Javier Pérez-Castells

Short-Term Monotherapy in HIV-Infected Patients with a Virus Entry Inhibitor Against the gp41 Fusion Peptide

An optimized derivative of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide shows antiviral potency and minimal side effects in a Phase I/II clinical trial. Anchors Away: Blocking HIV Entry Combination antiretroviral therapy has been very successful for treating infection with the human immunodeficiency virus (HIV-1), which causes AIDS. However, drug resistance is emerging and there is a need to develop new antiretroviral drugs that work earlier in the virus life cycle, for example, by preventing HIV-1 from entering host cells. Two such virus entry inhibitors, maraviroc and T-20, are in clinical use, but both have drawbacks. Forssmann, Kirchhoff and their colleagues have now developed a new virus entry inhibitor called VIRIP (VIRus-Inhibitory Peptide), a 20-peptide fragment of α1-antitrypsin, an abundant circulating serine protease inhibitor. VIRIP and its optimized derivative VIR-576 are so-called anchoring inhibitors because they prevent the gp41 fusion peptide of HIV-1 from inserting itself into the host cell membrane. This then blocks the next step in the virus life cycle, which is fusion of the virion envelope with the host cell membrane. Forssmann and co-workers now report on a Phase I/II clinical trial in which 18 HIV-1–infected patients who were not on any other antiretroviral therapy were treated for 10 days with three different doses of VIR-576 (0.5, 1.5, 5.0 g/day). They show that VIR-576 reduced the viral load in the plasma of patients on the highest dose by an order of magnitude and that the drug was well tolerated. Previous studies have shown that the gp41 fusion peptide is essential for HIV-1 entry into host cells, and suggest that it may be difficult for HIV-1 to develop resistance to VIR-576 because the fusion peptide is highly conserved and hardly tolerates changes without loss of function. This anchoring inhibitor, unlike other HIV entry inhibitors, is also active against many different HIV strains and has a different target (the gp41 fusion peptide). Thus, VIR-576 represents a potential new class of HIV entry inhibitor. However, VIR-576 does have some drawbacks too. Because VIR-576 is a peptide, it will be costly and time-consuming to produce and it must be administered intravenously. This has prompted Kirchhoff and colleagues to start searching for a small molecule that would block the gp41 fusion peptide in the same way as VIR-576 but would have the advantage that it could be made cheaper and given orally. To infect host cells, most enveloped viruses must insert a hydrophobic fusion peptide into the host cell membrane. Thus, fusion peptides may be valuable targets for developing drugs that block virus entry. We have shown previously that a natural 20-residue fragment of α1-antitrypsin, designated VIRus-Inhibitory Peptide (VIRIP), that binds to the gp41 fusion peptide of HIV-1 prevents the virus from entering target cells in vitro. Here, we examine the efficacy of 10-day monotherapy with the optimized VIR-576 derivative of VIRIP in treatment-naïve, HIV-1–infected individuals with viral RNA loads of ≥10,000 copies per ml. We report that at the highest dose (5.0 grams per day), intravenous infusion of VIR-576 reduced the mean plasma viral load by 1.23 log10 copies per ml without causing severe adverse effects. Our results are proof of concept that fusion peptide inhibitors suppress viral replication in human patients, and offer prospects for the development of a new class of drugs that prevent virus particles from anchoring to and infecting host cells.

Breaking Pseudo-Symmetry in Multiantennary Complex N-Glycans Using Lanthanide-Binding Tags and NMR Pseudo-Contact Shifts†

Controlling NMR shifts by lanthanides tagged to a symmetrical N-glycan reveals individual resonances for the residues of the otherwise identical A and Bu2005arms. This method provides a global perspective of conformational features and interactions in solution.

Structure of micelle-bound adrenomedullin: A first step toward the analysis of its interactions with receptors and small molecules†‡

Adrenomedullin (AM) is a regulatory peptide which plays many physiological roles including vasodilatation, bronchodilatation, hormone secretion regulation, growth, apoptosis, angiogenesis, and antimicrobial activities, among others. These regulatory activities make AM a relevant player in the pathophysiology of important diseases such as cardiovascular and renal conditions, cancer, and diabetes. Therefore, molecules that target the AM system have been proposed as having therapeutic potential. To guide the design and characterization of such molecules, we elucidated the three-dimensional structure of AM in a membrane mimicking medium using NMR spectroscopy methods. Under the employed experimental conditions, the structure can be described as composed by a central α-helical region, spanning about one third of its total length, flanked by two disordered segments at both N- and C-termini. The structure of AM in water is completely disordered. The 22-34 region of AM has a general tendency to adopt a helical structure under the employed experimental conditions. Furthermore, the study of the interaction of AM with two of its modulators has also been performed by using chemical shift perturbation analysis NMR methods with two-dimensional (2D)-TOCSY experiments, assisted with molecular modeling protocols. We expect these results will help in better understanding the interactions of AM with its receptor and binding proteins/molecules and in the development of novel modulators of AM activities.

