Sequence-activity mapping via depletion reveals striking mutational tolerance and elucidates functional motifs in Tur1a antimicrobial peptide.

Proline-rich antimicrobial peptides (PrAMPs) are attractive antibiotic candidates that target gram-negative bacteria ribosomes. We elucidated the sequence-function landscape of 43 000 variants of a recently discovered family member, Tur1a, using the validated SAMP-Dep platform that measures intracellular AMP potency in a high-throughput manner via self-depletion of the cellular host. The platform exhibited high replicate reproducibility (ρ = 0.81) and correlation between synonymous genetic variants (R2 = 0.93). Only two segments within Tur1a exhibited stringent mutational requirements to sustain potency: residues 9YLP11 and 19FP20. This includes the aromatic residue in the hypothesized binding domain but not the PRP domain. Along with unexpected mutational tolerance of PRP, the data contrast hypothesized importance of the 1RRIR4 motif and arginines in general. In addition to mutational tolerance of residue segments with presumed significance, 77% of mutations are functionally neutral. Multimutant performance mainly shows compounding effects from removed combinations of prolines and arginines in addition to the two segments of residues showing individual importance. Several variants identified as active from SAMP-Dep were externally produced and maintained activity when applied to susceptible species exogenously.

Sequence-function mapping of proline-rich antimicrobial peptides.

Antimicrobial peptides (AMPs) are essential elements of natural cellular combat and candidates as antibiotic therapy. Elevated function may be needed for robust physiological performance. Yet, both pure protein design and combinatorial library discovery are hindered by the complexity of antimicrobial activity. We applied a recently developed high-throughput technique, sequence-activity mapping of AMPs via depletion (SAMP-Dep), to proline-rich AMPs. Robust self-inhibition was achieved for metalnikowin 1 (Met) and apidaecin 1b (Api). SAMP-Dep exhibited high reproducibility with correlation coefficients 0.90 and 0.92, for Met and Api, respectively, between replicates and 0.99 and 0.96 for synonymous genetic variants. Sequence-activity maps were obtained via characterization of 26,000 and 34,000 mutants of Met and Api, respectively. Both AMPs exhibit similar mutational profiles including beneficial mutations at one terminus, the C-terminus for Met and N-terminus for Api, which is consistent with their opposite binding orientations in the ribosome. While Met and Api reside with the family of proline-rich AMPs, different proline sites exhibit substantially different mutational tolerance. Within the PRP motif, proline mutation eliminates activity, whereas non-PRP prolines readily tolerate mutation. Homologous mutations are more tolerated, particularly at alternating sites on one 'face' of the peptide. Important and consistent epistasis was observed following the PRP domain within the segment that extends into the ribosomal exit tunnel for both peptides. Variants identified from the SAMP-Dep platform were produced and exposed toward Gram-negative species exogenously, showing either increased potency or specificity for strains tested. In addition to mapping sequence-activity space for fundamental insight and therapeutic engineering, the results advance the robustness of the SAMP-Dep platform for activity characterization.

Systematic Evaluation of Protein-Small Molecule Hybrids on the Yeast Surface.

Protein-small molecule hybrids are structures that have the potential to combine the inhibitory properties of small molecules and the specificities of binding proteins. However, achieving such synergies is a substantial engineering challenge with fundamental principles yet to be elucidated. Recent work has demonstrated the power of the yeast display-based discovery of hybrids using a combination of fibronectin-binding domains and thiol-mediated conjugations to introduce small-molecule warheads. Here, we systematically study the effects of expanding the chemical diversity of these hybrids on the yeast surface by investigating a combinatorial set of fibronectins, noncanonical amino acid (ncAA) substitutions, and small-molecule pharmacophores. Our results show that previously discovered thiol-fibronectin hybrids are generally tolerant of a range of ncAA substitutions and retain binding functions to carbonic anhydrases following click chemistry-mediated assembly of hybrids with diverse linker structures. Most surprisingly, we identified several cases where replacement of a potent acetazolamide warhead with a substantially weaker benzenesulfonamide warhead still resulted in the assembly of multiple functional hybrids. In addition to these unexpected findings, we expanded the throughput of our system by validating a 96-well plate-based format to produce yeast-displayed hybrid conjugates in parallel. These efficient explorations of hybrid chemical diversity demonstrate that there are abundant opportunities to expand the functions of protein-small molecule hybrids and elucidate principles that dictate their efficient discovery and design.

