Antonello Pessi

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 × 107β-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors μ, δ, and κ and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human κ-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [35S]guanosine 5′-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for κ-receptors. To fine-tune the potency and selectivity of these κ-peptides we replaced their turn-forming template by other turn mimetic molecules. This “turn-scan” process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.

A Universal Influenza B Peptide Vaccine

Paolo Ingallinella, Elisabetta Bianchi, Xiaoping Liang, Marco Finotto, Michael Chastain, Jiang Fan, Tong-Ming Fu, Hong Chang Song, Melanie Horton, Daniel Freed, Walter Manger, Emily Wen, Li Shi, Roxana Ionescu, Colleen Price, Marc Wenger, Emilio Emini, Riccardo Cortese, Gennaro Ciliberto, John Shiver and Antonello Pessi IRBM P.Angeletti, Pomezia (Rome), Italy; Merck Research Laboratories, West Point, PA, USA

Covalent Trimeric Coiled Coils of the HIV gp41 HR1 Region are Extremely Potent and Broadly Neutralizing Inhibitors of Viral Infection

Introduction The key players in HIV viral entry are the envelope glycoprotein receptor-binding gp120 and transmembrane fusogenic gp41 subunits. The mechanism of fusion involves two helical regions of gp41, an N-terminal heptad repeat (HR1) and a Cterminal heptad repeat (HR2). The HR1 and HR2 helical regions form a fusogenic structure, a six-helix-bundle, in which three α-helices formed by HR2 peptides pack in an antiparallel manner against a central three stranded coiled coil formed by the HR1 peptides. It is generally accepted that fusion progresses via the formation of a fusion intermediate, in which both the HR1 coiled coil and the HR2 regions are exposed. The fusion intermediate is the target of both synthetic Cand N-peptides, that inhibit viral infection by preventing formation of the 6-helix bundle. HR2 peptides are potent inhibitors of viral fusion, and the peptide DP178 has become the first fusion inhibitor approved as a human therapeutic [1]. Peptides from the HR1 region of gp41 protein can also inhibit viral fusion, but their potency is limited by the low tendency to form a trimeric coil-coil. Accordingly, chimeric peptides, consisting of a designed trimeric coiled coil (IZ) fused to gp41 HR1 sequences are potent inhibitors of fusion as reported for IZN17 [2] (Fig. 1). Based on the hypothesis that antiviral potency of IZN17 is limited by the selfassociation equilibrium, we designed a new construct, CCIZN17, with the aim of stabilizing the homotrimeric coiled coil structure to a covalent trimer via formation of intermolecular disulfide bonds. To this purpose, two cysteine residues were introduced at the N-terminus of the IZN17 sequence (Fig. 1). Also, two glycine residues were added between the pair of cysteines and the IZ scaffold sequence to ensure a high conformational freedom of the cysteines from the coiled coil domain and thus to enhance the likelihood of spontaneous formation of the intermolecular disulfide bridges among the three peptide chains.

Identification of a Novel HIV-1 Neutralizing Antibody Using Synthetic Peptides that Mimic a GP41 Fusion Intermediate

Introduction HIV-entry into cells is mediated by the envelope glycoprotein receptor-binding gp120 and fusogenic gp41 subunits. During the fusion process gp41 undergoes a series of conformational changes that culminate in formation of the fusogenic structure: a 6-helix bundle, where three α-helices formed by the heptad repeat region 2 (HR2) pack in an antiparallel manner against a central three-stranded coiled coil formed by the heptad repeat region 1 (HR1). Viral fusion progresses via formation of an intermediate, which transiently exposes the HR1 coiled coil and the HR2 peptides. By targeting these regions with peptides derived from HR1, HR2, it is possible to prevent formation of the 6-helix bundle, and block viral infectivity. We investigated if antibodies could also block HIV-1 entry by the same mechanism, since this would open the pathway to a vaccine targeting the fusion intermediate.

Structural studies of peptide inhibitors bound to hepatitis C virus protease yield insights into the mechanism of action of the enzyme

Much effort for a therapy against hepatitis C virus (HCV) is devoted to the search of inhibitors of the virally-encoded protease NS3, which is required for maturation of HCV polyprotein [1]. In order to cleave its substrates NS3 must form a complex with the 54residue viral cofactor protein NS4A, whose activity is mimicked by a synthetic peptide (Pep4A) corresponding to amino acids 21-34 [1]. Binding of Pep4A was shown to induce important tertiary structure changes in the enzyme [2]. We have recently developed substrate-derived hexapeptide inhibitors of NS3 with affinities in the low nanomolar range [3]. Structure-activity relationships, molecular modelling, site-directed mutagenesis and most recently NMR have been used to gain knowledge about the salient features of inhibitor binding [3-5]. In the present work we have further studied the interaction between NS3, NS4A and the inhibitors, and found that major conformational changes take place in the enzyme upon binary and ternary complex formation.

Engineering and chemical synthesis of the HCV protease transmembrane protein cofactor NS4A

In the course of our studies on the obligatory cofactor protein, NS4A, of the serine protease NS3 of Hepatitis C virus (HCV), we had to address two problems common to the study of transmembrane (TM) proteins: synthetic difficulties and poor solubility. NS3 is an HCV-encoded enzyme required for maturation of the viral polyprotein; to do so however, it must form a non-covalent complex [1] with the 54 residue protein NS4A (Table 1), which has been predicted to be a type I TM protein. So far any attempt at preparing NS4A by recDNA failed, and to date no protein is available for study.

Structural Studies of the Complex Between Decapeptide Inhibitors and the Serine Protease NS3/4A of Hepatitis C Virus

One of the most promising approaches to anti-HCV therapy is the development of inhibitors of the viral NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors have been described [1], all taking advantage of binding to the S site of the enzyme. By studying the interaction of these inhibitors with NS3 [2], we showed that binding occurs according to an induced-fit mechanism. In the absence of a cofactor, multiple binding modes are apparent, while in the presence of a cofactor all P-inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by CD spectroscopy. Inhibitor complex formation is associated with stabilization of the enzyme, as highlighted by limited proteolysis experiments.

Prime Site Binding Inhibitors of the Hepatitis C Virus NS3/4A Serine Protease

Infection by hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer. Much effort for a therapy is currently devoted to the search of inhibitors of the serine protease NS3/4A, which is required for maturation of viral polyprotein.