RESUMEN
This review summarizes the biology of the proton-coupled folate transporter (PCFT). PCFT was identified in 2006 as the primary transporter for intestinal absorption of dietary folates, as mutations in PCFT are causal in hereditary folate malabsorption (HFM) syndrome. Since 2006, there have been major advances in understanding the mechanistic roles of critical amino acids and/or domains in the PCFT protein, many of which were identified as mutated in HFM patients, and in characterizing transcriptional control of the human PCFT gene. With the recognition that PCFT is abundantly expressed in human tumors and is active at pHs characterizing the tumor microenvironment, attention turned to exploiting PCFT for delivering novel cytotoxic antifolates for solid tumors. The finding that pemetrexed is an excellent PCFT substrate explains its demonstrated clinical efficacy for mesothelioma and non-small cell lung cancer, and prompted development of more PCFT-selective tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that derive their cytotoxic effects by targeting de novo purine nucleotide biosynthesis.
Asunto(s)
Antagonistas del Ácido Fólico/uso terapéutico , Ácido Fólico/metabolismo , Neoplasias/tratamiento farmacológico , Transportador de Folato Acoplado a Protón/genética , Transportador de Folato Acoplado a Protón/metabolismo , Transporte Biológico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Antagonistas del Ácido Fólico/metabolismo , Antagonistas del Ácido Fólico/farmacología , Glutamatos/metabolismo , Glutamatos/farmacología , Glutamatos/uso terapéutico , Guanina/análogos & derivados , Guanina/metabolismo , Guanina/farmacología , Guanina/uso terapéutico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Terapia Molecular Dirigida , Neoplasias/metabolismo , PemetrexedRESUMEN
Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [³H]THF cofactors derived from [³H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.
Asunto(s)
Antineoplásicos/farmacología , Antagonistas del Ácido Fólico/farmacología , Transportador de Folato Acoplado a Protón/metabolismo , Pirimidinas/farmacología , Pirroles/farmacología , Proteína Portadora de Folato Reducido/metabolismo , Tiofenos/farmacología , Animales , Antineoplásicos/metabolismo , Transporte Biológico , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Espacio Extracelular/metabolismo , Femenino , Antagonistas del Ácido Fólico/metabolismo , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Ácidos Pteroilpoliglutámicos/metabolismo , Pirimidinas/metabolismo , Pirroles/metabolismo , Relación Estructura-Actividad , Tetrahidrofolatos/metabolismo , Tiofenos/metabolismo , Microambiente TumoralRESUMEN
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or ß, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward ß-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.
Asunto(s)
Antineoplásicos/síntesis química , Receptor 1 de Folato/metabolismo , Receptor 2 de Folato/metabolismo , Antagonistas del Ácido Fólico/síntesis química , Glutamatos/síntesis química , Fosforribosilglicinamida-Formiltransferasa/antagonistas & inhibidores , Transportador de Folato Acoplado a Protón/metabolismo , Pirimidinas/síntesis química , Pirimidinonas/síntesis química , Proteína Portadora de Folato Reducido/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Glutamatos/química , Glutamatos/farmacología , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinonas/química , Pirimidinonas/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Trasplante Heterólogo , XenopusRESUMEN
The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-L-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}*-L-glutamic acid (compound 3), respectively) were inhibitory, with 2 â« 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [(3)H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by â¼50 and 75%, respectively. By an in situ GARFTase assay, 2 was â¼20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.
Asunto(s)
Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Antagonistas del Ácido Fólico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Purinas/biosíntesis , Pirimidinas/administración & dosificación , Pirroles/administración & dosificación , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Células CHO , Cricetinae , Cricetulus , Femenino , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/metabolismo , Células HeLa , Humanos , Neoplasias/metabolismo , Transportador de Folato Acoplado a Protón , Purinas/antagonistas & inhibidores , Pirimidinas/química , Pirimidinas/metabolismo , Pirroles/química , Pirroles/metabolismo , Xenopus laevisRESUMEN
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and four to six carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to alpha-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FR alpha, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.