A covalent inhibitor targeting the papain-like protease from SARS-CoV-2 inhibits viral replication.

Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 µM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1-2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.

A self-immolative linker that releases thiols detects penicillin amidase and nitroreductase with high sensitivity via absorption spectroscopy.

This article reports the synthesis and characterization of a novel self-immolative linker, based on thiocarbonates, which releases a free thiol upon activation via enzymes. We demonstrate that thiocarbonate self-immolative linkers can be used to detect the enzymes penicillin G amidase (PGA) and nitroreductase (NTR) with high sensitivity using absorption spectroscopy. Paired with modern thiol amplification technology, the detection of PGA and NTR were achieved at concentrations of 160 nM and 52 nM respectively. In addition, the PGA probe was shown to be compatible with both biological thiols and enzymes present in cell lysates.

Predicting the stability of homotrimeric and heterotrimeric collagen helices.

Robust methods for predicting thermal stabilities of collagen triple helices are critical for understanding natural structure and stability in the collagen family of proteins and also for designing synthetic peptides mimicking these essential proteins. In this work, we determine the relative stability imparted on the collagen triple helix by single amino acids and interactions between amino acid pairs. Using this analysis, we create a comprehensive algorithm, SCEPTTr, for predicting melting temperatures of synthetic triple helices. Critically, our algorithm is compatible with every natural amino acid, can evaluate both homotrimers and heterotrimers, and accounts for all possible helix compositions and registers, including non-canonically staggered helices. We test and optimize our algorithm against 431 published collagen triple helices to demonstrate the quality of our predictive system. Finally, we use this algorithm to successfully guide the design of an ABC heterotrimer possessing high assembly specificity.

Advances in Imaging Reactive Oxygen Species.

Reactive oxygen species (ROS) play a pivotal role in many cellular processes and can be either beneficial or harmful. The design of ROS-sensitive fluorophores has allowed for imaging of specific activity and has helped elucidate mechanisms of action for ROS. Understanding the oxidative role of ROS in the many roles it plays allows us to understand the human body. This review provides a concise overview of modern advances in the field of ROS imaging. Indeed, much has been learned about the role of ROS throughout the years; however, it has recently been shown that using nanoparticles, rather than individual small organic fluorophores, for ROS imaging can further our understanding of ROS.

A pH-sensitive eosin-block copolymer delivers proteins intracellularly.

There is great interest in developing strategies to deliver proteins into the cytoplasm of cells. We report here a PEG-poly-eosin block copolymer (PEG-pEosin) that can encapsulate proteins and release them in active form under mildly acidic conditions. A PEG-pEosin formulation composed of Cre and the endosomolytic protein LLO efficiently performed gene editing in cells and in the brains of mice after an intracranial injection.

Covalent Capture of Collagen Triple Helices Using Lysine-Aspartate and Lysine-Glutamate Pairs.

Collagen mimetic peptides (CMPs) self-assemble into a triple helix reproducing the most fundamental aspect of the collagen structural hierarchy. They are therefore important for both further understanding this complex family of proteins and use in a wide range of biomaterials and biomedical applications. CMP self-assembly is complicated by a number of factors which limit the use of CMPs including their slow rate of folding, relatively poor monomer-trimer equilibrium, and the large number of competing species possible in heterotrimeric helices. All of these problems can be solved through the formation of isopeptide bonds between lysine and either aspartate or glutamate. These amino acids serve two purposes: they first direct self-assemble, allowing for composition and register control within the triple helix, and subsequently can be covalently linked, fixing the composition and register of the assembled structure without perturbing the triple helical conformation. This self-assembly and covalent capture are demonstrated here with four different triple helices. The formation of an isopeptide bond between lysine and glutamate (K-E) is shown to be a faster and higher yielding reaction than lysine with aspartate (K-D). Additionally, K-E amide bonds increase the thermal stability, improve the refolding capabilities, and enhance the triple helical structure as compared to K-E supramolecular interactions, observed by circular dichroism. In contrast, covalent capture of triple helices with K-D amide bonds occurs slower, and the captured triple helices do not have enhanced helical structure. The crystal structure of a triple helix captured through the formation of three K-E isopeptide bonds unequivocally demonstrates the connectivity of the amide bonds formed while also confirming the preservation of the canonical triple helix. The rate of reaction and yield for covalently captured K-E triple helices along with the excellent preservation of triple helical structure demonstrate that this approach can be used to effectively capture and stabilize this important biological motif for biological and biomedical applications.

