Peptide Engineering Research Articles

Future applications of cyclic antimicrobial peptides in drug delivery.

Cyclic antimicrobial peptides (CAMPs) are gaining attention as promising candidates in advanced drug delivery systems due to their structural stability, resistance to proteolytic degradation, and versatile therapeutic potential. Their unique properties enable applications that extend beyond combating multidrug-resistant (MDR) pathogens. Their amphipathic and cell-penetrating properties allow them to efficiently transport drugs across cellular membranes. This review explores the structural advantages and mechanisms of action of CAMPs, emphasizing their role in drug delivery. The literature analysis (2010-2024) from PubMed, Scopus, and Web of Science highlights developments in CAMP-conjugated therapies, liposomal formulations, and encapsulation systems. The review also examines their antimicrobial potency, amphipathic and cell-penetrating properties, and integration into nanocarrier technologies to enhance drug stability, bioavailability, and precision targeting. Challenges such as toxicity, scalability, and cost are also discussed. CAMPs have the potential to revolutionize drug delivery through their robustness and multifunctionality, particularly in precision medicine. Future advancements in peptide engineering, nanotechnology, and AI-driven design are expected to enhance CAMPs' therapeutic specificity, reduce toxicity, and broaden their applications, including oncology and gene therapy, paving the way for their integration into next-generation therapeutics.

Structure-guided engineering of a mutation-tolerant inhibitor peptide against variable SARS-CoV-2 spikes

Pathogen mutations present an inevitable and challenging problem for therapeutics and the development of mutation-tolerant anti-infective drugs to strengthen global health and combat evolving pathogens is urgently needed. While spike proteins on viral surfaces are attractive targets for preventing viral entry, they mutate frequently, making it difficult to develop effective therapeutics. Here, we used a structure-guided strategy to engineer an inhibitor peptide against the SARS-CoV-2 spike, called CeSPIACE, with mutation-tolerant and potent binding ability against all variants to enhance affinity for the invariant architecture of the receptor-binding domain (RBD). High-resolution structures of the peptide complexed with mutant RBDs revealed a mechanism of mutation-tolerant inhibition. CeSPIACE bound major mutant RBDs with picomolar affinity and inhibited infection by SARS-CoV-2 variants in VeroE6/TMPRSS2 cells (IC50 4 pM to 13 nM) and demonstrated potent in vivo efficacy by inhalation administration in hamsters. Mutagenesis analyses to address mutation risks confirmed tolerance against existing and/or potential future mutations of the RBD. Our strategy of engineering mutation-tolerant inhibitors may be applicable to other infectious diseases.

Revisiting the potential of natural antimicrobial peptides against emerging respiratory viral disease: a review.

This review assesses the antiviral capabilities of antimicrobial peptides (AMPs) against SARS-CoV-2 and other respiratory viruses, focussing on their therapeutic potential. AMPs, derived from natural sources, exhibit promising antiviral properties by disrupting viral membranes, inhibiting viral entry, and modulating host immune responses. Preclinical studies demonstrate that peptides such as defensins, cathelicidins, and lactoferrin can effectively reduce SARS-CoV-2 replication and inhibit viral spread. In addition, AMPs have shown potential in enhancing the host's antiviral immunity. Despite these promising outcomes, several challenges require assessments before transforming into clinical translation. Several issues related to peptide stability, cytotoxicity, and efficient delivery systems pose significant limitations to their therapeutic application. Recent advancements in peptide engineering, nanotechnology-based delivery systems, and peptide conjugation strategies have improved AMPs stability and bioavailability; however, further optimization is essential. Moreover, whilst AMPs are safe, their effects on host cells and tissues need a thorough investigation to minimise potential adverse reactions. This review concludes that whilst AMPs present a promising route for antiviral therapies, particularly in targeting SARS-CoV-2, extensive clinical trials and additional studies are required to overcome current limitations. Future research should focus on developing more stable, less toxic AMPs formulations with enhanced delivery mechanisms, aiming to integrate AMPs into viable therapeutic options for respiratory viral diseases, including COVID-19 and other emerging infections.

Harnessing Non-Antibiotic Strategies to Counter Multidrug-Resistant Clinical Pathogens with Special Reference to Antimicrobial Peptides and Their Coatings.