Hyperstable Synthetic Mini-Proteins as Effective Ligand Scaffolds.

Small, single-domain protein scaffolds are compelling sources of molecular binding ligands with the potential for efficient physiological transport, modularity, and manufacturing. Yet, mini-proteins require a balance between biophysical robustness and diversity to enable new functions. We tested the developability and evolvability of millions of variants of 43 designed libraries of synthetic 40-amino acid ßαßß proteins with diversified sheet, loop, or helix paratopes. We discovered a scaffold library that yielded hundreds of binders to seven targets while exhibiting high stability and soluble expression. Binder discovery yielded 6-122 nM affinities without affinity maturation and Tms averaging ≥78 °C. Broader ßαßß libraries exhibited varied developability and evolvability. Sheet paratopes were the most consistently developable, and framework 1 was the most evolvable. Paratope evolvability was dependent on target, though several libraries were evolvable across many targets while exhibiting high stability and soluble expression. Select ßαßß proteins are strong starting points for engineering performant binders.

Determinants of Developability and Evolvability of Synthetic Miniproteins as Ligand Scaffolds.

Binding ligands empower molecular therapeutics and diagnostics. Despite an array of protein scaffolds engineered for binding, the biophysical elements that drive developability and evolvability are not fully understood. In particular, engineering novel function while maintaining biophysical integrity within the context of small, single-domain proteins is challenged by integration of the structural framework and the evolved binding site. Miniproteins present a challenge to our limits of protein engineering capability and provide advantages in physiological targeting, modularity for multi-functional constructs, and unique binding modes. Herein, we evaluate the ability of hyperstable synthetic miniproteins, originally designed for foldedness, to function as binding scaffolds. We synthesized 45 combinatorial libraries, with 109 variants, systematically varied across two topologies, each with five starting frameworks and four or five diverse, structurally distinct paratopes, to elucidate their impact on evolvability and developability. We evaluated evolvability with yeast display binding selections against four targets. High-throughput assays -stability via yeast display and soluble expression via split-GFP in E. coli - measured developability. The comprehensive, robust dataset demonstrates how protein topology, parental framework, and paratope structure and location all impact scaffold performance. A hyperstable framework and localized diversity are not sufficient for an effective scaffold, but several designs of these elements within synthetic miniproteins designed solely for stability result in scaffold libraries with effective evolvability and developability. Engineered variants were well-folded, thermally stable, and bound target with single-digit nanomolar affinity. Thus, hyperstable synthetic miniproteins can serve as precursors to developable, evolvable mini-scaffolds with unique potential for physiological transport, modularity, and binding modes.

Discovery of Kinetic Trapping of Poloxamers inside Liposomes via Thermal Treatment.

Poloxamers, a class of biocompatible, commercially available amphiphilic block polymers (ABPs) comprising poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks, interact with phospholipid bilayers, resulting in altered mechanical and surface properties. These block copolymers are useful in a variety of applications including therapeutics for Duchenne muscular dystrophy, as cell membrane stabilizers, and for drug delivery, as liposome surface modifying agents. Hydrogen bonding between water and oxygen atoms in PEO and PPO units results in thermoresponsive behavior because the bound water shell around both blocks dehydrates as the temperature increases. This motivated an investigation of poloxamer-lipid bilayer interactions as a function of temperature and thermal history. In this study, we applied pulsed-field-gradient NMR spectroscopy to measure the fraction of chains bound to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) liposomes between 10 and 50 °C. We measured an (11 ± 3)-fold increase in binding affinity at 37 °C relative to 27 °C. Moreover, following incubation at 37 °C, it takes weeks for the system to re-equilibrate at 25 °C. Such slow desorption kinetics suggests that at elevated temperatures polymer chains can pass through the bilayer and access the interior of the liposomes, a mechanism that is inaccessible at lower temperatures. We propose a molecular mechanism to explain this effect, which could have important ramifications on the cellular distribution of ABPs and could be exploited to modulate the mechanical and surface properties of liposomes and cell membranes.

