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A surrogate-based approach for post-genomic partner identification.

Pillutla RC, Hsiao K, Brissette R, Eder PS, Giordano T, Fletcher PW, Lennick M, Blume AJ, Goldstein NI - BMC Biotechnol. (2001)

Bottom Line: In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences.Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches.Modified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: DGI BioTechnologies, Inc, Edison NJ, USA. pillutla@dgibt.com

ABSTRACT

Background: Modern drug discovery is concerned with identification and validation of novel protein targets from among the 30,000 genes or more postulated to be present in the human genome. While protein-protein interactions may be central to many disease indications, it has been difficult to identify new chemical entities capable of regulating these interactions as either agonists or antagonists.

Results: In this paper, we show that peptide complements (or surrogates) derived from highly diverse random phage display libraries can be used for the identification of the expected natural biological partners for protein and non-protein targets. Our examples include surrogates isolated against both an extracellular secreted protein (TNFbeta) and intracellular disease related mRNAs. In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences. Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches.

Conclusions: Modified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery.

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surrogate Maturation. One clone for each of the three targets – APP, HCV, and IGF was identified for generation of secondary libraries. Residues that were selected for after four rounds of panning are indicated in bold and underlined.
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Figure 3: surrogate Maturation. One clone for each of the three targets – APP, HCV, and IGF was identified for generation of secondary libraries. Residues that were selected for after four rounds of panning are indicated in bold and underlined.

Mentions: In one series of experiments, biotinylated oligonucleotides comprising the UTRs (untranslated regions) of four mRNAs were synthesized (Table 2). The oligonucleotides were heat denatured and allowed to anneal at room temperature to allow the appropriate re-folding. All of the mRNAs were subjected to 4 rounds of panning using both 40 mer and 20 mer random libraries under similar but not equivalent conditions. Individual phage clones from rounds three and four were amplified, tested for binding to the specific and a non-specific mRNA and sequenced. Table 3 shows the overall results that were obtained from each of the pannings. On average, about 8% of the surrogates were found to be specific for each target when compared to a control RNA (RRE). For each RNA target, the predicted amino acid sequences of the peptide binders were analyzed in terms of both overall amino acid content and the occurrence of known RNA-binding motifs and consensus domains. Two motifs were observed for the APP and HCV RNAs (Figure 1 and see below). RNA binding proteins are known to have an overall abundance of certain amino acid residues [8,9]. Table 4 shows a comparison of the specific amino acid composition of peptide binders with regard to their average frequency of occurrence seen within the original unpanned library. All of the peptide binders showed enrichment of arginine residues, as would be expected for RNA binding proteins. Also, tryptophan, serine, and glycine residues were enriched. In addition, several peptide binders showed the presence of the RGG box (Figure 2A) and one sequence was found that contained the KH motif (Figure 2B), both of which are known RNA-binding motifs [8,9]. The isolation of surrogates containing generic RNA binding motifs is not unexpected and probably results from enhanced binding and concomitant enrichment of these peptides during the panning process. In addition, an additional consensus motif was identified among peptides isolated by panning on RRE RNA [10]. This motif [K/R] LRRR, aligns with a region on the expected natural partner, the Rev peptide (Figure 3).


A surrogate-based approach for post-genomic partner identification.

Pillutla RC, Hsiao K, Brissette R, Eder PS, Giordano T, Fletcher PW, Lennick M, Blume AJ, Goldstein NI - BMC Biotechnol. (2001)

surrogate Maturation. One clone for each of the three targets – APP, HCV, and IGF was identified for generation of secondary libraries. Residues that were selected for after four rounds of panning are indicated in bold and underlined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC57814&req=5

Figure 3: surrogate Maturation. One clone for each of the three targets – APP, HCV, and IGF was identified for generation of secondary libraries. Residues that were selected for after four rounds of panning are indicated in bold and underlined.
Mentions: In one series of experiments, biotinylated oligonucleotides comprising the UTRs (untranslated regions) of four mRNAs were synthesized (Table 2). The oligonucleotides were heat denatured and allowed to anneal at room temperature to allow the appropriate re-folding. All of the mRNAs were subjected to 4 rounds of panning using both 40 mer and 20 mer random libraries under similar but not equivalent conditions. Individual phage clones from rounds three and four were amplified, tested for binding to the specific and a non-specific mRNA and sequenced. Table 3 shows the overall results that were obtained from each of the pannings. On average, about 8% of the surrogates were found to be specific for each target when compared to a control RNA (RRE). For each RNA target, the predicted amino acid sequences of the peptide binders were analyzed in terms of both overall amino acid content and the occurrence of known RNA-binding motifs and consensus domains. Two motifs were observed for the APP and HCV RNAs (Figure 1 and see below). RNA binding proteins are known to have an overall abundance of certain amino acid residues [8,9]. Table 4 shows a comparison of the specific amino acid composition of peptide binders with regard to their average frequency of occurrence seen within the original unpanned library. All of the peptide binders showed enrichment of arginine residues, as would be expected for RNA binding proteins. Also, tryptophan, serine, and glycine residues were enriched. In addition, several peptide binders showed the presence of the RGG box (Figure 2A) and one sequence was found that contained the KH motif (Figure 2B), both of which are known RNA-binding motifs [8,9]. The isolation of surrogates containing generic RNA binding motifs is not unexpected and probably results from enhanced binding and concomitant enrichment of these peptides during the panning process. In addition, an additional consensus motif was identified among peptides isolated by panning on RRE RNA [10]. This motif [K/R] LRRR, aligns with a region on the expected natural partner, the Rev peptide (Figure 3).

Bottom Line: In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences.Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches.Modified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: DGI BioTechnologies, Inc, Edison NJ, USA. pillutla@dgibt.com

ABSTRACT

Background: Modern drug discovery is concerned with identification and validation of novel protein targets from among the 30,000 genes or more postulated to be present in the human genome. While protein-protein interactions may be central to many disease indications, it has been difficult to identify new chemical entities capable of regulating these interactions as either agonists or antagonists.

Results: In this paper, we show that peptide complements (or surrogates) derived from highly diverse random phage display libraries can be used for the identification of the expected natural biological partners for protein and non-protein targets. Our examples include surrogates isolated against both an extracellular secreted protein (TNFbeta) and intracellular disease related mRNAs. In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences. Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches.

Conclusions: Modified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery.

Show MeSH