Antimicrobial resistance is a critical global challenge in the 21st century, validating Sir Alexander Fleming's warning about the misuse of antibiotics leading to resistant microbes. With a dwindling arsenal of effective antibiotics, it is imperative to concentrate on alternative antimicrobial strategies. Previous studies have not comprehensively discussed the advantages and limitations of various strategies, including bacteriophage therapy, probiotics, immunotherapies, photodynamic therapy, essential oils, nanoparticles and antimicrobial peptides (AMPs) within a single review. This review addresses that gap by providing an overview of these various non-antibiotic antimicrobial strategies, highlighting their pros and cons, with a particular emphasis on antimicrobial peptides (AMPs). We explore the mechanism of action of AMPs against bacteria, viruses, fungi and parasites. While these peptides hold significant promise, their application in mainstream drug development is hindered by challenges such as low bioavailability and potential toxicity. However, advancements in peptide engineering and chemical modifications offer solutions to enhance their clinical utility. Additionally, this review presents updates on strategies aimed at improving the cost, stability and selective toxicity of AMPs through the development of peptidomimetics. These molecules have demonstrated effective activity against a broad range of pathogens, making them valuable candidates for integration into surface coatings to prevent device-associated infections. Furthermore, we discuss various approaches for attaching and functionalising these peptides on surfaces. Finally, we recommend comprehensive in vivo studies to evaluate the efficacy of AMPs and their mimetics, investigate their synergistic combinations with other molecules and assess their potential as coatings for medical devices.

Improvement in XIa Selectivity of Snake Venom Peptide Analogue BF9-N17K Using P2' Amino Acid Replacements.

Coagulation factor XIa is a new serine-protease family drug target for next-generation anticoagulants. With the snake venom Kunitz-type peptide BF9 as the scaffold, we obtained a highly active XIa inhibitor BF9-N17K in our previous work, but it also inhibited the hemostatic target plasmin. Here, in order to enhance the selectivity of BF9-N17K toward XIa, four mutants, BF9-N17K-L19A, BF9-N17K-L19S, BF9-N17K-L19D, and BF9-N17K-L19K, were further designed using the P2' amino acid classification scanning strategy. The anticoagulation assay showed that the four P2' single-point mutants still had apparent inhibitory anticoagulation activity that selectively inhibited the human intrinsic coagulation pathway and had no influence on the extrinsic coagulation pathway or common coagulation pathway, which indicated that the single-point mutants had minimal effects on the anticoagulation activity of BF9-N17K. Interestingly, the enzyme inhibitor assay experiments showed that the XIa and plasmin inhibitory activities were significantly changed by the P2' amino acid replacements. The XIa inhibitory activity of BF9-N17K-L19D was apparently enhanced, with an IC50 of 19.28 ± 2.53 nM, and its plasmin inhibitory was significantly weakened, with an IC50 of 459.33 ± 337.40 nM. BF9-N17K-L19K was the opposite to BF9-N17K-L19D, which had enhanced plasmin inhibitory activity and reduced XIa inhibitory activity. For BF9-N17K-L19A and BF9-N17K-L19S, no apparent changes were found in the serine protease inhibitory activity, and they had similar XIa and plasmin inhibitory activities to the template peptide BF9-N17K. These results suggested that the characteristics of the charge of the P2' site might be associated with the drug selectivity between the anticoagulant target XIa and hemostatic target plasmin. In addition, according to the molecular diversity and sequence conservation, a common motif GR/PCR/KA/SXIP-XYGGC is proposed in the XIa-inhibitory Kunitz-type peptides, which might provide a new clue for further peptide engineering. In conclusion, through P2' amino acid classification scanning with the snake venom Kunitz-type peptide scaffold, a new potent and selective XIa inhibitor, BF9-N17K-L19D, was discovered, which provides a new XIa-targeting lead drug template for the treatment of thrombotic-related diseases.

Rational engineering of an antimalarial peptide with enhanced proteolytic stability and preserved parasite invasion inhibitory activity.

