Alessandro Angelini

Peptide Ligands Stabilized by Small Molecules

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

Structural basis for chemokine recognition and activation of a viral G protein–coupled receptor

Molecular “go” signals reveal their secrets Chemokines are proteins that direct how cells move within the body. For instance, chemokines help immune cells locate invading pathogens and ensure that cells position themselves correctly within a developing organ. Cells detect chemokines through G protein–coupled receptors on their surface; however, the molecular details of how these proteins interact remain unclear (see the Perspective by Standfuss). Qin et al. solved the crystal structure of the chemokine receptor CXCR4 bound to the viral chemokine vMIP-II. Burg et al. solved the crystal structure of a viral chemokine receptor bound to the chemokine domain of CX3CL1. Given the role of chemokines in a number of diseases, these results may help in future drug design. Science, this issue p. 1117, p. 1113; see also p. 1071 The crystal structure of a viral chemokine receptor bound to the chemokine CX3CL1 provides insights into chemokine recognition. [Also see Perspective by Standfuss] Chemokines are small proteins that function as immune modulators through activation of chemokine G protein–coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state–like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

Bicyclic peptide inhibitor reveals large contact interface with a protease target

From a large combinatorial library of chemically constrained bicyclic peptides we isolated a selective and potent (K(i) = 53 nM) inhibitor of human urokinase-type plasminogen activator (uPA) and crystallized the complex. This revealed an extended structure of the peptide with both peptide loops engaging the target to form a large interaction surface of 701 Å(2) with multiple hydrogen bonds and complementary charge interactions, explaining the high affinity and specificity of the inhibitor. The interface resembles that between two proteins and suggests that these constrained peptides have the potential to act as small protein mimics.

Structurally Diverse Cyclisation Linkers Impose Different Backbone Conformations in Bicyclic Peptides

Combinatorial libraries of structurally diverse peptide macrocycles offer a rich source for the development of high‐affinity ligands to targets of interest. In this work we have developed linkers for the generation of genetically encoded bicyclic peptides and tested whether the peptides cyclised by them have significant variations in their backbone conformations. Two new cyclisation reagents, each containing three thiol‐reactive groups, efficiently and selectively cyclised linear peptides containing three cysteine moieties. When the mesitylene linker of the bicyclic peptide PK15, a potent inhibitor of plasma kallikrein (Ki=2 nM), was replaced by the new linkers, its inhibitory activity dropped by a factor of more than 1000, suggesting that the linkers impose different conformations on the peptide. Indeed, structural analysis by solution‐state NMR revealed different NOE constraints in the three bicyclic peptides, indicating that these relatively small linkers at the centres of bicyclic peptide structures significantly influence the conformations of the peptides. These results demonstrate the prominent structural role of linkers in peptide macrocycles and suggest that application of different cyclisation linkers in a combinatorial fashion could be an attractive means to generate topologically diverse macrocycle libraries.

Boosting the Sensitivity of Ligand−Protein Screening by NMR of Long-Lived States

A new NMR method for the study of ligand-protein interactions exploits the unusual lifetimes of long-lived states (LLSs). The new method provides better contrast between bound and free ligands and requires a protein-ligand ratio ca. 25 times lower than for established T(1ρ) methods, thus saving on costly proteins. The new LLS method was applied to the screening of inhibitors of urokinase-type plasminogen activator (uPA), which is a prototypical target of cancer research. With only 10 μM protein, a dissociation constant (K(D)) of 180 ± 20 nM was determined for the strong ligand (inhibitor) UK-18, which can be compared with K(D) = 157 ± 39 nM determined by the established surface plasmon resonance method.

Expression of Helicobacter pylori CagA domains by library-based construct screening

Highly pathogenic strains of Helicobacter pylori use a type IV secretion system to inject the CagA protein into human gastric cells. There, CagA associates with the inner side of the membrane and is tyrosine‐phosphorylated at EPIYA motifs by host kinases. The phosphorylation triggers a series of interactions between CagA and human proteins that result in a dramatic change of cellular morphology. Structural and functional analyses of the protein have proved difficult, due to the proteolytically sensitive nature of the recombinant protein. To circumvent these difficulties, we applied ESPRIT, a library‐based construct screening method, to generate a comprehensive set of 5′‐randomly deleted gene fragments. Screening of 18 432 constructs for soluble expression resulted in a panel of 40 clones, which were further investigated by large‐scale purification. Two constructs of approximately 25 and 33 kDa were particularly soluble and were purified to near homogeneity. CagA fragments larger than 40 kDa were prone to heavy proteolysis at the C‐terminus, with a favoured cleavage site near the first EPIYA motif. Thus, these well‐expressed recombinant constructs isolated are likely to be similar to those observed following natural proteolysis in human cells, and open the way for structural and functional studies requiring large amounts of purified material.

