Nucleobase Transporter Research Articles

Exploring novel dilazep derivatives as hENT1 inhibitors and potentially covalent molecular tools.

The human equilibrative nucleoside transporter 1 (SLC29A1, hENT1) is a solute carrier that modulates the passive transport of nucleosides and nucleobases, such as adenosine. This nucleoside regulates various physiological processes, such as vasodilation and -constriction, neurotransmission and immune defense. Marketed drugs such as dilazep and dipyridamole have proven useful in cardiovascular afflictions, but the application of hENT1 inhibitors can be beneficial in a number of other diseases. In this study, 39 derivatives of dilazep's close analogue ST7092 were designed, synthesized and subsequently assessed using [3H]NBTI displacement assays and molecular docking. Different substitution patterns of the trimethoxy benzoates of ST7092 reduced interactions within the binding pocket, resulting in diminished hENT1 affinity. Conversely, [3H]NBTI displacement by potentially covalent compounds 14b, 14c, and 14d resulted in high affinities (Ki values between 1.1 and 17.5 nM) for the transporter, primarily by the ability of accommodating the inhibitors in various ways in the binding pocket. However, any indication of covalent binding with amino acid residue C439 remained absent, conceivably as a result of decreased nucleophilic residue reactivity. In conclusion, this research introduces novel dilazep derivatives that are active as hENT1 inhibitors, along with the first high affinity dilazep derivatives equipped with an electrophilic warhead. These findings will aid the rational and structure-based development of novel hENT1 inhibitors and pharmacological tools to study hENT1's function, binding mechanisms, and its relevance in (patho)physiological conditions.

Assessment of uric acid-lowering activity, safety and stress tolerance of Lactiplantibacillus plantarum MC14 based on whole gene sequencing and phenotyping experiments

With lifestyle changes, hyperuricemia has become a global epidemic. Recent studies have indicated that some probiotic strains may help control serum uric acid. This study aimed to screen probiotic strains with uric acid-lowering potential and subsequently analyze the safety and stress tolerance of the strain in food applications, utilizing phenotyping experiments and genome sequencing. Initially, we isolated a strain of Lactiplantibacillus plantarum MC14 with the best in vitro uric acid-lowering activity. The inosine and guanosine degradation rates of MC14 were 5.00 and 4.11 mg/L·min, respectively, and the XOD inhibition rate was 81.56% ± 3.87%. Gene analysis revealed that the MC14 strain encodes three nucleoside hydrolases and six nucleoside and nucleobase transporter proteins. Subsequently, safety assessments of the MC14 strain in food applications revealed no nitroreductase, amino decarboxylase, or tryptophanase activities, absence of hemolysis, and no antibiotic resistance gene transfer risk. Finally, stress tolerance tests on MC14 indicated robust tolerance to gastrointestinal fluids and bile salts, with survival rates exceeding 98% after 3 h in artificial gastric fluids and approximately 100.49% ± 0.15% after 8 h in artificial intestinal fluids. Additionally, MC14 demonstrated effective antibacterial properties and antioxidant capacity, and its genome was annotated with numerous stress tolerance-related genes, including 18 acid-base stress, 4 bile salt tolerance, 11 heat stress, 3 cold stress, 6 osmotic stress, and 28 oxidative stress. Our findings highlighted MC14 as a promising candidate for probiotic and food applications.

Mapping the transporter-substrate interactions of the Trypanosoma cruzi NB1 nucleobase transporter reveals the basis for its high affinity and selectivity for hypoxanthine and guanine and lack of nucleoside uptake

Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (Ki) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG0. Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens.

Insight into the mechanism of H+-coupled nucleobase transport

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

Lactiplantibacillus plantarum enables blood urate control in mice through degradation of nucleosides in gastrointestinal tract