We describe rational chemical engineering to enhance the proteolytic stability of a chimeric peptide using a combination of unique strategies that involve the incorporation of a series of d-amino acids into the parent l-peptide sequence and restricting the conformational freedom of the peptide by covalent stitching. We hypothesize that replacing a stretch of sequence of an unstructured peptide motif with d-amino acids would increase its proteolytic stability without significantly affecting its affinity to the target protein. Also, considering the Cβ-Cβ distances, replacing an appropriate pair of residues with cysteine to form an additional disulfide bond in the molecule would provide additional stability to the engineered peptide. To verify this hypothesis, we have implemented these strategies to a previously reported peptidic inhibitor RR, against P. falciparum invasion into red blood cells (RBCs) and designed two novel heterochiral chimeric peptides, RR-I and RR-II. We have demonstrated that these peptides exhibit remarkable inhibitory activity with dramatically enhanced proteolytic stability. Finally, we have designed a cyclic analog, RR-III, to enhance the stability of the peptide against endopeptidases. The RR-III peptide exhibits the same inhibitory activity as RR-II while demonstrating impressive resistance to enzymatic degradation and prolonged stability in human plasma. These developments hold promise for a new generation of peptide-based therapeutics, showcasing the potential of residue selection for tailored modifications, as demonstrated in this work.

Enhanced pullulanase production through expression system optimization and biofilm-immobilized fermentation strategies.

Pullulanase (PUL) plays a crucial role in breaking down α-1,6-glycosidic bonds in starch, a key process in starch processing and conversion. Based on PulB with high enzymatic activity, the expression of PUL in Bacillus subtilis was enhanced by plasmid screening, double promoter optimization, and signal peptide engineering. Furthermore, we innovatively employed a mussel foot protein to enhance the cell adhesion to carriers and utilized biofilm-based cell immobilization technology to optimize the fermentation process and stimulate biofilm formation. This approach led to a notably elevated enzyme activity, reaching 2233.56 U mL-1. The PUL crude enzyme solution, capable of generating high glucose syrup and resistant starch, paves the way for new avenues of exploration and advancement in research and industrial biotechnology.

An efficient and easily obtainable butelase variant for chemoenzymatic ligation and modification of peptides and proteins

The expanding field of site-specific ligation of proteins and peptides has catalyzed the development of novel methods that enhance molecular modification. Among these methods, enzymatic strategies have emerged as dominant due to their specificity and efficiency in modifying proteins under mild conditions. Asparaginyl endopeptidase is a group of cyclotide-producing cysteine proteases from plants. These plant cysteine proteases, known for their specificity, effectively recognize the tripeptide motif (Asx-Xaa-Yaa) and cleave at the C-terminal side of Asx residues, forming acyl-enzyme intermediates that facilitate transpeptidation. Butelase 1 stands out as the most efficient AEP for protein engineering, yet challenges in its expression and purification limit its accessibility for widespread research and industrial use. To address these challenges, we engineered a new, catalytically efficient variant of Butelase 1, Butelase AY, by mutating the gatekeeping residues Val237Ala and Thr238Tyr within the LAD-1 region. These modifications significantly enhanced the stability and yield of Butelase AY, allowing for successful application in various peptide and protein engineering tasks. Butelase AY was tested on the peptide GLGKY, the globular protein GFP, and the intrinsically disordered protein α-synuclein, effectively labeling them with a fluorescent probe. Notably, Butelase AY maintained its efficiency with substrates containing unnatural amino acids, making it a promising candidate for biorthogonal applications. Importantly, the mutations did not compromise the enzyme’s specificity, as it continued to process model peptides and native protein substrates with N-term NHV recognition motifs effectively. In conclusion, Butelase AY presents a novel recombinant tool for diverse protein labeling and modifications, particularly in biorthogonal strategies. This innovation has the potential to expand applications in biotechnology and therapeutic development, ultimately revolutionizing protein engineering and its utility in synthetic biology.

Modular Tunable α‐Helical Peptide Hydrogels for Neuronal Cells

AbstractThe assembly of synthetic modules into user‐defined hydrogels for cell culture and clinical applications carries significant advantages over naturally derived materials. Modularity is exploited in synthetic‐polymer‐based hydrogels to alter their physical properties, and spatiotemporally incorporate signaling peptides and growth factors. By contrast, the design of self‐assembling peptide hydrogels has focused largely on assessing cellular responses to unmodified hydrogels and introducing small cell‐adhesive ligands. In short, the modularity of peptide hydrogels is yet to be fully exploited. Previously, hydrogelating self‐assembling fibers (hSAFs) are reported in which two de novo designed α‐helical peptides form gels when mixed. Here, rational peptide design and engineering are used to alter the elastic properties of these hydrogels, to introduce adhesion peptides and growth factors, and to spatially pattern proteins within them. Analysis of the viability and morphology of primary rat neurons cultured on these hydrogels indicates that the modularity of the hSAF system provides a flexible platform for manipulating cell behavior, with potential future applications for modeling natural systems in vitro and regenerative medicine in vivo.

