Structural Consequences of Introducing Bioactive Domains to Designer ß-Sheet Peptide Self-Assemblies.

We applied solid- and solution-state nuclear magnetic resonance spectroscopy to examine the structure of multidomain peptides composed of self-assembling ß-sheet domains linked to bioactive domains. Bioactive domains can be selected to stimulate specific biological responses (e.g., via receptor binding), while the ß-sheets provide the desirable nanoscale properties. Although previous work has established the efficacy of multidomain peptides, molecular-level characterization is lacking. The bioactive domains are intended to remain solvent-accessible without being incorporated into the ß-sheet structure. We tested for three possible anticipated molecular-level consequences of introducing bioactive domains to ß-sheet-forming peptides: (1) the bioactive domain has no effect on the self-assembling peptide structure; (2) the bioactive domain is incorporated into the ß-sheet nanofiber; and (3) the bioactive domain interferes with self-assembly such that nanofibers are not formed. The peptides involved in this study incorporated self-assembling domains based on the (SL)6 motif and bioactive domains including a VEGF-A mimic (QK), an IGF-mimic (IGF-1c), and a de novo SARS-CoV-2 binding peptide (SBP3). We observed all three of the anticipated outcomes from our examination of peptides, illustrating the unintended structural effects that could adversely affect the desired biofunctionality and biomaterial properties of the resulting peptide hydrogel. This work is the first attempt to evaluate the structural effects of incorporating bioactive domains into a set of peptides unified by a similar self-assembling peptide domain. These structural insights reveal unmet challenges in the design of highly tunable bioactive self-assembling peptide hydrogels.

Injectable Peptide Hydrogels Loaded with Murine Embryonic Stem Cells Relieve Ischemia In Vivo after Myocardial Infarction.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide, especially in aging and metabolically unhealthy populations. A major target of regenerative tissue engineering is the restoration of viable cardiomyocytes to preserve cardiac function and circumvent the progression to heart failure post-MI. Amelioration of ischemia is a crucial component of such restorative strategies. Angiogenic ß-sheet peptides can self-assemble into thixotropic nanofibrous hydrogels. These syringe aspiratable cytocompatible gels were loaded with stem cells and showed excellent cytocompatibility and minimal impact on the storage and loss moduli of hydrogels. Gels with and without cells were delivered into the myocardium of a mouse MI model (LAD ligation). Cardiac function and tissue remodeling were evaluated up to 4 weeks in vivo. Injectable peptide hydrogels synergized with loaded murine embryonic stem cells to demonstrate enhanced survival after intracardiac delivery during the acute phase post-MI, especially at 7 days. This approach shows promise for post-MI treatment and potentially functional cardiac tissue regeneration and warrants large-scale animal testing prior to clinical translation.

Self-Assembling Peptides with Insulin-Like Growth Factor Mimicry.

Growth factor (GF) mimicry involves recapitulating the signaling of larger molecules or cells. Although GF mimicry holds considerable promise in tissue engineering and drug design applications, difficulties in targeting the signaling molecule to the site of delivery and dissociation of mimicking peptides from their target receptors continue to limit its clinical application. To address these challenges, we utilized a self-assembling peptide (SAP) platform to generate synthetic insulin-like growth factor (IGF)-signaling, self-assembling GFs. Our peptide hydrogels are biocompatible and bind target IGF receptors in a dose-dependent fashion, activate proangiogenic signaling, and facilitate formation of angiogenic microtubules in vitro. Furthermore, infiltrated hydrogels are stable for weeks to months. We conclude that the enhanced targeting and long-term stability of our SAP/GF mimicry implants may improve the efficacy and safety of future GF mimic therapeutics.

Scalable Inhibitors of the Nsp3-Nsp4 Coupling in SARS-CoV-2.

