Molecular mechanisms of the adsorption of a model protein (human serum albumin) on poly(methylidene malonate 2.1.2) nanoparticles.

PURPOSE: To understand the molecular mechanisms involved in protein-methylidene malonate 2.1.2 polymer interactions. METHODS: To assess the importance of electrostatic forces in polymer-protein interactions use was made of HSA and its derivatives, which were anionized by succinylation and aconitylation. Surface plasmon resonance measurements, using the three HSA molecules as immobilized ligands and polymer nanoparticles as analytes in the liquid phase, allowed the determination of initial kinetic constants and affinity constants at equilibrium at two different temperatures. RESULTS: Saturation of binding for the three proteins occurred at approximately 900 protein molecules/nanoparticle. The apparent affinity decreased with increasing electronegativity of the proteins. Surface plasmon resonance measurement of proteins, covalently linked to the chip matrix, showed a high affinity for the nanoparticles (K(A) approximately 10(10) M(-1) and confirmed the moderate decrease of affinity with increasing electronegativity of the modified albumins. Measurements at 25 and 37 degrees C showed no significant increase in the albumin-nanoparticle interactions. Dissociation of the proteins from the nanoparticles could only be realized with chaotropic salt solutions. CONCLUSIONS: These results suggest the molecular forces initiating the protein-nanoparticle interactions are mainly of electrostatic nature followed by stabilization by hydrophobic forces. The high affinity confirms the nanoparticles as excellent carriers for protein delivery.

Peptidomimetic inhibitors of the human cytomegalovirus protease.

The development of peptidomimetic inhibitors of the human cytomegalovirus (HCMV) protease showing sub-micromolar potency in an enzymatic assay is described. Selective substitution of the amino acid residues of these inhibitors led to the identification of tripeptide inhibitors showing improvements in inhibitor potency of 27-fold relative to inhibitor 39 based upon the natural tetrapeptide sequence. Small side chains at P1 were well tolerated by this enzyme, a fact consistent with previous observations. The S2 binding pocket of HCMV protease was very permissive, tolerating lipophilic and basic residues. The substitutions tried at P3 indicated that a small increase in inhibitor potency could be realized by the substitution of a tert-leucine residue for valine. Substitutions of the N-terminal capping group did not significantly affect inhibitor potency. Pentafluoroethyl ketones, alpha,alpha-difluoro-beta-keto amides, phosphonates and alpha-keto amides were all effective substitutions for the activated carbonyl component and gave inhibitors which were selective for HCMV protease. A slight increase in potency was observed by lengthening the P1' residue of the alpha-keto amide series of inhibitors. This position also tolerated a variety of groups making this a potential site for future modifications which could modulate the physicochemical properties of these molecules.

Potent HIV protease inhibitors containing a novel (hydroxyethyl)amide isostere.

A series of HIV protease inhibitors containing a novel (hydroxyethyl)amidosuccinoyl core has been synthesized. These peptidomimetic structures inhibit viral protease activity at low nanomolar concentrations (IC50 < 10 nM for HIV-1 protease). The inhibition constant (Ki) for inhibitor 19 was determined to be 7.5 pM against HIV-1 and 1.2 nM against HIV-2 proteases, respectively. Several compounds (19-24) inhibited HIV-1 replication in cell culture assays with 50% effective concentrations (EC50) = 3.7-35 nM. This series of inhibitors was found to exhibit poor bioavailability (< 10%) in the rat, following oral administration. The synthesis and biological properties of these compounds are discussed. In addition, an X-ray structure of one of these inhibitors (23) in complex with HIV-2 protease provides insight into the binding mode of this novel class of HIV protease inhibitors.