Program:

Antifolates
Antimetabolites
Enzymology
Drug Resistance

Fundamental biochemical and molecular studies are designed to elucidate aspects of folate metabolism relevant to cancer therapeutics. Two areas are currently of greatest interest. One area is the potentiation of transport mediated by the reduced folate carrier (RFC) that occurs after exposure of cells to a natural nucleoside 5-amino-4-imidazolecarboxamide riboside (Z), which is a precursor of an intermediate in de novo purine synthesis. Elucidation of the mechanism of this potentiation should be relevant to use of antifolates such as methotrexate (MTX). A second area of interest is the synthesis, regulation, and function of poly(g-glutamyl) metabolites of folates and antifolates in proliferation and in resistance. Folylpolyglutamates are essential for normal folate metabolism and hence for cell viability. Thus, agents interfering with folylpolyglutamate synthesis and function may be therapeutically useful and could also be used as probes of their function. Folylpolyglutamate synthetase (FPGS) is being characterized from a recombinant human source, from human tumor cell lines, and from normal murine intestinal epithelium to provide a rational basis for design of selective FPGS inhibitors. Agents designed based on these studies are synthesized by collaborating chemists (Dr. J. K. Coward, University of Michigan, Ann Arbor and Dr. A. Gangjee, Duquesne University). Three classes of agents have been designed and synthesized to date: (1) direct FPGS inhibitors; (2) folate analogs that are nonsubstrates for FPGS; and (3) agents with enhanced ability to form short chain length polyglutamates. Biochemical and pharmacological properties of new agents are examined in detail and the results are used to refine the structures. The subcellular location of FPGS and factors regulating FPGS expression are also of interest. These studies should provide important information about the role of subcellular compartmentalization of folate metabolism. 

Biochemical and molecular studies of drug sensitivity and resistance may lead to increased efficacy in clinical antifolate use. The mechanism of action of methotrexate (MTX) and other “classical” antifolates and their metabolism to poly(g-glutamates) are being explored in antifolate-sensitive and antifolate-resistant human tumor cell lines. These studies may identify factors relevant to natural and acquired MTX resistance as well as provide data allowing more judicious clinical use of these agents.

Progress       

Transport is required before reduced folates and anticancer antifolates (e.g., MTX) can exert their physiological functions or cytotoxic effects. Although two transport systems mediate influx of folates and antifolates, the carrier with the widest tissue distribution and greatest apparent activity is the reduced folate carrier (RFC). Although the RFC has been extensively characterized, there is little evidence that RFC-mediated influx can be post-transcriptionally regulated. We have now shown that influx, but not efflux, of [³H]MTX in CCRF-CEM human childhood T-cell leukemia cells can be potentiated up to 6-fold by exogenous 5-amino-4-imidazolecarboxamide riboside (Z) in a Z- and MTX-concentration-dependent manner. Metabolism to the more biologically active polyglutamate forms is also potentiated for MTX and other antifolates. That potentiation of influx by Z is mediated by an effect on the RFC is supported by analyses ±Z showing: (a) the similarity of and magnitude of kinetic constants for [³H]MTX uptake; (b) the similarity of inhibitory potency by known alternate RFC substrates; (c) that no potentiation occurs in a CCRF-CEM subline that specifically does not express the RFC; and (d) the similarity of time- and temperature-dependence. Potentiation in CCRF-CEM cells occurs rapidly (<10 min) and does not require new protein synthesis. The effect of specific inhibitors of folate metabolism and the time and sequence of Z incubation with cells suggest that both inhibition of dihydrofolate reductase and metabolism of Z are essential for potentiation. Preincubation of CCRF-CEM with Z-related metabolites (e.g., adenosine, adenine, inosine, hypoxanthine) and under conditions of acute folate deficiency does not initiate potentiation; Z-aglycone does initiate potentiation, however. Z increases the growth inhibitory potency of MTX and aminopterin against CCRF-CEM cells when both Z and antifolate are present for the first 24 hr of a 120 hr growth period. Z represents the first small molecule that can regulate RFC activity.

Mutational deletion of FPGS activity is lethal, thus FPGS is a potential target for cancer chemotherapy. We pioneered a two-track strategy to design FPGS inhibitors. One track identifies changes in the acceptor Glu substructure of folate analogs that: (a) directly impart FPGS inhibition; or (b) inform about the acceptor Glu subsite of the FPGS active site. The second track is to define other structural requirements, generally in the pteroate/extended heterocycle moiety, for increasing potency and/or specificity of lead inhibitors identified in the first track; this involves extensive studies of human FPGS substrate specificity. Progress on both tracks has been expedited by a baculovirus shuttle vector containing the cDNA for human cytosolic FPGS (cFPGS; below; Dr. B. Shane; UC Berkeley).

