Efficiency in Drug Discovery: Liver S9 Fraction Assay As a Screen for Metabolic Stability.

BACKGROUND: A rapid and comprehensive metabolic stability screen at the top of a drug discovery flow chart serves as an effective gate in eliminating low value compounds. This imparts a significant level of efficiency and saves valuable resources. While microsomes are amenable to high throughput automation and are cost effective, their enzymatic make-up is limited to that which is contained in endoplasmic reticulum, thereby informing only on Phase I metabolism. Lack of Phase II metabolism data can become a potential liability later in the process, adversely affecting discovery projects' timelines and budget. Hepatocytes offer a full complement of metabolic enzymes and retain their cellular compartments, better representing liver metabolic function. However, hepatocyte screens are relatively expensive, labor intensive, and not easily automatable. Liver S9 fractions include Phase I and II metabolic enzymes, are relatively inexpensive, easy to use, and amenable to automation, making them a more appropriate screening system. We compare the data from the three systems and present the results. RESULTS: Liver S9 and hepatocyte stability assays binned into the same category 70-84% of the time. Microsome and hepatocyte data were in agreement 73-82% of the time. The true rate for stability versus plasma clearance was 45% for hepatocytes and 43% for S9. CONCLUSION: In our opinion, replacing liver microsome and hepatocyte assays with S9 assay for high throughput metabolic screening purposes provides the combined benefit of comprehensive and high quality data at a reasonable expense for drug discovery programs.

Mutagenesis and cysteine scanning of transmembrane domain 10 of the human dipeptide transporter.

PURPOSE: The human dipeptide transporter (hPEPT1) facilitates transport of dipeptides and drugs from the intestine into the circulation. The role of transmembrane domain 10 (TMD10) of hPEPT1 in substrate translocation was investigated using cysteine-scanning mutagenesis with 2-Trimethylammonioethyl methanethiosulfonate (MTSET). METHODS: Each amino acid in TMD10 was mutated individually to cysteine, and transport of [(3)H]Gly-Sar was evaluated with and without MTSET following transfection of each mutant in HEK293 cells. Similar localization and expression levels of wild type (WT) hPEPT1 and all mutants were confirmed by immunostaining and biotinylation followed by western blot analysis. RESULTS: E595C- and G594C-hPEPT1 showed negligible Gly-Sar uptake. E595D-hPEPT1 showed similar uptake to WT-hPEPT1, but E595K- and E595R-hPEPT1 did not transport Gly-Sar. Double mutations E595K/R282E and E595R/R282E did not restore uptake. G594A-hPEPT1 showed similar uptake to WT-hPEPT1, but G594V-hPEPT1 eliminated uptake. Y588C-hPEPT1 showed uptake of 20% that of WT-hPEPT1. MTSET modification supported a model of TMD10 with an amphipathic helix from I585 to V600 and increased solvent accessibility from T601 to F605. CONCLUSIONS: Our results suggest that G594 and E595 in TMD10 of hPEPT1 have key roles in substrate transport and that Y588 may have an important secondary mechanistic role.

Ethanol inhibits functional activity of the human intestinal dipeptide transporter hPepT1 expressed in Xenopus oocytes.

