Mechanistic Insights into PEPT1-Mediated Transport of a Novel


Jul 10, 2012 - azide (Na+/K+-ATPase inhibitor) on the flux rate of NP-647 was ... indicate high affinity of NP-647 toward PEPT1 binding site as compar...
4 downloads 0 Views 625KB Size


Subscriber access provided by DUKE UNIV

Article

Mechanistic Insights into PEPT1-Mediated Transport of a Novel Antiepileptic, NP-647 Kailas S Khomane, Prajwal P Nandekar, Banrida Wahlang, Pravin Bagul, Naeem Shaikh, Yogesh B Pawar, Chhuttan Lal Meena, Abhay T. Sangamwar, Rahul Jain, K. Tikoo, and Arvind K. Bansal Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp200672d • Publication Date (Web): 10 Jul 2012 Downloaded from http://pubs.acs.org on July 11, 2012

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

Mechanistic Insights into PEPT1-Mediated Transport of a Novel Antiepileptic, NP-647 Kailas S. Khomanea, Prajwal P. Nandekarb, Banrida Wahlanga,Pravin Bagula, Naeem Shaikhb, Yogesh B. Pawara, Chhuttan Lal Meenac, Abhay T. Sangamwarb, Rahul Jainc, K. Tikood, Arvind K. Bansala* a

Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER),

Sector-67, S. A. S Nagar, Mohali, Punjab, India b

Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research

(NIPER), Sector-67, S.A.S. Nagar, Mohali, Punjab, India c

Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research

(NIPER), Sector-67, S.A.S. Nagar, Mohali, Punjab, India d

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and

Research (NIPER), Sector-67, S.A.S. Nagar, Mohali, Punjab, India

*

Corresponding author:

Mailing address: Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER) S.A.S. Nagar, Mohali, Punjab- 160 062, India Tel.: -91-172-2214682-2126; fax: +91-172-2214692 Email: [email protected]

ACS Paragon Plus Environment

1

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

Graphical Abstract

ACS Paragon Plus Environment

2

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

Abstract Present study in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and in particular, understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters: Km = 1.2 mM and Vmax= 2.2 µM/min. The effect of pH, sodium ion (Na+), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na+/K+-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (PappCaco-2) of NP-647 (32.5x10-6 cm/s) was found to be four times higher than that of TRH. Results indicate that, NP-647 is transported into Caco-2 cells by means of a carried-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and together, these evidences suggest that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 towards PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity towards PEPT1 by interacting with hydrophobic pocket of transporter through hydrophobic forces. Keywords: TRH analogues, Caco-2 cells, Peptide transporters, PEPT1 (SLC15A1), Intestinal oligopeptide transporter, Homology modeling, Docking studies, Molecular dynamics simulation

Abbreviations

ACS Paragon Plus Environment

3

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

TRH, thyrotropin releasing hormone; PEPT1, intestinal oligopeptide transporter, (SLC15A1); human peptide transporter 1; HBSS, Hank’s balanced salts solutions; MD, molecular dynamic; ER, efflux ratio. Introduction Epilepsy is one of the most common chronic neurological disorders, affecting 1% of the world population1. A range of first line and newer antiepileptic drugs are available in the market; however, 15-35% patients do not respond to conventional treatment.1 This particular condition wherein the patient remains non-responsive to the epileptic drugs is called intractable epilepsy. Therefore, epilepsy remains a significant therapeutic challenge despite current advances in the treatment. The use of thyrotropin releasing hormone (TRH) in the treatment of intractable epilepsy such as infantile spasms, Lennox-Gastaut syndrome, myoclonic seizures and refractory partial seizures, is clinically well known.2,

3

However, TRH has poor oral bioavailability and

short plasma half life which limiting its clinical utility.4, 5 Various TRH analogues synthesized by substituting C2 position of the imidazole ring with alkyl group of varying size, have been reported.6, 7 Among them NP-647 (L-pGlu-(2-propyl)–LHis–L-ProNH2) [Figure 1] was found to be 12 times more selective towards TRH R2 receptors, that are responsible for neuropharmacological activity.7, 8 NP-647 was found to be more stable than TRH in plasma and had potent antiepileptic activity in various animal models.9-11 Despite being a peptide, it was found to be stable in the gut environment and did not undergo first pass metabolism.12 Safety pharmacological studies of NP-647 in the central nervous system (CNS), the cardiovascular system (CVS) and the endocrine system suggested that it is more selective for neuropharmacological activity and devoid of hormonal and cerebrovascular system effects.9 NP647 possesses advantage over other reported TRH analogues like Azetirelin and MK-771, which

ACS Paragon Plus Environment

4

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

were withdrawn from clinical trials because of poor oral absorption.13-15 Evidence exists that TRH is transported through intestinal oligopeptide transporter, PEPT1 (SLC15A1) mediated transporter.5, 16, 17 H N O

N H

CONH 2

O N

O N

NH

Figure 1. Chemical structure of NP-647

In addition to its physiological substrates, PEPT1 transports number of pharmaceuticals that include class of β-lactam antibiotics, angiotensin-converting enzyme (ACE) inhibitors and other drugs and prodrugs.18 Molecular interactions between these drugs and PEPT1 are under intense investigation to support the development of new drug substrates.19-25 In the present work authors aim to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells. The effect of pH, Gly-Sar and amoxicillin (well-known transported substrates of PEPT1) on the uptake of NP-647, and the effect of NP-647 on the uptake of Gly-Sar were studied to elucidate whether PEPT1 is implicated. Transport study was also carried out in absence of sodium ions (Na+) to determine whether and to which extent, uptake of NP-647 into Caco-2 cells, is sodium dependent. Transport in presence of verapamil was also carried out to rule out the possibility of efflux mechanism by P-gp efflux. Furthermore, in-silico methodologies were employed to gain further insights into the properties of PEPT1-mediated transport of NP-647. In absence of crystal structure in the protein data bank,

