Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases.

Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compounds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-characterized Escherichia coli enzyme, to resolutions of 2.2-3.0 A. The 2.9 A crystal structure of a complex of human TS with one of the inhibitors, the multi-targeted antifolate LY231514, demonstrates that this compound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate. This structure is one of the first liganded human TS structures, and its solution was aided by mutation to facilitate crystallization. Structures of three other pyrrolo(2,3-d)pyrimidine-based antifolates in complex with Escherichia coli TS confirm the orientation of this class of inhibitors in the active site. Specific interactions between the polyglutamyl moiety and a positively charged groove on the enzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d)pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo by the cellular enzyme folyl polyglutamate synthetase.

Enzyme inhibition, polyglutamation, and the effect of LY231514 (MTA) on purine biosynthesis.

The pyrrolopyrimidine-based antifolate, N-¿4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]benzoyl¿glutamic acid, LY231514 (MTA) has demonstrated antitumor activity in a broad array of human tumors, including breast cancer, colon cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, and other solid tumors. The biochemical basis of this activity was explored by measuring activation of MTA by polyglutamation and the activity of MTA to inhibit several folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). The enzyme folylpolyglutamate synthase (FPGS) activated MTA very efficiently. Using FPGS from two different species, Km values below 2 micromol/L and high relative first order rate constants, k' (Vmax/Km) of 6.4 and 13.7 compared with another substrate, lometrexol, were obtained. The formation of polyglutamates of several antifolates were compared in vitro at high and low substrate concentrations. At low MTA concentrations, tetraglutamated and pentaglutamated MTA were the predominant forms identified after a 24-hour incubation period. In contrast, only diglutamyl methotrexate and a mixture triglutamylated, tetraglutamylated, and pentaglutamylated forms of the GARFT inhibitor lometrexol were formed under the same conditions. At higher substrate concentrations (20 micromol/L, 24 hours), greater amounts of each product were formed. The major metabolites, however, were triglutamated MTA or triglutamated lometrexol, while only diglutamyl methotrexate was recovered. Thus, MTA was an excellent substrate for FPGS and it was efficiently metabolized to highly polyglutamated species by this enzyme. The activity of MTA and its polyglutamated metabolites to inhibit several folate-dependent enzymes was measured. In vitro, MTA and its polyglutamates were potent, tight-binding inhibitors of several folate-dependent enzymes, including thymidylate synthase, dihydrofolate reductase, and GARFT. Preliminary cell-based assays (CCRF-CEM) demonstrated inhibition of the purine de novo pathway by MTA, consistent with its multitargeted mechanism of action against tumor cells. The combined effects of activation of MTA to highly polyglutamated metabolites and the potency of these polyglutamates to inhibit multiple folate-dependent enzymes provide a mechanistic basis for understanding the broad antitumor activity of this compound against many human tumor types.

The synthesis and biological activity of a series of 2,4-diaminopyrido[2,3-d]pyrimidine based antifolates as antineoplastic and antiarthritic agents.

A new series of 2,4-diaminopyrido[2,3-d]pyrimidine based antifolates 1-3 were synthesized through an efficient conversion of 2-pivaloyl-4-oxo-6-ethynylpyrido[2,3-d]pyrimidine 5 to the corresponding 4-amino analog 7 via the activated 1,2,4-triazole intermediate 6. Compound 7 was used as the key intermediate for the preparation of the final products. The detailed biological evaluation of these compounds both as antineoplastic and antiarthritic agents will be discussed.

The antiproliferative and cell cycle effects of 5,6,7, 8-tetrahydro-N5,N10-carbonylfolic acid, an inhibitor of methylenetetrahydrofolate dehydrogenase, are potentiated by hypoxanthine.

5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid (LY354899) has been demonstrated to inhibit the dehydrogenase activity of C1-tetrahydrofolate synthase. This compound was only moderately antiproliferative toward CCRF-CEM lymphocytic leukemia cells in culture, but induced apoptosis after long incubation times. Slightly greater potency was observed in CEM cells adapted to grow in low folate media. Cell cycle alterations induced by LY354899 were unique relative to antifolates that inhibit either the purine or thymidine de novo biosynthetic pathways. Based on the observed changes in DNA content, we hypothesized that inhibition of the dehydrogenase resulted in two temporally distinct events: the first was a purineless-like effect and the second was a thymineless-like effect that resulted in apoptosis. To test this hypothesis, we combined LY354899 with the purine salvage metabolite, hypoxanthine. This combination resulted in an earlier and more dramatic apoptotic response, indicating that the thymineless effect had been potentiated. Biochemical analysis of ribo- and deoxyribonucleoside triphosphates confirmed that inhibition of the dehydrogenase activity initially resulted in decreased pools of deoxypurines and deoxypyrimidines, followed 16 hr later by an increase in deoxyadenosine triphosphate (dATP) and a further decrease in deoxythymidine triphosphate (dTTP). These studies demonstrate that the inhibition of the dehydrogenase activity of C1-tetrahydrofolate synthase may represent a viable target for the development of novel antifolates. The results are discussed in terms of deoxypurine and deoxypyrimidine biosynthesis.