BACKGROUND: The pathological effects of high alcohol (ethanol) consumption on gastrointestinal and hepatic systems are well recognized. However, the effects of ethanol intake on gastric and intestinal absorption and transport systems remain unclear. The present study investigates the effects of ethanol on the human peptide transporter 1 (hPepT1) which mediates the transport of di-and tripeptides as well as several orally administered peptidomimetic drugs such as beta-lactam antibiotics (e.g., penicillin), angiotensin-converting enzyme inhibitors, the anti-neoplastic agent bestatin, and prodrugs of acyclovir. METHODS: Xenopus oocytes were injected with hPepT1 cRNA and incubated for 3 to 10 days. Currents induced by glycyl-sarcosine (Gly-Sar), Ala-Ala (dipeptides), penicillin and enalapril measured in the presence or absence of ethanol were determined using an 8-channel 2-electrode voltage clamp system, with a membrane potential of -70 mV and 11 voltage steps of 100 milliseconds (from +50 mV to -150 mV in -20 mV increments). RESULTS: Ethanol (200 mM) inhibited Gly-Sar and Ala-Ala currents by 42 and 30%, respectively, with IC(50)s of 184 and 371 mM, respectively. Ethanol reduced maximal transport capacity (I(max)) of hPepT1 for Gly-Sar without affecting Gly-Sar binding affinity (K(0.5) and Hill coefficient). Penicillin- and enalapril-induced currents were significantly less than those induced by dipeptides and were not inhibited by ethanol. CONCLUSION: Ethanol significantly reduced transport of dipeptides via a reduction in transport capacity, rather than competing for binding sites in hPepT1. Ethanol inhibition or alteration of transport function may be a primary causative factor contributing to both the nutritional deficits as well as the immunological deficiencies that many alcoholics experience including alcohol liver disease and brain damage.

Cysteine scanning of transmembrane domain three of the human dipeptide transporter: implications for substrate transport.

The human intestinal dipeptide transporter (hPepT1) transports dipeptides and pharmacologically active drugs from the intestine to the blood. The role of transmembrane domain 3 (TMD3) of hPepT1 was studied using cysteine-scanning mutagenesis and methane thiosulfonate (MTS) cysteine modification. Each amino acid in TMD3 was individually mutated to a cysteine and Gly-Sar uptake by each mutated and modified transporter was determined relative to wild-type hPepT1. Uptake data for mutated transporters modified with the lipid-insoluble cysteine-modifying reagent MTSET suggested tilting of TMD3 relative to the substrate translocation pathway; the extracellular region of TMD3 showed little MTSET reactivity, indicative of solvent inaccessibility, whereas the intracellular part of TMD3 was relatively solvent accessible. Modification at 10 positions of TMD3 with MTSEA, a lipid-soluble cysteine-modifying reagent, gave unusual and statistically significant increases in Gly-Sar uptake relative to untreated mutants. We interpret these data in terms of the spatial properties of the hPepT1 substrate translocation channel and possible interactions of TMD3 with other transmembrane domains.

A charge pair interaction between Arg282 in transmembrane segment 7 and Asp341 in transmembrane segment 8 of hPepT1.

PURPOSE: To determine whether R282 in transmembrane segment 7 (TMS7) of hPepT1 forms a salt bridge with D341 in TMS8. METHODS: Mutated hPepT1 transporters containing point mutations at R282 and/or D341 were transiently transfected into HEK293 cells. Their steady state expression and functional activity were measured using immunoprecipitation and 3H-gly-sar uptake, respectively. Gly-sar uptake by cysteine mutants (R282C and D341C) was also measured in the presence and absence of cysteine-modifying MTS reagents. RESULTS: The reverse-charge mutants R282D-hPepT1 and D341R-hPepT1 showed significantly reduced gly-sar uptake, but the double mutant (R282D/D341R-hPepT1) has functionality comparable to that of wild-type hPepT1. Gly-sar uptake by R282C-hPepT1 is reduced, but pre-incubation with 1 mM MTSET, a positively charged cysteine-modifying reagent, restored function to wild-type levels. Similarly, pre-incubation of D341C-hPepT1 with 10 mM MTSES, a negatively charged cysteine-modifying reagent, increased gly-sar uptake compared to unmodified D341C-hPepT1. In contrast, MTSET modification of D341C-hPepT1 (giving a positive charge at position 341) resulted in significant reduction in gly-sar uptake, compared to D341C-hPepT1. CONCLUSION: Our results are consistent with a salt bridge between R282 and D341 in hPepT1, and we use these and other data to propose a role for the R282-D341 charge pair in the hPepT1 translocation mechanism.

Ethanol differentially affects ATP-gated P2X(3) and P2X(4) receptor subtypes expressed in Xenopus oocytes.

