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Published on: May 4, 2018
Genetic selection for peptide inhibitors of angiogenin
Bryan D Smith1, Ronald T Raines
1Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA.
Abstract:
The improper regulation of angiogenesis is implicit in a variety of diseases, including cancer. Angiogenin is unique among angiogenic factors in having ribonucleolytic activity. Inhibitors of this activity could serve as chemotherapeutics. The ribonucleolytic activity of angiogenin is toxic to the Origami strain of Escherichia coli. Herein, this cytotoxicity was used to identify inhibitors from a random nonapeptide library tethered to the C-terminus of human angiogenin. The selected sequences fell into three classes: (i) extremely hydrophobic, (ii) putative protease (ClpXP) substrates and (iii) slightly anionic. Two peptides corresponding to sequences in the last class were synthesized chemically and found to inhibit the ribonucleolytic activity of human angiogenin in vitro with micromolar values of Ki. Both peptides also inhibit bovine pancreatic ribonuclease, a homolog of angiogenin, though one exhibits selectivity for angiogenin. The affinity and selectivity of these peptides are comparable with the best known inhibitors of angiogenin. Moreover, the strategy used to identify them is general and could be applied to other cytotoxins.
Insights
Researchers identified novel peptide inhibitors of angiogenin by using its toxic ribonucleolytic activity against E. coli. These peptides show promising affinity and selectivity, offering potential as new cancer chemotherapeutics.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Improper regulation of angiogenesis is implicated in diseases like cancer.
- Angiogenin, a unique angiogenic factor, possesses ribonucleolytic activity.
- Inhibiting angiogenin's ribonucleolytic activity presents a potential chemotherapeutic strategy.
Purpose of the Study:
- To identify novel inhibitors of human angiogenin's ribonucleolytic activity.
- To utilize the cytotoxicity of angiogenin against Escherichia coli for inhibitor screening.
- To evaluate the affinity and selectivity of identified peptide inhibitors.
Main Methods:
- Screening a random nonapeptide library tethered to human angiogenin.
- Employing the ribonucleolytic activity-induced cytotoxicity in Origami E. coli as a selection pressure.
- Synthesizing and characterizing lead peptide inhibitors in vitro.
- Assessing inhibition constants (Ki) and selectivity against angiogenin and bovine pancreatic ribonuclease.
Main Results:
- Identified three classes of peptide inhibitors: hydrophobic, protease substrates, and anionic.
- Synthesized two anionic peptides that inhibit human angiogenin ribonucleolytic activity with micromolar Ki values.
- Demonstrated that both peptides inhibit bovine pancreatic ribonuclease, with one showing selectivity for angiogenin.
- Achieved affinity and selectivity comparable to existing top angiogenin inhibitors.
Conclusions:
- Developed a generalizable strategy using cytotoxicity to discover enzyme inhibitors.
- Identified potent and selective peptide inhibitors of angiogenin with therapeutic potential.
- The identified peptides and screening strategy offer a promising avenue for cancer drug development.
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