Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Variation of the bridge linking the heterocyclic ring and p-aminobenzoyl-L-glutamate portions of our previously described classical 2,4-diaminofuro[2,3-d]pyrimidines 1 and 2 are reported as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and as antitumor agents. Specifically -CH2CH2- and -CH2NHCH2- bridged analogues, N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) ethyl]benzoyl]-L-glutamic acid (3) and N-[4-[[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) methyl]amino]methyl]benzoyl]-L-glutamic acid (4), respectively, were synthesized. Compound 3 was obtained via a Wittig reaction of the tributylphosphonium salt of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine (5) and methyl 4-formylbenzoate (6) followed by reduction and coupling with the diethyl ester of L-glutamic acid. Compound 4 was synthesized by the nucleophilic displacement of 5 with diethyl N-[4-(aminomethyl)benzoyl]-L-glutamate (15) and saponification. Both analogues were evaluated in vitro as inhibitors of DHFRs from (recombinant) human, human CCRF-CEM cells, and Lactobacillus casei. Compound 3 showed moderate activity (IC50 10(-6)-10(-7) M). Compound 4 was essentially inactive (IC50 10(-5) M, CCRF-CEM). The compounds were also evaluated against TS from (recombinant) human and L. casei and were of low activity (IC50 10(-5) M). The three-atom-bridged analogue 4 was somewhat more inhibitory to human TS than methotrexate (MTX). Compound 3 inhibited the growth of tumor cells in culture (IC50 10(-7) M) while 4 showed a low level of growth inhibitory activity. The inhibition of the growth of leukemia CCRF-CEM cells by both compounds parallels their inhibition of CCRF-CEM DHFR. Analogue 3 was a good substrate for human folylpolyglutamate synthetase (FPGS) derived from CCRF-CEM cells (Km 8.5 microM). Further evaluation of the growth inhibitory activity of 3 against the MTX-resistant subline of CCRF-CEM cells (R30dm) with decreased FPGS indicated that poly-gamma-glutamylation was important for its action. Protection studies with 3 in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin [(6R,S-5-formyltetrahydrofolate] or by a combination of thymidine and hypoxanthine, suggesting an antifolate effect directed at DHFR.
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PMID:Effect of bridge region variation on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines. 756 10

It was previously shown that amino acids 609-613 (REDLK) at the C-terminus of Pseudomonas exotoxin (PE) are necessary for cytotoxicity, presumably by directing the toxin to the endoplasmic reticulum (ER) [Chaudhary, Jinno, FitzGerald and Pastan (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 308-312]. Using the anti-[interleukin 2 receptor (IL2R)] immunotoxin anti-Tac(Fv)-PE38 (AT-PE38REDLK), it was found that removing the terminal lysine did not alter the activity, but replacing REDL with KDEL, the most common ER retention sequence, increased activity. To determine which amino acid in KDEL was responsible for the increase in activity, we tested eight C-terminal mutants of AT-PE38REDLK. Using IL2R-bearing MT-1 cells, we found that the glutamate residue of KDEL was required for high activity, as the cytotoxicity of AT-PE38 ending in KDEL, RDEL, KEEL or REEL was much greater than that of AT-PE38 ending in REDL, KEDL, RDDL or KDDL. Using freshly isolated lymphocytic leukaemia cells, AT-PE38 ending in KDEL, REEL or RDEL was more than 100-fold more cytotoxic than AT-PE38 ending in KEDL, REDL, RDDL or the native sequence REDLK. The RDEL sequence also improved the cytotoxic activity of an interleukin 4-PE38 toxin fusion protein. Improved cytotoxic activity correlated with improved binding of the C-termini to the KDEL receptor on rat Golgi membranes. These data indicate that the glutamate residue of KDEL improves the cytotoxicity of PE by increasing binding to a sorting receptor which transports the toxin from the transreticular Golgi apparatus to the ER, where it is translocated to the cytosol and inhibits protein synthesis.
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PMID:Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. 771 88

