The Molecular Basis
of
5-fluorouracil, Methotrexate, Aminopterin, Trimetrexate, Lometrexol, Pemetrexed, Leucovorin, and Thymitaq, a novel lipophilic TS inhibitor
by
Suzann Yazd Pershing
Medical University of South Carolina
Medical Scientist Training Program
November 20, 2002
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Keywords: folic acid, folic acid antagonists, thymidylate synthase, dihydrofolate reductase, 5-phosphoribosylglycinamide formyltransferase, cancer, drug therapy, 5-fluorouracil, methotrexate, aminopterin, trimetrexate, lometrexol, LY231514, Pemetrexed, multitargeted antifolate, leucovorin, folinic acid, 5-formyltetrahydrofolate, thymitaq, AG337, and nolatrexed dihydrochloride.
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Table of
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N5, N10-Methylenetetrahydrofolate |
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Abstract
Folate antagonists are a group of compounds frequently used as part of a regimen for cancer treatment, and their role as such is examined here. Intended to prevent the occurrence of normal cellular processes in which folate or folate derivatives play a role, folate antagonists hamper thymidylate synthesis through inhibition of thymidylate synthase and dihydrofolate reductase, and reduce de novo purine synthesis through inhibition of 5-phosphoribosylglycinamide formyltransferase and/or aminoimidazole carboxamide ribonucleotide formyltransferase. Several examples of such drugs are presented with respect to their modes of action, effects, and mechanisms of resistance &emdash; 5-fluorouracil, Methotrexate, Aminopterin, Trimetrexate, Lometrexol, Pemetrexed, Leucovorin, and Thymitaq &emdash; and future possibilities are briefly discussed.
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Introduction
Folate antagonists were originally developed as antileukemic agents, but are now being used and/or investigated in the treatment of a wide range of cancerous and non-cancerous diseases, including, but not limited to, head and neck carcinomas, breast tumors, germ cell tumors, non-Hodgkin’s lymphoma, colorectal cancers, and gastric cancers. After folate was discovered to be vital to many cellular metabolic processes, the folate antagonists aminopterin (AMT) and methotrexate (MTX) were synthesized to interfere with these processes. They were shown to have anticancer activity in 1948 and the use of antifolates in cancer therapy became an area of interest. In addition to MTX and AMT, some original folate antagonists that are in use today include 5-fluorouracil (5-FU) and leucovorin (LV). The latter is not technically a folate antagonist, but rather a derivative of folate &emdash; 5-formyltetrahydrofolate, the most stable biological form of reduced folate &emdash; used to modulate the effects of many other folate antagonists. (1)
I. 5-fluorouracil
The antagonist 5-fluorouracil acts to indirectly inhibit the enzyme thymidylate synthase. Thymidylate synthase (TS) catalyzes the reaction of 5,10-methylenetetrahydrofolate (5,10-MTHF) ® 7,8 dihydrofolate (DHF), which converts deoxyuridylate (dUMP) to deoxythymidylate (dTMP). The primary mechanism by which 5-FU inhibits TS is that it is converted to fluorouridylate (F-UMP), which is then converted to F-UDP. The F-UDP can take either of two routes. It can become F-UTP and be incorporated into RNA or it can be reduced to deoxy-fluorouridylate dinucleotide (F-dUDP) and cleaved to form F-dUMP, which inhibits thymidylate synthase via the formation of a covalent ternary complex with TS and 5,10-MTHF (binds to dUMP binding site on TS). A less common mechanism by which 5-FU inhibits TS is through the direct formation of F-dUMP via fluorodeoxyuridine (FUDR). The application of this mechanism is limited because the cosubstrate for FUDR formation, deoxyribose 1-phosphate, is usually not present in sufficient amounts for the reaction to take place. It is therefore understandable that FUDR displays greater antitumor activity than 5-FU, since it is not associated with incorporation into RNA but rather results in longer retained TS inhibition. (2 and 3).