Predicting and Interpreting Protein Developability Via Transfer of Convolutional Sequence Representation.

Engineered proteins have emerged as novel diagnostics, therapeutics, and catalysts. Often, poor protein developability─quantified by expression, solubility, and stability─hinders utility. The ability to predict protein developability from amino acid sequence would reduce the experimental burden when selecting candidates. Recent advances in screening technologies enabled a high-throughput (HT) developability dataset for 105 of 1020 possible variants of protein ligand scaffold Gp2. In this work, we evaluate the ability of neural networks to learn a developability representation from a HT dataset and transfer this knowledge to predict recombinant expression beyond observed sequences. The model convolves learned amino acid properties to predict expression levels 44% closer to the experimental variance compared to a non-embedded control. Analysis of learned amino acid embeddings highlights the uniqueness of cysteine, the importance of hydrophobicity and charge, and the unimportance of aromaticity, when aiming to improve the developability of small proteins. We identify clusters of similar sequences with increased recombinant expression through nonlinear dimensionality reduction and we explore the inferred expression landscape via nested sampling. The analysis enables the first direct visualization of the fitness landscape and highlights the existence of evolutionary bottlenecks in sequence space giving rise to competing subpopulations of sequences with different developability. The work advances applied protein engineering efforts by predicting and interpreting protein scaffold expression from a limited dataset. Furthermore, our statistical mechanical treatment of the problem advances foundational efforts to characterize the structure of the protein fitness landscape and the amino acid characteristics that influence protein developability.

Protein engineering via sequence-performance mapping.

Discovery and evolution of new and improved proteins has empowered molecular therapeutics, diagnostics, and industrial biotechnology. Discovery and evolution both require efficient screens and effective libraries, although they differ in their challenges because of the absence or presence, respectively, of an initial protein variant with the desired function. A host of high-throughput technologies-experimental and computational-enable efficient screens to identify performant protein variants. In partnership, an informed search of sequence space is needed to overcome the immensity, sparsity, and complexity of the sequence-performance landscape. Early in the historical trajectory of protein engineering, these elements aligned with distinct approaches to identify the most performant sequence: selection from large, randomized combinatorial libraries versus rational computational design. Substantial advances have now emerged from the synergy of these perspectives. Rational design of combinatorial libraries aids the experimental search of sequence space, and high-throughput, high-integrity experimental data inform computational design. At the core of the collaborative interface, efficient protein characterization (rather than mere selection of optimal variants) maps sequence-performance landscapes. Such quantitative maps elucidate the complex relationships between protein sequence and performance-e.g., binding, catalytic efficiency, biological activity, and developability-thereby advancing fundamental protein science and facilitating protein discovery and evolution.

Rapid restitution of contractile dysfunction by synthetic copolymers in dystrophin-deficient single live skeletal muscle fibers.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, a cytoskeletal protein essential for the preservation of the structural integrity of the muscle cell membrane. DMD patients develop severe skeletal muscle weakness, degeneration, and early death. We tested here amphiphilic synthetic membrane stabilizers in mdx skeletal muscle fibers (flexor digitorum brevis; FDB) to determine their effectiveness in restoring contractile function in dystrophin-deficient live skeletal muscle fibers. After isolating FDB fibers via enzymatic digestion and trituration from thirty-three adult male mice (9 C57BL10, 24 mdx), these were plated on a laminin-coated coverslip and treated with poloxamer 188 (P188; PEO75-PPO30-PEO75; 8400 g/mol), architecturally inverted triblock (PPO15-PEO200-PPO15, 10,700 g/mol), and diblock (PEO75-PPO16-C4, 4200 g/mol) copolymers. We assessed the twitch kinetics of sarcomere length (SL) and intracellular Ca2+ transient by Fura-2AM by field stimulation (25 V, 0.2 Hz, 25 °C). Twitch contraction peak SL shortening of mdx FDB fibers was markedly depressed to 30% of the dystrophin-replete control FDB fibers from C57BL10 (P < 0.001). Compared to vehicle-treated mdx FDB fibers, copolymer treatment robustly and rapidly restored the twitch peak SL shortening (all P < 0.05) by P188 (15 µM = + 110%, 150 µM = + 220%), diblock (15 µM = + 50%, 150 µM = + 50%), and inverted triblock copolymer (15 µM = + 180%, 150 µM = + 90%). Twitch peak Ca2+ transient from mdx FDB fibers was also depressed compared to C57BL10 FDB fibers (P < 0.001). P188 and inverted triblock copolymer treatment of mdx FDB fibers increased the twitch peak Ca2+ transient (P < 0.001). This study shows synthetic block copolymers with varied architectures can rapidly and highly effectively enhance contractile function in live dystrophin-deficient skeletal muscle fibers.

