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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.

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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 ϳ40% 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.The human dipeptide transporter, hPepT1, is a 708-amino acid protein containing 12 ␣-helical transmembrane segments.

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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.

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.

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 [3H]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.