Chemical functionality of multidomain peptide hydrogels governs early host immune response.

Multidomain Peptide (MDP) hydrogels are nanofibrous materials with many potential biomedical applications. The peptide sequence design of these materials offers high versatility and allows for the incorporation of various chemical functionalities into the nanofibrous scaffold. It is known that host response to biomaterials is strongly affected by factors such as size, shape, stiffness, and chemistry. However, there is a lack of fundamental understanding of the host response to different MDP hydrogels. In particular, it is unknown what effect the chemical functionality displayed on the nanofiber has on biological activity. Here we evaluated the early inflammatory host response to four MDP hydrogels displaying amines, guanidinium ions, and carboxylates in a subcutaneous injection model. While all the studied peptide materials possess similar nanostructure and physical properties, they trigger markedly different inflammatory responses. These were characterized by immunophenotyping of the cellular infiltrate using multi-color flow cytometry. The negatively-charged peptides elicit minimal inflammation characterized by tissue-resident macrophage infiltration, fast remodeling, and no collagen deposition or blood vessel formation within the implants. In contrast, the positively-charged peptides are highly infiltrated by immune cells, are remodeled at a slower rate, promote angiogenesis, and result in a high degree of collagen deposition. The presence of dynamic cell phenotypes characterizes the inflammation caused by the lysine-based peptide, including inflammatory monocytes, macrophages, and lymphoid cells, which is seen to be resolving over time. The arginine-based hydrogel shows higher inflammatory response with a persistent and significant infiltration of polymorphonuclear myeloid-derived cells, even ten days after implantation. This understanding of the immune response to peptide biomaterials improves our ability to design effective materials and to tailor their use for specific biomedical applications.

Self-Assembling Multidomain Peptides: Design and Characterization of Neutral Peptide-Based Materials with pH and Ionic Strength Independent Self-Assembly.

Self-assembly of peptides is a powerful method of preparing nanostructured materials. These peptides frequently utilize charged groups as a convenient switch for controlling self-assembly in which pH or ionic strength determines the assembly state. Multidomain peptides have been previously designed with charged domains of amino acids, which create molecular frustration between electrostatic repulsion and a combination of supramolecular forces including hydrogen bonding and hydrophobic packing. This frustration is eliminated by the addition of multivalent ions or pH adjustment, resulting in a self-assembled hydrogel. However, these charged functionalities can have profound, unintended effects on the properties of the resulting material. Access to neutral self-assembled nanostructured hydrogels may allow for distinct biological properties that are not available to highly charged analogues. Here, we designed a series of peptides to determine if self-assembly could be mediated by the steric interactions created by neutral hydroxyproline (O) domains, eliminating the need for charged residues and creating a neutral peptide hydrogel. The series of peptides, O n (SL)6O n , was studied to determine the effect of oligo-hydroxyproline on peptide self-assembly and nanostructure. We show that peptide solubility and nanofiber length increase with a higher number of hydroxyproline residues. Within this series, O5(SL)6O5 displayed the optimal properties for self-assembly and hydrogelation. In vitro, this hydrogel supports cell viability of fibroblasts, while in vivo it is infiltrated with cells and easily degraded over time without promoting a strong inflammatory response. This neutral self-assembling peptide hydrogel shows promising properties for biomedical, cell preservation, and tissue regeneration applications.

Covalent Capture of a Heterotrimeric Collagen Helix.

Stabilizing the three-dimensional structure of supramolecular materials can be accomplished through covalent capture of the assembled system. The lysine-aspartate charge pairs designed to direct the self-assembly of a collagen triple helix were subsequently used to covalently capture the helix through proximity-directed amide bond formation using EDC/HOBT activation. The triple helix thus stabilized maintains its folded structure and can now be used for applications previously inaccessible due to problematic folding equilibria.

Multidomain Peptide Hydrogel Accelerates Healing of Full-Thickness Wounds in Diabetic Mice.

