JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila.

Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. Here, we performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. We identified that Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. Our observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.

ATB0,+-targeted delivery of triptolide prodrugs for safer and more effective pancreatic cancer therapy.

Triptolide (TP) is a diterpene epoxide component extracted from Tripterygium wilfordii and has been shown to possess an impressive anticancer effect. However, TP has not yet entered any clinic trials due to the severe adverse effects that resulted from the off-target absorption and distribution found in animal studies. In this study, we designed and synthesized three amino acids (tryptophan, valine, and lysine) based TP prodrugs to target ATB0,+ which are highly expressed in pancreatic cancer cells for more effective pancreatic cancer therapy. The stability, uptake profiles, uptake mechanism, and cancer-killing ability were studied in vitro. All three prodrugs showed increased uptake and enhanced cytotoxicity in pancreatic cancer cells, but not in normal pancreatic cells. The difference in killing effect on normal and cancer cells was attributed to pancreatic cancer over-expressed ATB0,+-mediated uptake. Specifically, tryptophan-conjugated TP prodrug (TP-Trp) showed the highest uptake and the best cancer cell killing effect, considered as the best candidate. The present study provided the proof-of-concept of exploiting TP prodrug to target ATB0,+ for pancreatic cancer-selective delivery and treatment.

Oncology Therapeutics Targeting the Metabolism of Amino Acids.

Amino acid metabolism promotes cancer cell proliferation and survival by supporting building block synthesis, producing reducing agents to mitigate oxidative stress, and generating immunosuppressive metabolites for immune evasion. Malignant cells rewire amino acid metabolism to maximize their access to nutrients. Amino acid transporter expression is upregulated to acquire amino acids from the extracellular environment. Under nutrient depleted conditions, macropinocytosis can be activated where proteins from the extracellular environment are engulfed and degraded into the constituent amino acids. The demand for non-essential amino acids (NEAAs) can be met through de novo synthesis pathways. Cancer cells can alter various signaling pathways to boost amino acid usage for the generation of nucleotides, reactive oxygen species (ROS) scavenging molecules, and oncometabolites. The importance of amino acid metabolism in cancer proliferation makes it a potential target for therapeutic intervention, including via small molecules and antibodies. In this review, we will delineate the targets related to amino acid metabolism and promising therapeutic approaches.

Amino Acid Transporters as Potential Therapeutic Targets in Thyroid Cancer.

Thyroid cancer cells have a high amino acid demand for proliferation, invasion, and metastasis. Amino acids are taken up by thyroid cancer cells, both thyroid follicular cell and thyroid parafollicular cells (commonly called "C-cells"), via amino acid transporters. Amino acid transporters up-regulate in many cancers, and their expression level associate with clinical aggressiveness and prognosis. This is the review to discuss the therapeutic potential of amino acid transporters and as molecular targets in thyroid cancer.

Synergism between SLC6A14 blockade and gemcitabine in pancreactic cancer: a 1H-NMR-based metabolomic study in pancreatic cancer cells.

Gemcitabine is the first-line chemotherapy for pancreatic cancer. To overcome the often-acquired gemcitabine resistance, other drugs are used in combination with gemcitabine. It is well-known that cancer cells reprogram cellular metabolism, coupled with the up-regulation of selective nutrient transporters to feed into the altered metabolic pathways. Our previous studies have demonstrated that the amino acid transporter SLC6A14 is markedly up-regulated in pancreatic cancer and that it is a viable therapeutic target. α-Methyltryptophan (α-MT) is a blocker of SLC6A14 and is effective against pancreatic cancer in vitro and in vivo. In the present study, we tested the hypothesis that α-MT could synergize with gemcitabine in the treatment of pancreatic cancer. We investigated the effects of combination of α-MT and gemcitabine on proliferation, migration, and apoptosis in a human pancreatic cancer cell line, and examined the underlying mechanisms using 1H-NMR-based metabolomic analysis. These studies examined the intracellular metabolite profile and the extracellular metabolite profile separately. Combination of α-MT with gemcitabine elicited marked changes in a wide variety of metabolic pathways, particularly amino acid metabolism with notable alterations in pathways involving tryptophan, branched-chain amino acids, ketone bodies, and membrane phospholipids. The metabolomic profiles of untreated control cells and cells treated with gemcitabine or α-MT were distinctly separable, and the combination regimen showed a certain extent of overlap with the individual α-MT and gemcitabine groups. This represents the first study detailing the metabolomic basis of the anticancer efficacy of gemcitabine, α-MT and their combination.