The human Betacoronavirus SARS-CoV-2 is a novel pathogen claiming millions of lives and causing a global pandemic that has disrupted international healthcare systems, economies, and communities. The virus is fast mutating and presenting more infectious but less lethal versions. Currently, some small-molecule therapeutics have received FDA emergency use authorization for the treatment of COVID-19, including Lagevrio (molnupiravir) and Paxlovid (nirmaltrevir/ritonavir), which target the RNA-dependent RNA polymerase and the 3CLpro main protease, respectively. Proteins downstream in the viral replication process, specifically the nonstructural proteins (Nsps1-16), are potential drug targets due to their crucial functions. Of these Nsps, Nsp4 is a particularly promising drug target due to its involvement in the SARS-CoV viral replication and double-membrane vesicle formation (mediated via interaction with Nsp3). Given the degree of sequence conservation of these two Nsps across the Betacoronavirus clade, their protein-protein interactions and functions are likely to be conserved as well in SARS-CoV-2. Through AlphaFold2 and its recent advancements, protein structures were generated of Nsp3 and 4 lumenal loops of interest. Then, using a combination of molecular docking suites and an existing library of lead-like compounds, we virtually screened 7 million ligands to identify five putative ligand inhibitors of Nsp4, which could present an alternative pharmaceutical approach against SARS-CoV-2. These ligands exhibit promising lead-like properties (ideal molecular weight and log P profiles), maintain fixed-Nsp4-ligand complexes in molecular dynamics (MD) simulations, and tightly associate with Nsp4 via hydrophobic interactions. Additionally, alternative peptide inhibitors based on Nsp3 were designed and shown in MD simulations to provide a highly stable binding to the Nsp4 protein. Finally, these therapeutics were attached to dendrimer structures to promote their multivalent binding with Nsp4, especially its large flexible luminal loop (Nsp4LLL). The therapeutics tested in this study represent many different approaches for targeting large flexible protein structures, especially those localized to the ER. This study is the first work targeting the membrane rearrangement system of viruses and will serve as a potential avenue for treating viruses with similar replicative function.

How to Design Peptides.

Novel design of proteins to target receptors for treatment or tissue augmentation has come to the fore owing to advancements in computing power, modeling frameworks, and translational successes. Shorter proteins, or peptides, can offer combinatorial synergies with dendrimer, polymer, or other peptide carriers for enhanced local signaling, which larger proteins may sterically hinder. Here, we present a generalized method for designing a novel peptide. We first show how to create a script protocol that can be used to iteratively optimize and screen novel peptide sequences for binding a target protein. We present a step-by-step introduction to utilizing file repositories, data bases, and the Rosetta software suite. RosettaScripts, an .xml interface that allows for sequential functions to be performed, is used to order the functions for repeatable performance. These strategies may lead to more groups venturing into computational design, which may result in synergies from artificial intelligence/machine learning (AI/ML) to phage display and screening. Importantly, the beginner is expected to be able to design their first peptide ligand and begin their journey in peptide drug discovery. Generally, these peptides potentially could be used to interact with any enzyme or receptor, for example, in the study of chemokines and their interactions with glycosoaminoglycans and their receptors.

Comparison of SGLT1, SGLT2, and Dual Inhibitor biological activity in treating Type 2 Diabetes Mellitus.

Diabetes Mellitus Type 2 (T2D) is an emerging health burden in the USand worldwide, impacting approximately 15% of Americans. Current front-line therapeutics for T2D patients include sulfonylureas that act to reduce A1C and/or fasting blood glucose levels, or Metformin that antagonizes the action of glucagon to reduce hepatic glucose production. Next generation glucomodulatory therapeutics target members of the high-affinity glucose transporter Sodium-Glucose-Linked-Transporter (SGLT) family. SGLT1 is primarily expressed in intestinal epithelium, whose inhibition reduces dietary glucose uptake, whilst SGLT2 is highly expressed in kidney - regulating glucose reabsorption. A number of SGLT2 inhibitors are FDA approved whilst SGLT1 and dual SGLT1 & 2 inhibitor are currently in clinical trials. Here, we discuss and compare SGLT2, SGLT1, and dual inhibitors' biochemical mechanism and physiological effects.

iPSC-derived cranial neural crest-like cells can replicate dental pulp tissue with the aid of angiogenic hydrogel.

