David Zanuy

Nanoparticle-induced vascular blockade in human prostate cancer

The tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) specifically homes to tumors by binding to fibrin and fibrin-associated clotted plasma proteins in tumor vessels. Previous results show that CREKA-coated superparamagnetic iron oxide particles can cause additional clotting in tumor vessels, which creates more binding sites for the peptide. We have used this self-amplifying homing system to develop theranostic nanoparticles that simultaneously serve as an imaging agent and inhibit tumor growth by obstructing tumor circulation through blood clotting. The CREKA nanoparticles were combined with nanoparticles coated with another tumor-homing peptide, CRKDKC, and nanoparticles with an elongated shape (nanoworms) were used for improved binding efficacy. The efficacy of the CREKA peptide was then increased by replacing some residues with nonproteinogenic counterparts, which increased the stability of the peptide in the circulation. Treatment of mice bearing orthotopic human prostate cancer tumors with the targeted nanoworms caused extensive clotting in tumor vessels, whereas no clotting was observed in the vessels of normal tissues. Optical and magnetic resonance imaging confirmed tumor-specific targeting of the nanoworms, and ultrasound imaging showed reduced blood flow in tumor vessels. Treatment of mice with prostate cancer with multiple doses of the nanoworms induced tumor necrosis and a highly significant reduction in tumor growth.

Short Peptide Amyloid Organization: Stabilities and Conformations of the Islet Amyloid Peptide NFGAIL☆

Experimentally, short peptides have been shown to form amyloids similar to those of their parent proteins. Consequently, they present useful systems for studies of amyloid conformation. Here we simulate extensively the NFGAIL peptide, derived from the human islet amyloid polypeptide (residues 22-27). We simulate different possible strand/sheet organizations, from dimers to nonamers. Our simulations indicate that the most stable conformation is an antiparallel strand orientation within the sheets and parallel between sheets. Consistent with the alanine mutagenesis, we find that the driving force is the hydrophobic effect. Whereas the NFGAIL forms stable oligomers, the NAGAIL oligomer is unstable, and disintegrates very quickly after the beginning of the simulation. The simulations further identify a minimal seed size. Combined with our previous simulations of the prion-derived AGAAAAGA peptide, AAAAAAAA, and the Alzheimer Abeta fragments 16-22, 24-36, 16-35, and 10-35, and the solid-state NMR data for Abeta fragments 16-22, 10-35, and 1-40, some insight into the length and the sequence matching effects may be obtained.

The sequence dependence of fiber organization. A comparative molecular dynamics study of the islet amyloid polypeptide segments 22-27 and 22-29.

Amyloid fiber formation and the possible polymorphism of molecular arrangements depend on the polypeptide length and composition. Here, we seek the chemical clues underlying these processes. Our starting point is based on the experimental observation that some short peptide segments are able to develop fibers that are very similar to those of their original parent proteins. We focus our study on the NFGAILSS peptide, derived from the human islet amyloid polypeptide (residues 22-29). This peptide turned out to be a perfect example, illustrating the fact that the amyloid microscopic organization is highly complex, rather than simply involving hydrogen bond formation. Furthermore, obtaining a reliable molecular model has allowed us to analyze the differences between the amyloid structure we have obtained for this peptide and that obtained for the previously studied, two residues shorter, segment (residues 22-27, NFGAIL). This comparative study yields some clues about chemical events that govern the aggregation of proteins into oriented fibers, such as molecular packing between sheets and the degree of interaction specificity. We characterize the important role played by the hydrophobic and aromatic residues in the inter-sheet association and present new approaches toward the understanding of the nature of events that are likely to take place during fibril formation. These include analysis of interaction patterns derived from specific sheet-associated packing.

Sequence dependence of C-end rule peptides in binding and activation of neuropilin-1 receptor

Neuropilin-1 (NRP-1) is a hub receptor that plays an essential role in angiogenesis and vascular permeability. It is over-expressed in the new blood vessels grown by tumor cells and is a target for anti-tumor treatments. Peptides that expose the consensus sequence R/K/XXR/K at the C-terminus (C-end rule or CendR peptides) bind to NRP-1 and are internalized into the cell. We used peptide phage display binding assays and molecular dynamics (MD) simulations to study the potential role of the central residues of CendR peptides in binding and activation of the NRP-1 receptor. The high stability of RPAR-receptor domain complex stems from the formation of a characteristic pattern of three hydrogen bonds between the peptide C-terminus and the residues in the NRP-1 loop III. Any changes in the peptide structure that fail to preserve this triad result in a less-stable complex. We performed a systematic study of RXXR mutants, where X=A/D/S/R/P, in order to test the effect of replacement of A or P on the binding capabilities. Our results, both experimental and computational, show that RRAR, RDAR, RPDR, RPRR and RPPR are capable of binding NRP-1. However, only RPPR and RPRR segments form an optimal organization around loop III with low potential energy. In other analogs, the absence of these stabilizing interactions always results in higher potential energy of the complexes. The binding of RPAR analogs does not guarantee receptor activation; only stable complexes that are properly stabilized via loop III appear able to trigger NRP-1 activation.

