Paolo Ingallinella

Structural Analysis of the Epitope of the Anti-HIV Antibody 2F5 Sheds Light into Its Mechanism of Neutralization and HIV Fusion

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I beta turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.

Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency

Peptides derived from the heptad repeat 2 (HR2) region of the HIV fusogenic protein gp41 are potent inhibitors of viral infection, and one of them, enfuvirtide, is used for the treatment of therapy-experienced AIDS patients. The mechanism of action of these peptides is binding to a critical intermediate along the virus–cell fusion pathway, and accordingly, increasing the affinity for the intermediate yields more potent inhibitors. We took a different approach, namely to increase the potency of the HR2 peptide inhibitor C34 by targeting it to the cell compartment where fusion occurs, and we show here that a simple, yet powerful way to accomplish this is attachment of a cholesterol group. C34 derivatized with cholesterol (C34-Chol) shows dramatically increased antiviral potency on a panel of primary isolates, with IC90 values 15- to 300-fold lower than enfuvirtide and the second-generation inhibitor T1249, making C34-Chol the most potent HIV fusion inhibitor to date. Consistent with its anticipated mechanism of action, the antiviral activity of C34-Chol is unusually persistent: washing target cells after incubation with C34-Chol, but before triggering fusion, increases IC50 only 7-fold, relative to a 400-fold increase observed for C34. Moreover, derivatization with cholesterol extends the half-life of the peptide in vivo. In the mouse, s.c. administration of 3.5 mg/kg C34-Chol yields a plasma concentration 24 h after injection >300-fold higher than the measured IC90 values. Because the fusion machinery targeted by C34-Chol is similar in several other enveloped viruses, we believe that these findings may be of general utility.

Universal Influenza B Vaccine Based on the Maturational Cleavage Site of the Hemagglutinin Precursor

ABSTRACT Conventional influenza vaccines can prevent infection, but their efficacy depends on the degree of antigenic “match” between the strains used for vaccine preparation and those circulating in the population. A universal influenza vaccine based on invariant regions of the virus, able to provide broadly cross-reactive protection, without requiring continuous manufacturing update, would solve a major medical need. Since the temporal and geographical dominance of the influenza virus type and/or subtype (A/H3, A/H1, or B) cannot yet be predicted, a universal vaccine, like the vaccines currently in use, should include both type A and type B influenza virus components. However, while encouraging preclinical data are available for influenza A virus, no candidate universal vaccine is available for influenza B virus. We show here that a peptide conjugate vaccine, based on the highly conserved maturational cleavage site of the HA0 precursor of the influenza B virus hemagglutinin, can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive influenza B virus lineages. We demonstrate that protection by the HA0 vaccine is mediated by antibodies, probably through effector mechanisms, and that a major part of the protective response targets the most conserved region of HA0, the P1 residue of the scissile bond and the fusion peptide domain. In addition, we present preliminary evidence that the approach can be extended to influenza A virus, although the equivalent HA0 conjugate is not as efficacious as for influenza B virus.

Structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus S2 fusion protein

A coronavirus (CoV) has recently been identified as the causative agent of the severe acute respiratory syndrome (SARS) in humans. CoVs enter target cells through fusion of viral and cellular membranes mediated by the viral envelope glycoprotein S. We have determined by x-ray crystallography the structure of a proteolytically stable core fragment from the heptad repeat (HR) regions HR1 and HR2 of the SARS-CoV S protein. We have also determined the structure of an HR1-HR2 S core fragment, containing a shorter HR1 peptide and a C-terminally longer HR2 peptide that extends up to the transmembrane region. In these structures, three HR1 helices form a parallel coiled-coil trimer, whereas three HR2 peptides pack in an oblique and antiparallel fashion into the coiled-coil hydrophobic grooves, adopting mixed extended and α-helical conformations as in postfusion paramyxoviruses F proteins structures. Our structure positions a previously proposed internal fusion peptide adjacent to the N-terminus of HR1. Peptides from the HR2 region of SARS-CoV S have been shown to inhibit viral entry and infection in vitro. The structures presented here can thus open the path to the design of small-molecule inhibitors of viral entry and candidate vaccine antigens against this virus.

Influenza virus vaccine

The present invention provides vaccines against disease caused by infection with influenza virus, and methods of vaccination. The vaccines comprise peptides derived from the M2 and/or HA proteins of influenza virus conjugated to a carrier protein.

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.

Development of a neuromedin U–human serum albumin conjugate as a long‐acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half‐life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half‐life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA–NMU displayed long‐lasting, potent anorectic, and glucose‐normalizing activity. When compared side by side with a previously described PEG conjugate, HSA–NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU‐based therapeutics are promising candidates for the treatment of obesity and diabetes. Copyright

A New Method for Chemoselective Conjugation of Unprotected Peptides to Dauno- and Doxorubicin

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.

The effect of prime-site occupancy on the hepatitis C virus NS3 protease structure

We recently reported a new class of inhibitors of the chymotrypsin‐like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S′ site of the protease, which is not generally used by the natural substrates. The effect of prime‐site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis‐mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S′ site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S′ site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N‐terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P‐P′ inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistance.