Microphase Behavior and Enhanced Wet-Cohesion of Synthetic Copolyampholytes Inspired by a Mussel Foot Protein.

Numerous attempts have been made to translate mussel adhesion to diverse synthetic platforms. However, the translation remains largely limited to the Dopa (3,4-dihydroxyphenylalanine) or catechol functionality, which continues to raise concerns about Dopa's inherent susceptibility to oxidation. Mussels have evolved adaptations to stabilize Dopa against oxidation. For example, in mussel foot protein 3 slow (mfp-3s, one of two electrophoretically distinct interfacial adhesive proteins in mussel plaques), the high proportion of hydrophobic amino acid residues in the flanking sequence around Dopa increases Dopa's oxidation potential. In this study, copolyampholytes, which combine the catechol functionality with amphiphilic and ionic features of mfp-3s, were synthesized and formulated as coacervates for adhesive deposition on surfaces. The ratio of hydrophilic/hydrophobic as well as cationic/anionic units was varied in order to enhance coacervate formation and wet adhesion properties. Aqueous solutions of two of the four mfp-3s-inspired copolymers showed coacervate-like spherical microdroplets (ϕ ≈ 1-5 μm at pH ∼4 (salt concentration ∼15 mM). The mfp-3s-mimetic copolymer was stable to oxidation, formed coacervates that spread evenly over mica, and strongly bonded to mica surfaces (pull-off strength: ∼17.0 mJ/m(2)). Increasing pH to 7 after coacervate deposition at pH 4 doubled the bonding strength to ∼32.9 mJ/m(2) without oxidative cross-linking and is about 9 times higher than native mfp-3s cohesion. This study expands the scope of translating mussel adhesion from simple Dopa-functionalization to mimicking the context of the local environment around Dopa

Microphase Behavior and Enhanced Wet-Cohesion of Synthetic Copolyampholytes Inspired by a Mussel Foot Protein

Numerous attempts have been made to translate mussel adhesion to diverse synthetic platforms. However, the translation remains largely limited to the Dopa (3,4-dihydroxyphenylalanine) or catechol functionality, which continues to raise concerns about Dopa’s inherent susceptibility to oxidation. Mussels have evolved adaptations to stabilize Dopa against oxidation. For example, in mussel foot protein 3 <i>slow</i> (mfp-3s, one of two electrophoretically distinct interfacial adhesive proteins in mussel plaques), the high proportion of hydrophobic amino acid residues in the flanking sequence around Dopa increases Dopa’s oxidation potential. In this study, copolyampholytes, which combine the catechol functionality with amphiphilic and ionic features of mfp-3s, were synthesized and formulated as coacervates for adhesive deposition on surfaces. The ratio of hydrophilic/hydrophobic as well as cationic/anionic units was varied in order to enhance coacervate formation and wet adhesion properties. Aqueous solutions of two of the four mfp-3s-inspired copolymers showed coacervate-like spherical microdroplets (ϕ ≈ 1–5 μm at pH ∼4 (salt concentration ∼15 mM). The mfp-3s-mimetic copolymer was stable to oxidation, formed coacervates that spread evenly over mica, and strongly bonded to mica surfaces (pull-off strength: ∼17.0 mJ/m²). Increasing pH to 7 after coacervate deposition at pH 4 doubled the bonding strength to ∼32.9 mJ/m² without oxidative cross-linking and is about 9 times higher than native mfp-3s cohesion. This study expands the scope of translating mussel adhesion from simple Dopa-functionalization to mimicking the context of the local environment around Dopa

Marine Bioinspired Underwater Contact Adhesion

Marine mussels and barnacles are sessile biofouling organisms that adhere to a number of surfaces in wet environments and maintain remarkably strong bonds. Previous synthetic approaches to mimic biological wet adhesive properties have focused mainly on the catechol moiety, present in mussel foot proteins (mfps), and especially rich in the interfacial mfps, for example, mfp-3 and -5, found at the interface between the mussel plaque and substrate. Barnacles, however, do not use Dopa for their wet adhesion, but are instead rich in noncatecholic aromatic residues. Due to this anomaly, we were intrigued to study the initial contact adhesion properties of copolymerized acrylate films containing the key functionalities of barnacle cement proteins and interfacial mfps, for example, aromatic (catecholic or noncatecholic), cationic, anionic, and nonpolar residues. The initial wet contact adhesion of the copolymers was measured using a probe tack testing apparatus with a flat-punch contact geometry. The wet contact adhesion of an optimized, bioinspired copolymer film was ∼15.0 N/cm² in deionized water and ∼9.0 N/cm² in artificial seawater, up to 150 times greater than commercial pressure-sensitive adhesive (PSA) tapes (∼0.1 N/cm²). Furthermore, maximum wet contact adhesion was obtained at ∼pH 7, suggesting viability for biomedical applications

Reversible covalent direct thrombin inhibitors.

INTRODUCTION:In recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high. METHODS:We have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1. RESULTS:We demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay. CONCLUSION:Compound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles

Structural model of the thrombin active site.

<p>The structural model of the thrombin active site is derived from X-ray crystallography of thrombin modified by Compound 1. The S195 in the active site required for thrombin enzymatic activity is modified by the 2-methoxybenzoyl group, rendering it inactive. In the monomer shown on the right, continuous electron density is found supporting the existence of a covalent linkage. In the monomer shown on the left, electronic density for the methoxyphenyl ring indicates blockage of the active site, but covalent linkage cannot be conclusively determined due to missing electron density (red).</p

Kinetic parameters for the inhibition of serine proteases.

<p>Kinetic parameters for the inhibition of serine proteases.</p

Enzyme inhibition IC₅₀s for thrombin and other serine proteases.

<p>Enzyme inhibition IC₅₀s for thrombin and other serine proteases.</p

Reversible covalent direct thrombin inhibitors - Fig 3

<p><i>Panel A</i>: Fluorescence intensity vs time for the incubation of Compound 2 with thrombin, thrombin mutant S195A, and thrombin with PPACK. After 3 min (arrow), the dansyl-labeled thrombin inhibitor Compound 2 was added to a solution of thrombin WT (black), thrombin preincubated with an excess of the irreversible active site inhibitor PPACK (magenta), or the active site mutant thrombin S195A (green) and incubated at room temperature for 2 h. The samples were monitored by fluorescence (excitation 280 nm, emission 340 nm). The observed differences in fluorescence quenching suggest that Compound 1 targets S195. <i>Panel B</i>: Compound 2 incubated with WT thrombin was analyzed by SDS-PAGE. Two samples are shown at time 0 and after 30 min. In the Coomassie-stained image (top), the thrombin band is visible irrespective of compound incubation. In contrast, when viewed under UV light (bottom), the thrombin band fluoresces only after incubation, indicating the incorporation of (parts of) Compound 2 into the enzyme.</p

Interaction of Compound 1 with thrombin.

<p>Compound 1 at 1 μM was incubated with excess thrombin at 5 μM at room temperature for 20 min. The reaction was then quenched and monitored by LC-MS for Compound 1 and Compound 3. Note that quantitation levels were normalized to Compound 1 without thrombin. Stochiometric conversion of Compound 1 to Compound 3 was observed.</p