A paradigm for drug discovery using a conformation from the crystal structure of a presentation scaffold

Zhao, Baoguang; Helms, Larry R.; DesJarlais, Renée L.; Abdel-Meguid, Sherin S.; Wetzel, Ronald

doi:10.1038/nsb1295-1131

Cited by 17 publications

(14 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, bacterial cell surface display or mRNA display might also have been successful as these technologies have previously been used to present folded EETI-II variants for high-throughput screening. 29,33,43 …”

Section: Discussionmentioning

confidence: 99%

“…32,43,49,50 However, in some cases these studies did not generate proteins with sufficient integrin binding affinities or biological activities for therapeutic applications, 32,50 presumably because simple loop grafting did not result in optimal presentation of residues within the RGD-containing loop to integrin receptors. When high affinity was achieved, the ligand was shown to bind promiscuously to α v β 3 and α iib β 3 integrins.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

et al. 2009

View full text Add to dashboard Cite

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI-II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin-binding agents. We generated yeast-displayed libraries of EETI-II by substituting its 6-amino acid trypsin binding loop with 11-amino acid loops containing the Arg-Gly-Asp (RGD) integrin binding motif and randomized flanking residues. These libraries were screened in a high-throughput manner by fluorescence-activated cell sorting to identify mutants that bound to αvβ3 integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half-maximal inhibitory concentration (IC50) values of 10-30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both αvβ3 and αvβ5 integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to αvβ3, αvβ5, and α5β1 integrins. This finding has important clinical implications since all three of these receptors can be co-expressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI-II as a scaffold for protein engineering, and highlight the development of unique integrin-binding peptides with potential for translational applications in cancer.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

et al. 2009

View full text Add to dashboard Cite

show abstract

“…The pharmacophoric activity can be expected from the RGD ligand towards the platelet receptor α IIb β 3 only when the distances R C α ‐D C α and R C β ‐D C β are ≥ 0.7 nm and 1.0 nm, respectively . In our study, the peptides RGD and PRGD showed the sampling of two conformations, that is extended and compact, irrespective of the temperature variation.…”

Section: Discussionmentioning

confidence: 48%

Molecular Dynamics Simulations of Certain RGD‐Based Peptides from Kistrin Provide Insight into the Higher Activity of REI‐RGD34 Protein at Higher Temperature

Upadhyay

2014

Chem Biol Drug Des

View full text Add to dashboard Cite

To determine the bioactive conformation required to bind with receptor aIIbb3, the peptide sequence RIPRGDMP from Kistrin was inserted into CDR 1 loop region of REI protein, resulting in REI-RGD34. The activity of REI-RGD34 was observed to increase at higher temperature towards the receptor aIIbb3. It could be justified in either way: the modified complex forces the restricted peptide to adapt bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. Here, we model the conformational preference of RGD sequence in RIPRGDMP at 25 and 42 °C using multiple MD simulations. Further, we model the peptide sequence RGD, PRGD and PRGDMP from kistrin to observe the effect of flanking residues on conformational sampling of RGD. The presence of flanking residues around RGD peptide greatly influenced the conformational sampling. A transition from bend to turn conformation was observed for RGD sequence at 42 °C. The turn conformation shows pharmacophoric parameters required to recognize the receptor aIIbb3. Thus, the temperaturedependent activity of RIPRGDMP when inserted into the loop region of REI can be explained by the presence of the turn conformation. This study will help in designing potential antagonist for the receptor aIIbb3.

show abstract

“…Only the monomeric form of this Ig fragment was shown to be active and to recognize an antigen expressed by human cells of the melanocytic lineage. In an approach to controlling and elucidating bioactive conformations of peptides, the dimeric Ig V L domain REI was successfully utilized as a presentation scaffold (Zhao et al, 1995). In an approach to controlling and elucidating bioactive conformations of peptides, the dimeric Ig V L domain REI was successfully utilized as a presentation scaffold (Zhao et al, 1995).…”

Section: Single Ig and Ig-like Domainsmentioning

confidence: 99%

Engineered protein scaffolds for molecular recognition

Skerra

2000

J. Mol. Recognit.

209

View full text Add to dashboard Cite

The use of so‐called protein scaffolds has recently attracted considerable attention in biochemistry in the context of generating novel types of ligand receptors for various applications in research and medicine. This development started with the notion that immunoglobulins owe their function to the composition of a conserved framework region and a spatially well‐defined antigen‐binding site made of peptide segments that are hypervariable both in sequence and in conformation. After the application of antibody engineering methods along with library techniques had resulted in first successes in the selection of functional antibody fragments, several laboratories began to exploit other types of protein architectures for the construction of practically useful binding proteins. Properties like small size of the receptor protein, stability and ease of production were the focus of this work. Hence, among others, single domains of antibodies or of the immunoglobulin superfamily, protease inhibitors, helix‐bundle proteins, disulphide‐knotted peptides and lipocalins were investigated. Recently, the scaffold concept has even been adopted for the construction of enzymes. However, it appears that not all kinds of polypeptide fold which may appear attractive for the engineering of loop regions at a first glance will indeed permit the construction of independent ligand‐binding sites with high affinities and specificities. This review will therefore concentrate on the critical description of the structural properties of experimentally tested protein scaffolds and of the novel functions that have been achieved on their basis, rather than on the methodology of how to best select a particular mutant with a certain activity. An overview will be provided about the current approaches, and some emerging trends will be identified. Copyright © 2000 John Wiley & Sons, Ltd.

show abstract

A paradigm for drug discovery using a conformation from the crystal structure of a presentation scaffold

Cited by 17 publications

References 45 publications

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

Molecular Dynamics Simulations of Certain RGD‐Based Peptides from Kistrin Provide Insight into the Higher Activity of REI‐RGD34 Protein at Higher Temperature

Engineered protein scaffolds for molecular recognition

Contact Info

Product

Resources

About