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An In Vivo Approach to Structure Activity Relationship Analysis of Peptide Ligands

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Abstract

Purpose

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.

Materials and Methods

The blood pharmacokinetics of filamentous and T7 phage were evaluated to choose the optimal platform. A randomized peptide library with a motif CX10C 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.

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Abbreviations

CFDA:

carboxyfluorescein diacetate succinimidyl ester

GST:

glutathione S-transferase

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

KSAR:

kinetic structure activity relationship

LPS:

Lipopolysaccharide

MPS:

monophagocytic system

PEG:

polyethylene glycol

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Acknowledgments

The authors thank Jie Li and Clara Polizzi for their technical assistance with the intravital microscopy studies and Zhe Li, Shaan Tolani, and Zaid Yusufi for technical assistance with the phage display work. The intravital microscopy studies were supported in part by NIH grant A140667-06 to HvdH.

Author information

Authors and Affiliations

  1. Targeted Molecules Corporation, Chromos Molecular System Inc, 8081 Lougheed Highway, Burnaby, BC, V5A 1W9, Canada

    Elias Lazarides

  2. AvantGen, Inc., 9924 Mesa Rim Rd, San Diego, California, 92121, USA

    Xiaomin Fan & Catherine M. Woods

  3. BioIT Xperts, 9011 Mira Mesa Blvd #216, San Diego, California, 92126, USA

    Ruben Venegas

  4. Isis Pharmaceuticals, Inc., 1896 Rutherford Road, Carlsbad, California, 92008, USA

    Robert Fey

  5. La Jolla Bioengineering Institute, 505 Coast Blvd, Suite 405, San Diego, California, 92037, USA

    Henri van der Heyde

  6. Bayer HealthCare Pharmaceuticals, 800 Dwight Way, PO Box 1986, Berkeley, California, 94701, USA

    Mark A. Bernard

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  1. Xiaomin Fan
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  2. Ruben Venegas
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Corresponding author

Correspondence to Elias Lazarides.

Additional information

Targeted Molecules Corp. is now a wholly owned subsidiary of Chromos Molecular Systems Inc, 8081 Lougheed Highway, Burnaby, BC, V5A 1W9, Canada.

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Fan, X., Venegas, R., Fey, R. et al. An In Vivo Approach to Structure Activity Relationship Analysis of Peptide Ligands. Pharm Res 24, 868–879 (2007). https://doi.org/10.1007/s11095-007-9238-z

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  • DOI: https://doi.org/10.1007/s11095-007-9238-z

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