ACS Paragon Plus Environment

5

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

homology model of human PEPT1 was developed and validated. Molecular docking of TRH and NP-647 was carried out using this validated homology model. Molecular dynamics simulation study was carried out to gain a more detailed insight. Experimental Section Chemicals NP-647 was synthesized in Department of Medicinal Chemistry, NIPER, SAS Nagar, India using solution phase peptide synthesis7. Dulbecco’s modified Eagle’s medium (DMEM), Hank’s balanced salts solutions (HBSS), lucifer yellow (LY), dimethyl sulfoxide (DMSO), antipyrine (ANT), TRH and glycylsarcosine (Gly-Sar) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum- heat inactivated (FBS), non-essential amino acids (NEAA) and Trypsin-ethylenediamine tetra acetic acid (Trypsin-EDTA) solutions were purchased from GIBCO, Invitrogen Corporation (NY, USA). Penicillin-Streptomycin-Amphotericin solution, 2[4-(2-hydroxyethyl)-1-piperazinyl]

ethanesulphonic

acid

(HEPES),

2-(N-morpholino)

ethanesulphonic acid (MES), phosphate-buffered saline (PBS), 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) and sodium azide were acquired from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Verapamil was a gift from Nicholas Piramal India Ltd. (Mumbai, India). Absolute ethanol was procured from Hong Yang Chemical Co. Ltd (Jiangsu, China). Milli-Q grade water purified by a Milli-Q UV Purification System (Millipore, Bedford, MA, USA) was used.

Cell culture

ACS Paragon Plus Environment

6

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

The Caco-2 cell line was obtained from American Type Culture Collection (ATCC, Virginia, USA) at passage no. 18. Cells were grown in DMEM supplemented with 15% of FBS, 1% Penicillin-Streptomycin-Amphotericin solution and 1% NEAA solution. Cells were cultured in T-75 cm2 tissue culture flasks obtained from Cellstar®, Greiner Bio-One (Frickenhausen, Germany). The cell cultures were maintained at 37 °C in a carbon dioxide (CO2) incubator, water jacketed with high-efficiency particulate air (HEPA) Class 100 (Forma Series II, Thermo electron Corporation, OH, USA). The incubator has an atmosphere of 95% air/5% CO2 and 95% humidity. The cells became 80-85% confluent in 4-7 days after which they were harvested with Trypsin-EDTA prior to seeding. The cells were grown on polycarbonate filters of 0.4 µm pore size (Millicell® 24-well Cell culture plate, Millipore, MA, USA) at a seeding density of 75,000 cells per well for 21-22 days to achieve a consistent monolayer. The growth media was changed and the transepithelial electrical resistance (TEER) value was measured every alternate day. It is known that Caco-2 cells of low passage number express carrier transporter to a lesser extent. Walter et al. demonstrated the heterogeneity in transport characteristics depending on the passage number of cells. TRH shows a carrier-mediated transport at higher passage number.17 Hence, cells from passage number 70-72 were used for the experiments. Methods Chromatographic conditions HPLC analysis was carried out using a reversed phase LiChrospher®100 C8 analytical column (4.6 × 200 mm, 5 µm particles; Merck, KgaA, Darmstadt, Germany), maintained at 40 °C. The column was preceded by a 3 mm × 4 mm C8 guard column (LichroCART®, Merck). Mobile phase consisted of a mixture of organic phase [acetonitrile: methanol (1:1)]: and aqueous phase (0.05% octansulfonic acid sodium salt, pH 2.2 adjusted with phosphoric acid) in the ratio of

ACS Paragon Plus Environment

7

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

72:28 respectively. The flow rate and detection wavelength (λmax) were 0.5 ml min-1 and 202 nm, respectively. Stability in HBSS solution NP-647 solution (1 mM) was incubated in HBSS kept at pH 6.5 ± 0.02 for 3 h at 37 ºC and 60 rpm. Samples were withdrawn at specific time intervals and analyzed using stability indicating HPLC method. Metabolic stability Caco-2 cells were homogenized in phosphate buffer of pH 7.4 (10% w/v) using an ULTRATUREXhomogenizer (Staufen, Germany) at 4 ºC. The homogenates were centrifuged (700 g, 4 ºC, 10 min) and supernatants were used for stability studies. TRH and NP-647 solutions (1 mM) were incubated in the protein solutions at 37 ºC, 60 rpm for 4 h.26 Samples were withdrawn at particular interval and deproteinized with 0.1 M zinc sulphate, centrifuged and supernatant was analyzed using HPLC method. Caco-2 cell monolayer permeability study MTT cytotoxicity assay was performed for NP-647 using a concentration range of 0.01-5.0 mM.27 Permeability experiments were performed under pH-gradient condition (apical pH 6.5, basolateral pH 7.4) in a shaker incubator maintained at 37 °C and 60 rpm. The transepithelial electrical resistance (TEER) value was measured with a Millipore ERS voltameter. For apical to basolateral transport studies (A→B), 400 µl of the NP-647 solution and TRH (1mM each) were added to apical side (A) and 800 µl of the blank transport buffer was added to the basolateral side (B). The basolateral to apical transport studies (B→A) were also carried out wherein the initial NP-647 solution was added to the receiver compartment (B) and the concentration in the donor compartment (A) was measured. Aliquots of 700 µl were withdrawn

ACS Paragon Plus Environment

8

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

from the respective compartment (B) at 20, 40, 60, 80, 100 and 120 min and the volume withdrawn was replaced with blank transport buffer. The apparent permeability coefficients, Papp (cm/s), for both A→B [Papp (AB)] and B→A [Papp (BA)] studies were calculated using Eq. (1). Papp = (dQ/dt) / (C0.A)

(1)

where dQ/dt is the cumulative transport rate (µM/min) defined as the slope obtained by linear regression of cumulative transported amount as a function of time (min), A is the surface area of the filters or inserts (0.7 cm2 in 24-wells), C0 is the initial concentration of the compounds on the donor side (µM). The efflux ratio (ER) was calculated from the following Eq. (2)ER = Papp (AB)/ Papp (BA)

(2)

