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Biomedical subjects

J B Hynes

Publications and source records attributed to J B Hynes.

At least 19 recordsLinked to original sources
PubMed1996

Endocytosis of GPI-linked membrane folate receptor-alpha.

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

Biological Transport↗
PubMed1995

Studies on the antitumor effects of analogues of 5,8-dideazaisofolic acid and 5,8-dideazaisoaminopterin.

Six new analogues of 5,8-dideazaisofolic acid and 5,8-dideazaisoaminopterin were synthesized in an effort to obtain enhanced antitumor activity. The modifications included the replacement of the 2-amino group by hydrogen or methyl as well as the inclusion of a methyl substituent at position 9. Based upon activity against L1210 leukemia cells in culture, three of the new analogues together with one compound described previously were evaluated for cytotoxicity in vitro using three human tumor cell lines (Colo 320 DM, Hep G2 and HL-60). The most effective compound was 2-desamino-N9-methyl-5,8-dideazaisoaminopterin (2c) with the HL-60 cells being the most sensitive to its cytotoxic effects. These analogues were evaluated in vitro as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) from human as well as bacterial (Lactobacillus casei) sources. All four of the 4-amino analogues were most effective toward L. casei DHFR compared with human DHFR, with 2-desamino-2-methyl-5,8-dideazaisoaminopterin (2d) and its 9-methyl derivative (2e) having 818- and 430-fold greater selectivity (L. casei/human). Most of the compounds studied were found to be only modest inhibitors of human TS (I50 values = 1.5 to 20 microM) and were therefore at least 40-fold less inhibitory than 10-propargyl-5,8-dideazafolic acid. Nevertheless, reversal of cytotoxicity studies with thymidine, hypoxanthine and folinic acid using the HL-60 cell line suggested that TS is the primary target for these analogues.

Aminopterin↗
PubMed1995

Structure-activity and structure-selectivity studies on diaminoquinazolines and other inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Twenty-eight 2,4-diaminopteridines with alkyl and aralkyl groups at the 6- and 7-positions, five 1,3-diamino-7,8,9,10-tetrahydropyrimido [4,5-c]isoquinolines with an alkyl, alkylthio, or aryl group at the 6-position, and nine 4,6-diamino-1,2-dihydro-s-triazines with one or two alkyl groups at the 2-position and a substituted phenyl or naphthyl group at the 1-position were evaluated as inhibitors of dihydrofolate reductase enzymes from Pneumocystis carinii, Toxoplasma gondii, and rat liver. Halogen substitution at the 5- or 6-position of 2,4-diaminoquinazoline favored selective binding to the P. carinii enzyme but not the T. gondii enzyme. For example, the 50% inhibitory concentrations of 2,4-diamino-6-chloroquinazoline as an inhibitor of P. carinii, T. gondii, and rat liver dihydrofolate reductase were 3.6, 14 and 29 microM, respectively, corresponding to 12-fold selectivity for the P. carinii enzyme but only marginal selectivity for the T. gondii enzyme. Greater than fivefold selectivity for P. carinii but not T. gondii dihydrofolate reductase was also observed for the 2,4-diaminoquinazolines with 5-methyl, 5-fluoro, 5- and 6-bromo, 6-chloro, and 5-chloro-6-bromo substitution. In contrast, alkyl and aralkyl substitution at the 6- and 7-positions of 2,4-diaminopteridines was found to be a favorable feature for selective inhibition of the T. gondii enzyme and, in two cases, for both enzymes. Nine of the fifty-one compounds tested against P. carinii dihydrofolate reductase and four of the thirty compounds tested against T. gondii dihydrofolate reductase displayed fivefold or greater selectivity for the microbial enzyme versus the rat liver enzyme. The most selective against both enzymes was 2,4-diamino-6,7-bis(cyclohexylmethyl) pteridine, with a selectivity ratio 2 orders of magnitude greater than the value reported for trimetrexate and piritrexim. Since substitution at the 7-position is generally considered to be detrimental to the binding of 2,4-diaminop-teridines and related compounds to mammalian dihydrofolate reductase, the selectivity observed in this study with the 6,7-bis(cyclohexylmethyl) analog may represent a useful approach to enhancing selective inhibition of the enzyme from nonmammalian species.

