Unraveling hidden regulatory sites in structurally homologous metalloproteases.

Monitoring enzymatic activity in vivo of individual homologous enzymes such as the matrix metalloproteinases (MMPs) by antagonist molecules is highly desired for defining physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous enzymes often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Yet, the challenge remains to identify such regulatory alternative sites that are often hidden and scattered over different locations on the protein's surface. We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers share the same chemical nature, providing versatile nonspecific binding reactivity that allows to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. This approach includes diverse experimental analysis, such as structural spectroscopic analyses, NMR, and protein crystallography combined with computational prediction of effector binding sites. We demonstrate how our approach works by analyzing members of the MMP family that possess a unique set of such sites. Our work provides a proof of principle for using ligand effectors to unravel hidden regulatory sites specific to members of the structurally homologous MMP family. This approach may be exploited for the design of novel molecular effectors and therapeutic agents affecting protein catalytic function via interactions with structure-specific regulatory sites.

Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential.

Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.

Toward iron sensors: bioinspired tripods based on fluorescent phenol-oxazoline coordination sites.

In the quest for fast throughput metal biosensors, it would be of interest to prepare fluorophoric ligands with surface-adhesive moieties. Biomimetic analogues to microbial siderophores possessing such ligands offer attractive model compounds and new opportunities to meet this challenge. The design, synthesis, and physicochemical characterization of biomimetic analogues of microbial siderophores from Paracoccus denitrificans and from the Vibrio genus are described. The (4S,5S)-2-(2-hydroxyphenyl)-5-methyl-4,5-dihydro-1,3-oxazole-4-carbonyl group (La), noted here as an HPO unit, was selected for its potential dual properties, serving as a selective iron(III) binder and simultaneously as a fluorophore. Three tripodal symmetric analogues cis-Lb, cis-Lc, and trans-Lc, which mainly differ in the length of the spacers between the central carbon anchor and the ligating sites, were synthesized. These ferric-carriers were built from a tetrahedral carbon as an anchor, symmetrically extended by three converging iron-binding chains, each bearing a terminal HPO. The fourth chain could contain a surface-adhesive function (Lc). A combination of absorption and emission spectrophotometry, potentiometry, electrospray mass spectrometry, and electrochemistry was used to fully characterize the corresponding ferric complexes and to determine their stability. The quenching mechanism is consistent with an intramolecular static process and is more efficient for the analogue with longer arms. Detection limits in the low nanogram per milliliter range, comparable with the best chemosensors based on natural peptide siderophores, have been determined. These results clearly demonstrate that these tris(phenol-oxazoline) ligands in a tripodal arrangement firmly bind iron(III). Due to their fluorescent properties, the coordination event can be easily monitored, while the fourth arm is available for surface-adhesive moieties. The tripodal system is therefore an ideal candidate for integration with solid-state materials for the development of chip-based devices and analytical methodologies.

Branched coordination multilayers on gold.

A C3-symmetric tridentate hexahydroxamate ligand molecule was specially synthesized and used for coordination self-assembly of branched multilayers on Au surfaces precoated with a self-assembled monolayer (SAM) of ligand anchors. Layer-by-layer (LbL) growth of multilayers via metal-organic coordination using Zr4+ ions proceeds with high regularity, adding one molecular layer in each step, as shown by ellipsometry, wettability, UV-vis spectroscopy, and atomic force microscopy (AFM). The branched multilayer films display improved stiffness, as well as a unique defect self-repair capability, attributed to cross-linking in the layers and lateral expansion over defects during multilayer growth. Transmetalation, i.e., exposure of Zr4+-based assemblies to Hf4+ ions, was used to evaluate the cross-linking. Conductive atomic force microscopy (AFM) was used to probe the electrical properties of the multilayers, revealing excellent dielectric behavior. The special properties of the branched layers were emphasized by comparison with analogous multilayers prepared similarly using linear (tetrahydroxamate) ligand molecules. The process of defect annihilation by bridging over defective areas, attributed to lateral expansion via the excess bishydroxamate groups, was demonstrated by introduction of artificial defects in the anchor monolayer, followed by assembly of two layers of either the linear or the branched molecule. Analysis of selective binding of Au nanoparticles (NPs) to unblocked defects emphasized the superior repair mechanism in the branched layers with respect to the linear ones.

Synthesis and biological evaluation of lipophilic iron chelators as protective agents from oxidative stress.

Lipophilic Fe(III) chelators were synthesized and shown to protect oligodendrial cells from oxidative damage induced by Fe(III) and hydrogen peroxide.

Preparative manipulation of gold nanoparticles by reversible binding to a polymeric solid support.

A preparative scheme is presented for controlled modification of gold nanoparticles (NPs) by using reversible binding to a polymeric solid support through boronic acid chemistry. Octanethiol-capped Au NPs were bound to a boronic acid functionalized resin by custom-synthesized bifunctional linker molecules. The NPs were chemically released from the resin to the solution, with one (or a few) linker molecules embedded in their capping layer. This was confirmed by rebinding the linker-derivatized NPs to a boronic resin, exploiting the reversibility of the boronic acid/diol chemistry. The same scheme was employed to demonstrate a new method for affinity separation of NPs by means of a solid-phase reaction. The use of boronic acid provides versatility and chemical reversibility, while the polymeric solid support affords the separation and preparative aspects. The method presented here may be useful in various facets of NP handling, manipulation, and separation.