BackgroundLactobacillus species in gut microbiota shows great promise in alleviation of metabolic diseases. However, little is known about the molecular mechanism of how Lactobacillus interacts with metabolites in circulation. Here, using high nucleoside intake to induce hyperuricemia in mice, we investigated the improvement in systemic urate metabolism by oral administration of L. plantarum via different host pathways.ResultsGene expression analysis demonstrated that L. plantarum inhibited the activity of xanthine oxidase and purine nucleoside phosphorylase in liver to suppress urate synthesis. The gut microbiota composition did not dramatically change by oral administration of L. plantarum over 14 days, indicated by no significant difference in α and β diversities. However, multi-omic network analysis revealed that increase of L. plantarum and decrease of L. johnsonii contributed to a decrease in serum urate levels. Besides, genomic analysis and recombinant protein expression showed that three ribonucleoside hydrolases, RihA–C, in L. plantarum rapidly and cooperatively catalyzed the hydrolysis of nucleosides into nucleobases. Furthermore, the absorption of nucleobase by intestinal epithelial cells was less than that of nucleoside, which resulted in a reduction of urate generation, evidenced by the phenomenon that mice fed with nucleobase diet generated less serum urate than those fed with nucleoside diet over a period of 9-day gavage.ConclusionCollectively, our work provides substantial evidence identifying the specific role of L. plantarum in improvement of urate circulation. We highlight the importance of the enzymes RihA–C existing in L. plantarum for the urate metabolism in hyperuricemia mice induced by a high-nucleoside diet. Although the direct connection between nucleobase transport and host urate levels has not been identified, the lack of nucleobase transporter in intestinal epithelial cells might be important to decrease its absorption and metabolization for urate production, leading to the decrease of serum urate in host. These findings provide important insights into urate metabolism regulation.BbgstkDZoHsJm_QsUh-oZNVideo

Inborn Errors of Nucleoside Transporter (NT)-Encoding Genes (SLC28 and SLC29).

The proper regulation of nucleotide pools is essential for all types of cellular functions and depends on de novo nucleotide biosynthesis, salvage, and degradation pathways. Despite the apparent essentiality of these processes, a significant number of rare diseases associated with mutations in genes encoding various enzymes of these pathways have been already identified, and others are likely yet to come. However, knowledge on genetic alterations impacting on nucleoside and nucleobase transporters is still limited. At this moment three gene-encoding nucleoside and nucleobase transporter proteins have been reported to be mutated in humans, SLC29A1, SLC29A3, and SLC28A1, impacting on the expression and function of ENT1, ENT3, and CNT1, respectively. ENT1 alterations determine Augustine-null blood type and cause ectopic calcification during aging. ENT3 deficiency translates into various clinical manifestations and syndromes, altogether listed in the OMIM catalog as histiocytosis-lymphoadenopathy plus syndrome (OMIM#602782). CNT1 deficiency causes uridine-cytidineuria (URCTU) (OMIM#618477), a unique type of pyrimidineuria with an as yet not well-known clinical impact. Increasing knowledge on the physiological, molecular and structural features of these transporter proteins is helping us to better understand the biological basis behind the biochemical and clinical manifestations caused by these deficiencies. Moreover, they also support the view that some metabolic compensation might occur in these disturbances, because they do not seem to significantly impact nucleotide homeostasis, but rather other biological events associated with particular subtypes of transporter proteins.

Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells’ ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

Impact of SLC43A3/ENBT1 Expression and Function on 6-Mercaptopurine Transport and Cytotoxicity in Human Acute Lymphoblastic Leukemia Cells.

6-Mercaptopurine (6-MP) is used extensively in the treatment of acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases. Our laboratory determined previously, using a recombinant HEK293 cell model, that the SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1) transports 6-MP into cells and significantly impacts the cytotoxicity of 6-MP in that model. To further investigate the clinical relevance of this finding, we now extend this work to an analysis of the impact of SLC43A3/ENBT1 expression and function on 6-MP uptake and cytotoxicity in leukemic lymphoblasts, the therapeutic target of 6-MP in ALL. A panel of ALL cell lines was assessed for SLC43A3/ENBT1 expression, ENBT1 function, and sensitivity to 6-MP. There was a significant difference in SLC43A3 expression among the cell lines that positively correlated with the rate of ENBT1-mediated 6-MP uptake. Cells with the lowest expression of SLC43A3 (SUP-B15: Vmax = 22± 5 pmol/µl per second) were also significantly less sensitive to 6-MP-induced cytotoxicity than were the highest expressing cells (ALL-1: Vmax = 69 ± 10 pmol/µl per second). Furthermore, knockdown of ENBT1 using short hairpin RNA interference (shRNAi) in RS4;11 cells caused a significant decrease in ENBT1-mediated 6-MP uptake (Vmax: RS4;11 = 40 ± 4 pmol/µl per second; RS4;11 shRNAi = 26 ± 3 pmol/µl per Second) and 6-MP cytotoxicity (EC50: RS4;11 = 0.58 ± 0.05 µM; RS4;11 shRNAi =1.44 ± 0.59 µM). This study showed that ENBT1 is a major contributor to 6-MP uptake in leukemia cell lines and may prove to be a biomarker for the therapeutic efficacy of 6-MP in patients with ALL. SIGNIFICANCE STATEMENT: This study shows that SLC43A3-encoded equilibrative nucleobase transporter 1 is responsible for the transport of 6-mercaptopurine (6-MP) into leukemia cells and that its level of expression can impact the cytotoxicity of 6-MP. Further studies are warranted to investigate the therapeutic implications in patient populations.