Augmentation of Cystic Fibrosis Transmembrane Conductance Regulator Function in Human Bronchial Epithelial Cells via SLC6A14-Dependent Amino Acid Uptake. Implications for Treatment of Cystic Fibrosis.

SLC6A14-mediated l-arginine transport has been shown to augment the residual anion channel activity of the major mutant, F508del-CFTR, in the murine gastrointestinal tract. It is not yet known if this transporter augments residual and pharmacological corrected F508del-CFTR in primary airway epithelia. We sought to determine the role of l-arginine uptake via SLC6A14 in modifying F508del-CFTR channel activity in airway cells from patients with cystic fibrosis (CF). Human bronchial epithelial (HBE) cells from lung explants of patients without CF (HBE) and those with CF (CF-HBE) were used for H3-flux, airway surface liquid, and Ussing chamber studies. We used α-methyltryptophan as a specific inhibitor for SLC6A14. CFBE41o-, a commonly used CF airway cell line, was employed for studying the mechanism of the functional interaction between SLC6A14 and F508del-CFTR. SLC6A14 is functionally expressed in CF-HBE cells. l-arginine uptake via SLC6A14 augmented F508del-CFTR function at baseline and after treatment with lumacaftor. SLC6A14-mediated l-arginine uptake also increased the airway surface liquid in CF-HBE cells. Using CFBE41o cells, we showed that the positive SLC6A14 effect was mainly dependent on the nitric oxide (NO) synthase activity, nitrogen oxides, including NO, and phosphorylation by protein kinase G. These finding were confirmed in CF-HBE, as inducible NO synthase inhibition abrogated the functional interaction between SLC6A14 and pharmacological corrected F508del-CFTR. In summary, SLC6A14-mediated l-arginine transport augments residual F508del-CFTR channel function via a noncanonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be used for the development of companion therapy in CF.

Acetaminophen reduces the protein levels of high affinity amino acid permeases and causes tryptophan depletion.

In yeast, toxicity of acetaminophen (APAP), a frequently used analgesic and antipyretic drug, depends on ubiquitin-controlled processes. Previously, we showed a remarkable overlap in toxicity profiles between APAP and tyrosine, and a similarity with drugs like rapamycin and quinine, which induce degradation of the amino acid permease Tat2. Therefore, we investigated in yeast whether APAP reduced the expression levels of amino acid permeases. The protein levels of Tat2, Tat1, Mup1 and Hip1 were reduced, while the expression of the general permease Gap1 was increased, consistent with a nutrient starvation response. Overexpression of Tat1 and Tat2, but not Mup1, Hip1 and Gap1 conferred resistance to APAP. A tryptophan auxotrophic strain trp1Δ was more sensitive to APAP than wild-type and addition of tryptophan completely restored the growth restriction of trp1∆ upon APAP exposure, while tyrosine had an additive effect on APAP toxicity. Furthermore, intracellular aromatic amino acid concentrations were reduced upon APAP exposure. This effect was less prominent in ubiquitin-deficient yeast strains that were APAP resistant and showed a reduced degradation of high affinity amino acid permeases. APAP-induced changes in intracellular amino acid concentrations were also detected in hepatoma HepG2 cells indicating significance for humans.

Transport of BMAA into Neurons and Astrocytes by System xc.

The study of the mechanism of ß-N-methylamino-L-alanine (BMAA) neurotoxicity originally focused on its effects at the N-methyl-D-aspartate (NMDA) receptor. In recent years, it has become clear that its mechanism of action is more complicated. First, there are certain cell types, such as motor neurons and cholinergic neurons, where the dominate mechanism of toxicity is through action at AMPA receptors. Second, even in cortical neurons where the primary mechanism of toxicity appears to be activation of NMDA receptors, there are other mechanisms involved. We found that along with NMDA receptors, activation of mGLuR5 receptors and effects on the cystine/glutamate antiporter (system xc-) were involved in the toxicity. The effects on system xc- are of particular interest. System xc- mediates the transport of cystine into the cell in exchange for releasing glutamate into the extracellular fluid. By releasing glutamate, system xc- can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and in this way may protect cells against oxidative stress. We have previously published that BMAA inhibits cystine uptake leading to GSH depletion and had indirect evidence that BMAA is transported into the cells by system xc-. We now present direct evidence that BMAA is transported into both astrocytes and neurons through system xc-. The fact that BMAA is transported by system xc- also provides a mechanism for BMAA to enter brain cells potentially leading to misincorporation into proteins and protein misfolding.

