ACS Chemical Biology

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Tools to interrogate glycoconjugate-protein interactions in the context living cells are highly attractive for the identification of critically important functional binding partners of glycan-binding proteins. These interactions are challenging to interrogate due to low affinity and rapid dissociation rates of glycan-protein binding events. The use of photo-crosslinkers to capture glycan-protein interaction complexes has shown great promise for identifying binding partners involved in these interactions. Current methodologies use metabolic oligosaccharide engineering (MOE) to incorporate photo-crosslinking sugars. However, these MOE strategies are not amenable to all cell types and can result in low incorporation and cell-surface display of the photo-crosslinking probe, limiting their utility for studying many types of interactions. We describe here an exo-enzymatic strategy for selectively introducing photo-crosslinking probes into cell-surface glycoconjugates using the recombinant human sialyltransferase ST6GAL1 and a diazirine-linked CMP-Neu5Ac derivative. Probe introduction is highly efficient, amenable to different cell types and resulted in improved crosslinking when compared to MOE. This exo-enzymatic labeling approach can selectively introduce the photo-crosslinking sugar on to specific glycan epitopes and subclasses by harnessing the specificity of the sialyltransferase employed, underscoring its potential as a tool to interrogate and identify glycoconjugate ligands for diverse glycan-binding proteins.

Glycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of D-arabinofuranose (D-Araf), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including Mycobacterium tuberculosis. The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of D-Araf residues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.

Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH, while demonstrating a potentiated inflammatory response when exposed to lipopolysaccharides, corresponding with a rise in histone lactoylation. Interestingly, our data demonstrate that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state, however, upon stimulation, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is a primary contributing factor facilitating the inflammatory response.

Cyclic peptides represent a burgeoning area of interest in therapeutic and biotechnological research. In opposition to their linear counterparts, cyclic peptides, such as certain ribosomally synthesized and post-translationally modified peptides (RiPPs), are more conformationally constrained and less susceptible to proteolytic degradation. The lanthipeptide RiPP cytolysin L forms a covalently enforced helical structure that may be used to disrupt helical interactions at protein-protein interfaces. Herein, an expression system is reported to produce lanthipeptides and structurally diverse cytolysin L derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide inhibitors of native protein-protein interactions. Table of contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/563208v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@aafac8org.highwire.dtl.DTLVardef@13687caorg.highwire.dtl.DTLVardef@10d16caorg.highwire.dtl.DTLVardef@533398_HPS_FORMAT_FIGEXP M_FIG C_FIG

Excessive degradation of the colonic mucin layer by Bacteroides within the human gut microbiota drives inflammatory bowel disease in mice. Bacterial carbohydrate sulfatases are key enzymes in gut colonization, as they are elevated in human inflammatory bowel disease and correlate with disease severity. Selective inhibitors of carbohydrate sulfatases could function as sulfatase-selective drugs, allowing precise control of sulfatase activity while preserving these otherwise beneficial bacteria. Arylsulfamates are covalent inhibitors that target a catalytic formylglycine residue of steroid sulfatases, a residue that is also conserved in carbohydrate sulfatases. Here, we find that a library of aryl- and carbohydrate sulfamates is ineffective against Bacteroides carbohydrate sulfatases, yet can inhibit human gut microbiota species grown on sulfated glycans. Leveraging thermal proteome profiling, we identify a lipid kinase as the target responsible for these effects. This work highlights the imperative for developing specific inhibitors targeting carbohydrate sulfatases and reveals the adverse effects that arylsulfamates have on Bacteroides species of the human gut microbiota. Significance statementArylsulfamates are currently the only effective class of sulfatase inhibitors available and offer a potential strategy to treat inflammatory bowel disease driven by gut microbiota carbohydrate sulfatases. Although arylsulfamates inhibit the growth of microbiota Bacteroides species on sulfated glycans, this is not mediated through carbohydrate sulfatases but, via a conserved lipid kinase. Carbohydrate sulfatases are resistant to arylsulfamates whilst steroid sulfatases are susceptible despite a conserved active site. Finally, selected complex plant glycans confer a resistant/protective phenotype against the harmful effects of arylsulfamates. These data guide the future development of targeted carbohydrate sulfatase inhibitors and potential drug-prebiotic pairings.