Insights into the transport mechanisms The transport studies (A→B) were performed at different concentrations (0.1-10 mM) of NP647. The permeability studies for NP-647 (1 mM), in the presence of 15 mM Gly-Sar and 5 mM amoxicillin (PEPT1 substrates), 1 mM sodium azide (Na+/K+-ATPase inhibitor), and 1 mM verapamil [P-glycoprotein (P-gp) inhibitor] were also carried out to elucidate the mechanism of transport. Transport studies were also conducted in the cell monolayer without sodium i.e. by replacing sodium with potassium, to study the sodium dependence of transport. Inhibitory effect of NP-647 on Gly-Sar was studied using 5 and 10 mM of NP-647. NP-647 (inhibitor) was dissolved in transport medium containing 100 µM of Gly-Sar and applied to apical side of the Caco-2 monolayers. Inhibitory effect was calculated as % inhibition according to the Eq. (3). % Inhibition

            

x 100

(3)

Effect of pH on the transport of NP-647 was also studied using both isocratic (both apical and basolateral pH 7.4) and gradient condition (apical pH 6.5, basolateral pH 7.4). Monolayer

ACS Paragon Plus Environment

9

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

integrity was tested by quantifying lucifer yellow (LY) in the apical and basolateral compartments, at the end of the Caco-2 transport experiments. Recovery of NP-647 was calculated at the end of the every experiment using mass balance studies.28 Data analysis The kinetic parameters of saturable component were characterized by a modified MichaelisMenten equation reported previously (Eq. 4)29, 30. 

 ∗  ! # "$  ! %4' " #  !

where, V0 is initial transport rate, Vmax is the maximum transport rate, Km is a constant analogous to the Michaelis-Menten constant, S is the initial concentration of substrate and Kd is the first order rate constant for passive transport. The best fit curve of the Michaelis-Menten equation was determined using nonlinear regression program of GraphPad Prism (GraphPad Prism 5.0; GraphPad Software Inc., San Diego, CA). Statistical analysis Statistical significance for Papp values were compared using the paired t-test assuming equal variances (SigmaStat version 3.5, San Jose, California, USA). The test was considered to be statistically significant if P 95.0%. As shown in Figure 3, NP-647 showed concentration dependent transport across Caco-2 cell monolayers. The saturable (active) transport component of NP-647 was calculated after subtracting the nonsaturable (passive) component from the total flux. The Km and Vmax values for NP-647 transport across Caco-2 cell monolayers were also determined and were found to be 1.2 ± 0.2 mM and 2.2 ± 0.1 µM/min respectively. 2.5

Flux rate (µM/min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

2.0 1.5 1.0 0.5 0.0 0

2

4

6

Concentration (mM) Figure 3. Concentration dependence of NP-647 transport across Caco-2 cell monolayers. Error bars represent standard deviation of the mean value for n = 3.

As shown in Table 1, presence of Gly-Sar and amoxicillin decreased transport rate of NP-647 to 86.7% and 85.5% respectively. Sodium azide, a Na+/K+-ATPase inhibitor had a significant inhibitory influence and decreased the flux of NP-647 by the largest extent (23.8%). Without sodium, flux rate of NP-647 decreased by an extent of 36.5%. This indicates sodium dependent

ACS Paragon Plus Environment

16

Page 17 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

transport mechanism of NP-647. Transport in presence of verapamil, a known P-gp inhibitor, gave similar Papp values. PEPT1 requires a proton gradient and hence, isocratic pH condition decreased the flux of NP-647 by almost 50% (Table 1). This is in accordance with findings reported by other research groups.17, 29, 30 Inhibitory effect of NP-647 on the transport of Gly-Sar was studied in the inhibition experiment. As shown in Figure 4, % inhibition increases with increase in concentration of NP-647. The uptake of Gly-Sar decreased by almost 50% and 70%, in presence of 5 and 10 mM of NP-647, respectively. 80

% Inhibition

60 40 20

m M 10

5

m M

0

C on tr ol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Figure 4. Inhibitory effect of NP-647 on the transport of Gly-Sar across Caco-2 monolayers. Permeability of GlySar was measured with and without NP-647 in the apical solution. Concentration of gly- sar (100 µM) and NP-647 (5 mM & 10 mM) was applied to the apical side. Inhibitory effect was calculated as % inhibition according to the equation (3) in the text. Error bars represent standard deviation of the mean value of at least three experiments.

Homology modelling The BLASTP search was performed to identify the suitable template from PDB. The top hit from BLASTP search having score 297 was crystal structure of POT family transporter (PDB ID 2XUT). It is prokaryotic oligopeptide-proton symporters PEPT1 and selected as template for homology modelling of hPEPT1. POT family transporter is a nearest congener of hPEPT1 and

ACS Paragon Plus Environment

17

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

has 12 transmembrane domains with resolution of 3.62 Å composed of 500 amino acids.51 The protein topology prediction servers were used to predict the topology of transmembrane protein PEPT1. According to consensus topology prediction servers, the protein is composed of 12 transmembrane (TM) helices, 7 cytoplasmic loops including N-terminal & C-terminal end of protein and 6 extra cellular loops acts as connecting bridge between 12 TM helices. It also showed presence of large extracellular loop from amino acid residue 378 to 582 as depicted in Figure 5. This may be because of unavailability of suitable template for amino acid residue 378 to 582. Similar results were observed in case of BlastP search against protein data bank. The target-template sequence alignment was done manually to obtain reported protein topology and amino acid orientations, as described in the section “Computational methodologies”. Homology modelling and loop refinement were performed using Modeller9v8 software. The final model obtained after loop modelling and structure refinement is depicted in Figure 6. The backbone dihedral distribution of all amino acid residues of hPEPT1 homology model was calculated by Ramachandran plot. Later demonstrated that 85.4% residues are in the most favored region, 13.2% in allowed region, 0.9% in generously allowed region and 0.5% residue in disallowed region (Figure 6). This indicates that the built homology model is reasonably accurate in terms of dihedral distribution and steric clashes. The overall quality of both models was found to be 79.694% in Errat plot, which further increases the confidence of homology model.