P2X receptors are cation-selective, ligand-gated ion channels activated by synaptically released, extracellular adenosine 5'-triphosphate (ATP). ATP-gated currents are inhibited by ethanol when tested in dorsal root ganglion and CA1 neurons. Recently, we reported differences in sensitivity to ethanol inhibition between homomeric P2X(2) and P2X(4) receptors expressed in Xenopus oocytes, which suggested that subunit composition of native P2X receptors determines their ethanol sensitivity. The present study extended the investigation to P2X(3) receptors. The effects of ethanol and zinc ions (Zn(2+)) were tested on homomeric P2X(3) and P2X(4) receptors expressed in Xenopus oocytes using two-electrode voltage clamp. Ethanol potentiated ATP-gated P2X(3) receptor currents in a concentration dependent manner. In contrast, ethanol inhibited P2X(4) receptor function. Ethanol did not directly alter receptor function, nor did it alter the Hill coefficient or maximal ATP response (E(max)) in either P2X(3) or P2X(4) receptors. Ethanol increased the maximal response to Zn(2+) ATP-gated currents in P2X3 receptors which suggests that ethanol and Zn(2+) act on different sites. The differences in ethanol response of P2X(3) and P2X(4) receptors set the stage for future investigations that will use chimeric P2X receptors or other molecular manipulations of P2X structure to investigate the molecular sites and mechanisms of action of ethanol.

Analysis of transmembrane segment 7 of the dipeptide transporter hPepT1 by cysteine-scanning mutagenesis.

To investigate the involvement of transmembrane segment 7 (TMS7) of hPepT1 in forming the putative central aqueous channel through which the substrate traverses, we individually mutated each of the 21 amino acids in TMS7 to a cysteine and analyzed the mutated transporters using the scanning cysteine accessibility method. Y287C- and M292C-hPepT1 did not express at the plasma membrane. Out of the remaining 19 transporters, three (F293C-, L296C-, and F297C-hPepT1) showed negligible glycyl-sarcosine (gly-sar) uptake activity and may play an important role in defining the overall hPepT1 structure. K278C-hPepT1 showed approximately 40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1. Gly-sar uptake for the 16 active transporters containing cysteine mutations was then measured in the presence of 2.5 mM 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 1 mM [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET). Gly-sar uptake was significantly inhibited for each of the 16 single cysteine mutants in the presence of 2.5 mM MTSEA. In contrast, significant inhibition of uptake was only observed for K278C-, M279C-, V280C-, T281C-, M284C-, L286C-, P291C-, and D298C-hPepT1 in the presence of 1 mM MTSET. MTSET modification of R282C-hPepT1 resulted in a significant increase in gly-sar uptake. To investigate this further, we mutated WT-hPepT1 to R282A-, R282E-, and R282K-hPepT1. R282E-hPepT1 showed a 43% reduction in uptake activity, whereas R282A- and R282K-hPepT1 had activities comparable with WT-hPepT1, suggesting a role for the Arg-282 positive charge in substrate translocation. Most of the amino acids that were MTSET-sensitive upon cysteine mutation, including R282C, are located toward the intracellular end of TMS7. Hence, our results suggest that TMS7 of hPepT1 is relatively solvent-accessible along most of its length but that the intracellular end of the transmembrane domain is particularly so. From a structure-function perspective, we speculate that the extracellular end of TMS7 may shift following substrate binding, providing the basis for channel opening and substrate translocation.

Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway.

This study is the first systematic attempt to investigate the role of transmembrane segment 5 of hPepT1, the most conserved segment across different species, in forming a part of the aqueous substrate translocation pathway. We used cysteine-scanning mutagenesis in conjunction with the sulfhydryl-specific reagents, MTSEA and MTSET. Neither of these reagents reduced wild-type-hPepT1 transport activity in HEK293 cells and Xenopus oocytes. Twenty-one single cysteine mutations in hPepT1 were created by replacing each residue within TMS5 with a cysteine. HEK293 cells were then transfected with each mutated protein and the steady-state protein level, [3H]Gly-Sar uptake activity, and sensitivity to the MTS reagents were measured. S164C-, L168C-, G173C-, and I179C-hPepT1 were not expressed on the plasma membrane. Y167C-, N171C-, and S174C-hPepT1 showed

Nucleotide-induced restoration of conjunctival chloride and fluid secretion in adenovirus type 5-infected pigmented rabbit eyes.