The dental pulp has irreplaceable roles in maintaining healthy teeth and its regeneration is a primary aim of regenerative endodontics. This study aimed to replicate the characteristics of dental pulp tissue by using cranial neural crest (CNC)-like cells (CNCLCs); these cells were generated by modifying several steps of a previously established method for deriving NC-like cells from induced pluripotent stem cells (iPSCs). CNC is the anterior region of the neural crest in vertebrate embryos, which contains the primordium of dental pulp cells or odontoblasts. The produced CNCLCs showed approximately 2.5-12,000-fold upregulations of major CNC marker genes. Furthermore, the CNCLCs exhibited remarkable odontoblastic differentiation ability, especially when treated with a combination of the fibroblast growth factors (FGFs) FGF4 and FGF9. The FGFs induced odontoblast marker genes by 1.7-5.0-fold, as compared to bone morphogenetic protein 4 (BMP4) treatment. In a mouse subcutaneous implant model, the CNCLCs briefly fated with FGF4 + FGF9 replicated dental pulp tissue characteristics, such as harboring odontoblast-like cells, a dentin-like layer, and vast neovascularization, induced by the angiogenic self-assembling peptide hydrogel (SAPH), SLan. SLan acts as a versatile biocompatible scaffold in the canal space. This study demonstrated a successful collaboration between regenerative medicine and SAPH technology.

Preclinical Efficacy of Pro- and Anti-Angiogenic Peptide Hydrogels to Treat Age-Related Macular Degeneration.

Pro-angiogenic and anti-angiogenic peptide hydrogels were evaluated against the standard of care wet age-related macular degeneration (AMD) therapy, Aflibercept (Eylea®). AMD was modeled in rats (laser-induced choroidal neovascularization (CNV) model), where the contralateral eye served as the control. After administration of therapeutics, vasculature was monitored for 14 days to evaluate leakiness. Rats were treated with either a low or high concentration of anti-angiogenic peptide hydrogel (0.02 wt% 8 rats, 0.2 wt% 6 rats), or a pro-angiogenic peptide hydrogel (1.0 wt% 7 rats). As controls, six rats were treated with commercially available Aflibercept and six with sucrose solution (vehicle control). Post lasering, efficacy was determined over 14 days via fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT). Before and after treatment, the average areas of vascular leak per lesion were evaluated as well as the overall vessel leakiness. Unexpectedly, treatment with pro-angiogenic peptide hydrogel showed significant, immediate improvement in reducing vascular leak; in the short term, the pro-angiogenic peptide performed better than anti-angiogenic peptide hydrogel and was comparable to Aflibercept. After 14 days, both the pro-angiogenic and anti-angiogenic peptide hydrogels show a trend of improvement, comparable to Aflibercept. Based on our results, both anti-angiogenic and pro-angiogenic peptide hydrogels may prove good therapeutics in the future to treat wet AMD over a longer-term treatment period.

Peptide-Based Inhibitors for SARS-CoV-2 and SARS-CoV.

The COVID-19 (coronavirus disease) global pandemic, caused by the spread of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus, currently has limited treatment options which include vaccines, anti-virals, and repurposed therapeutics. With their high specificity, tunability, and biocompatibility, small molecules like peptides are positioned to act as key players in combating SARS-CoV-2, and can be readily modified to match viral mutation rate. A recent expansion of the understanding of the viral structure and entry mechanisms has led to the proliferation of therapeutic viral entry inhibitors. In this comprehensive review, inhibitors of SARS and SARS-CoV-2 are investigated and discussed based on therapeutic design, inhibitory mechanistic approaches, and common targets. Peptide therapeutics are highlighted, which have demonstrated in vitro or in vivo efficacy, discuss advantages of peptide therapeutics, and common strategies in identifying targets for viral inhibition.

Self-assembling Peptide Hydrogels Facilitate Vascularization in Two-Component Scaffolds.