One of the major challenges in natural product discovery is the prioritization of compounds with useful activities from microbial sources. In particular, this is a challenge in genome mining for novel natural products, where the structures and activities of compounds produced by bioinformatically identified and uncharacterized biosynthetic gene clusters remain unknown. Here, we utilize a machine learning model to predict the antibacterial activity of a natural product from its biosynthetic gene cluster (BGC). We prioritized the strain Amycolatopsis azurea DSM 43854 which was predicted by machine learning to have the capacity to produce multiple natural products with antibacterial activity. Together with bioactivity-guided fractionation, we isolated dipyrimicins A and B from Amycolatopsis azurea DSM 43854 and, for the first time, linked them to their BGC. This dip BGC was predicted by our model to encode a product with 75% antibacterial probability and shares only 40-52% similarity with previously characterized BGCs. We confirmed the antimicrobial properties of the dipyrimicins against a few test strains and identified key tailoring enzymes, including an O-methyltransferase and amidotransferase, that differentiated them from other related 2,2-bipyridine biosynthetic pathways. Importantly, As the dip BGC was not in the training set of the model, our results demonstrate the ability of the model to generalize beyond its training set and the potential of machine learning to accelerate novel bioactive natural product discovery and deorphanization of biosynthetic gene clusters.

The thalidomide analog lenalidomide is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photo-lenalidomide, a lenalidomide probe with a photo-affinity label and enrichment handle, for target identification by chemical proteomics. After evaluating a series of lenalidomide analogs, we identified a specific amide linkage to lenalidomide that allowed for installation of the desired functionality, while preserving the substrate degradation profile, phenotypic anti-proliferative and immunomodulatory properties of lenalidomide. Photo-lenalidomide maintains these properties by enhancing binding interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using photo-lenalidomide, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells, and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by photolenalidomide and forms a complex with CRBN in the presence of lenalidomide, but is itself not ubiquitylated or degraded. These data point to the potentially broader array of substrates induced by ligands to CRBN that may or may not be degraded, which can be revealed by the highly translatable application of photo-lenalidomide and chemical proteomics in additional biological settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/452075v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1619364org.highwire.dtl.DTLVardef@113a07corg.highwire.dtl.DTLVardef@18d36acorg.highwire.dtl.DTLVardef@8e64_HPS_FORMAT_FIGEXP M_FIG C_FIG

Antimicrobial resistance (AMR) threatens global health; however, the molecular adaptations underlying resistance to emerging antibiotic classes remain poorly defined. Here, we applied long-read DNA and direct RNA nanopore sequencing and developed methods to deconvolute operon-specific epitranscriptomic changes. Together, this platform uncovered a previously unrecognized, operon-specific pathway of resistance in Staphylococcus aureus to the naphthyridone antibiotic A-692345. Genomic nanopore sequencing identified a single 23S rRNA mutation (T1732C) confined to one of the six rRNA operons (operon 2), which uniquely contains nine tRNA genes. RNA direct nanopore sequencing generated a comprehensive and updated rRNA modification map for S. aureus and revealed extensive remodeling of rRNA modifications in the resistant strain. Differentially incorporated modifications included pseudouridine, dihydrouridine, and 5-hydroxycytidine at functionally relevant positions within the ribosome. Upon mapping these epitranscriptomic changes, we noted they were operon specific. This likely gives rise to ribosome heterogeneity with potential for selective translation of stress-response genes that favor resistance. Collectively, these findings establish nanopore sequencing as a powerful platform for resolving coupled genomic and epitranscriptomic adaptations, providing molecular insight into how bacteria evolve resistance to antibiotics.

Antimicrobial peptides commonly act by disrupting bacterial membranes, but also frequently damage mammalian membranes. Deciphering the rules governing membrane selectivity is critical to understanding their function and enabling their therapeutic use. Past attempts to decipher these rules have failed because they cannot interrogate adequate peptide sequence variation. To overcome this problem, we develop deep mutational surface localized antimicrobial display (dmSLAY), which reveals comprehensive positional residue importance and flexibility across an antimicrobial peptide sequence. We apply dmSLAY to Protegrin-1, a potent yet toxic antimicrobial peptide, and identify thousands of sequence variants that positively or negatively influence its antibacterial activity. Further analysis reveals that avoiding large aromatic residues and eliminating disulfide bound cysteine pairs while maintaining membrane bound secondary structure greatly improves Protegrin-1 bacterial specificity. Moreover, dmSLAY datasets enable machine learning to expand our analysis to include over 5.7 million sequence variants and reveal full Protegrin-1 mutational profiles driving either bacterial or mammalian membrane specificity. Our results describe an innovative, high-throughput approach for elucidating antimicrobial peptide sequence-structure-function relationships which can inform synthetic peptide-based drug design.