Consequences of poly(ethylene oxide) and poloxamer P188 on transcription in healthy and stressed myoblasts.

Poly(ethylene oxide) (PEO) and poloxamers, a class of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, have many personal and medical care applications, including the stabilization of stressed cellular membranes. Despite the widespread use, the cellular transcriptional response to these molecules is relatively unknown. C2C12 myoblasts, a model muscle cell, were subjected to short-term Poloxamer 188 (P188) and PEO181 (8,000 g/mol) treatment in culture. RNA was extracted and sequenced to quantify transcriptomic impact. The addition of moderate concentrations (14 µM) of either polymer to unstressed cells caused substantial differential gene expression, including at least twofold modulation of 357 and 588 genes, respectively. In addition, evaluation of the transcriptome response to osmotic stress without polymer treatment revealed dramatic change in RNA expression. Interestingly, the addition of polymer to stressed cells-at concentrations that provide physiological protection-did not yield a significant difference in expression of any gene relative to stress alone. Genome-scale expression analysis was corroborated by single-gene quantitative real-time PCR. Changes in protein expression were measured via western blot, which revealed partial alignment with the RNA results. Collectively, the significant changes to expression of multiple genes and resultant protein translation demonstrates an unexpectedly broad biochemical response to these polymers in healthy myoblasts in vitro. Meanwhile, the lack of substantial transcriptional response to polymer treatment in stressed cells highlights the physical nature of that protective mechanism.

Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency That Does Not Affect TNFR2 Function.

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.

Molecular homing and retention of muscle membrane stabilizing copolymers by non-invasive optical imaging in vivo.

First-in-class membrane stabilizer Poloxamer 188 (P188) has been shown to confer membrane protection in an extensive range of clinical conditions; however, elements of the systemic distribution and localization of P188 at the organ, tissue, and muscle fiber levels in vivo have not yet been elucidated. Here we used non-invasive fluorescence imaging to directly visualize and track the distribution and localization of P188 in vivo. The results demonstrated that the Alx647 probe did not alter the fundamental properties of P188 to protect biological membranes. Distribution kinetics in mdx mice demonstrated that Alx647 did not interface with muscle membranes and had fast clearance kinetics. In contrast, the distribution kinetics for P188-Alx647 was significantly slower, indicating a dramatic depot and retention effect of P188. Results further demonstrated the significant retention of P188-Alx647 in the skeletal muscle of mdx mice, showing a significant genotype effect with a higher fluorescence signal in the mdx muscles over BL10 mice. High-resolution optical imaging provided direct evidence of P188 surrounding the sarcolemma of skeletal and cardiac muscle cells. Taken together, these findings provide direct evidence of muscle-disease-dependent molecular homing and retention of synthetic copolymers in striated muscles thereby facilitating advanced studies of copolymer-membrane association in health and disease.

Deep Antimicrobial Activity and Stability Analysis Inform Lysin Sequence-Function Mapping.

