Clark W. Smith

Production of an antibody to arterial fatty acid-binding protein (FABP) using a synthetic peptide as the antigen

1. A procedure is described for the preparation of an antibody to arterial FABP using a synthetic peptide as an antigen. In order to locate a highly conserved region located on the outer surface of FABP, computer analysis of primary and secondary structures of several proteins from the FABP family was undertaken and a 24 amino acid sequence beginning at the fifth position from the N-terminus of rat heart FABP was chosen. 2. The synthetic peptide consisted of eight replications of the 24 amino acid sequence individually attached to the alpha and epsilon amino groups of each terminal lysine on an octalysine branched peptide. 3. Antibody to the synthetic antigen was raised in New Zealand rabbits. Western analysis was conducted and detection was accomplished by using goat-anti-rabbit second antibody conjugated to alkaline phosphatase. 4. The antibody produced from the previously described peptide, recognized purified rat heart FABP and demonstrated a high positive correlation (r = 0.96) when known concentrations of purified hFABP were plotted against densitometric measurement of the bands. 5. Additionally, the antibody recognized FABP from the 104,000 g supernates of rat atrial and arterial tissue fractionated by a Sephadex G-75 column. 6. Therefore, the antibody produced from this particular protocol employing a synthetic peptide can be utilized qualitatively and quantitatively in the analysis of the heart and arterial FABP content.

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.

Cardiovascular Effects of a Renin Inhibitor in Relation to Posture in Nonhuman Primates

Orthostatic cardiovascular reflexes were evaluated in conscious cynomolgus monkeys during interruption of the renin-angiotensin system with the renin inhibitor: RIP (Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys-OH). RIP was synthesized via solid phase techniques and purified to homogeneity. In vitro studies indicated that it exhibited classical competitive inhibition of renin with a KI of 2.3 microM. In vivo, RIP at 2 mg/kg per min inhibited renin and angiotensin I pressor responses indicating that it was not a specific renin inhibitor at this dose. However, in spite of the nonspecificity, RIP did not affect the supine blood pressure of sodium-replete monkeys, but did evoke hypotension in supine sodium depleted monkeys. RIP did not elicit significant orthostatic hypotension in either sodium-replete or sodium depleted monkeys. The cardiovascular effects of RIP described in this study appear to be due to inhibition of the renin-angiotensin system.

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.

Reinvestigation of the 4-methoxy-2,6-dimethylbenzenesulphonyl (Mds) protecting group for the guanidino-function during peptide synthesis

The procedure for the synthesis of Z-Arg(Mds)-OH yields, in addition to the title compound, three products that have analogues of the Mds group on the guanidino-function making characterization of protected intermediate peptides difficult.