Ferrioxamine B analogues: targeting the FoxA uptake system in the pathogenic Yersinia enterocolitica.

A series of ferrioxamine B analogues that target the bacterium Yersinia enterocolitica were prepared. These iron carriers are composed of three hydroxamate-containing monomeric units. Two identical monomers consist of N-hydroxy-3-aminopropionic acid coupled with beta-alanine, and a third unit at the amino terminal is composed of N-hydroxy-3-aminopropionic acid and one of the following amino acids: beta-alanine (1a), phenylalanine (1b), cyclohexylalanine (1c), or glycine (1d). Thermodynamic results for representatives of the analogues have shown a strong destabilization (3-4 orders of magnitude) of the ferric complexes with respect to ferrioxamine B, probably due to shorter spacers and a more strained structure around the metal center. No significant effect of the variations at the N-terminal has been observed on the stability of the ferric complexes. By contrast, using in vivo radioactive uptake experiments, we have found that these modifications have a substantial effect on the mechanism of iron(III) uptake in the pathogenic bacteria Yersinia enterocolitica. Analogues 1a and 1d were utilized by the ferrioxamine B uptake system (FoxA), while 1b and 1c either used different uptake systems or were transported to the microbial cell nonspecifically by diffusion via the cell membrane. Transport via the FoxA system was also confirmed by uptake experiments with the FoxA deficient strain of Yersinia enterocolitica. A fluorescent marker, attached to 1a in a way that did not interfere with its biological activity, provided additional means to monitor the uptake mechanism by fluorescence techniques. Of particular interest is the observation that 1a was utilized by the uptake system of ferrioxamine B in Yersinia enterocolitica (FoxA) but failed to use the ferrioxamine uptake route in Pseudomonas putida. Here, we present a case in which biomimetic siderophore analogues deliberately designed for a particular bacterium can distinguish between related uptake systems of different microorganisms.

Chemical input multiplicity facilitates arithmetical processing.

We describe the design and function of a molecular logic system, by which a combinatorial recognition of the input signals is utilized to efficiently process chemically encoded information. Each chemical input can target simultaneously multiple domains on the same molecular platform, resulting in a unique combination of chemical states, each with its characteristic fluorescence output. Simple alteration of the input reagents changes the emitted logic pattern and enables it to perform different algebraic operations between two bits, solely in the fluorescence mode. This system exhibits parallelism in both its chemical inputs and light outputs.

Direct photo-induced DNA strand scission by a ruthenium bipyridyl complex.

Irradiation of plasmid DNA in the presence of Ru(II)-2, a modified tris(2,2'-bipyridyl)Ru(II) complex, in which two hydroxamic acid groups are attached to one of the three bipyridyl ligands, results in total fragmentation of the DNA. The photo-chemical reaction products were analyzed by gel electrophoresis, which revealed complete fragmentation. Further evidence for the complete degradation of the DNA was obtained by imaging the pre- and post-treated plasmid DNA using atomic force microscopy (AFM). A mechanism for the reaction is proposed. It initially involves the photo-chemical generation of Ru(III) ions and superoxide radicals, as corroborated by absorbance difference spectroscopy and electron paramagnetic resonance (EPR). Consequently, Ru(III) preferentially oxidizes guanine, liberating superoxide radicals that yield OH radicals. The OH radicals were identified by observing the spectral change at 532 nm of a 5'-dAdG substrate forming a colored adduct with thiobarbituric acid. These radicals are associated with the major non-specific damage exerted to DNA.

Stable room-temperature molecular negative differential resistance based on molecule-electrode interface chemistry.

We show reproducible, stable negative differential resistance (NDR) at room temperature in molecule-controlled, solvent-free devices, based on reversible changes in molecule-electrode interface properties. The active component is the cyclic disulfide end of a series of molecules adsorbed onto mercury. As this active component is reduced, the Hg-molecule contact is broken, and an insulating barrier at the molecule-electrode interface is formed. Therefore, the alignment of the molecular energy levels, relative to the Fermi levels of the electrodes, is changed. This effect results in a decrease in the current with voltage increase as the reduction process progresses, leading to the so-called NDR behavior. The effect is reproducible and repeatable over more than 50 scans without any reduction in the current. The stability of the system, which is in the "solid state" except for the Hg, is due to the molecular design where long alkyl chains keep the molecules aligned with respect to the Hg electrode, even when they are not bound to it any longer.

Synthesis and evaluation of iron chelators with masked hydrophilic moieties.

Synthetic iron chelators based on the natural siderophore ferrichrome have previously been shown to bind Fe(III) with high affinity (pKf > 27) and have shown no toxicity to mammalian cell cultures in vitro. A new class of lipophilic ferrichrome analogues carrying acetoxymethyl ester moieties has been synthesized. We have shown that these molecules penetrate rapidly through cell membranes and turn highly hydrophilic while inside the cells, upon esterase mediated hydrolysis of the lipophilic termini. The intracellular retention was visualized by labeling these analogues with a fluorescent naphthalic diimide probe. The prohydrophilic iron chelators have been shown to inhibit the metal-catalyzed intracellular formation of reactive oxygen species with high effectivity, and preliminary results suggest these molecules to be potent antimalarial agents.