Antibiotic-resistant infectious disease is a critical challenge to human health. Antimicrobial proteins offer a compelling solution if engineered for potency, selectivity, and physiological stability. Lysins, which lyse cells via degradation of cell wall peptidoglycans, have significant potential to fill this role. Yet, the functional complexity of antimicrobial activity has hindered high-throughput characterization for discovery and design. To dramatically expand knowledge of the sequence-function landscape of lysins, we developed a depletion-based assay for library-scale measurement of lysin inhibitory activity. We coupled this platform with a high-throughput proteolytic stability assay to assess the activity and stability of ∼5 × 104 lysin catalytic domain variants, resulting in the discovery of a variant with increased activity (70 ± 20%) and stability (7.2 ± 0.4 °C increased midpoint of thermal denaturation). Ridge regression of the resulting data set demonstrated that libraries with a higher average Hamming distance better informed pairwise models and that coupling activity and stability assays enabled better prediction of catalytically active lysins. The best models achieved Pearson's correlation coefficients of 0.87 ± 0.01 and 0.61 ± 0.04 for predicting catalytic domain stability and activity, respectively. Our work provides an efficient strategy for constructing protein sequence-function landscapes, drastically increases screening throughput for engineering lysins, and yields promising lysins for further development.

Effect of Bottlebrush Poloxamer Architecture on Binding to Liposomes.

Poloxamers─triblock copolymers consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)─have demonstrated cell membrane stabilization efficacy against numerous types of stress. However, the mechanism responsible for this stabilizing effect remains elusive, hindering engineering of more effective therapeutics. Bottlebrush polymers have a wide parameter space and known relationships between architectural parameters and polymer properties, enabling their use as a tool for mechanistic investigations of polymer-lipid bilayer interactions. In this work, we utilized a versatile synthetic platform to create novel bottlebrush analogues to poloxamers and then employed pulsed-field-gradient NMR and an in vitro osmotic stress assay to explore the effect of bottlebrush architectural parameters on binding to, and protection of, model phospholipid bilayers. We found that the binding affinity of a bottlebrush poloxamer (BBP) (B-E1043P515, Mn ≈ 26 kDa) is about 3 times higher than a linear poloxamer with a similar composition and number of PPO units (L-E93P54E93, Mn ≈ 11 kDa). Furthermore, BBP binding is sensitive to overall molecular weight, side-chain length, and architecture (statistical versus block). Finally, all tested BBPs exhibit a protective effect on cell membranes under stress at sub-µM concentrations. As the factors controlling membrane affinity and protection efficacy of bottlebrush poloxamers are not understood, these results provide important insight into how they adhere to and stabilize a lipid bilayer surface.

Affibody-mediated controlled release of fibroblast growth factor 2.

Protein therapeutics possess high target affinity and specificity, yet short residence times, which limit their broad utility. To overcome this challenge, we used affinity interactions to modulate protein release from a hydrogel delivery vehicle thereby prolonging therapeutic availability. Specifically, we designed an affibody-modified hyaluronan (HA)-based hydrogel as a delivery platform for fibroblast growth factor 2 (FGF2), a neuroprotective and neuroregenerative factor in the central nervous system (CNS). We identified a highly specific affibody binding partner with moderate affinity for FGF2 using yeast surface display and flow cytometry-based screening. Importantly, we demonstrated controlled release of bioactive FGF2 from the hydrogel by varying the ratio of affibody to protein and showed increased thermal stability of FGF2 in the presence of affibody. This versatile delivery platform will allow the distinct, simultaneous release of multiple proteins based on specific affinity interactions.

Directed Evolution Enables Simultaneous Controlled Release of Multiple Therapeutic Proteins from Biopolymer-Based Hydrogels.

With the advent of increasingly complex combination strategies of biologics, independent control over their delivery is the key to their efficacy; however, current approaches are hindered by the limited independent tunability of their release rates. To overcome these limitations, directed evolution is used to engineer highly specific, low affinity affibody binding partners to multiple therapeutic proteins to independently control protein release rates. As a proof-of-concept, specific affibody binding partners for two proteins with broad therapeutic utility: insulin-like growth factor-1 (IGF-1) and pigment epithelium-derived factor (PEDF) are identified. Protein-affibody binding interactions specific to these target proteins with equilibrium dissociation constants (KD ) between 10-7 and 10-8 m are discovered. The affibodies are covalently bound to the backbone of crosslinked hydrogels using click chemistry, enabling sustained, independent, and simultaneous release of bioactive IGF-1 and PEDF over 7 days. The system is tested with C57BL/6J mice in vivo, and the affibody-controlled release of IGF-1 results in sustained activity when compared to bolus IGF-1 delivery. This work demonstrates a new, broadly applicable approach to tune the release of therapeutic proteins simultaneously and independently and thus the way for precise control over the delivery of multicomponent therapies is paved.