A Mechanistic Rationale for Incretin-Based Therapeutics in the Management of Obesity.

Driven by increased caloric intake relative to expenditure, obesity is a major health concern placing economic and operational strain on healthcare and social care worldwide. Pharmacologically, one of the most effective avenues for the management of excess adiposity is the suppression of appetite. However, owing to the body's natural physiological defense to weight loss and tolerability issues that typically accompany anorectic agents, leveraging this approach to induce sustained weight loss is often easier said than done. As such, to address these challenges, researchers have coupled a thorough understanding of the gut-brain axis with advancements in peptide engineering to design therapeutics mimicking the actions of endocrine hormones to promote a negative energy balance. Indeed, multireceptor agonists targeting the GLP-1, GIP, and glucagon receptors produce meaningful weight loss in people with obesity. Herein, we provide a rationale for how activation of the GIP receptor in the brain and the glucagon receptor in the liver and adipose tissue functions to synergize with GLP-1 receptor agonism to curb the drive to feed and ignite the combustion of excess calories for providing next-generation weight loss.

Insight into Protein Engineering: From In silico Modelling to In vitro Synthesis.

Protein engineering alters the polypeptide chain to obtain a novel protein with improved functional properties. This field constantly evolves with advanced in silico tools and techniques to design novel proteins and peptides. Rational incorporating mutations, unnatural amino acids, and post-translational modifications increases the applications of engineered proteins and peptides. It aids in developing drugs with maximum efficacy and minimum side effects. Currently, the engineering of peptides is gaining attention due to their high stability, binding specificity, less immunogenic, and reduced toxicity properties. Engineered peptides are potent candidates for drug development due to their high specificity and low cost of production compared with other biologics, including proteins and antibodies. Therefore, understanding the current perception of designing and engineering peptides with the help of currently available in silico tools is crucial. This review extensively studies various in silico tools available for protein engineering in the prospect of designing peptides as therapeutics, followed by in vitro aspects. Moreover, a discussion on the chemical synthesis and purification of peptides, a case study, and challenges are also incorporated.

β-Silyl alkynoates: Versatile reagents for biocompatible and selective amide bond formation.

The study introduces a previously unidentified method for amide bond formation that addresses several limitations of conventional approaches. It uses the β-silyl alkynoate molecule, where the alkynyl group activates the ester for efficient amide formation, while the bulky TIPS (triisopropylsilane) group prevents unwanted 1,4-addition reactions. This approach exhibits high chemoselectivity for amines, making the method compatible with a wide range of substrates, including secondary amines, and targets the specific ε-amino group of lysine among the native amino ester's derivatives. It maintains stereochemistry during amide bond formation and TIPS group removal, allowing a versatile platform for postsynthesis modifications such as click reactions and peptide-drug conjugations. These advancements hold substantial promise for pharmaceutical development and peptide engineering, opening avenues for research applications.

Optimizing Nav1.7-Targeted Analgesics: Revealing Off-Target Effects of Spider Venom-Derived Peptide Toxins and Engineering Strategies for Improvement.

The inhibition of Nav1.7 is a promising strategy for the development of analgesic treatments. Spider venom-derived peptide toxins are recognized as significant sources of Nav1.7 inhibitors. However, their development has been impeded by limited selectivity. In this study, eight peptide toxins from three distinct spider venom Nav channel families demonstrated robust inhibition of hNav1.7, rKv4.2, and rKv4.3 (rKv4.2/4.3) currents, exhibiting a similar mode of action. The analysis of structure and function relationship revealed a significant overlap in the pharmacophore responsible for inhibiting hNav1.7 and rKv4.2 by HNTX-III, although Lys25 seems to play a more pivotal role in the inhibition of rKv4.2/4.3. Pharmacophore-guided rational design is employed for the development of an mGpTx1 analogue, mGpTx1-SA, which retains its inhibition of hNav1.7 while significantly reducing its inhibition of rKv4.2/4.3 and eliminating cardiotoxicity. Moreover, mGpTx1-SA demonstrates potent analgesic effects in both inflammatory and neuropathic pain models, accompanied by an improved in vivo safety profile. The results suggest that off-target inhibition of rKv4.2/4.3 by specific spider peptide toxins targeting hNav1.7 may arise from a conserved binding motif. This insight promises to facilitate the design of hNav1.7-specific analgesics, aimed at minimizing rKv4.2/4.3 inhibition and associated toxicity, thereby enhancing their suitability for therapeutic applications.

