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 proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 much more potently than 4. Compound 3 was more inhibitory than 4 toward β-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.

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.

The H + -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH.

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-, respectively) were inhibitory, with 2 Ͼ Ͼ 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 ϳ50 and 75%, respectively. By an in situ GARFTase assay, 2 was ϳ20-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.

In humans and terrestrial vertebrates, the kidney controls systemic pH in part by absorbing filtered bicarbonate in the proximal tubule via an electrogenic Na ؉ /HCO 3 ؊ cotransporter (NBCe1/SLC4A4). Recently, human genetics revealed that NBCe1 is the major renal contributor to this process. Homozygous point mutations in NBCe1 cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts (Igarashi, T., Inatomi, J., Sekine, T., Cha, S. H., Kanai, Y., Kunimi, M., Tsukamoto, K., Satoh, H., Shimadzu, M., Tozawa, F., Mori, T., Shiobara, M., Seki, G., and Endou, H. (1999) Nat. Genet. 23, 264 -266). We have identified and functionally characterized a novel, homozygous, missense mutation (S427L) in NBCe1, also resulting in pRTA and similar eye defects without mental retardation. To understand the pathophysiology of the syndrome, we expressed wild-type (WT) NBCe1 and S427L-NBCe1 in Xenopus oocytes. Function was evaluated by measuring intracellular pH (HCO 3 ؊ transport) and membrane currents using microelectrodes. HCO 3 ؊ -elicited currents for S427L were ϳ10% of WT NBCe1, and CO 2 -induced acidification was ϳ4-fold faster. Na ؉ -dependent HCO 3 ؊ transport (currents and acidification) was also ϳ10% of WT. Current-voltage (I-V) analysis reveals that S427L has no reversal potential in HCO 3 ؊ , indicating that under physiological ion gradient conditions, NaHCO 3 could not move out of cells as is needed for renal HCO 3 ؊ absorption and ocular pressure homeostasis. I-V analysis without Na ؉ further shows that the S427L-mediated NaHCO 3 efflux mode is depressed or absent. These experiments reveal that voltage-and Na ؉ -dependent transport by S427L-hkNBCe1 is unfavorably altered, thereby causing both insufficient HCO 3 ؊ absorption by the kidney (proximal RTA) and inappropriate anterior chamber fluid transport (glaucoma).

Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3(-) to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3(-) secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3(-) transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3(-) cotransporter) and Slc26a6A and Slc26a6B (putative Cl(-)/HCO3(-) exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3(-)-transporting activity as electrogenic Cl(-)/nHCO3(-) exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3(-) cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3(-) secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3(-) transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3(-) uptake (NBCe1) and apical HCO3(-) secretion (Slc26a6A and Slc26a6B).

The orphan cotransport protein expressed by the SLC5A8 gene has been shown to play a role in controlling the growth of colon cancers, and the silencing of this gene is a common and early event in human colon neoplasia. We expressed this protein in Xenopus laevis oocytes and have found that it transports small monocarboxylic acids. The electrogenic activity of the cotransporter, which we have named SMCT (sodium monocarboxylate transporter), was dependent on external Na + and was compatible with a 3 : 1 stoichiometry between Na + and monocarboxylates. A portion of the SMCT-mediated current was also Cl − dependent, but Cl − was not cotransported. SMCT transports a variety of monocarboxylates (similar to unrelated monocarboxylate transport proteins) and most transported monocarboxylates demonstrated K m values near 100 µM, apart from acetate and D-lactate, for which the protein showed less affinity. SMCT was strongly inhibited by 1 mM probenecid or ibuprofen. In the absence of external substrate, a Na + -independent leak current was also observed to pass through SMCT. SMCT activity was strongly inhibited after prolonged exposure to high external concentrations of monocarboxylates. The transport of monocarboxylates in anionic form was confirmed by the observation of a concomitant alkalinization of the cytosol. SMCT, being expressed in colon and kidney, represents a novel means by which Na + , short-chain fatty acids and other monocarboxylates are transported in these tissues. The significance of a Na + -monocarboxylate transporter to colon cancer presumably stems from the transport of butyrate, which is well known for having anti-proliferative and apoptosis-inducing activity in colon epithelial cells.

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K t at pH 5.5 for H247A was 28.4-fold. Although wild type (WT)-PCFT K i values varied among the folates, K i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His 247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser 172 . The folic acid influx K t for S172A-PCFT was decreased similar to H247A. His 281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ϳ12-fold1 in the folic acid influx K t . When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K t was unchanged, but the K t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT-and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His 247 to Ser 172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His 281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.This laboratory recently identified SLC46A1 as a protoncoupled folate transporter (PCFT) 2 that mediates transport of folates across the apical (brush border) membrane of enterocytes within the acidic microclimate at the absorptive surface of the proximal jejunum (1). The critical role that PCFT plays at this epithelium was confirmed with the demonstration by this laboratory that there are loss-of-function mutations in this gene in patients with the autosomal recessive disorder, hereditary folate malabsorption (Online Mendelian Inheritance in Man, 229050) (1, 2). Hereditary folate malabsorption is characterized by impaired intestinal folate absorption and impaired transport of folates into the central nervous system (3), the latter likely due to a defect in transport across the blood-choroid plexus-cerebrospinal fluid barrier. While operating most efficiently at low pH, PCFT contributes to the transport of folates and the pharmacological activity of the new generation antifolate, pemetrexed, even at neutral pH (4). PCFT may be even more important to the delivery of this, and possibly other antifolates, within the acidic interstitium of solid tumors (5, 6). PCFT may also play a role in the export of folates from acidified endosomes (7) during folate receptor-mediated endocytosis (8, 9). Although facilitative carriers in lower organisms com...