Animals↗
PubMed1995

Carrier- and receptor-mediated transport of folate antagonists targeting folate-dependent enzymes: correlates of molecular-structure and biological activity.

The transport properties and growth-inhibitory potential of 37 classic and novel antifolate compounds have been tested in vitro against human and murine cell lines expressing different levels of the reduced folate carrier (RFC), the membrane-associated folate binding protein (mFBP), or both. The intracellular targets of these drugs were dihydrofolate reductase (DHFR), glycinamide ribonucleotide transformylase (GARTF), folylpolyglutamate synthetase (FPGS), and thymidylate synthase (TS). Parameters that were investigated included the affinity of both folate-transport systems for the antifolate drugs, their growth-inhibitory potential as a function of cellular RFC/mFBP expression, and the protective effect of either FA or leucovorin against growth inhibition. Methotrexate, aminopterin, N10-propargyl-5,8-dideazafolic acid (CB3717), ZD1694, 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid (DDATHF), and 5-deazafolic acid (efficient substrate for FPGS) were used as the basic structures in the present study, from which modifications were introduced in the pteridine/quinazoline ring, the C9-N10 bridge, the benzoyl ring, and the glutamate side chain. It was observed that RFC exhibited an efficient substrate affinity for all analogues except CB3717, 2-NH2-ZD1694, and glutamate side-chain-modified FPGS inhibitors. Substitutions at the 2-position (e.g., 2-CH3) improved the RFC substrate affinity for methotrexate and aminopterin. Other good substrates included PT523 (N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine), 10-ethyl-10-deazaaminopterin, and DDATHF. With respect to mFBP, modifications at the N-3 and 4-oxo positions resulted in a substantial loss of binding affinity. Modifications at other sites of the molecule were well tolerated. Growth-inhibition studies identified a series of drugs that were preferentially transported via RFC (2,4-diamino structures) or mFBP (CB3717, 2-NH-ZD1694, or 5,8-dideazaisofolic acid), whereas other drugs were efficiently transported via both transport pathways (e.g., DDATHF, ZD1694, BW1843U89, or LY231514). Given the fact that for an increasing number of normal and neoplastic cells and tissue, different expression levels of RFC and mFBP are being recognized, this folate antagonist structure-activity relationship can be of value for predicting drug sensitivity and resistance of tumor cells or drug-related toxicity to normal cells and for the rational design and development of novel antifolates.

Acyltransferases↗
PubMed1992

Inhibition of hog liver folylpolyglutamate synthetase by 5-substituted 5,8-dideaza analogues of folic acid bearing a terminal L-ornithine residue.

Five new N alpha-(5,8-dideazapteroyl)-L-ornithines have been prepared using multistep synthetic sequences. These include N alpha-[5-(trifluoromethyl)-5,8-dideazapteroyl]-L-ornithine, 3, as well as N alpha-[5-(trifluoromethyl)-5,8-dideazaisopteroyl]-L-ornithine, 4, and its 5-fluoro and 5-chloro analogues. Both of the compounds containing a 5-(trifluoromethyl) group (3 and 4) were found to be excellent inhibitors of homogeneous hog liver folylpolyglutamate synthetase, having Ki values in the same range as N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, 2, (approximately 10 nM). However, the bridge-reversed isomer of 2 was 60-fold less inhibitory than 2.

Animals↗
PubMed1992

Synthesis and biological evaluation of N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine.