Co-targeting intracellular pH and essential amino acid uptake cooperates to induce cell death of T-ALL/LL cells.

Cancer cells reprogram their metabolism to optimize their growth and proliferation in the host microenvironment. For this purpose, they enhance the uptake of extracellular nutrients and deal with the metabolic waste products through the overexpression of numerous membrane proteins including amino-acid transporters (LAT1) and acid-base regulating enzymes, such as carbonic anhydrases (CAs). Here we describe the anti-tumoral effects of a new class of CAXII inhibitors, the glycosyl coumarins on T-ALL/LL cells. These effects appeared to be mediated through inhibition of mTOR/Akt pathway and c-myc downregulation. Interestingly, we show that the combined targeting of amino acid fluxes and pH regulators provides a promising therapeutic strategy in the future of T-ALL/LL management.

Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals.

The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na(+) and H(+), but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes.

The antifungal eugenol perturbs dual aromatic and branched-chain amino acid permeases in the cytoplasmic membrane of yeast.

Eugenol is an aromatic component of clove oil that has therapeutic potential as an antifungal drug, although its mode of action and precise cellular target(s) remain ambiguous. To address this knowledge gap, a chemical-genetic profile analysis of eugenol was done using ∼4700 haploid Saccharomyces cerevisiae gene deletion mutants to reveal 21 deletion mutants with the greatest degree of susceptibility. Cellular roles of deleted genes in the most susceptible mutants indicate that the main targets for eugenol include pathways involved in biosynthesis and transport of aromatic and branched-chain amino acids. Follow-up analyses showed inhibitory effects of eugenol on amino acid permeases in the yeast cytoplasmic membrane. Furthermore, phenotypic suppression analysis revealed that eugenol interferes with two permeases, Tat1p and Gap1p, which are both involved in dual transport of aromatic and branched-chain amino acids through the yeast cytoplasmic membrane. Perturbation of cytoplasmic permeases represents a novel antifungal target and may explain previous observations that exposure to eugenol results in leakage of cell contents. Eugenol exposure may also contribute to amino acid starvation and thus holds promise as an anticancer therapeutic drug. Finally, this study provides further evidence of the usefulness of the yeast Gene Deletion Array approach in uncovering the mode of action of natural health products.

Effects of the beta-lactam antibiotic ceftriaxone on nicotine withdrawal and nicotine-induced reinstatement of preference in mice.

RATIONALE: Several studies suggest that repeated nicotine administration causes alterations in glutaminergic transmission that may play an important role in developing and maintaining nicotine addiction. Chronic nicotine administration in rats decreases the expression of the glutamate transporter-1 (GLT-1) and cysteine-glutamate exchanger (system xC-) in the nucleus accumbens. We hypothesized that ceftriaxone, a GLT-1 and system xC- activator, would decrease murine behavioral aspects of nicotine dependence. OBJECTIVE: This study aimed to investigate the effect of repeated ceftriaxone administration on the behavioral effects of nicotine using mouse models of conditioned reward and withdrawal. METHOD: Using male ICR mice, the ability of repeated ceftriaxone injections to modulate the development and reinstatement of a nicotine-conditioned place preference (CPP) was evaluated. Additionally, nicotine withdrawal-associated signs were assessed. These included both physical (somatic signs and hyperalgesia) and affective (anxiety-related behaviors) withdrawal signs in mice. Finally, the effects of ceftriaxone on nicotine-induced antinociception and hypothermia after acute nicotine injection were measured. RESULT: Ceftriaxone had no effect on the development of nicotine preference but significantly attenuated nicotine-induced reinstatement of CPP. Furthermore, ceftriaxone reversed all nicotine withdrawal signs measured in mice. CONCLUSION: Altogether, these findings show that a ß-lactam antibiotic reduces nicotine withdrawal and nicotine-seeking behavior. Our results suggest that the documented efficacy of ceftriaxone against cocaine and morphine dependence-related behaviors effects extends to nicotine.