The evolution of vitamin C biosynthesis and transport in animals

BackgroundVitamin C (VC) is an indispensable antioxidant and co-factor for optimal function and development of eukaryotic cells. In animals, VC can be synthesized by the organism, acquired through the diet, or both. In the single VC synthesis pathway described in animals, the penultimate step is catalysed by Regucalcin, and the last step by l-gulonolactone oxidase (GULO). The GULO gene has been implicated in VC synthesis only, while Regucalcin has been shown to have multiple functions in mammals.ResultsBoth GULO and Regucalcin can be found in non-bilaterian, protostome and deuterostome species. Regucalcin, as here shown, is involved in multiple functions such as VC synthesis, calcium homeostasis, and the oxidative stress response in both Deuterostomes and Protostomes, and in insects in receptor-mediated uptake of hexamerin storage proteins from haemolymph. In Insecta and Nematoda, however, there is no GULO gene, and in the latter no Regucalcin gene, but species from these lineages are still able to synthesize VC, implying at least one novel synthesis pathway. In vertebrates, SVCT1, a gene that belongs to a family with up to five members, as here shown, is the only gene involved in the uptake of VC in the gut. This specificity is likely the result of a subfunctionalization event that happened at the base of the Craniata subphylum. SVCT-like genes present in non-Vertebrate animals are likely involved in both VC and nucleobase transport. It is also shown that in lineages where GULO has been lost, SVCT1 is now an essential gene, while in lineages where SVCT1 gene has been lost, GULO is now an essential gene.ConclusionsThe simultaneous study, for the first time, of GULO, Regucalcin and SVCTs evolution provides a clear picture of VC synthesis/acquisition and reveals very different selective pressures in different animal taxonomic groups.

Intracellular Superoxide Radical Formation Decreases 6‐Mercaptopurine Uptake by MOLT‐4 Leukemia Cells

BackgroundAcute lymphoblastic leukemia (ALL) is the most common cancer associated with childhood. 6‐Mercaptopurine (6‐MP) is a nucleobase analog drug used in the maintenance treatment phase of ALL. 6‐MP has numerous side‐effects associated with its use, which has both short‐term and long‐term implications which can impact patient compliance (especially in children). Our lab has established that SLC43A3, which encodes for the equilibrative nucleobase transporter 1 (ENBT1), is expressed in leukemia cells and transports 6‐MP. However, its modes of regulation are yet to be elucidated. A previous study in microvascular endothelial cells demonstrated that nucleobase uptake via ENBT1 was significantly decreased upon exposure to oxidative stress. Oxidative stress is very prevalent in cancer, including leukemia, suggesting that it could impact ENBT1 function and thus 6‐MP uptake and therapeutic activity.HypothesisENBT1 function will be reduced by oxidative stress and affect the cytotoxic efficacy of 6‐MP in leukemia cells.MethodsThe MOLT‐4 leukemia cell line was used for all experiments. Cell viability was assessed via the MTT assay. [14C]6‐MP was used to determine the functional kinetics of ENBT1 using an oil‐stop centrifugation assay. Compounds used for the studies on oxidative stress were tert‐butyl hydroperoxide (TBHP) and menadione (oxygen‐free radical generators). TBHP causes extracellular induction of oxidative stress, while menadione is primarily through an intracellular mechanism called redox cycling. Mn(III)tetrakis(1‐methyl‐4‐pyridyl)porphyrin (MnTMPyP) (superoxide dismutase mimetic) and N‐acetylcysteine (NAC) (antioxidant) were used to combat the effects caused by TBHP or menadione.ResultsIncubation of cells in TBHP and menadione for 30 min at 37℃ decreased cell viability only at the highest concentrations tested (100 ‐ 400 µM) (400 µM: 86 ± 4% and 72 ± 6%, respectively) Incubation with TBHP and menadione for 24 hours led to a greater loss of cell viability (Log EC50: ‐3.9 ± 0.1 and ‐4.3 ± 0.1, respectively). TBHP did not alter ENBT1‐mediated 6‐MP uptake. However, incubation with menadione at 100 µM for 30 min at 37℃ significantly decreased ENBT1‐mediated 6‐MP uptake (Vmax: Control – 77 ± 3 pmol/µL/sec; Menadione – 57 ± 3 pmol/µL/sec). The addition of the free radical scavenger MnTMPyP did not reverse the decrease caused by menadione. However, the addition of 1mM NAC caused a significant reversal of the reduction in 6‐MP uptake produced by menadione (Vmax: Menadione – 46 ± 2 pmol/µL/sec; Menadione + NAC – 55 ± 2 pmol/µL/sec).ConclusionsMenadione but not TBHP caused a significant decrease in ENBT1 functional activity, suggesting it was due to intracellular superoxide formation, thus supporting our hypothesis. The reduction caused by menadione could be reversed with NAC. This study confirms that ENBT1 function, and thus 6‐MP uptake by leukemia cells, is modified by oxidative stress, thus opening up potential therapeutic avenues in ALL treatment.

