Rapid Discovery of Potent and Selective Glycosidase-Inhibiting De Novo Peptides

Abstract

Human pancreatic α-amylase (HPA) is responsible for degrading starch to malto-oligosaccharides, thence to glucose, and is therefore an attractive therapeutic target for the treatment of diabetes and obesity. Here we report the discovery of a unique lariat nonapeptide, by means of the RaPID (Random non-standard Peptides Integrated Discovery) system, composed of five amino acids in a head-to-side-chain thioether macrocycle and a further four amino acids in a 3 helical C terminus. This is a potent inhibitor of HPA (K = 7 nM) yet exhibits selectivity for the target over other glycosidases tested. Structural studies show that this nonapeptide forms a compact tertiary structure, and illustrate that a general inhibitory motif involving two phenolic groups is often accessed for tight binding of inhibitors to HPA. Furthermore, the work reported here demonstrates the potential of this methodology for the discovery of de novo peptide inhibitors against other glycosidases.

A Case Study on the Keap1 Interaction with Peptide Sequence Epitopes Selected by the Peptidomic mRNA Display

“…Deep sequencing by a next‐generation sequencer : DNA sequencing of the cDNA library and the DNA library obtained after selection was conducted by using MiSeq (Illumina), as previously described . Briefly, two‐step PCR was performed to prepare samples for MiSeq sequencing.…”

“…DNA sequence analysis of the samples obtained by mRNA display : The data was analyzed with a program coded by Python, as previously described . Briefly, the DNA sequences that completely matched with T7g10M.F48 (forward primer) and the complement of amber3ad13D.R26 (reverse primer) were sought, and the sequences without these primers were removed.…”

Max‐Bergmann award lecture:A RaPID way to discover bioactive nonstandard peptides assisted by the flexizyme and FIT systems

“…We also solved the tertiary structures of some of the above thioether macrocyclic peptides by X‐ray crystallographic analyses (Figure A‐F). It is quite remarkable that we have witnessed a wide range of tertiary structures, eg, a ®‐sheet structure (PlexB1, Figure A; and KDM4A, Fig B), a macrocyclic structure containing an α‐helix motif (CmABCB1, Figure C), a macrocyclic structure containing a water molecule in the ring (SIRT2, Figure D), and a compact macrocyclic structure with a tail linear peptide (human α‐amylase; MATE, Figure E; iPGM, Figure F). The above results indicate how the RaPID system effectively samples a wide range of tertiary structures of macrocyclic peptides from the libraries.…”

“…The RaPID system has been utilized in our laboratory to discover potent thioether macrocyclic peptides against various targets. We found antagonists (inhibitors) against extracellular enzymes and membrane proteins, such as human α‐amylase, drug transporters (MATE and CmABCB1), and a receptor tyrosine kinase (PlexB1). Artificial agonists based on ligands against a receptor tyrosine kinase (cMET) were successfully devised .…”

“…The above thioether‐macrocyclic peptides discovered by the RaPID system displayed remarkable potencies, for which K D values and IC 50 values were in the range of low to sub‐nM in most cases. Their mode of actions turned out to be quite diverse, eg, competing with the substrates in the binding site (human α‐amylase, AKT2, SIRT2, and KDM4A), clogging the transporter site (MATE), targeting allosteric or allosteric‐like site (CmABCB1, PlexB1, and VP24), binding a unique remote‐inhibitory site (iPGM), and dimerizing the intercellular kinase domain via dimerization of the extracellular domain (cMET).…”