Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in <i>Pichia Pastoris</i> Transformants

Song, Feifeng; Hu, Yongjun; Jiang, Huidi; Smith, David Eugene

doi:10.1124/dmd.116.073320

Cited by 9 publications

(5 citation statements)

References 41 publications

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“…However, PEPT1 and PEPT2 show opposite affinities for GlySar, with mM values of K m for PEPT1 and μM values of K m for PEPT2. 38,39 Our kinetic analysis of GlySar uptake indicated hPHT1 was a low affinity transporter (K m = 1.69 ± 0.30 mM), whose transport activity was similar to PEPT1. However, Bhardwaj et al reported that the uptake of GlySar was not different in COS-7 transiently transfected with wildtype hPHT1 or vector.…”

Section: Molecular Pharmaceuticsmentioning

confidence: 94%

Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells

Song

Wang

et al. 2018

Mol. Pharmaceutics

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The proton-coupled oligopeptide transporter PHT1 (SLC15A4), which facilitates cross-membrane transport of histidine and small peptides from inside the endosomes or lysosomes to cytosol, plays an important role in intracellular peptides homeostasis and innate immune responses. However, it remains a challenge to elucidate functional properties of the PHT1 transporter because of its subcellular localization. The purpose of this study was to resort hPHT1 protein from the subcellular to outer cell membrane of MDCK cells stably transfected with human PHT1 mutants, and to characterize its functional activity in these cells. Using this model, the functional activity of hPHT1 was evaluated by cellular uptake studies with d-l-histidine, GlySar, and the bacterial peptidoglycan products MDP and Tri-DAP. We found that the disruption of two dileucine motifs was indispensable for hPHT1 transporter being preferentially targeting to plasma membranes. hPHT1 showed high affinity for d-l-histidine and low affinity for GlySar, with K values of 16.3 ± 1.9 μM and 1.60 ± 0.30 mM, respectively. Moreover, the bacterial peptidoglycan components MDP and Tri-DAP were shown conclusively to be hPHT1 substrates. The uptake of MDP by hPHT1 was inhibited by di/tripeptides and peptide-like drugs, but not by glycine and acyclovir. The functional activity of hPHT1 was also pH-dependent, with an optimal cellular uptake in buffer pH 6.5. Taken together, we established a novel cell model to evaluate the function of hPHT1 in vitro, and confirmed that MDP and Tri-DAP were substrates of hPHT1. Our findings suggest that PHT1 may serve as a potential target for reducing the immune responses and for drug treatment of inflammatory diseases.

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Section: Molecular Pharmaceuticsmentioning

confidence: 94%

Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells

Song

Wang

et al. 2018

Mol. Pharmaceutics

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“…Human data were obtained from the literature (Garrigues et al, 1991); hu, humanized PepT1 mice. species in drug capacity (V max ) and affinity (K m ) of related transporters (Hu et al, 2012;Song et al, 2017). Humanized mouse models have been developed in an attempt to improve the predictability of pharmacokinetics, metabolic contributions, drug toxicity, and receptor response when translating results from animals to human subjects (Katoh et al, 2004;Gonzalez and Yu, 2006;Scheer and Roland Wolf, 2013;Scheer and Wilson, 2016).…”

Section: Discussionmentioning

confidence: 99%

In Silico Prediction of the Absorption and Disposition of Cefadroxil in Humans using an Intestinal Permeability Method Scaled from Humanized PepT1 Mice

Smith

2018

Drug Metab Dispos

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It is difficult to predict the pharmacokinetics and plasma concentration-time profiles of new chemical entities in humans based on animal data. Some pharmacokinetic parameters, such as clearance and volume of distribution, can be scaled allometrically from rodents, mammals, and nonhuman primates with good success. However, it is far more challenging to predict the oral pharmacokinetics of experimental drug candidates. In the present study, we used in situ estimates of intestinal permeability, obtained in silico and from rat, wild-type (WT), and humanized PepT1 (huPepT1) mice, to predict the systemic exposure of cefadroxil, an orally administered model compound, under a variety of conditions. Using the GastroPlus simulation software program (Simulations Plus, Lancaster, CA), we found that the C max and area under the plasma concentration-time curve from time zero to the last measurable concentration of cefadroxil were better predicted using intestinal permeability estimates (both segmental and jejunal) from huPepT1 than from WT mice, and that intestinal permeabilities based on in silico and rat estimates gave worse predictions. We also observed that accurate predictions were possible for cefadroxil during oral dose escalation (i.e., 5, 15, and 30 mg/kg cefadroxil), a drug-drug interaction study (i.e., 5 mg/kg oral cefadroxil plus 45 mg/kg oral cephalexin), and an oral multiple dose study [i.e., 500 mg (6.7 mg/kg) cefadroxil every 6 hours]. Finally, the greatest amount of cefadroxil was absorbed in duodenal and jejunal segments of the small intestine after a 5 mg/kg oral dose. Thus, by combining a humanized mouse model and in silico software, the present study offers a novel strategy for better translating preclinical pharmacokinetic data to oral drug exposure during first-inhuman studies.

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“…[ 14 C]Oseltamivir was employed as well revealing it is not a substrate for PEPT1. [ 3 H]GlySar uptake and [ 3 H]Cefadroxil uptake were investigated for hPEPT2 [ 211 ].…”

Section: Current Methodology For Purification and Functional Studies ...mentioning

confidence: 99%

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

Pochini

Galluccio

2022

Life

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For more than 20 years, yeast has been a widely used system for the expression of human membrane transporters. Among them, more than 400 are members of the largest transporter family, the SLC superfamily. SLCs play critical roles in maintaining cellular homeostasis by transporting nutrients, ions, and waste products. Based on their involvement in drug absorption and in several human diseases, they are considered emerging therapeutic targets. Despite their critical role in human health, a large part of SLCs’ is ‘orphans’ for substrate specificity or function. Moreover, very few data are available concerning their 3D structure. On the basis of the human health benefits of filling these knowledge gaps, an understanding of protein expression in systems that allow functional production of these proteins is essential. Among the 500 known yeast species, S. cerevisiae and P. pastoris represent those most employed for this purpose. This review aims to provide a comprehensive state-of-the-art on the attempts of human SLC expression performed by exploiting yeast. The collected data will hopefully be useful for guiding new attempts in SLCs expression with the aim to reveal new fundamental data that could lead to potential effects on human health.

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Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in Pichia Pastoris Transformants

Cited by 9 publications

References 41 publications

Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells

Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells

In Silico Prediction of the Absorption and Disposition of Cefadroxil in Humans using an Intestinal Permeability Method Scaled from Humanized PepT1 Mice

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

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