A rigid, chiral, dendronized polymer with a thermally stable, right-handed helical conformation

First- and second-generation dendronized polymethacrylates PG1 and PG2 carrying chiral 4-aminoproline-based dendrons were obtained on the half-gram scale in high molar masses (PG1: M(n)=5 x 10(6) g mol(-1), PG2: M(n)=1x10(6) g mol(-1)) by spontaneous (radical) polymerization of the corresponding vinyl macromonomers. NMR spectroscopic studies on PG2 together with its unprecedented high glass transition temperature (T(g)>200 degrees C, decomp) and structural parameters provided by atomistic MD simulations show this polymer to be rather rigid. Optical rotation and CD measurements revealed that PG2 adopts a helical conformation that remains unchanged over wide ranges of temperature and solvent polarity. It is also retained when the polymer is deprotected (and thus positively charged, de-PG2) at its terminal amino groups, by which the mass and steric demand of the dendrons is reduced by roughly 50 %. Molecular dynamics simulations on models of PG2 reveal its helical conformation to be right-handed, irrespective of backbone tacticity, and initial results also indicate that de-PG2 retains the right-handedness.

Binding of a C-end rule peptide to the neuropilin-1 receptor: a molecular modeling approach.

Neuropilin-1 (NRP-1) is a receptor that plays an essential role in angiogenesis, vascular permeability, and nervous system development. Previous studies have shown that peptides with an N-terminal Arg, especially peptides with the four-residue consensus sequence R/K/XXR/K, bind to NRP-1 cell surfaces. Peptides containing such consensus sequences promote binding and internalization into cells, while blocking the C-terminal Arg (or Lys) prevents the internalization. In this study, we use molecular dynamics simulations to model the structural properties of the NRP-1 complex with a prototypic CendR peptide, RPAR. Our simulations show that RPAR binds NRP-1 through specific interactions of the RPAR C-terminus: three hydrogen bonds and a salt bridge anchor the ligand in the receptor pocket. The modeling results were used as the starting point for a systematic computational study of new RPAR analogues based on chemical modifications of their natural amino acids. Comparison of the structural properties of the new peptide-receptor complexes with the original organization suggests that some of the analogues can increase the binding affinity while reducing the natural sensitivity of RXXR to endogenous proteases.

Principles of Nanostructure Design With Protein Building Blocks

Currently there is increasing interest in nanostructures and their design. Nanostructure design involves the ability to predictably manipulate the properties of the self‐assembly of autonomous units. Autonomous units have preferred conformational states. The units can be synthetic material science‐based or derived from functional biological macromolecules. Autonomous biological building blocks with available structures provide an extremely rich and useful resource for design. For proteins, the structural databases contain large libraries of protein molecules and their building blocks with a range of shapes, surfaces, and chemical properties. The introduction of engineered synthetic residues or short peptides into these can expand the available chemical space and enhance the desired properties. Here we focus on the principles of nanostructure design with protein building blocks. Proteins 2007.

On the helical conformation of un-ionized poly(γ-d-glutamic acid)

Abstract The conformational preferences of the naturally occurring poly(γ- d -glutamic acid) in the un-ionized state were investigated using a combination of molecular dynamics and quantum mechanical calculations. Results indicated that a left-handed helix with 19-membered ring hydrogen bonds set between the CO of the amide group i and the NH of amide group i+3 is the most stable conformation for this poly(γ-amino acid). Weak intramolecular interactions between the oxygens of the carboxyl side groups and the NH of the backbone amide groups were detected. They are assumed to be responsible for the unexpected handedness exhibited by the helix with regards to the stereochemistry of the compound.

The Energy Landscape of a Selective Tumor-Homing Pentapeptide

Recently, a potentially powerful strategy based on phage-display libraries has been presented to target tumors via homing peptides attached to nanoparticles. The Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence has been identified as a tumor-homing peptide that binds to clotted plasmas proteins present in tumor vessels and interstitium. The aim of this work consists of mapping the conformational profile of CREKA to identify the bioactive conformation. For this purpose, a conformational search procedure based on modified simulated annealing combined with molecular dynamics was applied to three systems that mimic the experimentally used conditions: (i) the free peptide; (ii) the peptide attached to a nanoparticle; and (iii) the peptide inserted in a phage display protein. In addition, the free peptide was simulated in an ionized aqueous solution environment, which mimics the ionic strength of the physiological medium. Accessible minima of all simulated systems reveal a multiple interaction pattern involving the ionized side chains of Arg, Glu, and Lys, which induces a beta-turn motif in the backbone observed in all simulated CREKA systems.

DNA - Conducting polymer complexes: A computational study of the hydrogen bond between building blocks

Ab initio quantum mechanical calculations at the MP2 level were used for an extensive study concerning the stability of hydrogen-bonded complexes formed by pyrrole and thiophene, which are the most common building blocks of conducting polymers, and DNA bases. Results indicated that very stable complexes were formed with pyrrole, which shows a clear tendency to form specific hydrogen-bonding interactions with nucleic acid bases. Furthermore, the strength of such interactions depends significantly on the base, growing in the following order: thymine < adenine approximately equal to cytosine < guanine. On the contrary, thiophene formed complexes stabilized by nonspecific interactions between the pi-cloud of the ring and the N-H groups of the nucleic acid bases rather than specific hydrogen bonds. Overall, these results are fully consistent with experimental observations: polypyrrole is able not only to stabilize adducts with DNA but also to interact specifically, while the interactions of the latter with polythiophene and their derivatives are weaker and nonspecific.