Eight novel, nonclassical, antifolate 2,4-diamino-5-(anilinomethyl)pyrrolo[2,3-d]pyrimidines, 1-8, with 3',4',5'-trimethoxyphenyl, 3',4'-dimethoxyphenyl, 2',5'-dimethoxyphenyl, 4'-methoxyphenyl, 2',5'-diethoxyphenyl, 3',4'-dichlorophenyl, 1'naphthyl, and phenyl substituents were synthesized as potential inhibitors of dihydrofolate reductases (DHFRs). The classical analogue N-[4-[N-[(2,4-diaminopyrrolo[2,3-d]pyrimidin- 5-yl)methyl]amino]benzoyl]-L-glutamic acid (9) was also synthesized as an inhibitor of DHFR and an antitumor agent. The classical and nonclassical analogues were obtained via reductive condensations of the key intermediate 2,4-diamino-5-cyanopyrrolo[2,3-d]pyrimidine (12) with the appropriate substituted aniline or (p-aminobenzoyl)-L-glutamate followed by reduction of the intermediate Schiff bases with NaCNBH3. Compounds 1-9 were evaluated in vitro as inhibitors of rat liver (rl), Pneumocystis carinii (pc), and Toxoplasma gondii (tg) DHFRs. The nonclassical analogues were significantly selective against tgDHFR (vs rat liver DHFR), ranging from 7- to 92-fold. The inhibitory activity was lower in pcDHFR and rlDHFR (IC50s > 10(-5) M) than in tgDHFR (IC50s = 10(-6) M). The classical analogue had inhibitory activity similar to that of methotrexate (MTX) against the growth of human leukemia CCRF-CEM, A253, and FaDu squamous cell carcinoma (SCC) of the head and neck cell lines. Further evaluation of 9 against CCRF-CEM and its sublines having defined mechanisms of MTX resistance demonstrated that the analogue utilizes the reduced folate/MTX-transport system and primarily inhibits DHFR and poly-gamma-glutamylation plays a role in its mechanism of action. Compound 9 was found to be 3-fold more efficient than aminopterin as a substrate for human folylpolyglutamate synthetase.
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PMID:Novel 2,4-diamino-5-substituted-pyrrolo[2,3-d]pyrimidines as classical and nonclassical antifolate inhibitors of dihydrofolate reductases. 778 47

Methotrexate (MTX), one of the earliest cancer chemotherapy agents, continues to be used extensively in the treatment of leukemia and a variety of other tumors. The efficacy of this drug results from its facile uptake by cells, rapid polyglutamylation and virtually stoichiometric inhibition of dihydrofolate reductase (DHFR), a key enzyme in cell replication. From the work of a multitude of biochemists, molecular biologists, organic chemists and pharmacologists, much is known about the mode of action of MTX and the mechanisms by which tumors exhibit inherent or acquired resistance to this drug. MTX enters cells primarily by a carrier-mediated active transport system whose principal substrate is 5-methyltetrahydrofolate, and additional glutamates are added to the gamma-position of the parent glutamate moiety. The tight binding of MTX to DHFR is defined from NMR and X-ray crystallographic studies of the enzyme and its drug or substrate complexes, supplemented by site-directed mutagenesis to confirm specific interactions. Resistance to the drug, encountered in cell culture model systems or in cancer patients, can result from an increased level of DHFR (due to gene amplification), mutant DHFR with reduced affinity for MTX, or decreased uptake or polyglutamylation of the drug. Although DHFR is an extremely well-studied enzyme, there is still some uncertainty about its kinetics, mechanism for reduction of folate, multiple forms, and activation by a diverse group of agents. Prodrug forms of MTX, e.g., MTX alpha-phenylalanine, which can be activated by carboxypeptidase A-monoclonal antibody conjugates, offer promise for improved efficacy of the drug by selective targeting to tumors. The large body of information summarized above has aided in the development of other folate antagonists, provides a paradigm for assessing the status of other cancer chemotherapeutic agents in current use, and offers a platform from which to speculate about the future of the field.
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PMID:The methotrexate story: a paradigm for development of cancer chemotherapeutic agents. 794 84