Detailed knowledge of the enzyme mechanism of human FPGS will be useful in inhibitor design. In addition to natural folates, FPGS catalyzes the ATP-dependent ligation of glutamic acid to the anticancer drug 5,10-dideaza-5,6,7,8-tetrahydrofolate (Lometrexol™, (6R)-DDATHPteGlu₁), which is known to be readily converted to polyglutamates in vivo. Kinetic characteristics of the mono- and polyglutamate forms were determined so that the effects of the presence of polyglutamate products on FPGS activity might be understood. Synthesis of (6R)-DDATHPte[¹⁴C]Glu, and unlabeled mono- and various polyglutamates, DDATHPteGlu_n (6R, n=1-6; 6S, n=1-2) was effected from (6R)- or (6S)-5,10-dideazatetrahydropteroyl-azide and [¹⁴C]glutamic acid, glutamic acid, or H-Glu-g-Glu_n-g-Glu-OH (n=0-4), respectively. These compounds were evaluated as FPGS substrates to determine steady-state kinetic constants for subsequent mechanistic studies. Saturation kinetics were observed for (6R)-DDATHPteGlu₁, the isomer corresponding to natural tetrahydrofolate (H₄PteGlu), whereas marked substrate inhibition was observed for (6S)-DDATHPteGlu₁ and all DDATHPteGlu_n polyglutamate substrates, except for DDATHPteGlu₆. Multiple ligation of glutamate renders a quantitative analysis of these data difficult. However approximate values of K_m = 0.65 –1.6 µM and K_I = 144–417 µM for DDATHPteGlu_n were obtained using a simple kinetic model. Preliminary mechanistic studies, including time course, substrate trapping, and pulse-chase experiments, have provided strong evidence in favor of a processive, rather than distributive, mechanism of multiple glutamate ligations. The degree of processivity is dependent on the concentration of the folate substrate, thus suggesting a mechanism for regulation of folate polyglutamate synthesis in cells. 

With regard to track 1 of inhibitor design, we previously examined the FPGS inhibitory potency of two classes of phosphorous-containing pseudopeptide folate analogs (Tsukamoto et al. Arch. Biochem. Biophys. 355: 109-118, 1998; McGuire et al. Biochem. Pharmacol., 65: 315-318, 2003) synthesized by Dr. Coward that were designed to mimic the tetrahedral intermediate formed in the ATP-dependent reaction catalyzed by FPGS. To allow direct comparison, both classes were synthesized with the same heterocycle as MTX. Methotrexate-phosphinate (MTX-phosphinate; 4-amino-10-methyl-pteroyl-L-Glu-g-[Y{P(O)(OH)-CH₂}]glutarate) is a more highly potent (K_is, 3.1 nM), competitive inhibitor of recombinant human cFPGS than is MTX-phosphonate (4-amino-10-methyl-pteroyl-L-Glu-g-[Y{P(O)(OH)-O}]glutarate; K_is, 46 nM). For both inhibitors, FPGS inhibition is not time-dependent and preincubation of FPGS, inhibitor, and ATP does not potentiate inhibition, within experimental limits. These results suggest that slow phosphorylation to produce a more potent inhibitor form is not involved. Neither MTX-phosphinate nor MTX-phosphonate is growth inhibitory to human CCRF-CEM leukemia cells at 1 µM (70-fold above the concentration of MTX giving 50% growth inhibition), probably because of poor transport. Because of its exceedingly high potency as an FPGS inhibitor, MTX-phosphinate represents a lead structure from which cell-permeable analogs may be developed to test the hypothesis that FPGS inhibition is therapeutically efficacious. Further studies are aimed at determining: (a) the potency of the two isomers of the chiral phosphinate moiety; (b) the effect of the position of the phosphinate group in analogs of longer polyglutamates; and (c) the optimal heterocycle to ligate to the phosphinate to achieve potent and specific FPGS inhibition. 

With regard to track 2 of inhibitor design, a number of novel heterocycles and substituted variations synthesized by Dr. A. Gangjee (Duquesne U) have been evaluated for their FPGS substrate efficiency as leads for increasing potency and/or specificity of phosphinate inhibitors. Substrate efficiency, as we have shown, is a surrogate for inhibitory potency when the heterocycle is then appended to an inhibitory Glu analog (above).

