Linda L. Maggiora

The relationship between peptide structure and transport across epithelial cell monolayers

Abstract The successful development of orally bioavailable peptides and peptide-like substances as therapeutic agents will require an understanding of how structure influences absorption across the intestinal mucosa. In an attempt to define such relationships, homologous series of peptides were prepared which varied in lipophilicity, chain length and number of polar functionalities, and permeability studies conducted across Caco-2 cell monolayers as a model of the intestinal mucosa. The results suggested that the number of polar groups in the peptide, which presumably require desolvation before transfer of the peptide into the cell membrane, was a principal determinant of transport. Consistent with this hypothesis, two experimental methods of determining desolvation potential were found to correlate well with the observed permeability results for the peptides. The insights gained from these studies were used in an attempt to rationally modify a renin inhibitory peptide, in order to improve its permeability across the intestinal mucosa. Based on the results of this work, it is argued that a peptide must possess a delicate balance of affinity for the aqueous-membrane interface and a reasonably low desolvation energy in order for it to efficiently cross an epithelial cell membrane.

Role of complements C3 and C5 in the phagocytosis of liposomes by human neutrophils.

During the course of a previous investigation, we noticed that the uptake of liposomes by human polymorphonuclear neutrophils (PMNs) was significantly lower in the presence of heat-inactivated serum compared to that in intact whole serum (Scieszka et al., Pharm. Res. 5:352, 1988). This observation suggested the participation of heat-labile complement components in the phagocytic process. In this report we conclude that complement C3bi is the component responsible for opsonization of the liposome surface. Phagocytosis was not supported by C3-deficient serum, and phagocytosis in whole serum was blocked by the antibody to the receptor for C3bi (CR3) but not by the antibody to the receptor for C3b (CR1). We also found that with C5-deflcient serum the level of uptake was minimal but slightly higher than without any serum. When exogenous C5a was added along with C5-deficient serum, uptake levels similar in magnitude to those observed with intact serum were obtained. We conclude that C5a enhances phagocytosis of opsonized liposomes by activating the phagocytic capacity of CR3 on the PMN.

L-2-thiol-histidine : introduction of conformational constraints into peptides via thioether linkage

Abstract A synthetic strategy has been developed for a new type of conformational constraint in peptides, whereby two L-2-thiol-histidine (HisS) residues are bridged by a bis-thioether alkane linkage. The specificty of S-alkylation vs . N-alkylation in liquid ammonia would allow cyclization in the presence of nitrogen containing functional groups.

Conversion of uracil nucleotides into isotopically labelled 5-substituted uracil nucleotides: a convenient route to thymine nucleotides

Unprotected uracil nucleotides and nucleosides are converted into 5-formyluracil derivatives using a palladium(II) coupling reaction followed by oxidation of the intermediate styryl derivative.

Inhibitors of the bradykinin-degrading enzyme, aminopeptidase P

Aminopeptidase P inactivates bradykinin by hydrolyzing the bond [1]. This enzyme is a metallo-aminopeptidase with specificity for proline in the penultimate position. An inhibitor of this enzyme was synthesized and called apstatin (N-[(2S,3R)-3amino-2-hydroxy-4-phenylbutanoyl]-L-Pro-L-Pro-L-Ala-NH2) [2]. The N-terminal residue of apstatin was designed to chelate the active site through the amino and hydroxyl functions. The remaining residues were designed to accommodate the primary and secondary specificity requirements. Apstatin has an of for human membranebound aminopeptidase P. Apstatin together with an angiotensin converting enzyme inhibitor can completely block bradykinin degradation in the rat pulmonary and coronary circulations [2,3]. Apstatin can potentiate the vasodepressor response to intravenously-administered bradykinin and can reduce blood pressure in rats made hypertensive by aortic coarctation [4]. Apstatin can also significantly reduce cardiac ischemia/reperfusion damage as well as decrease reperfusion-induced ventricular fibrillation in the isolated rat heart [5]. The antihypertensive and cardioprotective effects of apstatin are blocked by a bradykinin receptor antagonist, suggesting that apstatin’s effects are due to potentiation of endogenously-formed bradykinin. In order to delineate the structural requirements for aminopeptidase P inhibition, 15 apstatin analogs were synthesized and tested for their ability to inhibit membrane-bound aminopeptidase P from human, monkey, rat, and bovine lung. Data for the human enzyme are described.

Design and Structure/Conformation-Activity Studies of a Prototypic Corticotropin-Releasing Factor (CRF) Antagonist: Multiple Alanine Substitutions of CRF12-41

The known physiological role(s) and proposed pathophysiological properties of the neuroendocrine peptide CRF have been previously described (for review, see 1), and CRF has been shown to exert a variety of CNS-mediated effects on behavior2,3, cardiovascular system4,5, reproduction6,7, gastrointestinal secretion8,9, motility10, and transit11. Of particular significance is that CRF may, therefore, be involved in stress stimuli-induced activation of neural/humoral pathways leading towards anxiety and depressive disorders (e.g.,depiession, panic and anorexia nervosa). Nevertheless, the molecular pharmacology and mechanisms which are involved in stress-induced behavioral, endocrine and metabolic activities are not well defined. The discovery and development of potent CRF antagonists may provide key molecular probes to investigate the biological activities of endogenous CRF in animal models as well as for studying the molecular pharmacology of CRF-receptor interactions. Such studies have been reported12,13 and have been primarily based upon synthetic modification of CRF; yet the emergence of a high affinity analog of low molecular mass (i.e., small peptide or peptidomimetic) has remained elusive to date. Nevertheless, studies14-18 on the blockade of endogenous CRF using CRF antiserum or prototypic CRF antagonists have probed the possible role that endogenous CRF may have on the effects of stress in different animal models. Of noteworthy contribution to such CRF research has been both structure-activity and structure-conformation studies12,13,19,20 to investigate CRFreceptor binding and functional properties (agonism/antagonism). These studies have culminated in the identification of prototypic CRF antagonists (or partial agonists) which were modified fragment analogs of the native peptide. Specifically, compound I (Fig. 1) has been advanced13 as a significant lead towards the development of high affinity CRF receptor antagonists. In this report we describe analogs of I to further explore the role of side-chain functionlization in CRF receptor binding using a strategy of multiple (iterative) Ala substitution with a particular focus on the central domain of this CRF analog corresponding to CRF22-31 In addition, the structure-conformation properties of these analogs were investigated by circular dichroism spectroscopy.

Exploiting the Molecular Template of Angiotensinogen in the Discovery and Design of Peptidyl, Pseudopeptidyl and Peptidemimetic Inhibitors of Human Renin: A Structure-Activity Perspective

The design of potent and pharmacologically effective, substrate-related inhibitors of renin has been the subject of intensive pharmaceutical discovery research for about one decade. Milestone achievements in synthetic tailoring of fragment analogs of angiotensinogen (ANG; Figure 1) have been documented in terms of identifying renin inhibitors of subnanomolar potency, sustained in vivo hypotensive activity, stability towards proteolytic degradation, and, more recently, oral bioavailability and decreased systemic clearance.1 By chemical modification of ANG-based derivatives, structure-activity analysis, and computer-assisted molecular modeling of peptidyl, pseudopeptidyl and peptidemimetic inhibitors using 3-D structural models of human renin, there currently exists a rather sophisticated wealth of information of relevance to the “rational” design of prototypic renin-targeted cardiovascular therapeutic agents. Such efforts have bridged biochemistry, medicinal chemistry, computational and biophysical chemistry, and in vivo pharmacology including, in a few cases, clinical evaluation in humans.