Patrick D. Bailey

How to Make Drugs Orally Active: A Substrate Template for Peptide Transporter PepT1

By building key structural features into hydrophilic drugs, they can be recognized by the PepT1 transporter system of the small intestine and rendered orally active. The model shown provides, for the first time, a 3D template for all known substrates of PepT1.

4‐Aminomethylbenzoic acid is a non‐translocated competitive inhibitor of the epithelial peptide transporter PepT1

1 4‐Aminomethylbenzoic acid, a molecule which mimics the spatial configuration of a dipeptide, competitively inhibits peptide influx in both Xenopus laevis oocytes expressing rabbit PepT1 and through PepT1 in rat renal brush border membrane vesicles. 2 This molecule is not translocated through PepT1 as measured both by direct HPLC analysis in PepT1‐expressing oocytes and indirectly by its failure to trans‐stimulate labelled peptide efflux through PepT1 in oocytes and in renal membrane vesicles. 3 However 4‐aminomethylbenzoic acid does reverse trans‐stimulation through expressed PepT1 of labelled peptide efflux induced by unlabelled peptide. Quantitatively this reversal is compatible with 4‐aminomethylbenzoic acid competitively binding to the external surface of PepT1. 4 4‐Aminomethylbenzoic acid (the first molecule discovered to be a non‐translocated competitive inhibitor of proton‐coupled oligopeptide transport) and its derivatives may thus be particularly useful as experimental tools.

Enhancing the yield and diastereoselectivity of the Pictet-Spengler reaction : a highly efficient route to cis-1,3-disubstituted tetrahydro-β-carbolines

Abstract Under conditions of kinetic control, the cis-diastereoselectivity of the Pictet-Spengler reaction between tryptophan esters and aldehydes can be controlled by varying the size of the ester group; the reaction proceeds in essentially quantitative yield with most aldehydes when conducted in chloroform in the presence of molecular sieves.

Substrate-charge dependence of stoichiometry shows membrane potential is the driving force for proton-peptide cotransport in rat renal cortex

The proton dependence of the transport of three labelled, hydrolysis-resistant synthetic dipeptides carrying a net charge of −1, 0 or +1 has been investigated in a brush border membrane vesicle preparation obtained from rat renal cortex. Cross-inhibition studies are consistent with the transport of all peptides studied being through a single system. The extent and time course of uptake in response to an inwardly directed electrochemical gradient of protons differed for each peptide. For the cationic peptide D-Phe-L-Lys this gradient did not stimulate the initial rate of uptake, while for the neutral dipeptide D-Phe-L-Ala and the anionic peptide D-Phe-L-Glu stimulation was observed. However, the effect on D-Phe-L-Glu was more marked than that on D-Phe-L-Ala and the proton activation differed for these two peptides. The calculated Hill coefficients for the two proton-dependent peptides were 1.14±0.16 and 2.15±0.10 for D-Phe-L-Ala and D-Phe-L-Glu, respectively, providing evidence that the stoichiometry of proton: peptide cotransport is different for each peptide (0∶1, 1∶1 and 2∶1 for D-Phe-L-Lys, D-Phe-L-Ala and D-Phe-L-Glu respectively); studies on energetics are compatible with this conclusion. The physiological and molecular implications of this model are discussed, as are the applicability of the conclusions to secondary active transport systems more generally.

