Biodegradable Tetra-PEG Hydrogels as Carriers for a Releasable Drug Delivery System

We have developed an approach to prepare drug-releasing Tetra-PEG hydrogels with exactly four cross-links per monomer. The gels contain two cleavable β-eliminative linkers: one for drug attachment that releases the drug at a predictable rate, and one with a longer half-life placed in each cross-link to control biodegradation. Thus, the system can be optimized to release the drug before significant gel degradation occurs. The synthetic approach involves placing a heterobifunctional connector at each end of a four-arm PEG prepolymer; four unique end-groups of the resultant eight-arm prepolymer are used to tether a linker-drug, and the other four are used for polymerization with a second four-arm PEG. Three different orthogonal reactions that form stable triazoles, diazines, or oximes have been used for tethering the drug to the PEG and for cross-linking the polymer. Three formats for preparing hydrogel–drug conjugates are described that either polymerize preformed PEG–drug conjugates or attach the drug postpolymerization. Degradation of drug-containing hydrogels proceeds as expected for homogeneous Tetra-PEG gels with minimal degradation occurring in early phases and sharp, predictable reverse gelation times. The minimal early degradation allows design of gels that show almost complete drug release before significant gel-drug fragments are released

Broad-Spectrum Allosteric Inhibition of Herpesvirus Proteases

Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds <b>2</b> and <b>3</b>) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound <b>2</b> has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) <i>J. Biol. Chem. 266</i>, 15591–15594] show DD2, compound <b>2</b>, and compound <b>3</b> inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions

A Hydrogel-Microsphere Drug Delivery System That Supports Once-Monthly Administration of a GLP‑1 Receptor Agonist

We have developed a chemically controlled very long-acting delivery system to support once-monthly administration of a peptidic GLP-1R agonist. Initially, the prototypical GLP-1R agonist exenatide was covalently attached to hydrogel microspheres by a self-cleaving β-eliminative linker; after subcutaneous injection in rats, the peptide was slowly released into the systemic circulation. However, the short serum exenatide half-life suggested its degradation in the subcutaneous depot. We found that exenatide undergoes deamidation at Asn²⁸ with an <i>in vitro</i> and <i>in vivo</i> half-life of approximately 2 weeks. The [Gln²⁸]­exenatide variant and exenatide showed indistinguishable GLP-1R agonist activities as well as pharmacokinetic and pharmacodynamic effects in rodents; however, unlike exenatide, [Gln²⁸]­exenatide is stable for long periods. Two different hydrogel-[Gln²⁸]­exenatide conjugates were prepared using β-eliminative linkers with different cleavage rates. After subcutaneous injection in rodents, the serum half-lives for the released [Gln²⁸]­exenatide from the two conjugates were about 2 weeks and one month. Two monthly injections of the latter in the Zucker diabetic fatty rat showed pharmacodynamic effects indistinguishable from two months of continuously infused exenatide. Pharmacokinetic simulations indicate that the delivery system should serve well as a once-monthly GLP-1R agonist for treatment of type 2 diabetes in humans

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

<div><p>Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound’s inhibitory activity is also dependent on the amino acid sequence and P1’ character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.</p> </div

Inhibitor potency and selectivity against caspase family members.

<p>(A) Schematic of divalent tetrapeptide substrate proteolysis to release R110 fluorophore. Removal of both tetrapeptides by caspases is required for signal generation at 535 nm. Concentration-response analysis of compound <b>3</b> (B) and VEID-CHO (C) against caspase-6 (green), caspase-3 (black or red) or caspase-7 (blue). The particular divalent R110 peptide substrate used with each enzyme is indicated in the figure key and assay specifics can be found in Experimental Procedures. Potency values for (B–C) can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s005" target="_blank">Table S2</a>. Concentration response curves were generated in duplicate and represent 1 of at least 2 experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. Data represent mean ± standard error of the mean.</p

Structure of the <i>N</i>-furoyl-phenylalanine screening hit (2) and the optimized analog 3.

<p>Potency values represent the inhibition of caspase-6 cleavage of (VEID)₂R110 substrate.</p

SPR detection of 3 binding to multiple caspase-6 surfaces confirms uncompetitive binding mode.

<p>(A) Catalytically inactive caspase-6 (green), apo-caspase-6 (blue) and caspase-6 saturated with VEID-FMK inhibitor (purple) were captured to chip surfaces and exposed to VEID-AMC, (VEID)₂R110 and/or <b>3</b> to qualitatively monitor binding. Cooperative binding of <b>3</b> and (VEID)₂R110 to C163 caspase-6 illustrate formation of the Michaelis-Menten complex. (B) Sensograms representing injections of escalating concentrations of <b>3</b> over VEID-FMK inhibitor-blocked caspase-6 surface (black). The inset represents similar injections of <b>3</b> over an unblocked apo-caspase-6 surface (blue). (C) Concentration-response analysis of data from (B) when compound <b>3</b> was injected over VEID-blocked caspase-6 surface (black) and apo-caspase-6 (blue) surfaces.</p

Docking models of caspase-6/VEID-R110/3 ternary complex explains fluorophore-dependent potency of this series of compounds.

<p>(A) Docking model of the Michaelis-Menten complex formed between caspase-6 (light blue), VEID-R110 (green sticks) and <b>3</b> (wheat sticks). (B) Docking model of the tetrahedral intermediate between caspase-6, VEID-R110 (green sticks) and <b>3</b> (wheat sticks) with substrate covalently bound to Cys163. (C) Depiction of monovalent VEID substrates with R110 or AMC fluorophores.</p

Kinetic caspase-6 enzymatic studies with compound 3 show uncompetitive mechanism of inhibition with (VEID)₂R110 substrate.

<p>(A) The initial enzyme velocity of caspase-6 was plotted against the indicated concentration of (VEID)₂R110 substrate in the presence of 0 nM (DMSO-black), 3 nM (red), 10 nM (orange), 30 nM (green) or 100 nM (blue) compound <b>3</b>. Double reciprocal plot of this data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s001" target="_blank">Figure S1</a> and Michaelis-Menten constants can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050864#pone.0050864.s006" target="_blank">Table S3</a>. (B) Concentration-response analysis of compound <b>3</b> when tested in the presence of 0.5 µM (red), 5 µM (black) or 20 µM (blue) (VEID)₂R110 substrate. Michaelis-Menten kinetic experiments were performed with single points while concentration-response curves were performed in duplicate. Each data set represents 1 of at least 3 experiments with similar results.</p

Crystal structure of caspase-6 ternary complex with 3 and covalently bound VEID inhibitor reveals the uncompetitive mechanism of this series of compounds.

<p>(A) Crystal structure of the ternary complex of caspase-6 with zVEID and compound <b>3</b> (PDB-ID 4HVA). The caspase-6 dimer is represented as cartoon with the A and B chains colored light blue and grey, respectively, and the L4 loop colored purple. The zVEID inhibitors are represented as sticks and are colored pink. Each inhibitor is covalently bound to the catalytic cysteine (Cys163) in both chain A and B. Two molecules of <b>3</b> are shown as ball and stick representation and colored orange. (B) Close up of the active site of chain A colored according to (A) with hydrogen bonds shown as black dashes. (C) Structural comparison of caspase-6 ternary complex with <b>3</b> bound (light blue) and caspase-6 binary complex with bound VEID-CHO (wheat) (PDB-ID 3OD5) illustrating the difference in the conformation of the tip of the L4 loop in the two crystal structures (residues 261–271).</p