One of the major constraints against using polymeric scaffolds as tissue-regenerative matrices is a lack of adequate implant vascularization. Self-assembling peptide hydrogels can sequester small molecules and biological macromolecules, and they can support infiltrating cells in vivo. Here we demonstrate the ability of self-assembling peptide hydrogels to facilitate angiogenic sprouting into polymeric scaffolds after subcutaneous implantation. We constructed two-component scaffolds that incorporated microporous polymeric scaffolds and viscoelastic nanoporous peptide hydrogels. Nanofibrous hydrogels modified the biocompatibility and vascular integration of polymeric scaffolds with microscopic pores (pore diameters: 100-250 µm). In spite of similar amphiphilic sequences, charges, secondary structures, and supramolecular nanostructures, two soft hydrogels studied herein had different abilities to aid implant vascularization, but had similar levels of cellular infiltration. The functional difference of the peptide hydrogels was predicted by the difference in the bioactive moieties inserted into the primary sequences of the peptide monomers. Our study highlights the utility of soft supramolecular hydrogels to facilitate host-implant integration and control implant vascularization in biodegradable polyester scaffolds in vivo. Our study provides useful tools in designing multi-component regenerative scaffolds that recapitulate vascularized architectures of native tissues.

Angiogenic hydrogels for dental pulp revascularization.

Angiogenesis is critical for tissue healing and regeneration. Promoting angiogenesis in materials implanted within dental pulp after pulpectomy is a major clinical challenge in endodontics. We demonstrate the ability of acellular self-assembling peptide hydrogels to create extracellular matrix mimetic architectures that guide in vivo development of neovasculature and tissue deposition. The hydrogels possess facile injectability, as well as sequence-level functionalizability. We explore the therapeutic utility of an angiogenic hydrogel to regenerate vascularized pulp-like soft tissue in a large animal (canine) orthotopic model. The regenerated soft tissue recapitulates key features of native pulp, such as blood vessels, neural filaments, and an odontoblast-like layer next to dentinal tubules. Our study establishes angiogenic peptide hydrogels as potent scaffolds for promoting soft tissue regeneration in vivo. STATEMENT OF SIGNIFICANCE: A major challenge to endodontic tissue engineering is the lack of in situ angiogenesis within intracanal implants, especially after complete removal of the dental pulp. The lack of a robust vasculature in implants limit integration of matrices with the host tissue and regeneration of soft tissue. We demonstrate the development of an acellular material that promotes tissue revascularization in vivo without added growth factors, in a preclinical canine model of pulp-like soft-tissue regeneration. Such acellular biomaterials would facilitate pulp revascularization approaches in large animal models, and translation into human clinical trials.

A 3D Bioprinted Material That Recapitulates the Perivascular Bone Marrow Structure for Sustained Hematopoietic and Cancer Models.

Translational medicine requires facile experimental systems to replicate the dynamic biological systems of diseases. Drug approval continues to lag, partly due to incongruencies in the research pipeline that traditionally involve 2D models, which could be improved with 3D models. The bone marrow (BM) poses challenges to harvest as an intact organ, making it difficult to study disease processes such as breast cancer (BC) survival in BM, and to effective evaluation of drug response in BM. Furthermore, it is a challenge to develop 3D BM structures due to its weak physical properties, and complex hierarchical structure and cellular landscape. To address this, we leveraged 3D bioprinting to create a BM structure with varied methylcellulose (M): alginate (A) ratios. We selected hydrogels containing 4% (w/v) M and 2% (w/v) A, which recapitulates rheological and ultrastructural features of the BM while maintaining stability in culture. This hydrogel sustained the culture of two key primary BM microenvironmental cells found at the perivascular region, mesenchymal stem cells and endothelial cells. More importantly, the scaffold showed evidence of cell autonomous dedifferentiation of BC cells to cancer stem cell properties. This scaffold could be the platform to create BM models for various diseases and also for drug screening.

In vivo Neuroprotective Effect of a Self-assembled Peptide Hydrogel.