Antimicrobial Peptides and Their Biomedical Applications: A Review.

The emergence of drug resistance genes and the detrimental health effects caused by the overuse of antibiotics are increasingly prominent problems. There is an urgent need for effective strategies to antibiotics or antimicrobial resistance in the fields of biomedicine and therapeutics. The pathogen-killing ability of antimicrobial peptides (AMPs) is linked to their structure and physicochemical properties, including their conformation, electrical charges, hydrophilicity, and hydrophobicity. AMPs are a form of innate immune protection found in all life forms. A key aspect of the application of AMPs involves their potential to combat emerging antibiotic resistance; certain AMPs are effective against resistant microbial strains and can be modified through peptide engineering. This review summarizes the various strategies used to tackle antibiotic resistance, with a particular focus on the role of AMPs as effective antibiotic agents that enhance the host's immunological functions. Most of the recent studies on the properties and impregnation methods of AMPs, along with their biomedical applications, are discussed. This review provides researchers with insights into the latest advancements in AMP research, highlighting compelling evidence for the effectiveness of AMPs as antimicrobial agents.

Discovery and engineering of a novel peptide, Temporin-WY2, with enhanced in vitro and in vivo efficacy against multi-drug resistant bacteria

Infections by drug-resistant microorganisms are a threat to global health and antimicrobial peptides are considered to be a new hope for their treatment. Temporin-WY2 was identified from the cutaneous secretion of the Ranidae frog, Amolops wuyiensis. It presented with a potent anti-Gram-positive bacterial efficacy, but its activity against Gram-negative bacteria and cancer cell lines was unremarkable. Also, it produced a relatively high lytic effect on horse erythrocytes. For further improvement of its functions, a perfect amphipathic analogue, QUB-1426, and two lysine-clustered analogues, 6K-WY2 and 6K-1426, were synthesised and investigated. The modified peptides were found to be between 8- and 64-fold more potent against Gram-negative bacteria than the original peptide. Additionally, the 6K analogues showed a rapid killing rate. Also, their antiproliferation activities were more than 100-fold more potent than the parent peptide. All of the peptides that were examined demonstrated considerable biofilm inhibition activity. Moreover, QUB-1426, 6K-WY2 and 6K-1426, demonstrated in vivo antimicrobial activity against MRSA and E. coli in an insect larvae model. Despite observing a slight increase in the hemolytic activity and cytotoxicity of the modified peptides, they still demonstrated a improved therapeutic index. Overall, QUB-1426, 6K-WY2 and 6K-1426, with dual antimicrobial and anticancer functions, are proposed as putative drug candidates for the future.

Efficiently directing differentiation and homing of mesenchymal stem cells to boost cartilage repair in osteoarthritis via a nanoparticle and peptide dual-engineering strategy

Mesenchymal stem cells (MSCs) are expected to be useful therapeutics in osteoarthritis (OA), the most common joint disorder characterized by cartilage degradation. However, evidence is limited with regard to cartilage repair in clinical trials because of the uncontrolled differentiation and weak cartilage-targeting ability of MSCs after injection. To overcome these drawbacks, here we synthesized CuO@MSN nanoparticles (NPs) to deliver Sox9 plasmid DNA (favoring chondrogenesis) and recombinant protein Bmp7 (inhibiting hypertrophy). After taking up CuO@MSN/Sox9/Bmp7 (CSB NPs), the expressions of chondrogenic markers were enhanced while hypertrophic markers were decreased in response to these CSB-engineered MSCs. Moreover, a cartilage-targeted peptide (designated as peptide W) was conjugated onto the surface of MSCs via a click chemistry reaction, thereby prolonging the residence time of MSCs in both the knee joint cavity of mice and human-derived cartilage. In a surgery-induced OA mouse model, the NP and peptide dual-modified W-CSB-MSCs showed an enhancing therapeutic effect on cartilage repair in knee joints compared with other engineered MSCs after intra-articular injection. Most importantly, W-CSB-MSCs accelerated cartilage regeneration in damaged cartilage explants derived from OA patients. Thus, this new peptide and NPs dual engineering strategy shows potential for clinical applications to boost cartilage repair in OA using MSC therapy.