In vivo, multidomain peptide (MDP) hydrogels undergo rapid cell infiltration and elicit a mild inflammatory response which promotes angiogenesis. Over time, the nanofibers are degraded and a natural collagen-based extracellular matrix is produced remodeling the artificial material into natural tissue. These properties make MDPs particularly well suited for applications in regeneration. In this work, we test the regenerative potential of MDP hydrogels in a diabetic wound healing model. When applied to full-thickness dermal wounds in genetically diabetic mice, the MDP hydrogel resulted in significantly accelerated wound healing compared to a clinically used hydrogel, as well as a control buffer. Treatment with the MDP hydrogel resulted in wound closure in 14 days, formation of thick granulation tissue including dense vascularization, innervation, and hair follicle regeneration. This suggests the MDP hydrogel could be an attractive choice for treatment of wounds in diabetic patients.

Nanofibrous peptide hydrogel elicits angiogenesis and neurogenesis without drugs, proteins, or cells.

The design of materials for regenerative medicine has focused on delivery of small molecule drugs, proteins, and cells to help accelerate healing. Additionally, biomaterials have been designed with covalently attached mimics of growth factors, cytokines, or key extracellular matrix components allowing the biomaterial itself to drive biological response. While the approach may vary, the goal of biomaterial design has often centered on promoting either cellular infiltration, degradation, vascularization, or innervation of the scaffold. Numerous successful studies have utilized this complex, multicomponent approach; however, we demonstrate here that a simple nanofibrous peptide hydrogel unexpectedly and innately promotes all of these regenerative responses when subcutaneously implanted into the dorsal tissue of healthy rats. Despite containing no small molecule drugs, cells, proteins or protein mimics, the innate response to this material results in rapid cellular infiltration, production of a wide range of cytokines and growth factors by the infiltrating cells, and remodeling of the synthetic material to a natural collagen-containing ECM. During the remodeling process, a strong angiogenic response and an unprecedented degree of innervation is observed. Collectively, this simple peptide-based material provides an ideal foundational system for a variety of bioregenerative approaches.

Covalent Capture of Aligned Self-Assembling Nanofibers.

A great deal of effort has been invested in the design and characterization of systems which spontaneously assemble into nanofibers. These systems are interesting for their fundamental supramolecular chemistry and have also been shown to be promising materials, particularly for biomedical applications. Multidomain peptides are one such assembler, and in previous work we have demonstrated the reversibility of their assembly under mild and easily controlled conditions, along with their utility for time-controlled drug delivery, protein delivery, cell encapsulation, and cell delivery applications. Additionally, their highly compliant criteria for sequence selection allows them to be modified to incorporate protease susceptibility and biological-recognition motifs for cell adhesion and angiogenesis. However, control of their assembly has been limited to the formation of disorganized nanofibers. In this work, we expand our ability to manipulate multidomain-peptide assembly into parallel-aligned fiber bundles. Albeit this alignment is achieved by the shearing forces of syringe delivery, it is also dependent on the amino acid sequence of the multidomain peptide. The incorporation of the amino acid DOPA (3,4-dihydroxyphenylalanine) allows the self-assembled nanofibers to form an anisotropic hydrogel string under modest shear stress. The hydrogel string shows remarkable birefringence, and highly aligned nanofibers are visible in scanning electronic microscopy. Furthermore, the covalent linkage induced by DOPA oxidation allows covalent capture of the aligned nanofiber bundles, enhancing their birefringence and structural integrity.

"Missing Tooth" Multidomain Peptide Nanofibers for Delivery of Small Molecule Drugs.

The clinical administration of many small molecule hydrophobic drugs is challenged by the insolubility of these drugs under physiological conditions. Because of this, the development of biocompatible scaffolds capable of effectively delivering hydrophobic drug molecules is of particular interest. Multidomain peptides (MDPs) provide biocompatible hydrogel scaffolds that are injectable and space-conforming, allowing for in situ delivery of a variety of drugs. Here we demonstrate that through manipulation of peptide primary sequence, a molecular cavity can be incorporated into the hydrophobic core of these peptide nanofibers allowing for encapsulation and delivery of small molecule drugs with poor water solubility. Using SN-38, daunorubicin, diflunisal, etodolac, levofloxacin, and norfloxacin, we demonstrate drug encapsulation and release from multidomain peptide fibers. Steady-state fluorescence and drug release studies show that hydrogels loaded with SN-38, diflunisal, and etodolac exhibit prolonged drug release profiles due to intrafibrillar drug encapsulation. This study establishes multidomain peptides as promising carriers for localized in situ delivery of small molecule drugs with poor water solubility.

Drug-triggered and cross-linked self-assembling nanofibrous hydrogels.