A novel folic acid analogue, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine, 3, was prepared via a multistep synthetic sequence. The key steps involved the conversion of 5-deazapteroic acid to its N10-formyl derivative followed by catalytic hydrogenation of the pyridine ring and subsequent heating in dilute sodium hydroxide to afford the new 5-deaza-5,6,7,8-tetrahydropteroic acid. After trifluoroacetylation, this compound was coupled to N delta-(tert-butyl-oxycarbonyl)-L-ornithine using conventional peptide bond forming conditions. Deprotection first in base and then in acid gave the title compound. Compound 3 was an effective inhibitor of hog liver folylpolyglutamate synthetase (Kis, estimated = 64 nM), and was shown to retard the formation of polyglutamates of a structurally related folic acid analogue in HCT-8 cells in vitro.

Adenocarcinoma↗
PubMed1991

In vitro metabolism of 5,8-dideazafolates and 5,8-dideazaisofolates by mammalian folylpoly-gamma-glutamate synthetase.

The in vitro metabolism of a variety of 5,8-dideazafolate and 5,8-dideazaisofolate analogues by pig liver folylpolyglutamate synthetase and the specificity of the enzyme for some polyglutamate derivatives of these analogues have been investigated. All 4-oxo-quinazoline analogues were metabolized to long chain polyglutamate derivatives, primarily the pentaglutamate, whereas 4-amino-quinazolines were metabolized to a lesser extent, with the accumulation of di- and triglutamate derivatives. This pattern of metabolism was consistent with the large drop in Vmax/Km and Vmax values for folylpolyglutamate synthetase observed with diglutamate derivatives of 4-aminofolate analogues. The extent of metabolism of the various analogues did not correlate with the relative substrate effectiveness of their parent monoglutamate derivatives. The 5-chloro and 5-methyl substitutions of quinazolines enhanced the addition of glutamate residues to 4-amino derivatives but markedly impaired the metabolism of 4-oxo derivatives.

Aminopterin↗
PubMed1991

Synthesis and biological evaluation of a fluorescent analogue of folic acid.

A fluorescein derivative of the lysine analogue of folic acid, N alpha-pteroyl-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (PLF), was synthesized as a probe for dihydrofolate reductase (DHFR) and a membrane folate binding protein (m-FBP). Excitation of PLF at 282 nm and at 497 nm gave a fluorescence emission maximum at 518 nm. Binding of PLF to human DHFR or human placental m-FBP results in approximately a 20-fold enhancement in the magnitude of the fluorescence emission, suggesting that the ligand interacts with a hydrophobic region on these proteins. Additional evidence suggests that an energy transfer may occur between the pteridine and the fluorescein moieties. PLF binds to the active site of human DHFR since methotrexate (MTX) competes stoichiometrically and the denatured enzyme in the presence of PLF did not exhibit fluorescent enhancement. The dissociation constant for the fluorescein derivative with respect to human DHFR is 115 nM as compared to 111 nM for folic acid. The Ki value for the competitive inhibition of human DHFR by the fluorescent analogue of folic acid is 2.0 microM compared to 0.48 microM for folic acid. PLF was reduced to N alpha-(7,8-dihydropteroyl)-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (H2PLF) and assayed by the enzymatic conversion to the tetrahydro derivative. The Km value for human DHFR for the dihydrofolate analogue is 2.0 microM. The KD value for H2PLF to human DHFR is 47 nM as compared to 44 nM for dihydrofolate. The KD values for both H2PLF and PLF indicate that the fluorescein moiety does not significantly affect folate binding in enzyme binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1991

Studies on the mechanism of antitumor action of 2-desamino-2-methyl-5,8-dideazaisofolic acid.