Bioavailability through PepT1: the role of computer modelling in intelligent drug design

In addition to being responsible for the majority of absorption of dietary nitrogen, the mammalian proton-coupled di- and tri-peptide transporter PepT1 is also recognised as a major route of drug delivery for several important classes of compound, including beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Thus there is considerable interest in the PepT1 protein and especially its substrate binding site. In the absence of a crystal structure, computer modelling has been used to try to understand the relationship between PepT1 3D structure and function. Two basic approaches have been taken: modelling the transporter protein, and modelling the substrate. For the former, computer modelling has evolved from early interpretations of the twelve transmembrane domain structure to more recent homology modelling based on recently crystallised bacterial members of the major facilitator superfamily (MFS). Substrate modelling has involved the proposal of a substrate binding template, to which all substrates must conform and from which the affinity of a substrate can be estimated relatively accurately, and identification of points of potential interaction of the substrate with the protein by developing a pharmacophore model of the substrates. Most recently, these two approaches have moved closer together, with the attempted docking of a substrate library onto a homology model of the human PepT1 protein. This article will review these two approaches in which computers have been applied to peptide transport and suggest how such computer modelling could affect drug design and delivery through PepT1.

Concise routes to pyrazolo[1,5-a]pyridin-3-yl pyridazin-3-ones

Cycloaddition of pyridine N-imine with 6-alkyl-4-oxohex-5-ynoates followed by condensation with hydrazine provides concise access to pharmacologically active 6-(pyrazolo[1,5-a]pyridin-3-yl)pyridazinones. For the first time alkynyl heterocycles are also shown to be effective dipolarophiles for pyridine N-imine, and analogous compounds can be accessed directly in modest yields through the reaction of 6-(alkyn-1-yl)pyridazin-3-one derivatives.

Spiro cyclisations of N-acyliminium ions involving an aromatic π-nucleophile

Spiro 2-pyrrolidin-5-ones were obtained from N-substituted succinimides by a two-step procedure, involving 5- or 6-endo-trig cyclisation of N-acyliminium ion intermediates with a tethered aromatic π-nucleophile.

The transmembrane tyrosines Y56, Y91 and Y167 play important roles in determining the affinity and transport rate of the rabbit proton-coupled peptide transporter PepT1

The mammalian proton-coupled peptide transporter PepT1 is widely accepted as the major route of uptake for dietary nitrogen, as well as being responsible for the oral absorption of a number of classes of drugs, including β-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors. Using site-directed mutagenesis and zero-trans transport assays, we investigated the role of conserved tyrosines in the transmembrane domains (TMDs) of rabbit PepT1 as predicted by hydropathy plots. All the individual TMD tyrosines were substituted with phenylalanine and shown to retain the ability to traffic to the plasma membrane of Xenopus laevis oocytes. These single substitutions of TMD tyrosines by phenylalanine residues did not affect the proton dependence of peptide uptake, with all retaining wild-type PepT1-like pH dependence. Individual mutations of four of the nine TMD residue tyrosines (Y64, Y287, Y345 and Y587) were without measurable effect on PepT1 function, whereas the other five (Y12, Y56, Y91, Y167 and Y345) were shown to result in altered transport function compared to the wild-type PepT1. Intriguingly, the affinity of Y56F-PepT1 was found to be dramatically increased (approximately 100-fold) in comparison to that of the wild-type rabbit PepT1. Y91 mutations also affected the substrate affinity of the transporter, which increased in line with the hydrophilicity of the substituted amino acid (F > Y > Q > R). Y167 was demonstrated to play a pivotal role in rabbit PepT1 function since Y167F, Y167R and Y167Q demonstrated very little transport function. These results are discussed with regard to a proposed mechanism for PepT1 substrate binding.

A concise, efficient route to fumitremorgins

Abstract Using the kinetically controlled Pictet–Spengler reaction, we have developed a three-step, diastereoselective and enantiospecific synthesis of the natural product demethoxy-fumitremorgin C in 21% overall yield.

Novel spiro cyclisations of N-acyliminium ions involving an aromatic π-nucleophile

Abstract Several spiro 2-pyrrolidin-5-ones were obtained by a two-step procedure from N -substituted succinimides, involving spiro cyclisation of N -acyliminium ion intermediates in refluxing trifluoroacetic acid; in all cases cyclisation utilised a tethered aromatic π-nucleophile, and ring-closure followed 5- or 6- exo-trig pathways.