Ornithine delta aminotransferase (OAT) is a nuclear-encoded mitochondrial matrix enzyme that catalyzes the reversible transamination of ornithine to glutamate semialdehyde. In humans, genetic deficiency of OAT results in gyrate atrophy of the choroid and retina, a blinding chorioretinal degeneration usually beginning in late childhood. This disorder has been shown to be autosomal recessive, and is often caused by missense, nonsense, and/or frameshift mutations in the OAT gene. With the view of applying gene therapy, a Moloney murine leukemia virus (MoMLV)-based recombinant retrovirus vector has been constructed. The human OAT cDNA was placed under the control of the enhancer-promoter regulatory elements derived from the MoMLV long terminal repeat (LTR). The construct was transfected into the retroviral packaging cell lines GP + E - 86 and psi CRIP to produce virus particles. Supernatant from these OAT retrovirus producer cell lines were used to transduce mouse C57B1/6 embryonal fibroblasts. We showed that the recombinant retrovirus transfers the OAT gene to the recipient cells, which produce an OAT RNA transcript when analyzed by Northern blot. Western blot analysis and enzymatic assays confirmed the presence of an OAT polypeptide that has a high enzymatic activity in the transduced cell lines, even after a long period of time in vitro.
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PMID:Retrovirus-mediated gene transfer and expression of human ornithine delta-aminotransferase into embryonic fibroblasts. 794 32

A highly cytotoxic macrocyclic lactone polyether has been isolated from a Spongia species and named spongistatin 1. With L1210 murine leukemia cells an IC50 value for cell proliferation of 20 pM was obtained, and an increase in the mitotic index concordant with the decrease in cell number was observed. Kangaroo rat kidney PtK1 cells were examined by indirect immunofluorescence with a spongistatin 1 concentration that caused 50% reduction in cellular protein (0.3 nM) and with a 10-fold higher concentration. These cells displayed mitotic and nuclear aberrations at both concentrations, and intracellular microtubules were reduced in number at the lower concentration and disappeared at the higher. Similar changes in PtK1 cells were observed after treatment with equivalent toxic concentrations of the antimitotic agents colchicine, vinblastine, halichondrin B, and dolastatin 10. Spongistatin 1 inhibited the glutamate-induced polymerization of purified tubulin (IC50 value of 3.6 microM versus 2.1 microM for dolastatin 10 and vinblastine and 5.2 microM for halichondrin B). Spongistatin 1 had no effect on the binding of colchicine to tubulin, but it was a potent inhibitor of the binding of vinblastine and GTP to tubulin. In initial experiments with 5 microM tubulin and 5 microM vinblastine, spongistatin 1 and dolastatin 10 both had IC50 values of 2 microM, whereas halichondrin B had an IC50 value of 5 microM. Spongistatin 1 thus represents a new member of the group of complex natural products that inhibit mitosis by binding in the Vinca alkaloid domain of tubulin.
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PMID:Spongistatin 1, a highly cytotoxic, sponge-derived, marine natural product that inhibits mitosis, microtubule assembly, and the binding of vinblastine to tubulin. 823 26

CCRF-CEM human leukemia sublines resistant to short-term methotrexate (MTX) exposure as a result of decreased folylpolyglutamate synthetase (FPGS) activity were examined for their response to other cytotoxic agents. The R3/7 and R30dm sublines display 25 and 1%, respectively, of the FPGS activity of CCRF-CEM cells as measured with MTX in vitro. Response to agents in outgrowth experiments was examined under both continuous exposure (120 h, where MTX resistance is not observed) and short-term (6-14.5 h) exposure. During continuous exposure to various classes of agents, cross-resistance of R3/7 and R30dm that correlated with FPGS level was not observed, although some minor (< or = 3-fold) stochastic variations in sensitivity were noted. These agents included actinomycin D, Adriamycin, etoposide, vincristine, cisplatin, cytosine arabinoside, 5-fluorouracil, and some other antifolates. Cross-resistance during continuous exposure that did correlate with FPGS level was noted, however, to glutamate-containing thymidylate synthase inhibitors (including ICI D1694) and, to a minor extent, to 6-mercaptopurine and 5-fluorodeoxyuridine. Slight collateral sensitivity during continuous exposure that apparently correlated with FPGS level was noted to the lipid-soluble antifolate trimetrexate and to 5,8-dideazapteroyl-L-ornithine, an FPGS-specific inhibitor. In short-term exposures (where MTX resistance of the sublines is observed), the resistant sublines displayed sensitivity or cross-resistance to each agent that was qualitatively similar to that observed for the same agent in continuous exposure. Because of the requirement for reduced folates in the anti-DNA mechanism of action of fluoropyrimidines and the current clinical use of leucovorin (LV) to enhance their effects, the interaction of LV and fluoropyrimidines was examined. The results suggest that even highly FPGS-deficient cells are as sensitive to the effects of LV modulation as are wild-type cells even at fluoropyrimidine exposure times as short as 4 h.
Leukemia 1993 Dec
PMID:Cross-resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines. 825 99