Functional Comparison of Human and Murine Equilibrative Nucleobase Transporter 1

BackgroundThiopurines are a key component for the immunosuppressive therapy of various leukemias, inflammatory bowels disease, and other autoimmune disorders. Thiopurine therapy discontinuation/interruption is associated with a higher risk of relapse, but is common due to the prevalence of severe and potentially fatal adverse events, such as hepatotoxicity.Drug transporter expression is a known factor for patient variability in drug response and toxicity. We have recently established that the SLC43A3‐encoded transporter, equilibrative nucleobase transporter 1 (ENBT1), is the primary mechanism by which the thiopurine, 6‐mercaptopurine (6‐MP) enters cells. ENBT1 is known to be highly expressed in human hepatocytes, however, the relationship between ENBT1 and thiopurine‐induced hepatotoxicity has not been explored in the literature.To investigate this paradigm, our lab has proposed developing a novel SLC43A3 knockout mouse model. However, the functional differences between human and murine ENBT1, in mediating 6‐MP transport, has equally not been explored. Evidence that suggests ENBT1 is functionally similar between species, would be an essential foundation to assist bridging the gap between our proposed animal model and clinical studies within the literature.HypothesisWe hypothesize that hSLC43A3‐encoded hENBT1 and mslc43a3‐encoded mENBT1 are functionally similar and will have non‐significantly different 6‐MP transport kinetics and resulting cytotoxicity.MethodsThe ENBT1‐deficient human embryonic kidney 293 (HEK293) cell line was stably transfected with either hSLC43A3 or mslc43a3. [3H]Adenine and [3H]6‐MP were used in an oil‐stop centrifugation assay to assess ENBT1‐mediated transport activity in wildtype and transfected HEK293 cell lines. To determine cytotoxicity, wildtype and transfected HEK293 cell lines were incubated for 48 hours with a range of 6‐MP concentrations (78 nM – 1.28 mM) before being assessed via MTT cell viability assay.ResultsOil stop centrifugation assay of ENBT1‐mediated [3H]adenine and [3H]6‐MP transport revealed that hENBT1 and mENBT1 have similar transport kinetics (Km and Vmax), where resulting Michaelis‐Menten curves were not significantly different (unpaired T‐test: Adenine ‐ t16=0.32, p=0.76, n=5 & 6‐MP ‐ t16=0.64, p=0.53, n=4). Adenine inhibition of ENBT1‐mediated [3H]6‐MP transport and 6‐MP inhibition of ENBT1‐mediated [3H]adenine transport also showed that hENBT1 and mENBT1 have concurring inhibition kinetics (Ki), inhibition curves were not significantly different (unpaired T‐test: Adenine: t17=0.29, p=0.77, n=5 & 6‐MP: t14=0.18, p=0.86, n=4). Subsequent MTT cell viability assay showed that wildtype HEK293 are relatively 6‐MP resistant, while the transfected‐HEK293s are 6‐MP sensitive and have non‐significantly different cell viability curves (unpaired T‐test: t28=0.13, p=0.89, n=5).ConclusionOur results show that hENBT1 and mENBT1 are functionally similar in regards to ENBT1‐mediated adenine and 6‐MP transport in a stably transfected cell line. Additional work is necessary to investigate whether this is the case in human and murine hepatic cell lines/primary hepatocytes that have endogenous SLC43A3/ENBT1 expression and a larger range of xenobiotic metabolizing enzymes that may alter the equilibrium of 6‐MP within cells.

