Mechanistic Insights into PEPT1-Mediated Transport of a Novel Antiepileptic, NP-647

Abstract

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.

Discovery of a low affinity thyrotropin-releasing hormone (TRH)-like peptide that exhibits potent inhibition of scopolamine-induced memory impairment in mice

“…TAL ([1-methyl-( S )-4,5-dihydroorotyl)]-L-His-L-ProNH 2 , TA-0910) produces CNS effects at about 100 times lower doses than TRH, and also showed eight times longer duration of antagonistic action on PB-induced sleep than TRH. 14–15,18 It is the first centrally acting TRH-like peptide that received approval in Japan for use as a drug for the treatment of adult spinal muscular atrophy under the trade name Ceredist ® 15 . Previously, Matsuoka et al 19 showed that TAL displayed higher activity in stimulating CNS effects than TRH in rodents.…”

“…These receptor-based results are consistent as found earlier, when analogue where ring NH of pGlu was replaced by a methylene group did not show binding affinity, confirming that the ring NH group of pGlu is necessary for the binding of the peptide to TRH receptors, while the modulation of the His residue at the N -1 position imparts selectivity toward TRH-R2. 14a–b …”

“…In recent studies, the susceptibility of TRH to degradation by TRH-DE is recognized to be a major factor undermining the investigation of the neurobiological functions and its therapeutic use, and a large number of TRH analogues and their various CNS applications are reported. 14–15 Recently Kelly et al reported a set of compounds that display ability to both inhibit TRH-DE and bind preferentially to central TRH receptors. This dual pharmacological activity within one molecular entity was found through selective manipulation of peptide stereochemistry.…”

Intestinal transport of TRH analogs through PepT1: the role ofin silicoandin vitromodeling

“…and Khomane et al . in earlier studies (Meredith and Price, ; Khomane et al ., ). The Glide docking score of the four analogs follows the order NP‐1894 (−8.03) < NP‐2378 (−8.06) < NP‐1896 (−8.45) < NP‐1895 (−8.65).…”

“…Transport studies of TRH analogs and Gly‐Sar were performed using the Caco‐2 cell model, and the permeability values are shown in Table . TRH analogs showed higher P app values indicating their improved permeability over TRH (9.17 × 10 −6 cm/s) (Khomane et al ., ). NP‐1895 had shown the highest P app (30.23 ± 0.50 × 10 −6 cm/s) among the four TRH analogs.…”