Sulfonimidamide analogs of oncolytic sulfonylureas.

A series of sulfonimidamide analogs of the oncolytic diarylsulfonylureas was synthesized and evaluated for (1) in vitro cytotoxicity against CEM cells, (2) in vivo antitumor activity against subaxillary implanted 6C3HED lymphosarcoma, and (3) metabolic breakdown to the o-sulfate of p-chloroaniline. The separated enantiomers of one sulfonimidamide analog displayed very different activities in the in vivo screening model. In general, several analogs demonstrated excellent growth inhibitory activity in the 6C3HED model when dosed orally or intraperitoneally. A correlative structure-activity relationship to the oncolytic sulfonylureas was not apparent.

LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes.

N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.

Dietary folate and folylpolyglutamate synthetase activity in normal and neoplastic murine tissues and human tumor xenografts.

The importance of polyglutamation for the activation of natural folates and classical antifolates and recent evidence for the role of dietary folate as a biochemical modulator of antifolate efficacy led us to investigate the influence of changes in dietary folate on folylpolyglutamate synthetase (FPGS) activity. Activities were measured using lometrexol (6R-5,10-dideazatetrahydrofolic acid) as a substrate for FPGS with extracts of murine tissues, murine tumors, and human tumor xenografts from mice on standard diet or low folate diet. Tissues and tumors from mice on standard diet exhibited a 6-fold range of FPGS activity. Kidney had the lowest activity (36 pmol/hr.mg protein), followed by the human xenograft PANC-1 pancreatic carcinoma (46 pmol/hr.mg protein), liver (109 pmol/hr.mg protein), murine C3H mammary tumor (112 pmol/hr.mg protein), and the human xenograft MX-1 mammary carcinoma (224 pmol/hr.mg protein). In response to restricted dietary folate, four out of five tissues had significantly increased (25-50%) FPGS activity. Only the tumor with highest FPGS activity under standard diet conditions (MX-1 mammary) did not respond to low folate diet. The results indicate that changes in dietary folate intake can modulate FPGS activity significantly in vivo and suggest that the tissue distribution and toxicities of classical antifolates requiring polyglutamation for activation and cellular retention will be influenced significantly by folate status of the host.

Characterization of folate receptor from normal and neoplastic murine tissue: influence of dietary folate on folate receptor expression.

Membrane-associated folate receptors (FRs) have been detected in many mammalian species, and multiple isoforms have been identified. The pharmacological properties of FRs from murine kidney, liver, and six murine tumors were characterized. Murine kidney expressed primarily folate-binding protein 1, analogous to human FR-alpha, whereas murine liver expressed predominantly folate-binding protein 2, analogous to human FR-beta. Five of six murine tumors expressed high-affinity FRs with pharmacological properties consistent with folate-binding protein 1 isoform expression. Restriction of dietary folate resulted in significant changes in the FR expression in most murine tissues. Kidney and tumor FRs showed a decreased affinity for folic acid, suggesting a change in isoform expression in response to a low folate diet. Density of the FR in the kidney decreased, and, in contrast, density of the FR in all tumors increased. The response of the liver to a low folate diet was unique in that there were no detectable changes in affinity or density of liver FR. Changes in dietary folate that modulate FR isoform expression may have relevance for cancer patients treated with antifolates.

The role of dietary folate in modulation of folate receptor expression, folylpolyglutamate synthetase activity and the efficacy and toxicity of lometrexol.