The primary effect of TS inhibition by 5-FU is a depletion of dTMP and dTTP levels, resulting in inhibition of DNA synthesis and “thymine-less death.” Thymine-less death occurs when a cell can still synthesize RNA and protein, but is unable to make DNA &emdash; resulting in cell overgrowth, and later, death. Another effect of TS inhibition is an accumulation of dUMP and, as inhibition progresses and the inhibitor approaches saturation of the enzyme, accumulation of F-dUMP. These nucleotide monophosphates convert to nucleotide triphosphates (dUTP and F-dUTP), and both can be incorporated into DNA. The nucleotides are subsequently excised, causing DNA strand breaks and eventually contributing to cell death. Prevention and repair of such damage is mediated by dUTPase and uracil-DNA glycosylase. The former degrades dUTP and F-dUTP to prevent their incorporation into DNA and the latter excises any dUTP or F-dUTP that is incorporated into DNA. (2)
Acquired resistance to 5-FU occurs mainly through an induction in TS levels brought on by 5-FU itself through deregulation of normal TS synthesis. Under normal conditions TS synthesis is regulated by the end product, TS, in a feedback mechanism. However, when bound in a ternary complex with F-dUMP and 5,10-MTHF, TS cannot regulate its synthesis, resulting in an increase in TS gene expression. (4) Resistance can also result from increased dUMP, decreased F-dUMP, decreased ternary complex stability (possibly due to unavailability of 5,10-MTHF), decreased accumulation of activated metabolites (via increased inactivation and decreased activation), increased incorporation into RNA, drug not reaching tumor, and/or increased drug elimination. (5)
II. Methotrexate
The antagonist Methotrexate competitively inhibits Dihydrofolate Reductase (DHFR &endash; major target) and, to a lesser degree, 5-phosphoribosylglycinamide formyltransferase (GARFT), aminoimidazole carboxamide ribonucleotide formyltransferase (AICARFT), and TS.
DHFR catalyzes the reduction of DHF to 5,6,7,8-tetrahydrofolate (THF) (5), which is converted to 5,10-MTHF, the substrate for TS. MTX binds tightly to DHFR (such that it might be mistaken for a noncompetitive inhibitor in normal kinetic analysis) (1) and inhibits it, causing accumulation of DHF and a lack of substrate for TS, indirectly inhibiting thymidylate synthesis. GARFT and AICARFT are inhibited directly by MTX as well as indirectly through the accumulated DHF. This inhibition causes decreased purine synthesis, affecting DNA and RNA synthesis so as to lead to cell death. However, thymidylate depletion is the primary effect of MTX. As in the case of 5-FU, it results in thymine-less death.
MTX exerts most of its pharmacological effect as a
polyglutamate. It can act on DHFR with or without polyglutamation,
but both MTX and DHF must be polyglutamated to act on GARFT and
AICARFT. The function of polyglutamation is to prevent efflux of the
drug from the cell and allow for a long-acting cellular repository.
Polyglutamates are formed from folate cofactors, glutamate residues,
and ATP by folylpolyglutamate synthetase (FPGS), with additional
glutamate residues added to the gamma carboxyl of the previous
glutamate residue. They are hydrolyzed in lysosomes by the enzyme
gamma-glutamyl hydrolase (GGH). (6
and 7).
Intrinsic resistance to MTX is primarily in the form of impaired long-chain polyglutamation. Studies have demonstrated that acute myeloid leukemia (AML &endash; intrinsically resistant to MTX) blasts, as opposed to acute lymphoblastic leukemia (ALL &endash; intrinsically sensitive to MTX) blasts, were less able to from long-chain polyglutamates. (8)
Acquired resistance to MTX occurs via two major mechanisms. One is decreased transport due to defects involving the reduced folate carrier (RFC) that transports MTX into the cell. That this was acquired was demonstrated by studies that showed that 13% of untreated ALL patients had impaired MTX transport while 71% of relapsed ALL transport had impaired transport. The molecular basis (via RFC) was deduced from comparisons of transport-defective ALL samples of RFC cDNA, the majority of which showed decreased RFC expression, as well as observations of increased mutations and polymorphisms (5 and 9).
Another mechanism of
acquired MTX resistance is via low-level DHFR gene amplification,
which occurs following exposure of tumor cells to increasing doses of
MTX and is sufficient to cause clinical resistance. It was shown that
approximately 30% of relapsed ALL patients had some DHFR
amplification, while only 10% of newly diagnosed ALL patients had
amplification (10 and 11).
III. Aminopterin
Although AMT, a DHFR inhibitor, was discovered prior to MTX, it was soon replaced by MTX due to studies which showed similar efficacy with decreased toxicity. (8). However, recent studies have suggested that AMT may have better uptake and polyglutamation than MTX, leading to renewed interest in the compound (12). Clinical trials have been initiated.
IV. Trimetrexate
Trimetrexate (TMQ) is a lipophilic analog of MTX, meaning that it does not depend on the RFC for entry into the cell, but rather enters by diffusion. In addition, it is unable to be polyglutamated because of the same lipophilic properties, although it is often retained at high levels intracellularly. (13). It has shown limited activity as a single agent in phase II clinical trials, but appears promising in combination with other agents, such as 5-FU and LV. (14)
V. Lometrexol
Lometrexol (LMTX) is a tight-binding inhibitor of GARFT, the first potent and selective such inhibitor to be discovered, with a broad antitumor spectrum. GARFT catalyzes the formation of purines from the reaction of 10-formyltetrahydrofolate (10-FTHF) ® THF. Its inhibition results in a depletion in intracellular purine levels, which in turn inhibits DNA and RNA synthesis &emdash; compromising the genetic machinery of the cell and resulting in cell death. (15).
