Kerstin Moehle

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.

Structure–Activity Studies in a Family of β-Hairpin Protein Epitope Mimetic Inhibitors of the p53–HDM2 Protein–Protein Interaction

Inhibitors of the interaction between the p53 tumor‐suppressor protein and its natural human inhibitor HDM2 are attractive as potential anticancer agents. In earlier work we explored designing β‐hairpin peptidomimetics of the α‐helical epitope on p53 that would bind tightly to the p53‐binding site on HDM2. The β‐hairpin is used as a scaffold to display energetically hot residues in an optimal array for interaction with HDM2. The initial lead β‐hairpin mimetic, with a weak inhibitory activity (IC50=125 μM), was optimized to afford cyclo‐(L‐Pro‐Phe‐Glu‐6ClTrp‐Leu‐Asp‐Trp‐Glu‐Phe‐D‐Pro) (where 6ClTrp=L‐6‐chlorotryptophan), which has an affinity almost 1000 times higher (IC50=140 nM). In this work, insights into the origins of this affinity maturation based on structure–activity studies and an X‐ray crystal structure of the inhibitor/HDM2(residues 17–125) complex at 1.4 Å resolution are described. The crystal structure confirms the β‐hairpin conformation of the bound ligand, and also reveals that a significant component of the affinity increase arises through new aromatic/aromatic stacking interactions between side chains around the hairpin and groups on the surface of HDM2.

A new family of β-hairpin mimetics based on a trypsin inhibitor from sunflower seeds

The ability of proteases to regulate many aspects of cell function and defense accounts for the considerable interest in the design of novel protease inhibitors. There are many naturally occurring proteinaceous serine protease inhibitors, one of which is a 14 amino acid cyclic peptide from sunflower seeds that shows both sequence and conformational similarity with the trypsin‐reactive loop of the Bowman–Birk family of serine protease inhibitors. This inhibitor adopts a β‐hairpin conformation when bound at the active site of bovine β‐trypsin. We illustrate here an approach to inhibitor design in which the β hairpin from the naturally occurring peptide is transplanted onto a hairpin‐inducing template. Two mimetics with the sequences RC*TKSIPPIC*F (where C*C* is a disulfide) and TKSIPPI are studied, each mounted onto a D‐Pro–L‐Pro template. NMR studies revealed a well‐defined β‐hairpin conformation for each mimetic in aqueous solution; this conformation is closely related to the trypsin‐bound conformation of the natural inhibitor and includes a cis‐Ile–Pro peptide bond. Both mimetics inhibit trypsin in the mid nanomolar range. An alanine scan revealed the importance for inhibitory activity of the specificity‐determining Lys residue and of the first but not the second Pro residue in the IPPI motif. Since these hairpin mimetics can be prepared by parallel combinatorial synthesis, this family of molecules may be a useful starting point for the discovery of other biologically or medicinally useful serine protease inhibitors.

Macrocyclic hairpin mimetics of the cationic antimicrobial peptide protegrin I: a new family of broad-spectrum antibiotics.

The problems associated with increasing antibiotic resistance have stimulated great interest in newly discovered families of naturally occurring cationic antimicrobial peptides. These include protegrin, tachyplesin, and RTD‐1, which adopt β‐hairpin‐like structures. We report here an approach to novel peptidomimetics based on these natural products. The mimetics were designed by transplanting the cationic and hydrophobic residues onto a β‐hairpin‐inducing template, either a D‐Pro‐L‐Pro dipeptide or a xanthene derivative. The mimetics have good antimicrobial activity against Gram‐positive and Gram‐negative bacteria (minimal inhibitory concentration≈6–25 μg mL−1). Analogues with improved selectivity for microbial rather than red blood cells (1 % hemolysis at 100 μg mL−1) were identified from a small library prepared by parallel synthesis. Thus, it is possible to separate the antimicrobial and hemolytic activities in this class of mimetics. NMR studies on one mimetic revealed a largely unordered structure in water, but a transition to a regular β‐hairpin backbone conformation in the presence of dodecylphosphocholine micelles. This family of mimetics may provide a starting point for the optimization of antimicrobial agents of potential clinical value in the fight against multiple‐drug‐resistant microorganisms.

Engineered synthetic virus-like particles and their use in vaccine delivery

Engineered nanoparticles have been designed based on the self‐assembling properties of synthetic coiled‐coil lipopeptide building blocks. The presence of an isoleucine zipper within the lipopeptide together with the aggregating effects of an N‐terminal lipid drives formation of 20–25 nm nanoparticles in solution. Biophysical studies support a model in which the lipid is buried in the centre of the nanoparticle, with 20–30 trimeric helical coiled‐coil bundles radiating out into solution. A promiscuous T‐helper epitope and a synthetic B‐cell epitope mimetic derived from the circumsporozoite protein of Plasmodium falciparum have been linked to each lipopeptide chain, with the result that 60–90 copies of each antigen are displayed over the surface of the nanoparticle. These nanoparticles elicit strong humoral immune responses in mice and rabbits, including antibodies able to cross‐react with the parasite, thereby, supporting the potential value of this delivery system in synthetic vaccine design.

β-Hairpin protein epitope mimetic technology in drug discovery

Epitopes involved in protein–protein and protein–nucleic acid interactions provide ideal starting points for rational structure-based inhibitor design. The process of design and optimization of epitope mimetics is now emerging as an innovative new approach in drug discovery. Although often derided as unsuitable for drug development, we provide examples to show how peptidomimetics can provide a new generation of drug candidates to tackle some of the most challenging targets in pharmaceutical research, and address some of the most pressing current threats to human health.

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

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.

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

ABSTRACT Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

Synthetic Virus-Like Particles and Conformationally Constrained Peptidomimetics in Vaccine Design

Conformationally constrained peptidomimetics could be of great value in the design of vaccines targeting protective epitopes on viral and bacterial pathogens. But the poor immunogenicity of small synthetic molecules represents a serious obstacle for their use in vaccine development. Here, we show how a constrained epitope mimetic can be rendered highly immunogenic through multivalent display on the surface of synthetic virus‐like nanoparticles. The target epitope is the V3 loop from the gp120 glycoprotein of HIV‐1 bound to the neutralizing antibody F425‐B4e8. The antibody‐bound V3 loop adopts a β‐hairpin conformation, which is effectively stabilized by transplantation onto a D‐Pro‐L‐Pro template. The resulting mimetic after coupling to synthetic virus‐like particles elicited antibodies in rabbits that recognized recombinant gp120. The elicited antibodies also blocked infection by the neutralization sensitive tier‐1 strain MN of HIV‐1, as well as engineered viruses with the V1V2 loop deleted; this result is consistent with screening of V3 by the V1V2 loop in intact trimeric viral gp120 spikes. The results provide new insights into HIV‐1 vaccine design based on the V3 loop, and illustrate how knowledge from structural biology can be exploited for the design of constrained epitope mimetics, which can be delivered to the immune system by using a highly immunogenic synthetic nanoparticle delivery system.