Traumatic brain injury (TBI) is associated with poor intrinsic healing responses and long-term cognitive decline. A major pathological outcome of TBI is acute glutamate-mediated excitotoxicity (GME) experienced by neurons. Short peptides based on the neuroprotective extracellular glycoprotein ependymin have shown the ability to slow down the effect of GME - however, such short peptides tend to diffuse away from target sites after in vivo delivery. We have designed a self-assembling peptide containing an ependymin mimic that can form nanofibrous matrices. The peptide was evaluated in situ to assess neuroprotective utility after an acute fluidpercussion injury. This biomimetic matrix can conform to the intracranial damaged site after delivery, due its shear-responsive rheological properties. We demonstrated the potential efficacy of the peptide for supporting neuronal survival in vitro and in vivo. Our study demonstrates the potential of these implantable acellular hydrogels for managing the acute (up to 7 days) pathophysiological sequelae after traumatic brain injury. Further work is needed to evaluate less invasive administrative routes and long-term functional and behavioral improvements after injury.

Implantable anti-angiogenic scaffolds for treatment of neovascular ocular pathologies.

The retinal physiology can accrue oxidative damage and inflammatory insults due to age and metabolic irregularities. Two notable diseases that involve retinal and choroidal neovascularization are proliferative diabetic retinopathy and wet age-related macular degeneration. Currently, these diseases are mainly treated with anti-VEGF drugs (VEGF = vascular endothelial growth factor), generally on a monthly dosage scheme. We discuss recent developments for the treatment of these diseases, including bioactive tissue-engineered materials, which may reduce frequency of dosage and propose a path forward for improving patient outcomes. Graphical abstract Development of materials for long-term intravitreal delivery for management of posterior segment diseases.

A self-assembled peptide hydrogel for cytokine sequestration.

Cytokine-directed monocyte infiltration is involved in multiple pathological processes. Immuno-isolating matrices that can sequester cell-released chemokines in a microenvironment may prolong the viability and functionality of implanted materials. We describe a self-assembling peptide-based hydrogel that can capture the cytokine CCL2 released in the extracellular space by immune cells and stromal cells. The shear-responsive matrix can absorb and retain this signaling molecule needed for the chemotaxis of the infiltrating monocytes and their differentiation into phagocytic macrophages. Such cytokine-sequestering biomaterials may be useful as adjunctive materials with the delivery of exogenous implants or cell suspensions for tissue regeneration, without the administration of systemic immunosuppressants. Our work highlights the versatility of nanofibrous peptide hydrogels for modulating the biological response in tissue niches.

Evaluation of Injectable Naloxone-Releasing Hydrogels.

The opioid epidemic in the United States is a serious public health crisis affecting over 1.7 million Americans. In the last two decades, almost 450 000 people have died from an opioid overdose, with nearly 20% of these deaths occurring in 2017 and 2018 alone. During an overdose, overstimulation of the µ-opioid receptor leads to severe and potentially fatal respiratory depression. Naloxone is a competitive µ-opioid-receptor antagonist that is widely used to displace opioids and rescue from an overdose. Here, we describe the development of a slow-release, subcutaneous naloxone formulation for potential management of opioid overdose, chronic pain, and opioid-induced constipation. Naloxone is loaded into self-assembling peptide hydrogels for controlled drug release. The mechanical, chemical, and structural properties of the nanofibrous hydrogel enable subcutaneous administration and slow, diffusion-based release kinetics of naloxone over 30 days in vitro. The naloxone hydrogel scaffold showed cytocompatibility and did not alter the ß-sheet secondary structure or thixotropic properties characteristic of self-assembling peptide hydrogels. Our results show that this biocompatible and injectable self-assembling peptide hydrogel may be useful as a vehicle for tunable, sustained release of therapeutic naloxone. This therapy may be particularly suited for preventing renarcotization in patients who refuse additional medical assistance following an overdose.

Challenges in Translating from Bench to Bed-Side: Pro-Angiogenic Peptides for Ischemia Treatment.

We describe progress and obstacles in the development of novel peptide-hydrogel therapeutics for unmet medical needs in ischemia treatment, focusing on the development and translation of therapies specifically in peripheral artery disease (PAD). Ischemia is a potentially life-threatening complication in PAD, which affects a significant percentage of the elderly population. While studies on inducing angiogenesis to treat PAD were started two decades ago, early results from animal models as well as clinical trials have not yet been translated into clinical practice. We examine some of the challenges encountered during such translation. We further note the need for sustained angiogenic effect involving whole growth factor, gene therapy and synthetic growth factor strategies. Finally, we discuss the need for tissue depots for de novo formation of microvasculature. These scaffolds can act as templates for neovasculature development to improve circulation and healing at the preferred anatomical location.