The major mechanism of resistance to LMTX is through the hydrolytic cleavage of polyglutamates by GGH. High levels of GGH have been shown to correlate with LMTX resistance. (16).
As an excellent substrate for FPGS (17), LMTX is very susceptible to polyglutamation, and once formed, long-chain polyglutamates turn over so slowly in the cell that they are virtually impossible to eliminate. This results in LMTX being trapped in the cell, which causes severe delayed toxicity (thrombocytopenia). (18). This toxicity is greater under conditions of folate depletion because LMTX binds tightly to the membrane folate binding protein (mFBP &emdash; facilitates folate absorption). Under low extracellular folate concentrations, mFBP is up-regulated to bring folate into the cell. The up-regulation also causes greater uptake, and hence toxicity, of circulating LMTX. (19).
Due to its high toxicity, LMTX was initially discontinued prior to phase II clinical trials, but recent studies have demonstrated that concurrent administration of folate modulates the cytotoxicity without eliminating antitumor activity. (20). It has also been shown that LV rescue will serve to temper the toxic effects of LMTX and allow a greater dosage to be administered. (21).
VI. Pemetrexed
The drug Pemetrexed (LY231514), developed through modification of LMTX, is known as multi-targeted antifolate (MTA) for its role in inhibiting all three of the major steps targeted by antifolates: inhibition of TS (thymidylate synthesis), DHFR (DHF reduction), and GARFT and AICARFT (purine synthesis). It also plays a role in inhibition of C1 tetrahydrofolate synthase (catalyzes incorporation of formyl group into 10-FTHF for purine synthesis, and incorporation of methylene group into 5,10-MTHF for thymidylate synthesis), but to a lesser degree. Like many of the previously discussed drugs, it is a tight-binding competitive inhibitor of the enzymes on which its acts. (22).
Inhibition of TS by MTA leads to dTMP depletion (and hence thymine-less death) and dUMP accumulation (incorrectly incorporates into DNA, causing strand breaks and loss of genetic material), which eventually results in cell death. The MTA inhibition of DHFR yields a decrease in substrate for TS (further inhibiting thymidylate synthesis) and increase in DHF levels. GARFT and AICARFT inhibition (by MTA and accumulated THF) leads to a decrease in de novo purine synthesis. Another effect of MTA, most likely due to GARFT inhibition, is to cause initial blockade of the cell cycle prior to the G1 checkpoint, followed by movement into the S stage under lethal concentrations.
MTA is transported by the RFC into the cell, where, as one of the best known substrates for FPGS, it is highly polyglutamated. (23). Polyglutamation of MTA results in a large increase in its affinity for TS and GARFT, but has little effect on its affinity for DHFR, as shown by studies comparing the effects of the unglutamated, triglutamated, and pentaglutamated forms. (24).
Having seen the importance of polyglutamation in MTA activity, it is understandable that an important mechanism of MTA resistance is through impaired polyglutamation. Also contributing to resistance is impaired MTA transport into the cell by the RFC. This was determined by studies of three human cell sublines with acquired MTA resistance. MTA was found not to be particularly sensitive to increased TS expression, as would be predicted given its other modes of action. (25).
VII. Leucovorin
LV is not a folate antagonist per se, but the folate derivative 5-formyltetrahydrofolate, which can act to prevent/reverse the toxic side effects of antifolates (allowing higher doses to be utilized) as well as improve antitumor activity in some cases (such as TS inhibitors &emdash; 5-FU). After transport through the membrane by the RFC, LV is metabolized to 5,10-MTHF, thus increasing the availability of 5,10-MTHF (substrate for TS &emdash; counters effects of TS inhibition). Through this mechanism, it modulates the actions of many folate antagonists. LV can also enhance the antitumor activity of some folate antagonists. Polyglutamated 5,10-MTHF enhances inhibition of TS, via the formation and stabilization of the ternary complex with TS and 5-FU. LV was also observed to prevent drug-induced TS induction, as in the case of 5-FU. (26).
VIII. Thymitaq, a Novel Nonclassical Lipophilic
Thymidylate Synthase Inhibitor
Thymitaq (TM), or Nolatrexed dichloride (also referred to as AG337), is a noncompetitive, high-affinity inhibitor (Ki = 11nM) of TS. A quinazoline derivative, it was designed using knowledge of the three-dimensional structure of the TS 5,10-MTHF binding site. (27). It was developed by Aguoron , but discontinued due to initial results showing that although effective for head and neck and pancreatic tumors, it was not promising enough to justify further development over existing therapeutics. In 1999, Aguoron licensed worldwide rights to Zarix (now Eximias), and it was discovered that many patients realized disease stabilization with TM treatment. (28).