The Helicobacter pylori CagD (HP0545, Cag24) protein is essential for CagA translocation and maximal induction of interleukin-8 secretion.

Pathogenic strains of Helicobacter pylori use a type IV secretion system (T4SS) to deliver the toxin CagA into human host cells. The T4SS, along with the toxin itself, is coded into a genomic insert, which is termed the cag pathogenicity island. The cag pathogenicity island contains about 30 open-reading frames, for most of which the exact function is not well characterized or totally unknown. We have determined the crystal structure of one of the proteins coded by the cag genes, CagD, in two crystal forms. We show that the protein is a covalent dimer in which each monomer folds as a single domain that is composed of five beta-strands and three alpha-helices. Our data show that in addition to a cytosolic pool, CagD partially associates with the inner membrane, where it may be exposed to the periplasmic space. Furthermore, CagA tyrosine phosphorylation and interleukin-8 assays identified CagD as a crucial component of the T4SS that is involved in CagA translocation into host epithelial cells; however, it does not seem absolutely necessary for pilus assembly. We have also identified significant amounts of CagD in culture supernatants, which are not a result of general bacterial lysis. Since this localization was independent of the various tested cag mutants, our findings may indicate that CagD is released into the supernatant during host cell infection and then binds to the host cell surface or is incorporated in the pilus structure. Overall, our results suggest that CagD may serve as a unique multifunctional component of the T4SS that may be involved in CagA secretion at the inner membrane and may localize outside the bacteria to promote additional effects on the host cell.

Bicyclization and Tethering to Albumin Yields Long-Acting Peptide Antagonists

Proteolytically stable peptide architectures are required for the development of long-acting peptide therapeutics. In this work, we found that a phage-selected bicyclic peptide antagonist exhibits an unusually high stability in vivo and subsequently deciphered the underlying mechanisms of peptide stabilization. We found that the bicyclic peptide was significantly more stable than its constituent rings synthesized as two individual macrocycles. The two rings protect each other from proteolysis when linked together, conceivably by constraining the conformation and/or by mutually shielding regions prone to proteolysis. A second stabilization mechanism was found when the bicyclic peptide was linked to an albumin-binding peptide to prevent its rapid renal clearance. The bicyclic peptide conjugate not only circulated 50-fold longer (t(1/2) = 24 h) but also became entirely resistant to proteolysis when tethered to the long-lived serum protein. The bicyclic peptide format overcomes a limitation faced by many peptide leads and appears to be suitable for the generation of long-acting peptide therapeutics.

Phage display libraries of differently sized bicyclic peptides

Phage selections with combinatorial libraries of uniformly sized bicyclic peptides have recently yielded potent and selective binders of several protein targets. In this work we varied in a combinatorial fashion the ring sizes of bicyclic peptides in phage libraries, expecting that they would yield binders with higher affinities and/or more diverse binding motifs that could be affinity matured. 14 new phage peptide libraries of the format Cys-(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3, 4, 5 or 6) were generated and cyclized with tris-(bromomethyl)benzene. Affinity selections against the tumor-associated serine protease urokinase-type plasminogen activator yielded bicyclic peptide inhibitors with a large variety of consensus sequences. Several of the identified consensus sequences were exclusively found in bicyclic peptides having defined ring size combinations. Some of these peptides may bind in orientations that allow affinity maturation of non-conserved regions, while others do not. Having available multiple leads isolated from such bicyclic peptide libraries with variable ring sizes could therefore be a great asset for the generation of high affinity binders.

Protein Engineering and Selection Using Yeast Surface Display

Yeast surface display is a powerful technology for engineering a broad range of protein scaffolds. This protocol describes the process for de novo isolation of protein binders from large combinatorial libraries displayed on yeast by using magnetic bead separation followed by flow cytometry-based selection. The biophysical properties of isolated single clones are subsequently characterized, and desired properties are further enhanced through successive rounds of mutagenesis and flow cytometry selections, resulting in protein binders with increased stability, affinity, and specificity for target proteins of interest.