent from a marine sponge, is less susceptible than paclitaxel to multidrug resistance arising from overexpression of the P-glycoprotein efflux pump and is not affected by mutations that affect the taxoid binding site of beta-tubulin. In vitro studies with purified tubulin indicate that peloruside directly induces tubulin polymerization in the absence of microtubule-associated proteins. Competition for binding between peloruside, paclitaxel, and laulimalide revealed that peloruside binds to a different site on tubulin to paclitaxel. Moreover, laulimalide was able to displace peloruside, indicating that peloruside and laulimalide may compete for the same or overlapping binding sites. It was concluded that peloruside and laulimalide have binding properties that are distinct from other microtubule-stabilizing compounds currently under investigation.
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PMID:Peloruside A does not bind to the taxoid site on beta-tubulin and retains its activity in multidrug-resistant cell lines. 1528 5

HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [(3)H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [(14)C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major beta-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in alpha-tubulin that substitutes Ser for Ala(12) near the nonexchangeable GTP-binding site of alpha-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala(12) mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of alpha-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3.
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PMID:Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin. 1548 99

Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for P-glycoprotein, they are attractive candidates for cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of hemiasterlin, HTI-286 (HTI), was examined in cell populations derived from ovarian carcinoma (A2780/1A9) cells selected in HTI-286. 1A9-HTI-resistant cells (1A9-HTI(R) series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTI(R) cells corroborated the HTI cytotoxicity data. At 22 degrees C or 37 degrees C, in the absence of any drug, the percentage of polymerized microtubules for each of the 1A9-HTI(R) populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTI(R) populations were also more resistant to depolymerization at 4 degrees C and had more acetylated and detyrosinated (Glu-tubulin) alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTI(R) cell populations exhibited either a single nucleotide change in the M40 beta-tubulin isotype, S172A, or in two cell populations where no beta-tubulin mutation was detected, mutations in the Kalpha-1 alpha-tubulin isotype, S165P and R221H in one resistant cell population and I384V in another. Unlike reports of mutations resulting in reduced drug affinity, the experimental data and location of mutations are consistent with resistance to HTI-286 mediated by microtubule-stabilizing mutations in beta- or alpha-tubulin.
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PMID:Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analogue, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability. 1551 43

Taxanes (docetaxel and paclitaxel) as well as cisplatin (CDDP) are key chemotherapeutic agents in the treatment of non-small cell lung cancer (NSCLC). Although some indicators of taxane resistance, such as beta-tubulin mutations, P-glycoprotein (P-gp) and Bcl-2, have been reported in malignant cells, the mechanisms of taxane resistance in NSCLCs have yet to be fully elucidated. We evaluated in vitro chemosensitivity to docetaxel (DOC) and CDDP in 87 surgically-resected specimens of NSCLC by collagen gel-droplet embedded culture drug sensitivity test (CD-DST). Bcl-2 and P-gp expression in these specimens were also investigated by immunohistochemistry. We examined the association between Bcl-2 and P-gp expression and in vitro chemosensitivity to DOC and CDDP. Out of the 87 NSCLCs that were examined, Bcl-2 and P-gp were expressed in 32 (36.8%) and 28 (32.2%) of the tumors, respectively. Positive Bcl-2 expression was significantly associated with enhanced DOC sensitivity in NSCLCs (p=0.007) while no apparent association was observed between DOC sensitivity and P-gp expression. Interestingly, although DOC, but not CDDP has been reported to be a substrate of P-gp, P-gp expression was significantly inversely correlated with CDDP sensitivity in pulmonary adenocarcinomas (p=0.03). Positive Bcl-2 expression may be a promising indicator in determining in vitro taxane sensitivity in NSCLCs. On the other hand, positive P-gp expression may be an indicator of enhanced in vitro resistance to CDDP in pulmonary adenocarcinomas.
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PMID:Bcl-2 overexpression enhances in vitro sensitivity against docetaxel in non-small cell lung cancer. 1564 8

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.
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PMID:Two photoaffinity analogues of the tripeptide, hemiasterlin, exclusively label alpha-tubulin. 1586 30


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