Degradable hydrogels have been extensively used in biomedical applications such as drug delivery, and recent interest has grown in hydrogels that degrade in recognition of a cellular response. This contribution describes a poly(ethylene glycol) (PEG) hydrogel platform with human neutrophil elastase (HNE) sensitive peptide cross-links formed using thiol-ene photopolymerization rendering the gel degradable at sites of inflammation. Further, protein therapeutics can be physically entrapped within the network and selectively released upon exposure to HNE. HNE responsive hydrogels exhibited surface erosion where the degradation kinetics was influenced by changes in peptide k cat , concentration of HNE, and concentration of peptide within the gel. Using this platform, we were able to achieve controlled, zero-order release of bovine serum albumin (BSA) in the presence of HNE, and release was arrested in the absence of HNE. To further exploit the advantages of surface eroding delivery systems, a smaller protein (carbonic anhydrase) was delivered at the same rate as BSA and only dependent on gel formulation and environmental conditions. Also, protein release was predicted from a 3-layered hydrogel device using mass loss data. Lastly, the bioactivity of lysozyme was maintained above 90% following the exposure to thiol-ene photopolymerization conditions.
The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LFN-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LFN bound. At pH 7.5, the contributions from the three unstructured LFN lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region.
The advent of SARS-CoV-2, the causative agent of COVID-19, and its worldwide impact on global health, have provided the impetus for the development of effective countermeasures that can be deployed against the virus, including vaccines, monoclonal antibodies, and direct-acting antivirals (DAAs). Despite these efforts, the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection. SARS-CoV-2 3C-like protease (3CL pro ), an enzyme essential for viral replication, is a validated target for the discovery of SARS-CoV-2 therapeutics. In this report, we describe the structure-guided utilization of the cyclopropane moiety in the design of highly potent inhibitors of SARS-CoV-2 3CL pro , SARS-CoV-1 3CL pro , and MERS-CoV 3CL pro . High-resolution cocrystal structures were used to identify the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and unravel the mechanism of action. Aldehydes 5c and 11c inhibited SARS-CoV-2 replication with EC 50 values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts 5d and 11d were equipotent with EC 50 values of 13 and 12 nM, respectively. The safety index (SI) values for compounds 5c/11c and 5d/11d ranged between 7692 and 9090. Importantly, aldehydes 5c/11c and bisulfite adducts 5d/11d potently inhibited MERS-CoV 3CL pro with IC 50 values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds 5c/11c and 5d/11d inhibited SARS-CoV-1 with IC 50 values of 960 and 350 nM and 790 and 240 nM, respectively. Taken together, these studies suggest that the inhibitors described herein have low cytotoxicity and high potency and are promising candidates for further development as broad-spectrum direct-acting antivirals against highly pathogenic coronaviruses.
Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins.
The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states.
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