The new folate analogue, 2-desamino-2-methyl-5,8-dideazaisofolic acid, 2c, was synthesized and evaluated using a variety of biochemical and antitumor assays. For purposes of comparison, its 2-desamino, 2b, and 2-amino, 2a, counterparts, as well as N10-propargly-5,8-dideazafolic acid, 1a, and the corresponding 2-desamino, 1b, and 2-desamino-2-methyl, 1c, modifications were included in these studies. Compound 2c was found to be a potent inhibitor of the growth of L1210 and MCF-7 cells in culture, being only 2-fold and 5-fold less effective than 1c, respectively. However, although analogue 2c was 189-fold less inhibitory toward L1210 thymidylate synthase (TS) than 1c, its cytotoxicity was reversed completely by thymidine alone which suggests that the compound behaves as a TS inhibitor in cells. Enzymatically synthesized polyglutamates of 2c were substantially more inhibitory toward human TS than the parent compound. Compound 2c was the most efficient substrate for mammalian folyl-polyglutamate synthetase of the compounds studied having a Vmax/Km nearly 12-fold larger than 1c. Both 1c and 2c were effective inhibitors of the uptake of [3H]methotrexate into MOLT-4 cells, implying that each is efficiently transported into tumor cells. These results suggest that a weak inhibitor of TS in vitro can be a potent cytotoxic agent if it can readily gain entry into target cells and be converted to polyglutamated metabolites.

Animals↗
PubMed1991

Synthesis and biological evaluation of 5-deazaisofolic acid, 5-deaza-5,6,7,8-tetrahydroisofolic acid, and their N9-substituted analogues.

Prompted by recent disclosures concerning the potent antitumor activities of 5-deaza-5,6,7,8-tetrahydrofolic acid and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), we have prepared 5-deazaisofolic acid (3a) and 5-deaza-5,6,7,8-tetrahydroisofolic acid (4a). Reductive condensation of 2,6-diamino-3,4-dihydro-4- oxopyrido[2,3-d]pyrimidine with di-tert-butyl N-(4-formylbenzoyl)-L-glutamate and subsequent deprotection with trifluoroacetic acid yielded 5-deazaisofolic acid in good yield. Catalytic hydrogenation of this analogue then gave 4a. The 9-CH3 and 9-CHO modifications of 3a and the 9-CH3 derivative of 4a were also synthesized. Each of the new analogues was evaluated with a variety of folate-requiring enzymes as well as MCF-7 cells in culture. Compound 4a had an IC50 of ca. 1 microM against MCF-7 cells and was nearly 100-fold less potent than DDATHF in this regard. The three oxidized isofolate analogues were all poor inhibitors of tumor cell growth.

Animals↗
PubMed1989

Tricyclic 2,4-diaminopyrimidines with broad antifolate activity and the ability to inhibit Pneumocystis carinii growth in cultured human lung fibroblasts in the presence of leucovorin.

A selected number of 1,3-diaminobenzo[f]quinazolines and 1,3-diamino-5,6-dihydrobenzo[f]quinazolines, which may be viewed as tricyclic analogues of the lipid-soluble antifolates pyrimethamine (PM), metoprine (DDMP), and etoprine (DDEP), were tested as inhibitors of purified dihydrofolate reductase (DHFR) from WI-L2 lymphoblasts, and as inhibitors of the growth of Streptococcus faecium ATCC 8043 and L1210 murine leukemia cells in culture. In addition, these tricyclic compounds were tested for antimalarial activity against Plasmodium berghei in mice, and for the ability to inhibit the growth of Pneumocystis carinii trophozoites in WI-38 human lung fibroblast cultures in the presence of leucovorin (LV). The most potent analogues were those with chlorine substitution in the ring distal to the 2,4-diaminopyrimidine moiety. Fully aromatic compounds tended to be more active than those in which the 5,6-bond was reduced, suggesting that planarity favors binding to the DHFR active site and may be favorable for cellular uptake. Several of the 2,4-diaminopyrimidine analogues showed greater potency than PM, DDMP or DDEP, and were more nearly comparable to the bicyclic 2,4-diaminopyrimidine antifolates trimetrexate (TMQ) or piritrexim (BW301U), which are known to be selectively toxic to P. carinii in the presence of LV. Two of the tricyclic compounds, 1,3-diamino-8-chlorobenzo[f]quinazoline and 1,3-diamino-9-chlorobenzo[f]quinazoline, proved to have activity similar to TMQ and BW301U in this system.

Animals↗
PubMed1989

Comparison of the biological effects of selected 5,8-dideazafolate analogues with their 2-desamino counterparts.