In 2001, TM was granted orphan drug and Fast Track status by the U.S. Food and Drug Administration (FDA) for the treatment of unresectable hepatocellular carcinoma (HCC). (29). (Orphan Drug status designates drugs for the treatment of a rare disease affecting fewer than 200,000 individuals in the U.S., and Fast Track status is reserved for drugs that display potential to address unmet needs for fatal or life-threatening conditions). Recently (11/07/2002), a phase III study comparing TM to the drug doxorubicin in patients with unresectable HCC reached 50% recruitment, and marketing of TM has begun in Korea (11/13/2002). (30 and 31).
As an inhibitor of TS synthesis, TM (like most of the previously discussed folate antagonists) will cause dTMP depletion and dUMP accumulation. This will cause an inability to form new, correctly synthesized DNA, which results in thymine-less death. TM is not dependent on the cell cycle &emdash; high concentrations of TM failed to induce S phase arrest but still produced apoptosis. (32).
Although TM itself is lipophilic, it can be administered via intravenous infusion as a water-soluble dihydrochloride salt. (33). Due to its lipophilic properties, TM is able to enter cells by passive diffusion and is nonpolyglutamatable (not a substrate for FPGS) (34), such that it has no requirements for membrane transport or intracellular activation &emdash; causes of drug resistance for many folate antagonists. Because of this, many tumor cell lines that are resistant to most classical (polyglutamatable, enter cell by RFC or mFBP) antifolates will be sensitive to TM. (35).
Thymitaq resistance is primarily the result of TS gene
amplification and point mutations. (36).
As discussed, altering the RFC or decreasing FPGS activity would have
no effect on lipophilic TM. Studies have observed that TM causes
significant up-regulation of TS. (37). Point mutations to TS that affect
function would be expected to occur at the highly conserved Ile-108
and Phe-225 residues. It has been demonstrated that the mutation of
isoleucine to a polar, acidic, or other disrupting amino acid at
position 108 does confer resistance to TM, due to removal of the
necessary hydrophobic interaction at the binding site. However,
mutation to the Phe-225 residue does not have any noticeable effect
on resistance to TM. (38).
Iododeoxyuridine (IdU) is a halogenated thymidine analog that competes with thymidine in DNA biosynthesis. When combined, TM appears to aid in the incorporation of IdU into DNA, a potential antitumor effect. This is also potentially useful for imaging and in situ radiotherapy when radiolabeled IdU is utilized. (39 and 40).
The Kisliuk effect is a very pronounced synergistic action observed with the combination of a lipophilic DHFR inhibitor (i.e. TMQ) and a classical polyglutamatable TS, GARFT, or AICARFT inhibitor. The mechanism by which the synergy occurs is that the lipophilic DHFR inhibitor depletes intracellular THF polyglutamates, resulting in higher levels of polyglutamates of the classical antifolate. Therefore, TM, as a nonclassical TS inhibitor, would not be expected to display synergistic activity with TMQ in accordance with the Kisliuk effect. A study was done to test the combination of TMQ and TM, and they were found to be antagonistic. (41).
Conclusions and
Future Directions
Having summarized the action, effect, and resistance of some old and new folate antagonists used and being investigated in cancer therapy, it appears that the primary sites of inhibition occur in thymidylate synthesis (TS), DHF reduction (DHFR), and de novo purine synthesis (GARFT and AICARFT). Classical folate antagonists frequently act in the polyglutamated form, and thus resistance is often developed to them through defects in transport into the cell or in intracellular polyglutamation. This makes nonclassical, lipophilic folate antagonists of interest since they do not require a transport mechanism or polyglutamation, eliminating the possibility of such resistance. One new lipophilic antagonist is the noncompetitive TS inhibitor Thymitaq, which has demonstrated effectiveness in unresectable hepatocellular carcinomas.
It would appear that the future of antifolate cancer therapy lies in newer drugs that target several sites of inhibition (such as Pemetrexed), as well as the further development of lipophilic, nonclassical, drugs like Trimetrexate and Thymitaq. The latter particular drugs showed promise but did not appear to be widely effective. To achieve a balance &emdash; between the undesirable decreased intracellular retention (and hence activity), afforded by unregulated transport and lack of polyglutamation, and the desirable decreased drug resistance from the same &emdash; is a difficult task, but hopefully one that can and will be achieved. Other goals for the future include developing an alternative way to relieve cytotoxicity while maintaining a high degree of antitumor efficacy, and exploring new synergistic drug combinations. To conclude, the use of folate antagonists in cancer treatment is a fascinating field that is only just beginning to be truly explored and discovered, with numerous applications already known and even more ahead on the horizon.
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Acknowledgements:
I wish to thank Sergey
Krupenko and L. William Stillway for
their unselfish guidance and encouragement in the studies that led to
this paper.
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