High‐Throughput Screening (HTS) by NMR Guided Identification of Novel Agents Targeting the Protein Docking Domain of YopH

Abstract

Recently we described a novel approach, named HTS by NMR that allows the identification, from large combinatorial peptide libraries, of potent and selective peptide mimetics against a given target. Here we deployed the HTS by NMR approach for the design of novel peptoid sequences targeting the amino terminal domain of the Yersinia outer protein H (YopH-NT). We aimed at disrupting the protein-protein interactions between YopH-NT and its cellular substrates, with the goal of inhibiting indirectly YopH enzymatic function. These studies resulted in a novel agent of sequence Ac-F-pY-cPG-D-P-NH2 (pY = phosphotyrosine; cPG = cyclopentyl glycine) with a Kd value against YopH-NT of 310 nM. We demonstrated that such pharmacological inhibitor of YopH-NT resulted in the inhibition of the dephosphorylation of a cellular substrate by full length YopH. Hence, potentially this agent represents a valuable stepping stone for the development of novel therapeutics against Yersinia infections. The data reported further demonstrate the utility of the HTS by NMR approach in deriving novel peptide-mimetics targeting protein-protein interactions.

The Cell Surface Receptor CD44: NMR‐Based Characterization of Putative Ligands

“…As additional attempt to derive novel CD44 binding agents, we also carried out a screening campaign using the HTS by NMR approach [29, 31] . Hence, we prepared and tested 76 mixtures of a tetra-peptide combinatorial library composed by 19 natural amino acids (the 20 natural amino acids except cysteine) as building blocks [31a] .…”

“…Hence, we prepared and tested 76 mixtures of a tetra-peptide combinatorial library composed by 19 natural amino acids (the 20 natural amino acids except cysteine) as building blocks [31a] . In addition, we also prepared and tested a tri-peptide combinatorial library (see Methods).…”

“…In addition, we also prepared and tested a tri-peptide combinatorial library (see Methods). [31c] Each mixture was tested at 2 mM total concentration using 2D [ 1 H, 15 N]-sofast-HMQC spectra with 15 N-labaled hCD44(21–178). However, none of the mixtures tested produced a significant chemical shift perturbation in the 2D [ 1 H, 15 N]-sofast-HMQC spectra of the target under these experimental conditions.…”

“…[24] From the electron density it was possible only to identify a valine residue as the first amino acid of this unknown peptide located into the small binding pocket. Based on these observations we decided to perform a further screening campaign using the HTS by NMR approach [31a] . Aimed at identifying a possible CD44 binding peptide, we decided to screen a four-position combinatorial library composed by 19 natural amino acids (the 20 natural amino acids except cysteine) as building blocks.…”

Immunomodulatory Yersinia outer proteins (Yops)–useful tools for bacteria and humans alike

“…189 In times of recurring endemic outbreaks and an increasing awareness of potential bioterroristic attacks, YopH lately became a highly studied target for the treatment of especially Y. pestis infections through small molecule inhibitors of YopH. [190][191][192][193] Finally, recent data showed that -at least in pathogenic E. coli -bacterial proteins involved in the regulation of virulence, including type III secretion, are also activated by tyrosine phosphorylation -a mechanism that was long believed to be completely absent in bacteria. 194 Whether YopH might thus also play a regulatory role within the bacterial cell is an exciting topic for future research.…”