ACS Paragon Plus Environment

18

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

Figure 5. 12-TM model for hPEPT1 showing N-terminal and C-terminal at cytoplasmic side. Amino acid residues in substrate binding domain are highlighted in red color. Point of loop truncation are highlighted in green color. Other anchor group amino acid residues are highlighted in blue color.

Figure 6. a) Cartoon view of PEPT1 homology model (Rainbow color scheme). White color region indicates position of substrate binding site. B) Ramachandran plot for PEPT1 homology model.

The resultant cavities were visually inspected to detect the appropriate cavity where substrates bind, to get transported by PEPT1. The volume of active site cavity was found to be 1586Å3 in homology model of hPEPT1. Further analysis of homology model shows that, TM helices 1, 4,

ACS Paragon Plus Environment

19

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

5, 6, 7, 8, 10 are arranged diagonally to each other. The amino acid residue Tyr91 is present in TM3, His121 in TM4, while Tyr167, Asn171, Ser174 and Pro182 are in TM5. TM2 is actively involved in pore formation. Amino acid residues His57 and Gln594 are facing each other. The results obtained correlated well with the previously reported observations about 3D structure of hPEPT1.38, 40 Molecular docking Molecular docking of PEPT1 substrates, TRH and NP-647, in PEPT1 homology model was performed using Glide software. The Emodel score for TRH and NP-647 were -12.747 and 22.187 kcal/mol, respectively. The interaction profiles of homology model with TRH and propyl analogue of TRH have been illustrated in Figure 7. The results indicate that His57 is involved in hydrogen bonding interaction with carbonyl group of prolinamide part of TRH and NP-647, which is reported as specific interaction for PEPT1 substrate.38 The surrounding amino acid residues involved in interactions are Ile55, His57, Thr58, Ala61, Leu65, Thr66, Trp294, Val626, Val628, Gly629, Asn630, Val636, Ala637, Gly638, Ala639, Gly640, Lys644, Glu648, Tyr649, Leu651, Phe652, Leu655 and Leu661. Amongst which His57, Thr58, Val626, Gly629, Gly638, Ala639 and Glu648 are involved in hydrogen bonding interactions with tripeptide TRH and NP647. Phe652 is involved in pi-stacking interaction with pyroglutamyl group of tripeptide substrate, while pi-cationic interaction was observed between histidyl group of substrate and Lys644. These observations are consistent with the results earlier reported by Meredith et al.38 Two fold increase in Emodel score was observed in case of NP-647 as compared to TRH in PEPT1 active site. This suggests significant binding of NP-647 to PEPT1, thus making it a substrate and enabling its active transport through intestine. The effect probably occurs due to presence of propyl substitution at histidyl group of NP-647 and its interaction with hydrophobic

ACS Paragon Plus Environment

20

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

amino acid residues like Ile635, val636, Gly638, Leu651 and Phe652 in active site cavity. The above hypothesis was further confirmed by molecular dynamics simulation studies.

Figure 7. Molecular docking pose of TRH (a) and NP-647 (b) in PEPT1 active site, showing interacting amino acid residues

Molecular dynamics PEPT1-substarte complexes obtained from molecular docking were used as starting structure for 2ns MD simulation run. The 2ns long NpT simulation in TIP3P water box of PEPT1substarte complexes showed that the geometry of PEPT1-substrate complex was well maintained. The potential, total energy and RMSD plots have all clearly converged by the end of MD simulation. The total and potential energy plots show an initial decrease and then stabilization, implying that system has folded up to a state more stable than the starting linear structure. The binding free energy calculations were done using MM-PB/GBSA method, as well as per residue decomposition energy was calculated for 2ns MD simulation run. The five binding energy calculating models i.e. Glide Emodel docking score, MM-PB/GBSA VDWAALS, MMPBSA, and MM-GBSA binding free energy, were used to compare the PEPT1-substrate binding as shown in Figure 8. The graph shows that, the binding energy score obtained from Glide

ACS Paragon Plus Environment

21

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

docking for TRH and NP-645 were TRH -12.747 kcal/mol and -22.187 kcal/mol respectively. This indicates around two fold increase in binding energy with NP-647, as compared to TRH as PEPT1 substrate. Flexibility was incorporated in enzyme substrate complex using MD simulation and MM-PBSA/GBSA binding and van der Waals energy contributions were calculated. The total MM-PBSA ∆G binding energy for TRH and NP-647 were found to be 21.4701 kcal/mol and -22.4537 kcal/mol respectively, which again confirms comparatively more substrate binding of NP-647 with PEPT1. The van der Waals energy contribution to the total ∆G binding energy were estimated as -43.1497 kcal/mol and -53.8042 kcal/mol respectively, which indicates that van der Waals interaction energy is responsible for tight binding of substrates to hPEPT1. Human PEPT1 demonstrates a higher degree of affinity to NP-647, as compared to TRH in MD simulation. NP-647

MM-GBSA-DELTA G Binding

TRH MM-PBSA-DELTA G Binding MM-PBSA/GBSA VDWAALS Glide Emodel Docking -60

-40

-20

0

Binding free energy (kcal/mol) Figure 8. Graph showing comparison five binding energy calculating models i.e. Glide Emodel docking score, MMPB/GBSA VDWAALS, MM-PBSA and MM-GBSA binding free energy for NP-647 and TRH.