Membrane-Disrupting Nanofibrous Peptide Hydrogels.

Self-assembled peptide nanofibers can form biomimetic hydrogels at physiological pH and ionic strength through noncovalent and reversible interactions. Inspired by natural antimicrobial peptides, we designed a class of cationic amphiphilic self-assembled peptides (CASPs) that self-assemble into thixotropic nanofibrous hydrogels. These constructs employ amphiphilicity and high terminal charge density to disrupt bacterial membranes. Here, we focus on three aspects of the self-assembly of these hydrogels: (a) the material properties of the individual self-assembled nanofibers, (b) emergence of bulk-scale elasticity in the nanofibrous hydrogel, and (c) trade-off between the desirable material properties and antimicrobial efficacy. The design of the supramolecular nanofibers allows for higher-order noncovalent ionic cross-linking of the nanofibers into a viscoelastic network. We determine the stiffness of the self-assembled nanofibers via the peak force quantitative nanomechanical atomic force microscopy and the bulk-scale rheometry. The storage moduli depend on peptide concentration, ionic strength, and concentration of multivalent ionic cross-linker. CASP nanofibers are demonstrated to be effective against Pseudomonas aeruginosa colonies. We use nanomechanical analysis and microsecond-time scale coarse-grained simulation to elucidate the interaction between the peptides and bacterial membranes. We demonstrate that the membranes stiffen, contract, and buckle after binding to peptide nanofibers, allowing disruption of osmotic equilibrium between the intracellular and extracellular matrix. This is further associated with dramatic changes in cell morphology. Our studies suggest that self-assembled peptide nanofibrils can potentially acts as membrane-disrupting antimicrobial agents, which can be formulated as injectable hydrogels with tunable material properties.

Angiogenic Self-Assembling Peptide Scaffolds for Functional Tissue Regeneration.

Implantation of acellular biomimetic scaffolds with proangiogenic motifs may have exciting clinical utility for the treatment of ischemic pathologies such as myocardial infarction. Although direct delivery of angiogenic proteins is a possible treatment option, smaller synthetic peptide-based nanostructured alternatives are being investigated due to favorable factors, such as sustained efficacy and high-density epitope presentation of functional moieties. These peptides may be implanted in vivo at the site of ischemia, bypassing the first-pass metabolism and enabling long-term retention and sustained efficacy. Mimics of angiogenic proteins show tremendous potential for clinical use. We discuss possible approaches to integrate the functionality of such angiogenic peptide mimics into self-assembled peptide scaffolds for application in functional tissue regeneration.

Self-Assembly of a Dentinogenic Peptide Hydrogel.

Current standard of care for treating infected dental pulp, root canal therapy, retains the physical properties of the tooth to a large extent, but does not aim to rejuvenate the pulp tissue. Tissue-engineered acellular biomimetic hydrogels have great potential to facilitate the regeneration of the tissue through the recruitment of autologous stem cells. We propose the use of a dentinogenic peptide that self-assembles into ß-sheet-based nanofibers that constitute a biodegradable and injectable hydrogel for support of dental pulp stem cells. The peptide backbone contains a ß-sheet-forming segment and a matrix extracellular phosphoglycoprotein mimic sequence at the C-terminus. The high epitope presentation of the functional moiety in the self-assembled nanofibers may enable recapitulation of a functional niche for the survival and proliferation of autologous cells. We elucidated the hierarchical self-assembly of the peptide through biophysical techniques, including scanning electron microscopy and atomic force microscopy. The material property of the self-assembled hydrogel was probed though oscillatory rheometry, demonstrating its thixotropic nature. We also demonstrate the cytocompatibility of the hydrogel with respect to fibroblasts and dental pulp stem cells. The self-assembled peptide platform holds promise for guided dentinogenesis and it can be tailored to a variety of applications in soft tissue engineering and translational medicine in the future.