We evaluated the role of extracellular UTP and other nucleotides in the regulation of chloride (JCl) and fluid secretion (JCl) across the pigmented rabbit conjunctiva. Jv was determined in freshly excised conjunctival tissues mounted between two buffer reservoirs maintained in an enclosed environment at 37 degrees C. Short circuit current (Isc) and 36Cl flux were measured using modified Ussing-type chambers. Fluid flux measurements were made with a pair of capacitance probes. After observing the baseline for 15 to 30 min, fluid flux was measured in the presence of mucosally applied nucleotides (10 microM) for a period of 30 min. Mucosal application of 10 microM each of UTP, UDP, ATP, ADP, AMP, adenosine, and ATP-gamma-S transiently stimulated fluid secretion across the conjunctiva to a significant extent for 10 to 15 min. Other nucleotides did not show any significant effect. The stimulation of fluid secretion correlated well with the stimulation in Isc (r2 = 0.85). UTP (0.1-1000 microM) led to a maximal increase in fluid secretion by 11.72 +/- 0.48 microl/(h x cm2) with an EC50 value of 10.39 +/- 1.08 microM. ATP (0.1-1000 microM) caused a maximal increase in fluid secretion by 11.89 +/- 0.88 microl/(h x cm2) with an EC50 value of 17.23 +/- 2.63 microM. Adenovirus type 5 (Ad5) infection significantly decreased both net 36Cl secretion across the conjunctiva by approximately 56% and the rate of fluid secretion by approximately 56%. UTP (10 microM), but not 1 mM 8-bromo-cAMP, was able to elicit a normal stimulatory response in the Ad5-infected tissues. In conclusion, mucosal application of purinergic nucleotides may be therapeutically important in restoring ion and fluid secretion in the diseased conjunctiva.

Biophysical evidence for His57 as a proton-binding site in the mammalian intestinal transporter hPepT1.

PURPOSE: The objective of this study was to provide direct evidence of the relative importance of the His57 residue present in transmembrane domain 2 (TMD 2) and the His121 residue in TMD 4 as proton-binding sites in human PepT1 (hPepT1) by using a novel mutagenesis approach. METHODS: His57 and His121 in hPepT1 were each mutated to alanine, arginine, or lysine individually to obtain H57A-, H57R-, H57K-, H121A-, H121R-, and H121K-hPepT1. H7A-hPepT1 was used as a negative control. [3H]Glycylsarcosine (Gly-Sar) uptake was measured 72 h posttransfection using HEK293 cells individually transfected with these mutated proteins. Steady-state I/V curves (-150 mV to +50 mV, holding potential -70 mV) were obtained by measuring 5 mM Gly-Sar-induced currents in oocytes expressing H-57R- and H57K-hPepT1. Noninjected oocytes and wild-type hPepT1 (WT-hPepT1)-injected oocytes served as negative and positive controls, respectively. RESULTS: At pH 6.0, H57K-, H57R-, H121K-, and H121R-hPepT1 led to a 97%, 90%, 45%, and 75% decrease in [3H]Gly-Sar uptake into HEK293 cells, respectively. At pH 7.4, uptake in cells transfected with H57K- and H57R-hPepT1 was not significantly different from that at pH 6.0, whereas cells expressing H121R- and H121K-hPepT1 showed 56% and 65% decrease, respectively, compared to that at pH 6.0. In oocytes expressing H57R-hPepT1, steady-state currents induced by 5 mM Gly-Sar increased with increasing pH (I(max) = 300 nA at pH 8.5), suggesting the binding of protons to H57R. No such trend was observed in oocytes injected with H57K, H121R, and H121K cRNA. CONCLUSIONS: H57R-hPepT1 is able to bind protons at a relatively basic pH, resulting in facilitation of transport of Gly-Sar by hPepT1 at higher pH. Our novel approach provides direct evidence that His57 is a principal proton-binding site in hPepT1.