DCPIB, the proposed selective blocker of volume-regulated anion channels, inhibits several glutamate transport pathways in glial cells.

4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB) was identified as the selective blocker of volume-regulated anion channels (VRAC). VRAC are permeable to small inorganic and organic anions, including the excitatory neurotransmitter glutamate. In recent years DCPIB has been increasingly used for probing the physiologic and pathologic roles of VRAC and was found to potently suppress pathologic glutamate release in cerebral ischemia. Because ischemic glutamate release can be mediated by a plethora of mechanisms, in this study we explored the selectivity of DCPIB toward the majority of previously identified glutamate transporters and permeability pathways. l-[(3)H]glutamate, d-[(3)H]aspartate, and l-[(14)C]cystine were used to trace amino acid release and uptake. We found that in addition to its well-characterized effect on VRAC, DCPIB potently inhibited glutamate release via connexin hemichannels and glutamate uptake via the glutamate transporter GLT-1 in rat glial cells. In contrast, DCPIB had no direct effect on vesicular glutamate release from rat brain synaptosomes or the cystine/glutamate exchange in astrocytes. The compound did not affect the astrocytic glutamate transporter GLAST, nor did it block glutamate release via the P2X(7)/pannexin permeability pathway. The ability of DCPIB to directly block connexin hemichannels was confirmed using a gene-specific siRNA knockdown approach. Overall, our data demonstrate that DCPIB influences several glutamate transport pathways and that its effects on VRAC in vivo should be verified using additional pharmacological controls.

Estradiol inhibits the activity of proton-coupled amino acid transporter PAT1 expressed in Xenopus oocytes.

Estrogen has great potential as a therapeutic agent in focal ischemic brain injury. Amino acids as energy resources and neurotransmitters in the central nervous system are crucial for proper neuronal function and excitability. The proton-coupled amino acid transporter PAT1 has clear potential in drug absorption. In this paper, human brain PAT1 was cloned and expressed in Xenopus oocytes. The effects of estradiol on the activity of PAT1 were investigated. Glycine-induced membrane currents mediated by PAT1 were measured using the two-electrode voltage clamp technique. The amplitude of the glycine-elicited current was decreased progressively with increasing concentrations of ß-estradiol. A concentration-dependent outwards current of PAT1 was also detected by the presence of ß-estradiol. We conclude that estrogen attenuates the activity of PAT1 by directly closing PAT1 channel. Our results may provide an additional mechanism for estrogen on neurotransmission and neuronal metabolism during ischemic injury.

Peptides as inhibitors of the first phosphorylation step of the Streptomyces coelicolor phosphoenolpyruvate: sugar phosphotransferase system.

The phosphotransferase system (PTS) controls the use of sugars in bacteria. The PTS is ubiquitous in bacteria, but it does not occur in plants and animals; it modulates catabolite repression, intermediate metabolism, gene expression, and chemotaxis. Its uniqueness and pleiotropic function make the PTS an attractive target for new antibacterial drugs. The PTS is constituted of two general proteins, namely, enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI has two domains: the N-terminal domain (EIN), which binds to HPr, and the C-terminal domain (EIC), which contains the dimerization interface. In this work, we determined the binding affinities of peptides derived from EIN of Streptomyces coelicolor (EIN(sc)) against HPr of the same organism (HPr(sc)), by using nuclear magnetic resonance and isothermal titration calorimetry techniques. Furthermore, we measured the affinity of EIN(sc) for (i) a peptide derived from HPr(sc), containing the active-site histidine, and (ii) other peptides identified previously by phage display and combinatorial chemistry in Escherichia coli [Mukhija, S. L., et al (1998) Eur. J. Biochem. 254, 433-438; Mukhija, S., and Erni, B. (1997) Mol. Microbiol. 25, 1159-1166]. The affinities were in the range of ~10 µM, being slightly higher for the binding of EIN(sc) with peptides derived from HPr(sc), phage display, or combinatorial chemistry (K(D) ~ 5 µM). Because the affinity of intact EIN(sc) for the whole HPr(sc) is 12 µM, we suggest that the assayed peptides might be considered as good hit compounds for inhibiting the interaction between HPr(sc) and EIN(sc).