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Uniport, Not Proton-Symport, in a Non-Mammalian SLC23 Transporter

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.

Comparative de novo transcriptome analysis identifies salinity stress responsive genes and metabolic pathways in sugarcane and its wild relative Erianthus arundinaceus [Retzius] Jeswiet

Erianthus arundinaceus [Retzius] Jeswiet, a wild relative of sugarcane has a high biomass production potential and a reservoir of many genes for superior agronomic traits and tolerance to biotic and abiotic stresses. A comparative physiological, anatomical and root transcriptome analysis were carried out to identify the salt-responsive genes and metabolic pathways associated with salt-tolerant E. arundinaceus genotype IND99-907 and salinity-sensitive sugarcane genotype Co 97010. IND99-907 recorded growth of young leaves, higher proline content, higher relative water content, intact root anatomical structures and lower Na+/K+, Ca2+/K+ and Mg2+/K+ ratio as compared to the sugarcane genotype Co 97010. We have generated four de novo transcriptome assemblies between stressed and control root samples of IND99-907 and Co 97010. A total of 649 and 501 differentially expressed genes (FDR<0.01) were identified from the stressed and control libraries of IND99-907 and Co 97010 respectively. Genes and pathways related to early stress-responsive signal transduction, hormone signalling, cytoskeleton organization, cellular membrane stabilization, plasma membrane-bound calcium and proton transport, sodium extrusion, secondary metabolite biosynthesis, cellular transporters related to plasma membrane-bound trafficking, nucleobase transporter, clathrin-mediated endocytosis were highly enriched in IND99-907. Whereas in Co 97010, genes related to late stress-responsive signal transduction, electron transport system, senescence, protein degradation and programmed cell death, transport-related genes associated with cellular respiration and mitochondrial respiratory chain, vesicular trafficking, nitrate transporter and fewer secondary metabolite biosynthetic genes were highly enriched. A total of 27 pathways, 24 biological processes, three molecular functions and one cellular component were significantly enriched (FDR≤ 0.05) in IND99-907 as compared to 20 pathways, two biological processes without any significant molecular function and cellular components in Co 97010, indicates the unique and distinct expression pattern of genes and metabolic pathways in both genotypes. The genomic resources developed from this study is useful for sugarcane crop improvement through development of genic SSR markers and genetic engineering approaches.

Identification of New Specificity Determinants in Bacterial Purine Nucleobase Transporters based on an Ancestral Sequence Reconstruction Approach

The relation of sequence with specificity in membrane transporters is challenging to explore. Most relevant studies until now rely on comparisons of present-day homologs. In this work, we study a set of closely related transporters by employing an evolutionary, ancestral-reconstruction approach and reveal unexpected new specificity determinants. We analyze a monophyletic group represented by the xanthine-specific XanQ of Escherichia coli in the Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2 (NAT/NCS2) family. We reconstructed AncXanQ, the putative common ancestor of this clade, expressed it in E. coli K-12, and found that, in contrast to XanQ, it encodes a high-affinity permease for both xanthine and guanine, which also recognizes adenine, hypoxanthine, and a range of analogs. AncXanQ conserves all binding-site residues of XanQ and differs substantially in only five intramembrane residues outside the binding site. We subjected both homologs to rationally designed mutagenesis and present evidence that these five residues are linked with the specificity change. In particular, we reveal Ser377 of XanQ (Gly in AncXanQ) as a major determinant. Replacement of this Ser with Gly enlarges the specificity of XanQ towards an AncXanQ-phenotype. The ortholog from Neisseria meningitidis retaining Gly at this position is also a xanthine/guanine transporter with extended substrate profile like AncXanQ. Molecular Dynamics shows that the S377G replacement tilts transmembrane helix 12 resulting in rearrangement of Phe376 relative to Phe94 in the XanQ binding pocket. This effect may rationalize the enlarged specificity. On the other hand, the specificity effect of S377G can be masked by G27S or other mutations through epistatic interactions.