We previously identified human mitochondrial (mFPGS) and cytosolic (cFPGS) FPGS isoforms and others have shown that they are encoded by one gene. We also showed that the two forms have different electrophoretic mobilities in SDS-PAGE. Because of the potential role(s) of cFPGS and mFPGS isoforms in antifolate sensitivity and resistance, we have continued our study of these isoforms. In order to determine the submitochondrial location of mFPGS, we adapted literature methods to purify mitochondria from CCRF-CEM human leukemia cells in order to be able to isolate mFPGS free from cFPGS. Since there are no well-described, published methods for isolating pure human mitochondria, considerable effort was expended developing these methods. We are now able to obtain a good yield of intact human mitochondria from CCRF-CEM cells. In addition, methods for submitochondrial fractionation with digitonin, which are best described for rat liver where large quantities of mitochondria can be obtained, had to be adapted for human cell culture sources. Human mitochondria were fractionated with increasing concentrations of digitonin to successively extract the four submitochondrial compartments. Western analyses of the fractions using protein markers specific for each compartment suggest that mFPGS is distributed in the matrix and/or inner membrane compartments. Interaction of any FPGS with membranes has not previously been reported, although there is literature precedent for matrix proteins that are partially associated with the inner membrane. Further support for an interaction of mFPGS with the inner mitochondrial membrane is provided by localization of about half of the mFPGS in the mitochondrial membrane fraction obtained by freeze-thaw of intact mitochondria; the remaining mFPGS is located in the soluble fraction. Resistance of about half of the mFPGS in whole mitochondria to alkaline carbonate extraction suggests that its interaction with the inner membrane is more similar to an integral, than a peripheral, membrane protein. The data suggest that human mFPGS is at least in part strongly associated with the inner mitochondrial membrane. Because of the success of our method, we were able to collaborate with Dr. B.J. Dolnick of this center in his efforts to characterize the subcellular distribution of the rTSb, a protein involved in growth regulation that is translated from a transcript that is a partial antisense of the TMPS gene. Bioinformatics analysis indicated that some transcripts encoded N-terminal mitochondrial signaling sequences. Using our methods, we were able to show for the first time that rTSbis expressed both in the cytosol and in mitochondria. Further studies are planned to investigate the submitochondrial location of rTSb and its functional significance.

Selected Publications

Gangjee, A., Zeng, Y., McGuire, J.J., and Kisliuk, R.L. Synthesis of classical, four-carbon-bridged 5-substituted furo[2,3-d]pyrimidine and 6-substituted pyrrolo[2,3-d]pyrimidine analogues as antifolates. J. Med. Chem. 48: 5329-5336, 2005.

Liang, P., Nair, J.R., Song, L., McGuire, J.J., and Dolnick, B.J. Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene. BMC Genomics 6: 125, 2005 (14 September). http://www.biomedcentral.com/1471-2164/6/125

Jayakumar Nair, R. and McGuire, J.J. Submitochondrial localization of human mitochondrial folylpolyglutamate synthetase. Biochimica Biophysica Acta–Mol. Cell Res. 1746: 38-44, 2005. doi:10.1016/j.bbamcr.2005.08.004

Tomsho, J., McGuire, J.J., and Coward, J.K. Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-ý-glutamates: Kinetics of multiple glutamate ligations catalyzed by folylpoly-ý-glutamate synthetase. Org. Biomolec. Chem. 3: 3388-3398, 2005. On-line publication http://www.rsc.org/Publishing/Journals/OB/article.asp?doi=b505907k.

Gangjee, A., Lin, X., Kisliuk, R.L., and McGuire, J.J. Synthesis of N-{4-[2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid and N-{4-[2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-L-glutamic acid as dual inhibitors of dihydrofolate reductase and thymidylate synthase, and as potential antitumor agents. J. Med. Chem. 48: 7215-7222, 2005.

McGuire, J.J., Haile, W.H., and Yeh, C.-C. 5-Amino-4-imidazolecarboxamide riboside (Z) potentiates both transport of reduced folates and antifolates by the Reduced Folate Carrier (RFC) and their subsequent metabolism. Cancer Res. 66: 3836-3844, 2006.

Gangjee, A., Jain, H.D., Phan, J., Song, X., McGuire, J.J., and Kisliuk, R.L. Dual inhibitors of thymidylate synthase and dihydrofolate reductase as antitumor agents: Design, synthesis and biological evaluation of classical and nonclassical pyrrolo[2,3-d]pyrimidine antifolates. J. Med. Chem. 49: 1055-1065, 2006.

Gangjee, A., Yang, J., McGuire, J.J., and Kisliuk, R.L. Synthesis and evaluation of classical 2, 4-diamino-5-substituted-furo[2,3-d]pyrimidine and 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine as antifolates. Bioorg. Med. Chem. 14 (24): 8590-8598, 2006. (Epub 2006 Sep 20).