Three new 5,8-dideaza analogues of folic acid devoid of an amino group at position 2 have been prepared by using synthetic routes patterned after earlier methodologies. They were 2-desamino-5,8-dideazaisofolic acid, 2b, 2-desamino-10-thia-5,8-dideazafolic acid, 2c, and 2-desamino-10-oxa-5,8-dideazafolic acid, 2d. These compounds were found to be 4-6-fold more cytoxic toward L1210 leukemia cells than their 2-NH2 counterparts and to be poor inhibitors of mammalian thymidylate synthase. However, they were only 1.5-3-fold less inhibitory toward dihydrofolate reductase than the analogous compounds containing a 2-NH2 group. The known thymidylate synthase inhibitors 2-desamino-10-propargyl-5,8-dideazafolic acid and 10-propargyl-5,8-dideazafolic acid were included in this study for purposes of comparison.

Animals↗
PubMed1989

Inhibition of mammalian folylpolyglutamate synthetase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin bearing a terminal L-ornithine.

Six new 5,8-dideaza analogues of folic acid and aminopterin containing a terminal L-ornithine residue were prepared by using multistep synthetic sequences. Each was evaluated as an inhibitor of hog liver folylpolyglutamate synthetase and human dihydrofolate reductase. Structural modifications at positions 2, 4, 5, and 10 were included to help define structure-activity relationships for compounds of this type. The compound N alpha-(4-amino-4-deoxy-5-chloro-5,8-dideazapteroyl)-L-ornithine (3f) was identified as the most potent inhibitor of mammalian folylpolyglutamate synthetase reported thus far (Ki congruent to 2 nM). Its 4-oxy counterpart, N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, was only 5-fold less inhibitory than 3f toward folylpolyglutamate synthetase but was found to be a much weaker inhibitor of dihydrofolate reductase than 3f.

Aminopterin↗
PubMed1988

Substrate specificity of mammalian folylpoly-gamma-glutamate synthetase for 5,8-dideazafolates and 5,8-dideaza analogues of aminopterin.

The substrate specificity of pig liver folylpolyglutamate synthetase (tetrahydrofolate:L-glutamate gamma-ligase (ADP-forming), EC 6.3.2.17) for classical 5,8-dideaza analogues of folic acid, isofolic acid aminopterin and isoaminopterin has been investigated. 5,8-Dideazafolate and 5,8-dideazaaminopterin are very effective substrates with activities approaching those of the best reduced folate substrates. The analogous isofolate analogues are less effective substrates, but still better than folic acid. The 5-chloro substituent is the only modification that consistently increases the on rate, with 5-chloro-5,8-dideazaaminopterin being the most effective substrate found, thus far, for the enzyme. Methylation at positions 9 or 10 generally decreases binding, while 5-methylation increases the binding of 4-oxoquinazolines, but decreases the binding of their 4-amino counterparts. The presence of a formyl group at N9 or N10 has the opposite effect, decreasing the binding of 4-oxo analogues while increasing the rate for 4-amino derivatives. Increases in on rate with methyl, formyl or 4-amino substitutions are only significant when the parent compound is a poor substrate, suggesting that these groups do not interact directly with the enzyme but cause conformational changes in the structure of the substrate that influence binding to the enzyme.

Adenosine Triphosphate↗
PubMed1988

Inhibition of chicken liver 5-aminoimidazole-4-carboxamide ribonucleotide transformylase by 5,8-dideaza analogues of folic acid.

A series of fourteen 5,8-dideaza analogues of folic and pteroic acids was evaluated for inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR TFase) from chicken liver. Of the 5,8-dideaza folate derivatives studies, 10-oxa-5,8-dideazafolic acid was the most potent inhibitor. The addition of one L-glutamate moiety to the gamma-carboxyl group caused a 6- to 7-fold reduction in Ki in three instances. Two compounds devoid of an L-glutamate were 4- to 6-fold less inhibitory than their parent counterparts possessing one L-glutamate residue.

Acyltransferases↗
PubMed1988

Inhibition of murine thymidylate synthase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin.

A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.

Aminopterin↗