Transport Characteristics of 6-Mercaptopurine in Brain Microvascular Endothelial Cells Derived From Human Induced Pluripotent Stem Cells.

The likelihood of reoccurrence of acute lymphoblastic leukemia is influenced by the cerebral concentration of the therapeutic agent 6-mercaptopurine (6-MP) during treatment. Therefore, it is important to understand the blood-brain barrier (BBB) transport mechanism of 6-MP. The purpose of this study was to characterize this mechanism using human induced pluripotent stem cell-derived microvascular endothelial cells (hiPS-BMECs). The permeability coefficient of 6-MP across hiPS-BMECs monolayer in the basal-to-apical direction (B-to-A) was significantly greater than that in the opposite direction (A-to-B). The inhibition profiles of 6-MP transport in the A-to-B direction were different from those in the B-to-A direction. Transport in the A-to-B direction was mainly inhibited by adenine (an inhibitor of equilibrative nucleobase transporter 1; ENBT1), while transport in the B-to-A direction was significantly reduced by inhibitors of multidrug resistance-associated proteins (MRPs), especially zaprinast (an MRP5 inhibitor). Immunocytochemical analyses demonstrated the expression of ENBT1 and MRP5 proteins in hiPS-BMECs. We confirmed that the cellular uptake of 6-MP is decreased by ENBT1 inhibitors in hiPS-BMECs and by knockdown of ENBT1 in hCMEC/D3 cells. These results suggest that ENBT1 and MRP5 make substantial contributions to the transport of 6-MP in hiPS-BMECs and hCMEC/D3 cells.

Model Analysis of the Apparent Saturation Kinetics of Purine Nucleobase Uptake in Cells co-Expressing Transporter and Metabolic Enzyme.

This study aims to understand the effect of salvage enzyme activity on the saturable kinetics of facilitated cellular uptake of purine nucleobase by developing a cellular kinetic model incorporating equilibrative nucleobase transporter 1 (ENBT1) and adenine phosphoribosyltransferase (APRT), with adenine as a model nucleobase. A cellular kinetic model incorporating the functions of ENBT1 and APRT was developed using Napp software and employed for model-based analysis of the cellular disposition of adenine. Simulation analysis using the developed cellular kinetic model could account for the experimentally observed time-dependent changes in the Km(app) value of adenine for ENBT1-mediated uptake. At a long experimental time, the model shows that uptake of adenine is rate-limited by APRT, enabling determination of the Km value for APRT. At early time, the rate-limiting step for adenine uptake is ENBT1-mediated transport, enabling determination of the Km value for ENBT1. Further simulations showed that the effect of experimental time on the Km(app) value for ENBT1-mediated uptake is dependent on the APRT expression level. Our findings indicate that both enzyme expression levels and experimental time should be considered when using cellular uptake studies to determine the Km values of purine nucleobases for facilitated transporters.

Effect of Tyrosine Kinase Inhibitors on 6‐Mercaptopurine Cytotoxicity: Interactions with Equilibrative Nucleobase Transporter 1