The stereospecific syntheses of L-threo-gamma-fluoromethotrexate (1t) and L-threo-gamma-fluorofolic acid (3t) are reported. Compounds 1t and 3t have no substrate activity with folylpoly-gamma-glutamate synthetase isolated from CCRF-CEM human leukemia cells, and compound 1t inhibits human dihydrofolate reductase at similar levels as methotrexate. The synthesis of DL-3,3-difluoroglutamic acid (6) and its incorporation into DL-beta,beta-difluorofolic acid (4) are also reported. Compound 4 acts as a better substrate for human CCRF-CEM folylpoly-gamma-glutamate synthetase than folic acid (V/K = ca. 7-fold greater). Thus, replacement of the glutamate moiety of methotrexate and folic acid with 4-fluoroglutamic acid and 3,3-difluoroglutamic acid results in folates and antifolates with altered polyglutamylation activity.
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PMID:Synthesis and biological activity of folic acid and methotrexate analogues containing L-threo-(2S,4S)-4-fluoroglutamic acid and DL-3,3-difluoroglutamic acid. 856 27

We have analysed the cellular metabolism of a novel thymidylate synthase (TS) inhibitor, ZD1694, in MOLT-3 and K562 human leukaemia cell lines sensitive to or made resistant to ZD1694 by continuous exposure of the cells to ZD1694 with stepwise escalation of the drug concentration. The initial cellular uptake of [3H]ZD1694 was greater in K562 cells than in MOLT-3 cells and the drug accumulated approximately 3-fold more in the former cells following incubation with 0.1 microM ZD1694 at 37 degrees C for 24 h. TS and dihydrofolate reductase activities were not significantly different between the two cell lines. After a 30-min incubation with the drug at 37 degrees C, 85% of the total drug (2.3 pmol/mg protein) in K562 cells was found as tri- to pentaglutamates, whereas MOLT-3 cells accumulated less drug in this time (0.83 pmol/mg protein) and polyglutamates of chain length greater than triglutamate were not found to a significant extent. When the incubation time was extended to 24 h, the polyglutamate profile in K562 cells was progressively shifted towards those of long glutamate chain length and 59% of the total cellular drug (204 pmol/mg protein) was identified as the penta form. In contrast, even distribution between tri- and pentaglutamate was observed in MOLT-3 cells. Total cellular polyglutamates were approximately 3-fold higher in K562 cells than in MOLT-3 cells, and this may explain the 2.5-fold difference in the sensitivity to ZD1694 between the two cell lines. Continuous exposure of MOLT-3 and K562 cells to ZD1694 up to 1 microM or 0.1 microM resulted in 1600- and 4200-fold resistant sublines, respectively (MOLT-3/ZD1694.C and K562/ZD1694.C). The resistant MOLT-3 cells showed a markedly lower cellular accumulation and poor retention of [3H]ZD1694 with no significant change of initial drug uptake by 10 min and with a little increase of TS activity. HPLC analysis demonstrated that more than 90% of the 3H co-eluted with the monoglutamate (parent drug) in the resistant MOLT-3 cells, indicating extremely diminished polyglutamation in the cells. On the other hand, cellular uptake of [3H]ZD1694 was extensively impaired in K562/ZD1694.C cells and cellular accumulation of the drug was only 2.5% of that in the parent cells following 24 h incubation with the drug. Neither an increase of TS or dihydrofolate reductase activity nor a change in the polyglutamate formation profile was observed in the resistant K562 cells. These results indicate that the cellular ability to produce the polyglutamate metabolites of ZD1694 must influence the sensitivity of the tumour cells to this drug, and development of mechanisms involved in the ZD1694 resistance may relate to the intrinsic biochemical properties of the cells.
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PMID:Cellular pharmacokinetics of ZD1694 in cultured human leukaemia cells sensitive, or made resistant, to this drug. 857 77

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.
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PMID:Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase. 863 14


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