Discovery and characterization of an endogenous CXCR4 antagonist.

CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.

CgNa, a type I toxin from the giant Caribbean sea anemone Condylactis gigantea shows structural similarities to both type I and II toxins, as well as distinctive structural and functional properties 1

CgNa (Condylactis gigantea neurotoxin) is a 47-amino-acid- residue toxin from the giant Caribbean sea anemone Condylactis gigantea. The structure of CgNa, which was solved by 1H-NMR spectroscopy, is somewhat atypical and displays significant homology with both type I and II anemone toxins. CgNa also displays a considerable number of exceptions to the canonical structural elements that are thought to be essential for the activity of this group of toxins. Furthermore, unique residues in CgNa define a characteristic structure with strong negatively charged surface patches. These patches disrupt a surface-exposed cluster of hydrophobic residues present in all anemone-derived toxins described to date. A thorough characterization by patch-clamp analysis using rat DRG (dorsal root ganglion) neurons indicated that CgNa preferentially binds to TTX-S (tetrodotoxin-sensitive) voltage-gated sodium channels in the resting state. This association increased the inactivation time constant and the rate of recovery from inactivation, inducing a significant shift in the steady state of inactivation curve to the left. The specific structural features of CgNa may explain its weaker inhibitory capacity when compared with the other type I and II anemone toxins.

The conformational behaviour and P-selectin inhibition of fluorine-containing sialyl LeX glycomimetics

A combination of experimental J/NOE NMR data with molecular mechanics and dynamics calculations has been used to examine the conformational behaviour and assign the configuration of synthetically prepared epimeric 3-carboxymethyl-O-Gal-(1-->1)-alpha-Man-fluoro-C-glycosides. It is shown that the population distributions around the glycosidic linkages strongly depend on the configuration at the fluorinated carbon of the pseudoacetal residue. It is also shown that these compounds resemble the inhibition ability of sialyl LeX towards P-selectin.

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch‐Selective Interaction of a Tetra‐Antennary N‐Glycan with Lectins

Abstract The biological recognition of complex‐type N‐glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N‐glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra‐antennary N‐glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N‐acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N‐glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N‐acetyllactosamine‐binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).

Unraveling the Conformational Landscape of Ligand Binding to Glucose/Galactose-Binding Protein by Paramagnetic NMR and MD Simulations

Protein dynamics related to function can nowadays be structurally well characterized (i.e., instances obtained by high resolution structures), but they are still ill-defined energetically, and the energy landscapes are only accessible computationally. This is the case for glucose-galactose binding protein (GGBP), where the crystal structures of the apo and holo states provide structural information for the domain rearrangement upon ligand binding, while the time scale and the energetic determinants for such concerted dynamics have been so far elusive. Here, we use GGBP as a paradigm to define a functional conformational landscape, both structurally and energetically, by using an innovative combination of paramagnetic NMR experiments and MD simulations. Anisotropic NMR parameters induced by self-alignment of paramagnetic metal ions was used to characterize the ensemble of conformations adopted by the protein in solution while the rate of interconversion between conformations was elucidated by long molecular dynamics simulation on two states of GGBP, the closed-liganded (holo_cl) and open-unloaded (apo_op) states. Our results demonstrate that, in its apo state, the protein coexists between open-like (68%) and closed-like (32%) conformations, with an exchange rate around 25 ns. Despite such conformational heterogeneity, the presence of the ligand is the ultimate driving force to unbalance the equilibrium toward the holo_cl form, in a mechanism largely governed by a conformational selection mechanism.

New synthesis and promising neuroprotective role in experimental ischemic stroke of ONO-1714

In an experimental permanent stroke model, we report here the contribution of ONO-1714 to brain damage prevention. Daily drug administration, twenty-one days prior to and two days after an experimental infarct, was performed by using mini-osmotic pumps (ALZET). Infarct volumes were assessed by image analysis of sequential coronal brain 1 mm(3) sections stained following the 2,3,5-triphenyltetrazolium chloride histological staining technique. Results of this study provide evidence of a significant reduction of the brain lesion size, suggesting ONO-1714 as a potential neuroprotective agent in stroke patients. ONO-1714 was prepared in our laboratory following a procedure which resulted in the supply of the desired compound in an easy and excellent yield.