John A. Robinson

The design, structures and therapeutic potential of protein epitope mimetics.

Using a biologically relevant peptide or protein structure as a starting point for lead identification represents one of the most powerful approaches in modern drug discovery. Here, we focus on the protein epitope mimetic (PEM) approach, where folded 3D structures of peptides and proteins are taken as starting points for the design of synthetic molecules that mimic key epitopes involved in protein-protein and protein-nucleic acid interactions. By transferring the epitope from a recombinant to a synthetic scaffold that can be produced by parallel combinatorial methods, it is possible to optimize target affinity and specificity as well as other drug-like ADMET properties. The PEM technology is a powerful tool for target validation, and for the development of novel PEM-based drugs.

Crystal structure of an NPNA-repeat motif from the circumsporozoite protein of the malaria parasite Plasmodium falciparum

A crystal structure is reported of the peptide Ac-Ala-Asn-Pro-Asn-Ala-NH2, representing the immunodominant region of the major surface protein on the malaria parasite; the NPNA motif adopts a type-I beta-turn, which is stabilized by hydrogen bonding between the CO of Asn2 and the NH of Ala5 as well as between the O(delta) of Asn2 and the NH of Asn4.

Antibodies elicited by a virosomally formulated Plasmodium falciparum serine repeat antigen-5 derived peptide detect the processed 47 kDa fragment both in sporozoites and merozoites.

Serine repeat antigen-5 (SERA5) is a candidate antigen for inclusion into a malaria subunit vaccine. During merozoite release and reinvasion the 120 kDa SERA5 precursor protein (P120) is processed, and a complex consisting of an N-terminal 47 kDa (P47) and a C-terminal 18kDa (P18) processing product associates with the surface of merozoites. This complex is thought to be involved in merozoite invasion of and/or egress from host erythrocytes. Here we describe the synthesis and immunogenic properties of virosomally formulated synthetic phosphatidylethanolamine (PE)-peptide conjugates, incorporating amino acid sequence stretches from the N-terminus of Plasmodium falciparum SERA5. Choosing an appropriate sequence was crucial for the development of a peptide that elicited high titers of parasite cross-reactive antibodies in mice. Monoclonal antibodies (mAbs) raised against the optimized peptide FB-23 incorporating amino acids 57-94 of SERA5 bound to both P120 and to P47. Western blotting analysis proved for the first time the presence of SERA5 P47 in sporozoites. In immunofluorescence assays, the mAbs stained SERA5 in all its predicted localizations. The virosomal formulation of peptide FB-23 is suitable for use in humans and represents a candidate component for a multi-valent malaria subunit vaccine targeting both sporozoites and blood stage parasites.

Synthesis, solution structure and immune recognition of an epidermal growth factor-like domain from Plasmodium falciparum merozoite surface protein-1

The Plasmodium falciparum merozoite surface protein‐1 19 kDa fragment (MSP‐119) comprises two closely packed EGF‐like domains (EGF=epidermal growth factor), each stabilized by three disulfide bonds. The native conformation of this protein is important for eliciting P. falciparum growth inhibitory antibodies. Here we show that the N‐terminal EGF domain alone can be chemically synthesized and efficiently refolded to a native‐like state, as shown by its solution structure as determined by NMR spectroscopy. In order to study its immunogenicity, the domain was coupled through its N terminus to a phospholipid and incorporated into reconstituted influenza virus‐like particles (virosomes). When used to immunize mice, the peptide‐loaded virosomes elicited potent humoral immune responses that were shown by Western blots and immunofluorescence assays to cross‐react with native MSP‐1 on the surfaces of P. falciparum blood stage parasites. This opens the way for a medicinal chemistry‐oriented approach to the study and optimization of the antigenicity of the protein as a potential malaria vaccine candidate, whilst exploiting the immunopotentiating properties of influenza virosomes as a delivery vehicle.

Molecular Characterization of the NCoA-1–STAT 6 Interaction

Many protein–protein interactions involved in cell signalling, cell adhesion and regulation of transcription are mediated by short α‐helical recognition motifs with the sequence Leu‐Xaa‐Xaa‐Leu‐Leu (LXXLL, where Xaa is any amino acid). Originally observed in cofactors that interact with hormone‐activated nuclear receptors, LXXLL motifs are now known to occur in many transcription factors, including the STAT family, which transmit signals from activated cytokine receptors at the cell surface to target genes in the nucleus. STATu20096 becomes activated in response to IL‐4 and IL‐13, which regulate immune and anti‐inflammatory responses. Structural studies have revealed how an LXXLL motif located in 2.5 turns of an α‐helical peptide derived from STATu20096 provide contacts through the leucine side chains to the coactivator of transcription, NCoA‐1. However, since many protein–protein interactions are mediated by LXXLL motifs, it is important to understand how specificity is achieved in this and other signalling pathways. Here, we show that energetically important contacts between STATu20096 and NCoA‐1 are made in residues that flank the LXXLL motif, including the underlined residues in the sequence LLPPTEQDLTKLL. We also demonstrate how the affinity for NCoA‐1 of peptides derived from this region of STATu20096 can be significantly improved by optimising knobs‐into‐holes contacts on the surface of the protein. The results provide important new insights into the origins of binding specificity, and might be of practical value in the design of novel small‐molecule inhibitors of this important protein–protein interaction.

Inhibition and stabilization of protein-protein interactions. Preface.

Protein–protein interactions (PPIs) form the molecular basis for almost every phenomenon of life including signal perception and transduction, movement, gene expression, and cell cycle control, and hence play important roles in human health and disease. The estimated number of PPIs in the human body, in the range of 300,000 1 to 650,000, 2 by far exceeds the number of protein targets that can be addressed by classical small-molecule approaches, like active-site inhibition or modulation of cell surface receptors or ion channels. Consequently, pharmacological interventions of PPIs has increasingly attracted attention from chemical biology and bioorganic- and medicinal chemistry, and a number of excellent reviews on the topic have been published in recent years. 3–9 This Bioorganic and Medicinal Chemistry special issue on inhibition and stabilization of protein–protein interactions contains a collection of original papers and reviews covering important technological aspects of prominent PPI target classes. In a contribution by Doemling and co-workers, inhibition of the cancer-relevant Mdm2/p53 interaction by benzimidazolones is reported. Burke and colleagues describe the design, synthesis and activity of phosphomimetic-containing peptides targeting the polo-box domain (PDB) of Plk1. Ohkanda and colleagues demonstrate how peptidomimetic modification enhances membrane permeability and cellular activity of a farnesyl transferase