An in vivo approach to structure activity relationship analysis of peptide ligands.
OBJECTIVE
The goals in this study were several-fold. First, to optimize the in vivo phage display methodology by incorporating phage pharmacokinetic properties, to isolate peptides that target the brain microvasculature, and then to build focused libraries to obtain structure activity relationship information in vivo to identify the optimal targeting motif.


METHODS
The blood pharmacokinetics of filamentous and T7 phage were evaluated to choose the optimal platform. A randomized peptide library with a motif CX(10)C was constructed in T7 phage and used for in vivo panning. Focused peptide libraries around each structural element of the brain-specific peptide were constructed to perform kinetic structure activity relationship (kSAR) analysis in vivo. To determine potential function, sepsis was induced in mice by LPS administration and four hours later the effect of GST-peptide on adhesion of rhodamine-labelled lymphocytes or CFDA-labelled platelets to pial microvasculature was observed by intravital microscopy.


RESULTS
The blood phamacokinetics of T7 was rapid (half-life of 12 min) which aids the clearance of non-specific phage. In vivo panning in brain enriched for isolates expressing the motif CAGALCY. Kinetic analysis of focused libraries built around each structural element of the peptide provided for rapid pharmacophore mapping. The computer modeling data suggested the peptide showed similarities to peptide mimetics of adhesion molecule ligands. GST-CAGALCY but not GST control protein was able to inhibit the rolling and adhesion of labeled platelets to inflamed pial vasculature. GST-CAGALCY had no effect on lymphocyte adhesion.


CONCLUSIONS
Incorporating normal blood phamacokinetics of T7 phage into in vivo phage display improves the ability to recover targeting peptide motifs and allows effective lead optimization by kSAR. This approach led to the isolation of a brain-specific peptide, CAGALCY, which appears to function as an effective antagonist of platelet adhesion to activated pial microvasculature.
Fan
Xiaomin
X
AvantGen, Inc., 9924 Mesa Rim Rd, San Diego, California 92121, USA.
Venegas
Ruben
R
Fey
Robert
R
van der Heyde
Henri
H
Bernard
Mark A
MA
Lazarides
Elias
E
Woods
Catherine M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D001798:Blood Proteins / Q000494:pharmacology; D001921:Brain / Q000098:blood supply / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D002560:Cerebrovascular Circulation / Q000187:drug effects; D003198:Computer Simulation; D005260:Female; D006207:Half-Life; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000737:chemistry / Q000493:pharmacokinetics*; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D010841:Pia Mater / Q000098:blood supply / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D013329:Structure-Activity Relationship; D013678:Technology, Pharmaceutical / Q000379:methods
D001798:Blood Proteins; D015815:Cell Adhesion Molecules; D008024:Ligands; D008070:Lipopolysaccharides; D009842:Oligopeptides; D019151:Peptide Library; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; C487763:cyclic citrullinated peptide; C024875:platelet adhesion inhibitor