Discussion Model drugs having experimental Papp values of 14.0 x 10-6 cm/s in Caco-2 cell assay are considered highly permeable whereas Papp values lesser than 5.0 x 10-6 cm/s are characteristic for low permeability model drugs.52, 53 Based on these values, NP-647 can be classified as a highly

ACS Paragon Plus Environment

22

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

permeable compound. Sun et al. have reported a correlation between Caco-2 monolayer permeability coefficients and human intestinal permeability for carrier mediated drugs.54 According to in-vitro/in-vivo correlation equation (LogY = 0.542 LogX +0.06), NP-647 indicated a Phuman of 7.56 X 10-4 cm/s. NP-647 is a tri-peptide with a log P value of -1.07 ± 0.06 indicating its highly hydrophilic nature. Hence, a high Papp value for NP-647 was rather surprising. NP-647 is a tripeptide having three modified amino acids namely prolylamide, propyl histidine and pyroglutamate. It is well documented that absorption of tri-peptides in the human body takes place through the transporter called as a intestinal oligopeptide transporter, PEPT1.55, 56 PEPT1 is a proton coupled transporter which acts as a symporter for peptide and proton (H+). Sodium/proton exchanger (antiporter for Na+/H+) is responsible for maintaining a proton gradient. However, maintaining sodium flux is an energy driven process and Na+/K+-ATPase is responsible for it. Thus, PEPT1 indirectly depends upon the Na+/K+-ATPase and hence called as secondary active transporter. Results of permeability experiments showed decrease in flux rate of NP-647 in presence of Gly-Sar, amoxicillin and sodium azide. pH condition influenced the permeability of NP-647 and isocratic condition decreased the permeability of NP-647 by almost 50%. In inhibition experiment, NP-647 blocked the influx of Gly-Sar, a specific PEPT1 substrate. Moreover, concentration dependent transport of NP-647 is consistent with a carrier-mediated transport. Km value obtained for NP-647 (1.2 ± 0.2 mM) is in typical range, reported for small peptide drugs transported by PEPT1.18, 57 Thus, results indicate that, NP-647 is transported into Caco-2 cells by means of a carriedmediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-

ACS Paragon Plus Environment

23

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

647 inhibits the uptake of Gly-Sar into Caco-2 cells and together these evidences suggest that PEPT1 is involved in the process. However, other mechanisms can not be ruled out, as demonstrated by the fact that uptake of NP-647 into Caco-2 cells seems to be, at least in part, Na+ dependent and also exhibits non-mediated, diffusive component. Sodium azide decreased the flux rate of NP-647, however, it is reported not to inhibit Na+/K+ATPase directly. Sodium azide inhibits the cytochrome C oxidase (or complex IV) of the mitochondrial respiratory chain.58 As a consequence, the gradient of H+ between the inner and outer membranes of mitochondria is diminished and this decreases the rate of ATP synthesis by the ATP synthase. The subsequent decrease in cytosolic ATP results in potential decrease in basolateral Na+/K+-ATPase function. The decrease in cytosolic ATP may have other metabolic and physiological consequences and their indirect influence on the NP-647 influx cannot be ruled out. Higher flux rate in A→B direction as compared to B→A direction and similar Papp values in presence of verapamil ruled out the possibility of efflux mechanism by P-gp in transport of NP647. However, Caco-2 cell line does not significantly expresses all the efflux transporters and enzymes59 and hence role of any other efflux system cannot be rule out. NP-647 (Papp= 3.24±0.24) has higher intestinal permeability as compared to TRH (Papp= 0.83±0.26). Higher flux rate may be attributed to increased hydrophobicity of NP-647 compared to TRH [log PNP-647 = -1.07; log PTRH =-2.46].12,

60

Lin et al. have shown positive correlation

between hydrophobicity of ACE inhibitor and their affinities towards the peptide transporter.61 The increase in hydrophobicity may increase the drug’s affinity for the transporter, possibly by interacting with the hydrophobic pocket of the peptide transporter through hydrophobic forces. However, this hypothesis needs to be tested. Docking is most commonly used and accepted

ACS Paragon Plus Environment

24

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

methodology to study the ligand-receptor interactions. Hence, comparative docking assessment of NP-467 and TRH with PEPT1 can provide important insights and helps to delineate the mechanism involved. The preliminary molecular docking analysis of atomic level interactions between PEPT1 and substrate indicates that the substrate binding was influenced by presence of hydrophobic residues in active site cavity of PEPT1. Around two fold increase in Emodel docking score from -12.747 upto -22.187 kcal/mol for TRH and NP-647 respectively, indicates stronger binding of NP-647 with PEPT1 that would favour its transportation. Overall binding free energy for PEPT1substrate complexes shows that similar energy profile was observed for MM-PBSA as well as MM-GBSA method, when complex were simulated in physiological conditions. The estimated van der Waal’s energy contribution to total binding free energy showed approximately 10 kcal/mol increase in binding energy for NP-647 as compared to TRH. This confers greater contribution of van der Waal’s energy in substrate binding with PEPT1. The per residue decomposition energy62 for each amino acid residues were calculated to study the effect of interacting residue on substrate binding during 2ns MD simulation, as shown in Figure 9. Decomposition energy contribution mainly due to hydrophobic amino acid residues Leu51, Thr53, Ala61, Lys253, Ile635, Val636, Gly638, Leu651 and Phe652 were comparatively larger in NP-647 than in TRH. Decomposition energy contributions were similar in both substrates for hydrophilic residues Asp298, Asn630 and Lys644. The decomposition energy contribution of Leu51, Thr53, Lys253, Ile635, Val636, Gly638, Leu651 and Phe652 for NP-647:TRH are 0.52:0.21, 0.4:0.09, 0.42:0.16, 0.8:0.05, 0.63:0.17, 1.47:0.68, 1.04:0.42 and 0.74:0.38 kcal/mol respectively. It clearly indicates that interactions of hydrophobic amino acid residues are crucial for the substrate binding and ultimately transporter activity of PEPT1.

ACS Paragon Plus Environment

25

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

2.5

TGBTOT (kcal/mol)

NP-647 TRH

2.0 1.5 1.0 0.5

IL E6 35 VA L6 36 G LY 63 8 LY S6 44 LE U 65 1 PH E6 52

A

SN

63 0

29 8

A SP

25 3

61

LY S

A LA

53 TH R

51

0.0 LE U

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 33

Residue Number Figure 9. Residue wise decomposition energy for active site amino acid residues for NP-647 and TRH in PEPT1 homology model.

