Potent inhibition of MMP-9 by a novel sustained-release platform attenuates left ventricular remodeling following myocardial infarction.

Sustained drug-release systems prolong the retention of therapeutic drugs within target tissues to alleviate the need for repeated drug administration. Two major caveats of the current systems are that the release rate and the timing cannot be predicted or fine-tuned because they rely on uncontrolled environmental conditions and that the system must be redesigned for each drug and treatment regime because the drug is bound via interactions that are specific to its structure and composition. We present a controlled and universal sustained drug-release system, which comprises minute spherical particles in which a therapeutic protein is affinity-bound to alginate sulfate (AlgS) through one or more short heparin-binding peptide (HBP) sequence repeats. Employing post-myocardial infarction (MI) heart remodeling as a case study, we show that the release of C9-a matrix metalloproteinase-9 (MMP-9) inhibitor protein that we easily bound to AlgS by adding one, two, or three HBP repeats to its sequence-can be directly controlled by modifying the number of HBP repeats. In an in vivo study, we directly injected AlgS particles, which were bound to C9 through three HBP repeats, into the left ventricular myocardium of mice following MI. We found that the particles substantially reduced post-MI remodeling, attesting to the sustained, local release of the drug within the tissue. As the number of HBP repeats controls the rate of drug release from the AlgS particles, and since C9 can be easily replaced with almost any protein, our tunable sustained-release system can readily accommodate a wide range of protein-based treatments.

APPI-Derived Cyclic Peptide Enhances Aß42 Aggregation and Reduces Aß42-Mediated Membrane Destabilization and Cytotoxicity.

An amyloid precursor protein inhibitor (APPI) and amyloid beta 42 (Aß42) are both subdomains of the human transmembrane amyloid precursor protein (APP). In the brains of patients with Alzheimer's disease (AD), Aß42 oligomerizes into aggregates of various sizes, with intermediate, low-molecular-weight Aß42 oligomers currently being held to be the species responsible for the most neurotoxic effects associated with the disease. Strategies to ameliorate the toxicity of these intermediate Aß42 oligomeric species include the use of short, Aß42-interacting peptides that either inhibit the formation of the Aß42 oligomeric species or promote their conversion to high-molecular-weight aggregates. We therefore designed such an Aß42-interacting peptide that is based on the ß-hairpin amino acid sequence of the APPI, which exhibits high similarity to the ß-sheet-like aggregation site of Aß42. Upon tight binding of this 20-mer cyclic peptide to Aß42 (in a 1:1 molar ratio), the formation of Aß42 aggregates was enhanced, and consequently, Aß42-mediated cell toxicity was ameliorated. We showed that in the presence of the cyclic peptide, interactions of Aß42 with both plasma and mitochondrial membranes and with phospholipid vesicles that mimic these membranes were inhibited. Specifically, the cyclic peptide inhibited Aß42-mediated mitochondrial membrane depolarization and reduced Aß42-mediated apoptosis and cell death. We suggest that the cyclic peptide modulates Aß42 aggregation by enhancing the formation of large aggregates─as opposed to low-molecular-weight intermediates─and as such has the potential for further development as an AD therapeutic.

Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site.

Although myriad protein-protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein-protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex.

A Mechanism for the Inhibition of Tau Neurotoxicity: Studies with Artificial Membranes, Isolated Mitochondria, and Intact Cells.

It is currently believed that molecular agents that specifically bind to and neutralize the toxic proteins/peptides, amyloid ß (Aß42), tau, and the tau-derived peptide PHF6, hold the key to attenuating the progression of Alzheimer's disease (AD). We thus tested our previously developed nonaggregating Aß42 double mutant (Aß42DM) as a multispecific binder for three AD-associated molecules, wild-type Aß42, the tauK174Q mutant, and a synthetic PHF6 peptide. Aß42DM acted as a functional inhibitor of these molecules in in vitro assays and in neuronal cell-based models of AD. The double mutant bound both cytotoxic tauK174Q and synthetic PHF6 and protected neuronal cells from the accumulation of tau in cell lysates and mitochondria. Aß42DM also reduced toxic intracellular levels of calcium and the overall cell toxicity induced by overexpressed tau, synthetic PHF6, Aß42, or a combination of PHF6and Aß42. Aß42DM inhibited PHF6-induced overall mitochondrial dysfunction: In particular, Aß42DM inhibited PHF6-induced damage to submitochondrial particles (SMPs) and suppressed PHF6-induced elevation of the ζ-potential of inverted SMPs (proxy for the inner mitochondrial membrane, IMM). PHF6 reduced the lipid fluidity of cardiolipin/DOPC vesicles (that mimic the IMM) but not DOPC (which mimics the outer mitochondrial membrane), and this effect was inhibited by Aß42DM. This inhibition may be explained by the conformational changes in PHF6 induced by Aß42DM in solution and in membrane mimetics. On this basis, the paper presents a mechanistic explanation for the inhibitory activity of Aß42DM against Aß42- and tau-induced membrane permeability and cell toxicity and provides confirmatory evidence for its protective function in neuronal cells.