Molluscs toxin peptides in cardiovascular disease management

This letter emphasizes the potential of mollusc toxin peptides as innovative treatments for cardiovascular diseases (CVDs). Given the rising incidence of CVDs and the limitations of current therapies, new approaches are essential. Molluscs produce bioactive peptides that show promising anti-hypertensive, anti-thrombotic, and cardio-protective properties. For example, conotoxins from marine snails inhibit voltage-gated calcium channels, indicating their potential as anti-hypertensive agents. Advancements in peptide engineering can address challenges related to bioavailability and stability. Increased research and collaboration are needed to investigate these peptides’ mechanisms, therapeutic potential, and safety, which could lead to groundbreaking CVD treatments.

Proline Analogues.

Within the canonical repertoire of the amino acid involved in protein biogenesis, proline plays a unique role as an amino acid presenting a modified backbone rather than a side-chain. Chemical structures that mimic proline but introduce changes into its specific molecular features are defined as proline analogues. This review article summarizes the existing chemical, physicochemical, and biochemical knowledge about this peculiar family of structures. We group proline analogues from the following compounds: substituted prolines, unsaturated and fused structures, ring size homologues, heterocyclic, e.g., pseudoproline, and bridged proline-resembling structures. We overview (1) the occurrence of proline analogues in nature and their chemical synthesis, (2) physicochemical properties including ring conformation and cis/trans amide isomerization, (3) use in commercial drugs such as nirmatrelvir recently approved against COVID-19, (4) peptide and protein synthesis involving proline analogues, (5) specific opportunities created in peptide engineering, and (6) cases of protein engineering with the analogues. The review aims to provide a summary to anyone interested in using proline analogues in systems ranging from specific biochemical setups to complex biological systems.

A new mRNA structure prediction based approach to identifying improved signal peptides for bone morphogenetic protein 2

BackgroundSignal peptide (SP) engineering has proven able to improve production of many proteins yet is a laborious process that still relies on trial and error. mRNA structure around the translational start site is important in translation initiation and has rarely been considered in this context, with recent improvements in in silico mRNA structure potentially rendering it a useful predictive tool for SP selection. Here we attempt to create a method to systematically screen candidate signal peptide sequences in silico based on both their nucleotide and amino acid sequences. Several recently released computational tools were used to predict signal peptide activity (SignalP), localization target (DeepLoc) and predicted mRNA structure (MXFold2). The method was tested with Bone Morphogenetic Protein 2 (BMP2), an osteogenic growth factor used clinically for bone regeneration. It was hoped more effective BMP2 SPs could improve BMP2-based gene therapies and reduce the cost of recombinant BMP2 production.ResultsAmino acid sequence analysis indicated 2,611 SPs from the TGF-β superfamily were predicted to function when attached to BMP2. mRNA structure prediction indicated structures at the translational start site were likely highly variable. The five sequences with the most accessible translational start sites, a codon optimized BMP2 SP variant and the well-established hIL2 SP sequence were taken forward to in vitro testing. The top five candidates showed non-significant improvements in BMP2 secretion in HEK293T cells. All showed reductions in secretion versus the native sequence in C2C12 cells, with several showing large and significant decreases. None of the tested sequences were able to increase alkaline phosphatase activity above background in C2C12s. The codon optimized control sequence and hIL2 SP showed reasonable activity in HEK293T but very poor activity in C2C12.ConclusionsThese results support the use of peptide sequence based in silico tools for basic predictions around signal peptide activity in a synthetic biology context. However, mRNA structure prediction requires improvement before it can produce reliable predictions for this application. The poor activity of the codon optimized BMP2 SP variant in C2C12 emphasizes the importance of codon choice, mRNA structure, and cellular context for SP activity.

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Progress in the design and synthesis of nanostructured self-assembling systems has facilitated the realization of numerous nanoscale geometries, including fibers, ribbons, and sheets. A key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical macrostructures. Further, we demonstrate the ability to tune the nanostructure of macroscopic hydrogels through modulating phosphate buffer concentration during peptide self-assembly. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an extracellular matrix-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to decouple nanostructure from macrostructure and generate a gradient of anisotropic nanofibrous hydrogels. We anticipate that control of architecture at multiple length scales will be critical for a variety of applications, including the bottom-up tissue engineering explored here.