We have studied the molecular effects of a LFD in a murine model in order to better define the biochemical changes associated with folate deficiency. In addition, we have demonstrated the effect of a LFD on the pharmacokinetic profile and therapeutic activity and toxicity of lometrexol. These studies showed increased density of FR in tumors implanted in LFD mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. The higher density of this FR would provide greater capacity for garnering serum folate. FPGS activity increased in several tumors and liver and kidney of LFD mice. The increase in this enzyme activity would result in enhanced polyglutamation of folates and classical antifolates and thus increased cellular retention. Consistent with these changes in liver FPGS, mice injected i.v. with a single dose of lometrexol accumulated significantly more drug in liver and tumors of LFD animals compared to SD mice. Also, higher liver concentrations of lometrexol persisted longer in LFD mice. Polyglutamate analysis showed that longer polyglutamate forms appeared earlier in liver of LFD mice. After 7 days, longer polyglutamyl forms were recovered from liver of LFD mice (octa- and hepta-glutamyl lometrexol) compared to those on SD. A comparison of the efficacy and toxicity of lometrexol in C3H mammary tumor-bearing mice showed that in mice on LFD, lometrexol treatment produced a delayed toxicity with an LD50 of 0.1-0.3 mg/kg, a 3000-fold increase in lethality compared to SD mice. Supplementation of mice with folic acid restored anti-tumor activity and increased the therapeutic dose-range over which efficacy could be assessed. These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs.

Role of membrane-associated folate binding protein in the cytotoxicity of antifolates in KB, IGROV1, and L1210A cells.

Based on differential levels of membrane-associated folate binding protein (mFBP) expression, murine L1210 leukemia, human KB epidermoid carcinoma, and human IGROV1 ovarian carcinoma cells maintained under low (physiological) folate conditions (2 nM folinic acid) were used as model systems to investigate the potential role of mFBP in antifolate transport. In addition, L1210 parental cells were compared to a subline, L1210A, expressing high levels of mFBP and defective reduced folate carrier. Antifolates for which KB-derived mFBP has high affinity (5, 10-dideazatetrahydrofolic acid [DDATHF] and homo-DDATHF [0.24 and 0.78 respectively relative to folic acid]) and low affinity (methotrexate [0.002]) were chosen for this study. Protection against DDATHF/homo-DDATHF induced cytotoxicity was achieved preferentially by folic acid compared to folinic acid in IGROV1 and L1210A cells. In IGROV1 cells, cytotoxicity IC50s were increased 18- and 5.5-fold for DDATHF and homo-DDATHF respectively by 20 nM folic acid. Moreover, greater protection was observed in L1210A cells, where IC50s were increased 354- and 80-fold for these same compounds by 20 nM folic acid. Similar protection was not observed in KB cells, suggesting that KB mFBP was not functional in DDATHF transport. Although mFBP expression may be an important determinant in the cytotoxicity of antifolates for certain tumor cells, our data demonstrate a lack of correlation between levels of mFBP and function of mFBP for DDATHF transport in the models studied.

Effect of endogenous and exogenous polyamines on organic cation transport in rabbit renal plasma membrane vesicles.

The effect of several polyamines on the transport of organic cations [N1-[3H]methylnicotinamide (NMN), [3H]NMN and 3H-tetraethylammonium, ([3H]TEA)] in rabbit renal brush border (BBMV) and basolateral membrane vesicles (BLMV) was studied using a rapid filtration assay. Under pH-driven conditions in BBMV, [3H]NMN transport was cis inhibited approximately 30% by the naturally occurring polyamines (cadaverine, putrescine, spermine and spermidine) and nearly 90% by methylglyoxal bis(guanylhydrazone) (MGBG), a synthetic polyamine, mepiperphenidol and cimetidine, classical organic cation transport inhibitors. In the absence of a pH gradient, the capability of these agents to block [3H]NMN transport was diminished. The capability of these compounds to translocate the membrane was assessed by examining the phenomenon of counterflow. TEA, mepiperphenidol and MGBG produced trans stimulation of TEA uptake, whereas putrescine did not. Kinetic studies revealed that both putrescine and MGBG affected the Km value for TEA transport while having a minimal effect on the Vmax value. These results are most consistent with competitive inhibition. The specificity of polyamine inactivation of organic cation transport was assessed by examining what effect they had on the transport of another brush border transporter, D-glucose. These agents did not inhibit D-glucose transport. In addition, the effect of polyamines on the transport of organic cations in BLMV was studied. Only MGBG, cimetidine and mepiperphenidol inhibited organic cation transport. The results indicate that endogenous polyamines cis inhibited the organic cation transporter in BBMV competitively, whereas the exogenous polyamine MGBG cis inhibited competitively and produced trans stimulation. In contrast in BLMV, only MGBG was an effective inhibitor.