The observations from five interaction energy calculating models and per residue decomposition energy contribution suggest that, the hydrophobic substitution at histidyl amino acid in TRH analogue i. e. NP-647 leads to significant increase in its transport through PEPT1 as confirmed by in vitro experiments. Peptide suffers from the poor oral bioavailability as their passive transport is very less due to their hydrophilicity. Nevertheless, peptide can be transported through carrier mediated mechanism, if it has similar configuration as that of nature bioactives like TRH. However, peptides should have sufficient affinity towards binding site of the transporter to enable their carrier mediated transport. Docking studies with transporter can predict the binding affinity and hence extent of its transport across biological membrane. TRH and its few analogues are transported through PEPT1. Binding affinity towards PEPT1 is crucial parameter for their permeability. Present study showed that permeability of TRH analogue, NP-647 increase with increase in hydrophobicity. Thus hydrophobicity plays critical role in active transport of drugs.

ACS Paragon Plus Environment

26

Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

Conclusions NP-647, apart from other mechanisms, is transported through the PEPT1. It possesses higher permeability than TRH. Thus unlike other TRH analogues, no permeability related hurdles are expected during its oral delivery. Docking studies revealed that NP-647 has greater affinity towards transporter as compared to its parent compound, TRH. Results of molecular dynamics simulation confirm the higher affinity of NP-647 due to hydrophobic interaction of a propyl moiety with hydrophobic pocket of the PEPT1 binding site. Thus, present study highlights the importance of docking studies as a screening parameter for selection of lead from TRH analogues. Hence, in drug discovery of novel TRH analogues, critical tailoring of the TRH structure that retains the native conformation of parent at the same time increases its hydrophobicity, plays vital role in overcoming permeability hurdles. However, further experimental validation using exhaustive kinetic and functional characterization of the compound in a system, that allows for over expression of the PEPT1, such as Xenopus oocytes or mammalian cells, should to be performed before predicting the oral bioavailability. Acknowledgements We thank the reviewers for critical review of this manuscript and for helpful comments. Yogesh B. Pawar acknowledges Department of Science and Technology (DST), Govt. of India for providing Senior Research Fellowship.

ACS Paragon Plus Environment

27

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647

References 1.

Gupta, V.; Gupta, S. K.; Gupta, R.; Ie, I. E. Intractable epilepsy. JK Pract. 2004, 12 (1), 105-107.

2.

Kubek, M. J.; Garg, B. P. Thyrotropin-releasing hormone in the treatment of intractable epilepsy. Pediatr. Neurol. 2002, 26 (1), 9-17.

3.

Takeuchi, Y.; Tominaga, M.; Mitsufuji, N.; Yamazoe, I.; Kawase, S.; Nishimura, A.; Matsuo, S.; Sawada, T. Thyrotropin-releasing hormone in treatment of intractable epilepsy: neurochemical analysis of CSF monoamine metabolites* 1. Pediatr. Neurol. 1995, 12 (2), 139-145.

4.

Mitsuma, T.; Nogimori, T. Influence of the route of administration on thyrotropin-releasing hormone concentration in the mouse brain. Cell. Mol. Life Sci. 1983, 39 (6), 620-622.

5.

Yokohama, S.; Yamashita, K.; Toguchi, H.; Takeuchi, J.; Kitamori, N. Absorption of thyrotropin-releasing hormone after oral administration of TRH tartrate monohydrate in the rat, dog and human. J. Pharm. Dyn. 1984, 7, 101-111.

6.

Monga, V.; Meena, C. L.; Rajput, S.; Pawar, C.; Sharma, S. S.; Lu, X.; Gershengorn, M. C.; Jain, R. Synthesis, receptor binding, and CNS pharmacological studies of new Thyrotropin Releasing Hormone (TRH) analogues. Chem. Med. Chem. 2011, 6 (3), 531-543.

7.

Jain, R.; Kaur, N.; Monga, V. CNS effective thyrotropin releasing hormone analogs. WTO Indian Patent Application No. 2065/DEL/2006, 04/04/2008, 2006.

8.

Kaur, N.; Lu, X.; Gershengorn, M. C.; Jain, R. Thyrotropin-releasing hormone (TRH) analogues that exhibit selectivity to TRH receptor subtype. J. Med. Chem. 2005, 48 (19), 6162-6165.

9.

Rajput, S. K.; Krishnamoorthy, S.; Pawar, C.; Kaur, N.; Monga, V.; Meena, C. L.; Jain, R.; Sharma, S. S. Antiepileptic potential and behavioral profile of l-pGlu-(2-propyl)–l-His–l-ProNH2, a newer thyrotropinreleasing hormone analog. Epilepsy Behav. 2009, 14 (1), 48-53.

10.

Rajput, S. K.; Siddiqui, M. A.; Kumar, V.; Meena, C. L.; Pant, A. B.; Jain, R.; Sharma, S. S. Protective effects of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer Thyrotropin Releasing Hormone analog in in vitro and in vivo models of cerebral ischemia. Peptides 2011, 32 (6), 1225-1231.

11.

Sah, N.; Rajput, S. K.; Singh, J. N.; Meena, C. L.; Jain, R.; Sikdar, S. K.; Sharma, S. L-pGlu-(2-propyl)-LHis-L-ProNH2 attenuates 4-aminopyridine-induced epileptiform activity and sodium current: a possible action of new TRH analog for its anticonvulsant potential. Neuroscience 2011, 199, 74-85.

ACS Paragon Plus Environment

28

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647 12.

Khomane, K.; Kumar, L.; Meena, C.; Jain, R.; Bansal, A. NP-647, a novel TRH analogue: Investigating physicochemical parameters critical for its oral and parenteral delivery. Int. J. Pharm. 2011, 406, 21-30.

13.

Sasaki, I.; Fujita, T.; Murakami, M.; Yamamoto, A.; Nakamura, E.; Imasaki, H.; Muranishi, S. Intestinal absorption of azetirelin, a new thyrotropin-releasing hormone (TRH) analogue. I: possible factors for the low oral bioavailability in rats. Biol. Pharm. Bull. 1994, 17 (9), 1256-1261.

14.

Sasaki, I.; Tanaka, K.; Fujita, T.; Murakami, M.; Yamamoto, A.; Muranishi, S. Intestinal absorption of azetirelin, a new thyrotropin-releasing hormone (TRH) analogue. II: in situ and in vitro absorption characteristics of azetirelin from the rat intestine. Biol. Pharm. Bull. 1995, 18 (7), 976-979.