Lanthipeptides represent the largest group of ribosomally synthesized and post-translationally modified peptides (RiPPs). Lanthipeptides offer promising avenues for discovering new antibacterial and antifungal agents. Here, we identify and structurally analyze the product of the tla BGC, which encodes a class II lanthipeptide in the thermophilic bacterium Thermoactinomyces sp. DSM 45891. Heterologous co-expression of the lanthipeptide synthetase TlaM resulted in modification of the two precursor peptides TlaA1 and TlaA2, which share 58% identity. TlaA1 was dehydrated seven times and TlaA2 six times. In both peptides, four thioether rings were formed with two overlapping DL-(methyl)lanthionine rings at the C-terminus. Both peptides also contain two central and N-terminal non-overlapping DL-methyllanthionines. These findings demonstrate that these peptides deviate from the general rule of stereoselective LL-(methyl)lanthionine formation from a DhxDhxXxxXxxCys motif (Dhx = dehydroalanine or dehydrobutyrine). AspN-cleaved TlaM-modified TlaA1 displayed anti-microbial activity against a subset of bacteria including Gram-negative ESKAPE pathogens. We named the lantibiotic thermolanthin.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies of this strain led to the discovery of several novel compounds such as the hectochlorins and jamaicamides; however, bioinformatic analyses of its genome suggested that there were many more cryptic biosynthetic gene clusters yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was co-cultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that co-culture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/547815v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1b507d4org.highwire.dtl.DTLVardef@152376org.highwire.dtl.DTLVardef@1cb410dorg.highwire.dtl.DTLVardef@11bcabc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pathogenic fungi rely on the cell wall component, chitin, for critical structural and immunological functions. Here a chitin labeling method to visualize the hyphal pathogenic response was developed. The data show that filamentous fungi, Candida albicans, transport N-acetylglucosamine (NAG) bio-orthogonal probes and incorporate them into the cell wall, indicating the probes utility for in vivo study of the morphological, pathogenic switch. As yeast reside in complex microenvironments, The data show that the opportunistic microbe C. albicans, has developed processes to utilize surrounding bacterial cell wall fragments to initiate the morphogenic switch. The probes are utilized for visualization of growth patterns of pathogenic fungi, providing insights into novel mechanisms for the development of antifungals. Remodeling chitin in fungi using NAG derivatives will advance yeast pathogenic studies.

The industrial production of phosphochemicals is highly energy-intensive, involving the reduction of the phosphate mineral apatite to white phosphorus, a toxic and reactive intermediate with significant environmental risk. Phosphite, an activated form of phosphorus, is a potential alternative substrate for phosphochemical synthesis, yet direct reduction of phosphate to phosphite remains challenging. While environmental and microbial studies have suggested biochemical pathways for reducing phosphate to phosphite, these pathways have not been conclusively demonstrated in axenic culture. In this study, we characterize phosphite production by Streptomyces viridochromogenes and demonstrate that phosphite is an abiological product of phosphonoformyl-CMP decomposition, an intermediate in the biosynthesis of the herbicide phosphinothricin. The phosphonoformyl-CMP intermediate yields an "activated" phosphonoformate for decarboxylation, producing phosphite at biological temperatures and pH following phosphoanhydride hydrolysis. Using S. viridochromogenes spent media, we demonstrate a hybrid biotic-abiotic synthesis of the metal chelator aminotris(methylenephosphonate), illustrating a potential synthetic route to phosphochemicals from biogenic phosphite.

The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is therefore regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of [~]108 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a non-competitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest an unprecedented mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity. SIGNIFICANCE STATEMENTThousands of proteins within humans are modified by the monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAc regulates cellular physiology and is being pursued to create therapeutics. Remarkably, only one enzyme, O-GlcNAc transferase (OGT), installs O-GlcNAc and its regulation is poorly understood. By affinity selection using a vast peptide library, we uncover an amino acid sequence motif that drives binding of polypeptides to OGT. An OGT-peptide complex shows how this motif binds to an allosteric site proximal to the active site and inhibits OGT in an unprecedented manner. Given the distribution of this sequence motif within the human proteome proteins containing this motif likely regulate the activity of OGT, outlining a new mode by which OGT is controlled and opening new avenues for research.