The B°AT1 amino acid transporter from rat kidney reconstituted in liposomes: kinetics and inactivation by methylmercury.

The neutral amino acid transporter B°-like from rat kidney, previously reconstituted in liposomes, was identified as B°AT1 by a specific antibody. Collectrin was present in the brush-border extract but not in functionally active proteoliposomes, indicating that it was not required for the transport function. Neutral amino acids behaved as competitive inhibitors of the glutamine transport mediated by B°AT1 with half saturation constants ranging from 0.13 to 4.74mM. The intraliposomal half saturation constant for glutamine was 2.0mM. By a bisubstrate kinetic analysis of the glutamine-Na(+) cotransport, a random simultaneous mechanism was found. Methylmercury and HgCl(2) inhibited the transporter; the inhibition was reversed by dithioerythritol, Cys and, at a lower extent, N-acetylcysteine but not by S-carboxymethylcysteine. The IC(50) of the transporter for methylmercury and HgCl(2) was 1.88 and 1.75µM, respectively. The reagents behaved as non-competitive inhibitors toward both glutamine and Na(+) and no protection by glutamine or Na(+) was found for the two inhibitors.

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes.

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 µM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 µM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), ß-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.

Inhibition of L-type amino acid transporter modulates the expression of cell cycle regulatory factors in KB oral cancer cells.

The purpose of this study was to examine the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of L-type amino acid transporters, on the cell growth suppression in KB human oral cancer cells and to study the roles of cell cycle regulatory factors in the BCH-induced growth inhibition. The effect of BCH on cell growth suppression and the influence of BCH to cell cycle regulatory factors in KB cell growth inhibition were examined using cell cycle analysis, immunoblotting and immunoprecipitation. The BCH treatment induced cell cycle arrest at G1 phase in KB cells. The expression of cyclin D3 was remarkably decreased by BCH treatment. The BCH inhibited the expression of cyclin-dependent protein kinase 6 (CDK6) in a time-dependent manner. In addition, the expression of CDK inhibitor p27 was increased by BCH treatment in KB cells, but not CDK inhibitors p21 and p15. These results suggest that, in KB cells, the inhibition of LAT1 by BCH causes cell cycle arrest at G1 phase by inhibiting cyclin D3-CDK6 complex whereas increasing expression of a CDK inhibitor p27.

Downregulation of cystine transporter xc by irinotecan in human head and neck cancer FaDu xenografts.

BACKGROUND: The purpose of this study was: (1) to document the critical requirement of cystine for growth of human tumor cells in vitro, and (2) to determine the effect of the anticancer agent irinotecan on the cystine transporter x(c)(-) in head and neck FaDu xenografts. METHODS: Cell growth was measured by sulforhodamine B assay. xCT protein, glutathione (GSH) and DNA damage were determined using Western blot, spectrophotometry, and immunohistochemistry, respectively. RESULTS: Depletion of cystine from the medium inhibited tumor cell growth. Treatment of FaDu tumor with a therapeutic dose of irinotecan resulted in depression of xCT protein levels, leading to tumor growth retardation and downregulation of GSH with increased reactive oxygen species (ROS). The accumulation of ROS correlated with increased DNA damage as evidenced by increased H2AX. CONCLUSION: Depression of xCT protein by irinotecan resulted in downregulation of GSH and increase in ROS, which could be the other possible mechanisms of DNA damage by irinotecan.

Transport and signaling via the amino acid binding site of the yeast Gap1 amino acid transceptor.

Transporter-related nutrient sensors, called transceptors, mediate nutrient activation of signaling pathways through the plasma membrane. The mechanism of action of transporting and nontransporting transceptors is unknown. We have screened 319 amino acid analogs to identify compounds that act on Gap1, a transporting amino acid transceptor in yeast that triggers activation of the protein kinase A pathway. We identified competitive and noncompetitive inhibitors of transport, either with or without agonist action for signaling, including nontransported agonists. Using substituted cysteine accessibility method (SCAM) analysis, we identified Ser388 and Val389 as being exposed into the amino acid binding site, and we show that agonist action for signaling uses the same binding site as used for transport. Our results provide the first insight, to our knowledge, into the mechanism of action of transceptors. They indicate that signaling requires a ligand-induced specific conformational change that may be part of but does not require the complete transport cycle.