Self-assembly of multidomain peptides (MDP) can be tailored to carry payloads that modulate the extracellular environment. Controlled release of growth factors, cytokines, and small-molecule drugs allows for unique control of in vitro and in vivo responses. In this study, we demonstrate this process of ionic cross-linking of peptides using multivalent drugs to create hydrogels for sustained long-term delivery of drugs. Using phosphate, heparin, clodronate, trypan, and suramin, we demonstrate the utility of this strategy. Although all multivalent anions result in good hydrogel formation, demonstrating the generality of this approach, suramin led to the formation of the best hydrogels per unit concentration and was studied in greater detail. Suramin ionically cross-linked MDP into a fibrous meshwork as determined by scanning and transmission electron microscopy. We measured material storage and loss modulus using rheometry and showed a distinct increase in G' and G″ as a function of suramin concentration. Release of suramin from scaffolds was determined using UV spectroscopy and showed prolonged release over a 30 day period. Suramin bioavailability and function were demonstrated by attenuated M1 polarization of THP-1 cells compared to positive control. Overall, this design strategy has allowed for the development of a novel class of polymeric delivery vehicles with generally long-term release and, in the case of suramin, cross-linked hydrogels that can modulate cellular phenotype.

UV-activated multilayer nanomatrix provides one-step tunable carbohydrate structural characterization in MALDI-MS.

The structure-specific fragmentation of gas-phase ions in tandem mass spectrometry among other techniques provides an efficient analytical method for confirming unknown analytes or for elucidating chemical structures. Using concentration-dependent UV-absorbing matrix-functionalized magnetic nanoparticles and matrix-assisted laser desorption-ionization mass spectrometry (MALDI MS), we developed a single-step pseudo-MS/MS approach for tunable ionization and fragmentation to facilitate structure determination. Without chemical derivatization, we have demonstrated that this approach successfully distinguished isomeric sets of di-, tri- and tetrasaccharides. Low concentration of nanomatrix provided an enhanced signal for accurate mass determination of the intact molecular ions of analytes present in the sample. In contrast, high concentration of nanomatrix induced extensive and unique fragmentation, including high-energy facile bond breakage (A- and X-type cross-ring cleavages), which facilitated the linkage and sequence characterization of oligosaccharides without conventional tandem mass spectrometric instrumentation. The practicality of this approach for complex sample analysis was evaluated by an oligosaccharide mixture, wherein molecular ions are unambiguously observed and signature product ions are distinguishable enough for molecular identification and isomer differentiation by this simple tunable approach. By probing the roles of the multilayer nanomatrix components: matrix (energy absorption), silane-coating (energy pooling and dissipation) and core Fe3O4 (fragmentation), a plausible energy transfer mechanism was proposed based on a computational study and photoelectron experiments. The differentiation of tri- and tetra-oligosaccharide shown in this study not only demonstrated the first step toward glycan characterization by nanoparticle-assisted MALDI-MS, but also shed some insight on the nanoparticle-mediated energy transfer dynamics behind our approach.

Iron oxide nanomatrix facilitating metal ionization in matrix-assisted laser desorption/ionization mass spectrometry.

The significance and epidemiological effects of metals to life necessitate the development of direct, efficient, and rapid method of analysis. Taking advantage of its simple, fast, and high-throughput features, we present a novel approach to metal ion detection by matrix-functionalized magnetic nanoparticle (matrix@MNP)-assisted MALDI-MS. Utilizing 21 biologically and environmentally relevant metal ion solutions, the performance of core and matrix@MNP against conventional matrixes in MALDI-MS and laser desorption ionization (LDI) MS were systemically tested to evaluate the versatility of matrix@MNP as ionization element. The matrix@MNPs provided 20- to >100-fold enhancement on detection sensitivity of metal ions and unambiguous identification through characteristic isotope patterns and accurate mass (<5 ppm), which may be attributed to its multifunctional role as metal chelator, preconcentrator, absorber, and reservoir of energy. Together with the comparison on the ionization behaviors of various metals having different ionization potentials (IP), we formulated a metal ionization mechanism model, alluding to the role of exciton pooling in matrix@MNP-assisted MALDI-MS. Moreover, the detection of Cu in spiked tap water demonstrated the practicability of this new approach as an efficient and direct alternative tool for fast, sensitive, and accurate determination of trace metal ions in real samples.