Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.
The 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, to 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 K(m) = 1.2 mM and V(max) = 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 (P(appCaco-2)) of NP-647 (32.5 × 10(-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 carrier-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, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Mohali, Punjab, India.
Kailas S
KS
Khomane
Prajwal P
PP
Nandekar
Banrida
B
Wahlang
Pravin
P
Bagul
Naeem
N
Shaikh
Yogesh B
YB
Pawar
Chhuttan Lal
CL
Meena
Abhay T
AT
Sangamwar
Rahul
R
Jain
K
K
Tikoo
Arvind K
AK
Bansal
D000658:Amoxicillin / Q000494:pharmacology; D000927:Anticonvulsants / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D004151:Dipeptides / Q000494:pharmacology; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007408:Intestinal Absorption / Q000187:drug effects; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D000074011:Peptide Transporter 1; D012964:Sodium / Q000378:metabolism; D019810:Sodium Azide / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics
D000927:Anticonvulsants; D004151:Dipeptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C539979:pyroglutamyl-(2-propyl)histidyl-prolinamide; C004194:glycylsarcosine; D013973:Thyrotropin-Releasing Hormone; D000658:Amoxicillin; D019810:Sodium Azide; D012964:Sodium