Marco Finotto

DPP‐IV‐resistant, long‐acting oxyntomodulin derivatives

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon‐like peptide 1 (GLP‐1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose‐dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37‐amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP‐1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment‐associated nausea than GLP‐1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half‐life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP‐IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP‐IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon‐like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258–2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use. Copyright

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

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.