15.

Mahato, R. I.; Narang, A. S.; Thoma, L.; Miller, D. D. Emerging trends in oral delivery of peptide and protein drugs. Crit. Rev. Ther. Drug Carrier Syst. 2003, 20 (2-3), 153-214.

16.

Yokohama, S.; Yoshioka, T.; Yamashita, K.; Kitamori, N. Intestinal absorption mechanisms of thyrotropinreleasing hormone. J. Pharmacobio-Dyn. 1984, 7, 445-450.

17.

Walter, E.; Kissel, T. Transepithelial transport and metabolism of thyrotropin-releasing hormone (TRH) in monolayers of a human intestinal cell line (Caco-2): evidence for an active transport component? Pharm. Res. 1994, 11 (11), 1575-1580.

18.

Brandsch, M.; Knutter, I.; Bosse-Doenecke, E. Pharmaceutical and pharmacological importance of peptide transporters. J. Pharm. Pharmacol. 2008, 60 (5), 543-585.

19.

Sala-Rabanal, M.; Loo, D. D. F.; Hirayama, B. A.; Turk, E.; Wright, E. M. Molecular interactions between dipeptides, drugs and the human intestinal H+-oligopeptide cotransporter hPEPT1. J. Physiol. 2006, 574 (1), 149-166.

20.

Knutter, I.; Hartrodt, B.; Theis, S.; Foltz, M.; Rastetter, M.; Daniel, H.; Neubert, K.; Brandsch, M. Analysis of the transport properties of side chain modified dipeptides at the mammalian peptide transporter PEPT1. Eur. J. Pharm. Sci. 2004, 21 (1), 61-67.

21.

Biegel, A.; Knutter, I.; Hartrodt, B.; Gebauer, S.; Theis, S.; Luckner, P.; Kottra, G.; Rastetter, M.; Zebisch, K.; Thondorf, I. The renal type H+/peptide symporter PEPT2: structure-affinity relationships. Amino acids 2006, 31 (2), 137-156.

22.

Brandsch, M.; Leibach, F. H. The intestinal H+/peptide symporter PEPT1: structure-affinity relationships. Eur. J. Pharm. Sci. 2004, 21 (1), 53-60.

ACS Paragon Plus Environment

29

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647 23.

Daniel, H.; Rubio-Aliaga, I. An update on renal peptide transporters. Am. J. Physiol. Renal Physiol. 2003, 284 (5), F885-F892.

24.

Daniel, H.; Kottra, G. The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology. Pflugers Arch. 2004, 447 (5), 610-618.

25.

Fei, Y. J.; Liu, J. C.; Fujita, T.; Liang, R.; Ganapathy, V.; Leibach, F. H. Identification of a potential substrate binding domain in the mammalian peptide transporters PEPT1 and PEPT2 using PEPT1-PEPT2 and PEPT2PEPT1 chimeras. Biochem. Biophys. Res. Commun. 1998, 246 (1), 39-44.

26.

Moss, J.; Buur, A.; Bundgaard, H. Prodrugs of peptides. 8. in vitro study of intestinal metabolism and penetration of thyrotropin-releasing hormone (TRH) and its prodrugs. Int. J. Pharm. 1990, 66 (1-3), 183-191.

27.

Wahlang, B.; Pawar, Y. B.; Bansal, A. K. Identification of permeability-related hurdles in oral delivery of curcumin using the Caco-2 cell model. Eur. J. Pharm. Biopharm. 2011, 77, 275–282.

28.

Neuhoff, S.; Ungell, A. L.; Zamora, I.; Artursson, P. pH-dependent passive and active transport of acidic drugs across Caco-2 cell monolayers. Eur. J. Pharm. Sci. 2005, 25 (2-3), 211-20.

29.

Scow, J. S.; Madhavan, S.; Chaudhry, R. M.; Zheng, Y.; Duenes, J. A.; Sarr, M. G. Differentiating passive from transporter-mediated uptake by PepT1: a comparison and evaluation of four methods1. J. Surg. Res. 2011, 170, 17-23.

30.

Bhardwaj, R. K.; Herrera-Ruiz, D.; Sinko, P. J.; Gudmundsson, O. S.; Knipp, G. Delineation of human peptide transporter 1 (hPepT1)-mediated uptake and transport of substrates with varying transporter affinities utilizing stably transfected hPepT1/Madin-Darby canine kidney clones and Caco-2 cells. J. Pharmacol. Exp. Ther. 2005, 314 (3), 1093-1100.

31.

Apweiler, R.; Martin, M. J.; O'Donovan, C.; Magrane, M.; Alam-Faruque, Y.; Antunes, R.; Barrell, D.; Bely, B.; Bingley, M.; Binns, D. The universal protein resource (UniProt) in 2010. Nucleic Acids Res. 2010, 38, D142-D148.

32.

Liang, R.; Fei, Y. J.; Prasad, P. D.; Ramamoorthy, S.; Han, H.; Yang-Feng, T. L.; Hediger, M. A.; Ganapathy, V.; Leibach, F. H. Human intestinal H+/peptide cotransporter. J. Biol. Chem. 1995, 270 (12), 6456.

33.

Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 1990, 215 (3), 403-410.

ACS Paragon Plus Environment

30

Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647 34.

Nugent, T.; Jones, D. Transmembrane protein topology prediction using support vector machines. BMC Bioinformatics 2009, 10 (1), 159.

35.

Muller, S.; Croning, M. D. R.; Apweiler, R. Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 2001, 17 (7), 646-653.

36.

Nakanishi, Y.; Matsuda, N.; Aizawa, K.; Kashiyama, T.; Yamamoto, K.; Mimura, T.; Ikeda, M.; Maeshima, M. Molecular cloning and sequencing of the cDNA for vacuolar H+-pyrophosphatase from Chara corallina1. Biochim. Biophys. Acta 1999, 1418 (1), 245-250.

37.

Jones, D. T. Improving the accuracy of transmembrane protein topology prediction using evolutionary information. Bioinformatics 2007, 23 (5), 538-542.

38.