Background Acute lymphoblastic leukemia (ALL) is the most prevalent malignancy in children and adolescents in the developed world. The nucleobase analogue 6-mercaptopurine (6-MP) is a key component in the treatment of pediatric ALL, particularly for the maintenance of remission. While most patients tolerate 6-MP well, intra-individual variability between patients continues to complicate attainment of target drug efficacy with low off-target toxicities at standardized doses of 6-MP. Drug transporter expression is a known factor for patient variability in drug response/toxicity. We have recently established that the SLC43A3-encoded transporter, equilibrative nucleobase transporter 1 (ENBT1), is the primary mechanism by which 6-MP enters cells. ENBT1 expression levels in human leukemia cell lines directly correlate with the rate of 6-MP uptake and resulting cytotoxicity. Tyrosine kinase inhibitors (TKI) are used in combination with 6-MP for treating some subtypes of ALL, i.e., Philadelphia chromosome-positive ALL. Previous studies have indicated that TKI can interfere with nucleobase uptake by cells, and thus may compete for ENBT1. Therefore, it is important to establish the existence of potential drug-drug interactions between 6-MP and TKIs that may affect therapeutic efficacy. Hypothesis We hypothesized that TKI can inhibit ENBT1-mediated 6-MP transport and thereby reduce the therapeutic efficacy of 6-MP in ALL cell lines. Methods [3H]Adenine was used in an oil-stop centrifugation assay to assess ENBT1-mediated uptake activity in a SLC43A3-transfected human embryonic kidney 293 (HEK293) cell line in the presence and absence of various TKI. To determine impact on 6-MP toxicity, a well-established human ALL (MOLT-4) cell line was incubated for 48 hours with a range of 6-MP concentrations (78 nM – 1.28 mM), plus or minus a defined concentration of TKI and assessed for their cell viability via the MTT assay. Results An initial screen showed significant inhibition of ENBT1-mediated [3H]adenine uptake by various TKI at 10 μM: gefitinib >> imatinib = dasatinib (one-way ANOVA, F4,26=22, p<0.0001, n=6). Subsequent concentration-inhibition analyses showed a gefitinib IC50 of 5.5 ± 0.8 μM for inhibiting [3H]adenine uptake in MOLT-4 cells (n=5). Gefitinib alone also decreased MOLT-4 cell viability with an EC50 of 90 ± 68 μM (n=5). From these data, 3 μM gefitinib was selected as a concentration that did not affect MOLT-4 cell viability but did inhibit ENBT1-mediated adenine uptake by approximately 30%. Co-administration of 6-MP and 3 μM gefitinib in MOLT-4 cells resulted in a significant 2-fold increase in the cytotoxicity of 6-MP: Control EC50=1.5 ± 0.6 μM vs Gefitinib EC50= 0.72 ± 0.19 μM (unpaired T-test, t8=2.763, p=0.0245. n=5). Conclusion Our results show that TKI can significantly inhibit ENBT1-mediated transport of nucleobases by ENBT1. However, contrary to expectations, gefitinib increased the cytotoxicity of 6-MP. We hypothesize gefitinib inhibits xanthine oxidase, an enzyme that metabolizes 6-MP into an inactive and readily excreted metabolite. Additional work is needed to confirm the mechanism(s) underlying this synergistic effect of gefitinib on 6-MP cytotoxicity.

The genome of the gymnosperm Picea glauca encodes a single Nucleobase Cation Symporter 1 (PgNCS1) that displays a broad yet unique solute specificity profile

The Picea glauca genome contains a locus that encodes for a nucleobase cation symporter 1 (PgNCS1). As a gymnosperm, P. glauca belongs to a key taxonomic position for an ongoing evolution-function analysis of viridiplantae nucleobase cation symporter 1 proteins (NCS1). Here the solute transport and binding properties for PgNCS1 are determined through heterologous expression in Saccharomyces cerevisiae strains deficient in functional NCS1 loci. PgNCS1 displays a broad, yet unique, solute specificity profile –common with other plant NCS1. Yeast containing PgNCS1 transport adenine, guanine, hypoxanthine, xanthine and uracil and are sensitive to growth on 8-azaadenine. Neither cytosine nor 5 flourocytosine are transported by PgNCS1 but along with caffeine and uric acid, act as competitive inhibitors of [3H]-adenine and [3H]-hypoxanthine uptake. This transporter displays high affinity for adenine (Km = 2.67 μM), guanine (Ki = 1.71 μM) and hypoxanthine (Ki = 1.82 μM) but lesser affinity for xanthine (Ki = 5.36 μM). Arabidopsis plants that are deficient in their endogenous NCS1, yet carry PgNCS1, show significant uptake of [3H]-adenine. The results support previous studies and together confirm a broad nucleobase transport and binding pattern for plant NCS1 across the viridiplantae. PgNCS1 displays a broad yet unique nucleosbase transport and binding profile. It transports adenine, guanine, hypoxanthine, xanthine and uracil but only binds cytosine, caffeine, and uric acid.

An RNA-binding protein complex regulates the purine-dependent expression of a nucleobase transporter in trypanosomes.

Post-transcriptional regulation of gene expression is particularly important in trypanosomatid protozoa. RNA-binding proteins (RBPs) regulate mRNA stability and translation, yet information about how RBPs are able to link environmental cues to post-transcriptional control is scarce. In Trypanosoma brucei, we have previously characterized a short RNA stem-loop cis-element (PuRE, Purine Responsive Element) within the 3′-UTR of the NT8 nucleobase transporter mRNA that is necessary and sufficient to confer a strong repression of gene expression in response to purines. In this study, we have identified a protein complex composed of two RNA-binding proteins (PuREBP1 and PuREBP2) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNA interference results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression.