Wen-Jian Qian

Identification of high affinity polo-like kinase 1 (Plk1) polo-box domain binding peptides using oxime-based diversification.

In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.

Peptoid-Peptide hybrid ligands targeting the polo box domain of polo-like kinase 1.

We replaced the amino terminal Pro residue of the Plk1 polo‐box‐domain‐binding pentapeptide (PLHSpT) with a library of N‐alkyl‐Gly “peptoids”, and identified long‐chain tethered phenyl moieties giving greater than two‐orders‐of‐magnitude affinity enhancement. Further simplification by replacing the peptoid residue with appropriate amides gave low‐nanomolar affinity N‐acylated tetrapeptides. Binding of the N‐terminal long‐chain phenyl extension was demonstrated by X‐ray co‐crystal data.

Investigation of Unanticipated Alkylation at the N(π) Position of a Histidyl Residue Under Mitsunobu Conditions and Synthesis of Orthogonally Protected Histidine Analogues

We had previously reported that Mitsunobu-based introduction of alkyl substituents onto the imidazole N(π)-position of a key histidine residue in phosphothreonine-containing peptides can impart high binding affinity against the polo-box domain of polo-like kinase 1. Our current paper investigates the mechanism leading to this N(π)-alkylation and provides synthetic methodologies that permit the facile synthesis of histidine N(π)-modified peptides. These agents represent new and potentially important tools for biological studies.

Mono‐anionic phosphopeptides produced by unexpected histidine alkylation exhibit high plk1 polo‐box domain‐binding affinities and enhanced antiproliferative effects in hela cells

Binding of polo‐like kinase 1 (Plk1) polo‐box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)‐containing sequences is critical for the proper function of Plk1. Although high‐affinity synthetic pThr‐containing peptides provide starting points for developing PBD‐directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di‐anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on‐resin N(τ)‐alkylation of histidine residues already bearing a N(π)‐ alkyl group. This resulted in cationic imidazolium‐containing pThr peptides, several of which exhibit single‐digit nanomolar PBD‐binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio‐reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD‐binding peptides and peptide mimetics.

Effects on polo-like kinase 1 polo-box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position.

Protein-protein interactions (PPIs) mediated by the polo-box domain (PBD) of polo-like kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr β-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.

Non-proteinogenic amino acids in the pThr-2 position of a pentamer peptide that confer high binding affinity for the polo box domain (PBD) of polo-like kinase 1 (Plk1).

We report herein that incorporating long-chain alkylphenyl-containing non-proteinogenic amino acids in place of His at the pT-2 position of the parent polo-like kinase 1 (Plk1) polo box domain (PBD)-binding pentapeptide, PLHSpT (1a) increases affinity. For certain analogs, approximately two orders-of-magnitude improvement in affinity was observed. Although, none of the new analogs was as potent as our previously described peptide 1b, in which the pT-2 histidine imidazole ring is alkylated at its π nitrogen (N3), our current finding that the isomeric His(N1)-analog (1c) binds with approximately 50-fold less affinity than 1b, indicates the positional importance of attachment to the His imidazole ring. Our demonstration that a range of modified residues at the pT-2 position can enhance binding affinity, should facilitate the development of minimally-sized Plk1 PBD-binding antagonists.

Application of ring-closing metathesis to Grb2 SH3 domain-binding peptides.

Molecular processes depending on protein–protein interactions can use consensus recognition sequences that possess defined secondary structures. Left‐handed polyproline II (PPII) helices are a class of secondary structure commonly involved with cellular signal transduction. However, unlike α‐helices, for which a substantial body of work exists regarding applications of ring‐closing metathesis (RCM), there are few reports on the stabilization of PPII helices by RCM methodologies. The current study examined the effects of RCM macrocyclization on left‐handed PPII helices involved with the SH3 domain‐mediated binding of Sos1–Grb2. Starting with the Sos1‐derived peptide “Ac‐V1‐P2‐P3‐P4‐V5‐P6‐P7‐R8‐R9‐R10‐amide,” RCM macrocyclizations were conducted using alkenyl chains of varying lengths originating from the pyrrolidine rings of the Pro4 and Pro7 residues. The resulting macrocyclic peptides showed increased helicity as indicated by circular dichroism and enhanced abilities to block Grb2–Sos1 interactions in cell lysate pull‐down assays. The synthetic approach may be useful in RCM macrocyclizations, where maintenance of proline integrity at both ring junctures is desired.

Peptide-Based Inhibitors of Plk1 Polo-box Domain Containing Mono-anionic Phosphothreonine Esters and Their Pivaloyloxymethyl Prodrugs

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides may be used to disrupt PBD function, the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising in part from the di-anionic nature of the phosphoryl group. We report five-mer peptides containing mono-anionic pThr phosphoryl esters that exhibit single-digit nanomolar PBD binding affinities in extracellular assays and improved antimitotic efficacies in whole cell assays. The cellular efficacies of these peptides have been further enhanced by the application of bio-reversible pivaloyloxymethyl (POM) phosphoryl protection to a pThr-containing polypeptide. Our findings may redefine structural parameters for the development of PBD-binding peptides and peptide mimetics.

Neighbor-directed histidine N (τ)-alkylation: A route to imidazolium-containing phosphopeptide macrocycles.

Our recently discovered, selective, on‐resin route to N(τ)‐alkylated imidazolium‐containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring‐forming junctions. Interestingly, these cationic moieties subsequently serve to charge‐mask the phosphoamino acid group that directed their formation. Neighbor‐directed histidine N(τ)‐alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.

Design and synthesis of a reagent for solid-phase incorporation of the phosphothreonine mimetic (2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid (Pmab) into peptides in a bio-reversible phosphonyl-bis-pivaloyloxymethyl (POM) prodrug form

Abstract Reported herein are the synthesis and solid-phase peptide incorporation of N-Fmoc-(2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid bis-pivaloyloxymethyl phosphoryl ester [Fmoc-Pmab(POM)2-OH, 2] as a phosphatase-stable phosphothreonine (pThr) mimetic bearing orthogonal protection suitable for the synthesis of Pmab-containing peptides having bio-reversible protection of the phosphonic acid moiety. This represents the first report of a bio-reversibly protected pThr mimetic in a form suitable for facile solid-phase peptide synthesis.