Synthesis and Micellization of Bottlebrush Poloxamers.

Bottlebrush polymers are characterized by an expansive parameter space, including graft length and spacing along the backbone, and these features impact various structural and physical properties such as molecular diffusion and bulk viscosity. In this work, we report a synthetic strategy for making grafted block polymers with poly(propylene oxide) and poly(ethylene oxide) side chains, bottlebrush analogues of poloxamers. Combined anionic and sequential ring-opening metathesis polymerization yielded low dispersity polymers, at full conversion of the macromonomers, with control over graft length, graft end-groups, and overall molecular weight. A set of bottlebrush poloxamers (BBPs), with identical graft lengths and composition, was synthesized over a range of molecular weights. Dynamic light scattering and transmission electron microscopy were used to characterize micelle formation in aqueous buffer. The critical micelle concentration scales exponentially with overall molecular weight for both linear and bottlebrush poloxamers; however, the bottlebrush architecture shifts micelle formation to a much higher concentration at a comparable molecular weight. Consequently, BBPs can exist in solution as unimers at significantly higher molecular weights and concentrations than the linear analogues.

Engineered protein-small molecule conjugates empower selective enzyme inhibition.

Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

Concentration Threshold for Membrane Protection by PEO-PPO Block Copolymers with Variable Molecular Architectures.

Poloxamer 188, a poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer, protects cell membranes in several injury models. However, the nature of the copolymer/membrane interaction and the mechanism of membrane protection remain unknown. Systematic variations of the block copolymer architecture - including PPO-PEO-PPO triblocks and PPO-PEO diblocks - were used to probe the mechanism and evaluate the potential for alternative architectures to yield superior protection. To test the polymers, murine myoblasts were subjected to an osmotic stress, and membrane integrity was quantified by measuring lactate dehydrogenase (LDH) leakage. These experiments exposed a concentration threshold effect where all tested polymers reach 50% leakage of LDH compared to a non-treated buffer only control over a narrow concentration range of 0.8-4 µM. Differences in polymer protection at lower concentrations indicate that protection increases with the PPO-PEO-PPO molecular architecture and increasing hydrophobicity.

A Platform for Deep Sequence-Activity Mapping and Engineering Antimicrobial Peptides.

Developing potent antimicrobials, and platforms for their study and engineering, is critical as antibiotic resistance grows. A high-throughput method to quantify antimicrobial peptide and protein (AMP) activity across a broad continuum would be powerful to elucidate sequence-activity landscapes and identify potent mutants. Yet the complexity of antimicrobial activity has largely constrained the scope and mechanistic bandwidth of AMP variant analysis. We developed a platform to efficiently perform sequence-activity mapping of AMPs via depletion (SAMP-Dep): a bacterial host culture is transformed with an AMP mutant library, induced to intracellularly express AMPs, grown under selective pressure, and deep sequenced to quantify mutant depletion. The slope of mutant growth rate versus induction level indicates potency. Using SAMP-Dep, we mapped the sequence-activity landscape of 170 000 mutants of oncocin, a proline-rich AMP, for intracellular activity against Escherichia coli. Clonal validation supported the platform's sensitivity and accuracy. The mapped landscape revealed an extended oncocin pharmacophore contrary to earlier structural studies, clarified the C-terminus role in internalization, identified functional epistasis, and guided focused, successful synthetic peptide library design, yielding a mutant with 2-fold enhancement in both intracellular and extracellular activity. The efficiency of SAMP-Dep poises the platform to transform AMP engineering, characterization, and discovery.