Proteolysis targeting chimeras (PROTACs) are an emerging therapeutic modality that induces protein degradation by recruiting E3 ligases. Most reported PROTACs recruit ubiquitously expressed E3 ligases, such as cereblon and the von Hippel-Lindau tumor suppressor. Of the additional 600+ E3 ligases, recruiting those with tissue-restricted expression is attractive for increasing the specificity of PROTACs. To this end, tissue-specific E3 ligases or E3 ligase-associated proteins that can be recruited for targeted protein degradation need to be identified. This work describes the first reported PROTAC that recruits the tissue-specific E3 ligase scaffolding protein MAGEA11. As an initial demonstration, a library of bromodomain and extra-terminal domain (BET)-targeting PROTACs that recruit MAGEA11 was synthesized. The library was screened in osteosarcoma U2OS cells, identifying lead compound 105B. 105B potently degrades BET proteins in U2OS osteosarcoma cell lines (BRD4 DC50 = 0.130 nM, Dmax = 78%) and KYSE180 esophageal squamous cell carcinoma cell lines (DC50 = 40 nM, Dmax = 70%), but shows no degradation in non-cancerous, MAGEA11-deficient HEK293T cells. Mechanistic studies confirmed 105Bs dependence on the ubiquitin-proteasome system and engagement of both MAGEA11 and BRD4. 105B decreased levels of BET-regulated gene products c-Myc, RUNX2, and KRT14; however, improvements are still necessary to affect selective cytotoxicity. This work reports the first example of a PROTAC recruiting a tissue-specific E3 ligase for cancer-restricted degradation of BET proteins and highlights the need for further development of MAGEA11-recruiting degraders.

The methylenomycins are highly functionalized cyclopentanone antibiotics produced by Streptomyces coelicolor A3(2). A biosynthetic pathway to the methylenomycins has been proposed based on sequence analysis of the proteins encoded by the methylenomycin biosynthetic gene cluster and incorporation of labelled precursors. However, the roles played by putative biosynthetic enzymes remain experimentally uninvestigated. Here, the biosynthetic functions of enzymes encoded by mmyD, mmyO, mmyF and mmyE were investigated by creating in-frame deletions in each gene and investigating the effect on methylenomycin production. No methylenomycin-related metabolites were produced by the mmyD mutant, consistent with the proposed role of MmyD in an early biosynthetic step. The production of methylenomycin A, but not methylenomycin C, was abolished in the mmyF and mmyO mutants, consistent with the corresponding enzymes catalyzing epoxidation of methylenomycin C, as previously proposed. Expression of mmyF and mmyO in a S. coelicolor M145 derivative engineered to express mmr, which confers methylenomycin resistance, enabled the resulting strain to convert methylenomycin C to methylenomycin A, confirming this hypothesis. A novel metabolite (pre-methylenomycin C), which readily cyclizes to form the corresponding butanolide (pre-methylenomycin C lactone), accumulated in the mmyE mutant, indicating the corresponding enzyme is involved in introducing the exomethylene group into methylenomycin C. Remarkably, both pre-methylenomycin C and its lactone precursor were one to two orders of magnitude more active against various Gram-positive bacteria, including antibiotic-resistant Staphylococcus aureus and Enterococcus faecium isolates, than methylenomycins A and C, providing a promising starting point for the development of novel antibiotics to combat antimicrobial resistance.

Benzoxaboroles offer unusual reactivity and protein recognition for the development of small molecule drugs. Despite this potential, they are uncommon in drug discovery or in large fragment screening libraries. We synthesized a small series of structurally related benzoxaboroles containing a diazirine/alkyne tag to enable in-cell photoaffinity labeling (PAL) experiments. A subset of this library was found to have high selectivity for eukaryotic translation initiation factor 4E (eIF4E). The benzoxaborole-eIF4E interaction was found to be stereoselective in nature and competitive with the 7-methylguanosine cap of mRNA. Site of labeling experiments revealed that the benzoxaborole fragment interacts with the cap binding pocket of eIF4E. In silico modeling of the modified protein suggests that H-bonding interactions between the main chain of Trp102 and the side chain of Asn155 to the amide carbonyl and anionic boronate of the benzoxaborole, respectively, drive affinity for this challenging to drug pocket.

Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the relatively weak nature of the non-covalent aptamer-fluorogen interaction limits the utility of these systems in that multiple copies of the aptamer are often required, and in most cases the aptamer must be expressed on a second scaffold such as a transfer RNA. We propose that these limitations can be averted through covalent RNA labeling, and here we describe a photoaffinity approach in which the aptamer ligand is functionalized with a photoactivatable reactive group such that irradiation with UV light results in covalent attachment to the RNA of interest. In addition to the robustness of the covalent linkage, this approach benefits from the ability to temporally control RNA labeling. To demonstrate this method, we incorporated a photoaffinity linker onto malachite green and fused the malachite green aptamer to a specific mRNA reporter of interest. We observed markedly improved sensitivity for fixed cell imaging of mRNA using this approach compared to in situ hybridization. Additionally, we demonstrate visualization of RNA dynamics in live cells using an mRNA having only a single copy of the aptamer, minimizing perturbation of the structure and localization. Our initial biological application utilizes the photoaffinity labeling approach to monitor RNA stress granule dynamics and we envision future application of this method for a wide range of investigations into the cellular localization, dynamics, and protein binding properties of cellular RNAs.

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.