Meredith, D.; Price, R. A. Molecular modeling of PepT1 - towards a structure. J. Membr. Biol. 2006, 213 (2), 79-88.

39.

Wolf, S.; Buckmann, M.; Huweler, U.; Schlitter, J.; Gerwert, K. Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand binding site. FEBS Letters 2008, 582 (23-24), 33353342.

40.

Bolger, M. B.; Haworth, I. S.; Yeung, A. K.; Ann, D.; von Grafenstein, H.; Hamm†Alvarez, S.; Okamoto, C. T.; Kim, K. J.; Basu, S. K.; Wu, S. Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1. J. Pharm. Sci. 1998, 87 (11), 1286-1291.

41.

Eswar, N.; Webb, B.; Marti-Renom, M. A.; Madhusudhan, M. S.; Eramian, D.; Shen, M. Y.; Pieper, U.; Sali, A. Comparative protein structure modeling using Modeller. Curr. Protoc. Protein Sci. 2007, 50 (2.9), 1-2.9.

42.

NIH MBI Laboratory for Structural Genomics and Proteomics, Stuctural Analysis and Verification Server. http://nihserver.mbi.ucla.edu/SAVES_3/

43.

Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl.Crystallogr. 1993, 26 (2), 283-291.

44.

Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Daniel, T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 2004, 47 (7), 1739-1749.

ACS Paragon Plus Environment

31

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 33

Mechanistic Insights into PEPT1-Mediated Transport of NP-647 45.

Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. Evaluation and reparametrization of the OPLS-AA force field for proteins via comparison with accurate quantum chemical calculations on peptides. J. Phys. Chem. B 2001, 105 (28), 6474-6487.

46.

Case, D. A.; Cheatham Iii, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz Jr, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. The Amber biomolecular simulation programs. J. Comput. Chem. 2005, 26 (16), 1668-1688.

47.

Paesani, F.; Vanicek, J.; Wu, X.; Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V. AMBER 10. University of California: San Francisco 2008.

48.

Lindorff-Larsen, K.; Piana, S.; Palmo, K.; Maragakis, P.; Klepeis, J. L.; Dror, R. O.; Shaw, D. E. Improved side chain torsion potentials for the Amber ff99SB protein force field. Proteins 2010, 78 (8), 1950-1958.

49.

Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 1996, 14 (1), 3338.

50.

Swanson, J. M. J.; Henchman, R. H.; McCammon, J. A. Revisiting free energy calculations: a theoretical connection to MM/PBSA and direct calculation of the association free energy. Biophys. J. 2004, 86 (1), 6774.

51.

Newstead, S.; Drew, D.; Cameron, A. D.; Postis, V. L. G.; Xia, X.; Fowler, P. W.; Ingram, J. C.; Carpenter, E. P.; Sansom, M. S. P.; McPherson, M. J. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide proton symporters, PepT1 and PepT2. EMBO J. 2011, 30 (2), 417-426.

52.

Sun, D.; Yu, L. X.; Hussain, M. A.; Wall, D. A.; Smith, R. L.; Amidon, G. L. In vitro testing of drug absorption for drug 'developability' assessment: forming an interface between in vitro preclinical data and clinical outcome. Curr. Opin. Drug Discov. Devel. 2004, 7 (1), 75-85.

53.

Volpe, D. A.; Faustino, P. J.; Ciavarella, A. B.; Asafu-Adjaye, E. B.; Ellison, C. D.; Yu, L. X.; Hussain, A. S. Classification of drug permeability with a Caco-2 cell monolayer assay. Clin. Res. Reg. Affairs 2007, 24 (1), 39-47.

54.

Sun, D.; Lennernas, H.; Welage, L. S.; Barnett, J. L.; Landowski, C. P.; Foster, D.; Fleisher, D.; Lee, K. D.; Amidon, G. L. Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm. Res. 2002, 19 (10), 1400-1416.

ACS Paragon Plus Environment

32

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Mechanistic Insights into PEPT1-Mediated Transport of NP-647 55.

Addison, J. M.; Burston, D.; Dalrymple, J. A.; Matthews, D. M.; Payne, J. W.; Sleisenger, M. H.; Wilkinson, S. A common mechanism for transport of di-and tri-peptides by hamster jejunum in vitro. Clin. Sci. Mol. Med. 1975, 49 (4), 313-322.

56.

Walter, E.; Kissel, T.; Amidon, G. L. The intestinal peptide carrier: a potential transport system for small peptide derived drugs. Adv. Drug Deliv. Rev. 1996, 20 (1), 33-58.

57.

Friedman, D. I.; Amidon, G. L. Characterization of the intestinal transport parameters for small peptide drugs. J. Control Release 1990, 13 (2-3), 141-146.

58.

Karu, T. I.; Pyatibrat, L. V.; Kalendo, G. S. Photobiological modulation of cell attachment via cytochrome c oxidase. Photochem. Photobiol. Sci. 2004, 3 (2), 211-216.

59.

Maubon, N.; Le Vee, M.; Fossati, L.; Audry, M.; Le Ferrec, E.; Bolze, S.; Fardel, O. Analysis of drug transporter expression in human intestinal Caco-2 cells by real-time PCR. Fundam. Clin. Pharmacol. 2007, 21 (6), 659-663.

60.

Bundgaard, H.; Moss, J. Prodrugs of peptides. 6. bioreversible derivatives of thyrotropin-releasing hormone (TRH) with increased lipophilicity and resistance to cleavage by the TRH-specific serum enzyme. Pharm. Res. 1990, 7 (9), 885-92.

61.

Lin, C. J.; Akarawut, W.; Smith, D. E. Competitive inhibition of glycylsarcosine transport by enalapril in rabbit renal brush border membrane vesicles: interaction of ACE inhibitors with high-affinity H+/peptide symporter. Pharm. Res. 1999, 16 (5), 609-15.

62.

Ahmad, R.; Brandsdal, B. O.; Michaud-Soret, I.; Willassen, N. P. Ferric uptake regulator protein: Binding free energy calculations and per-residue free energy decomposition. Proteins 2009, 75 (2), 373-386.

ACS Paragon Plus Environment

33