Biology subjects

Pentelute, B. L.

Publications and source records attributed to Pentelute, B. L..

At least 19 recordsLinked to original sources

bioRxiv2026

High-throughput discovery of arginine-depleted peptides enables effective antisense delivery for Duchenne muscular dystrophy

Phosphorodiamidate morpholino oligomers (PMOs) are approved exon-skipping antisense therapeutics for Duchenne muscular dystrophy (DMD), but their clinical utility is limited by poor uptake in muscle tissue, necessitating frequent high-dose administration. Cell-penetrating peptides (CPPs) can enhance intracellular delivery of PMOs, yet conventional arginine-rich CPPs often cause dose-limiting toxicity, including renal damage, which hinders their clinical translation. To address this challenge, we developed a high-throughput, charge-based chromatographic enrichment platform capable of screening over 15,000 synthetic peptides, including sequences with noncanonical (abiotic) amino acids. This approach enabled de novo discovery of arginine-depleted CPPs with improved delivery profiles. Four lead candidates demonstrated efficient nuclear PMO delivery with ~10-fold lower in vitro toxicity compared to standard CPPs such as penetratin. The top-performing peptide, CXP1, showed robust splice-switching activity and favorable tolerability in both cellular and animal models. In dystrophic mdx mice, CXP1-PMO conjugates achieved greater exon skipping compared to PMOs conjugated to R6G at equivalent doses. Tissue levels of CXP1-PMO correlated with exon-skipping efficacy, establishing a clear pharmacokinetic-pharmacodynamic relationship. These findings highlight a mechanistically novel and translationally relevant discovery strategy, demonstrating the potential of high-throughput platforms to generate more effective CPP-based delivery vehicles for antisense therapeutics in DMD and related neuromuscular disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/730741v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@84b7f2org.highwire.dtl.DTLVardef@1487850org.highwire.dtl.DTLVardef@d2e263org.highwire.dtl.DTLVardef@10ce371_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Modulation of the human cathelicidin dimerization interface separates antimicrobial activity from mammalian membrane disruption

The human CAMP gene product LL-37 is a prototypical cationic host defense peptide with potent activity against gram-negative bacteria. The development of LL-37 and other membrane-active host defense peptides as antibiotics, however, is limited in part by their activity against mammalian membranes. To better understand the structural features underlying LL-37 functions, we reconceived of LL-37 as a small protein and systematically ablated the sidechain content of entire surfaces while preserving helical character. This approach revealed that selected modifications within surface m4 retain wild-type levels of activity against gram-negative bacteria while disrupting activity against mammalian membranes by >100-fold. Separation of antimicrobial and mammalian membrane activities mapped primarily to residues I24 and L28, and was attributable to a combination of disrupted oligomerization and decreased hydrophobic content within the dimerization interface. Modulation of the LL-37 dimerization interface thus constitutes a rational pathway by which to engineer derivatives with improved therapeutic potential.

biochemistry↗

bioRxiv2025

Carborane-NHS esters as versatile reagents for amine-selective small-molecule modification, bioconjugation, and peptide-polymer assembly

Carboranes - hydrophobic, boron-rich clusters with unique structural properties - offer broad potential in materials science and biomedicine. They can mimic carbocyclic frameworks for designing advanced materials such as polymers and MOFs, and when incorporated into biomolecules, reduce local polarity while enriching boron content: a feature attractive for boron neutron capture therapy (BNCT). Here, we report the development of organic carborane reagents based on N-hydroxysuccinimide (NHS) esters serving as versatile platform for selective conjugation to amine residues, enabling functionalization of small molecules, peptides, and antibodies. We further introduce bifunctional carborane reagents bearing two NHS ester sites, which mediate inter- and intramolecular coupling as well as peptide-driven polymerization. The resulting carborane-peptide polymers, composed of fragments with distinct physicochemical features, represent a new class of hybrid materials. To demonstrate biomedical utility, therapeutic antibodies (trastuzumab, cetuximab, and daratumumab) were conjugated with these reagents, attaching up to 13 carboranes per antibody. Linker length was found to be critical for maximizing loading efficiency. nLC-MS/MS mapping of trastuzumab conjugates identified 15 preferential lysine modification sites, located outside CDR regions. Importantly, trastuzumab-carborane conjugates retained native HER2-targeting activity in BT-474 cell assays. These structures, integrating therapeutic antibody efficacy with boron delivery capability, hold promise as multimodal agents for BNCT.

biochemistry↗

bioRxiv2025

PSMtags improve peptide sequencing and throughput in sensitive proteomics

Mass spectrometry-based proteomics enables comprehensive characterization of protein abundance, function, and interactions. Label-free approaches are simple to implement but challenging to scale to thousands of samples per day. Multiplexed techniques, such as plexDIA, can address these limitations but remain restricted by the lack of mass tags optimized for data-independent acquisition (DIA) workflows. Here, we present a systematic approach screening a library of 576 compounds that identifies several small molecules that, when conjugated to peptides, improve their detection and sequence identification by mass spectrometry. The lead molecule, PSMtag, substantially increases the detection of fragment b-ions, which increases the confidence of sequence identification and enhances de novo sequencing. PSMtags allow 9-plexDIA, using only stable isotopes of carbon, oxygen and nitrogen. As a result, it allows simultaneously increasing proteome coverage and sample throughput for plexDIA workflows without compromising quantitative accuracy. We demonstrate 240 samples-per-day with 9-plexDIA, while acquiring 28,359 protein data points in the same time label-free methods acquire 4,340. Our approach constitutes an expandable framework for designing mass tags to overcome existing limitations in multiplexed proteomics and provides plexDIA reagents capable of analyzing over 1,000 samples per day when using 10 minute runs. By facilitating higher throughput and improved identification, this innovation holds significant potential for accelerating proteomic studies across diverse biological and clinical applications.

bioengineering↗

bioRxiv2025

Separability of antibacterial and membranolytic activity in the human host defense peptide LL-37

The human CAMP gene product LL-37 is thought to exert direct antimicrobial activity against gram-negative bacteria via membrane disruption. In the course of structure-activity relationship studies of LL-37 aimed at developing peptidomimetic antibiotics, however, we incidentally noted mutations in LL-37 that globally inhibit membrane disruption in both mammalian and gram-negative bacterial cells. Despite their diminished capacity for membranolysis, these variants retained full antibacterial activity against gram-negative bacteria. While testing LL-37 and derivatives thereof against clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis, we further noted unusually high rates of elevated minimum inhibitory concentrations for LL-37. Further evaluation of these clinical isolates revealed that they are fully permeabilized by LL-37 without being killed. Thus, we have identified variants of LL-37 that kill gram-negative bacteria without permeabilizing, and gram-negative bacteria that are permeabilized by LL-37 without being killed. This may suggest the existence of one or more mechanisms other than membrane disruption by which LL-37 can kill gram-negative bacteria, which may open up new avenues for antibiotic development based on the naturally evolved, non-membrane targets of human host defense peptides.

microbiology↗

bioRxiv2025

Mimicry of the LL-37 N-terminus enhances the activity of short human cathelicidin derivatives

Issues such as potency and stability limit our ability to realize the potential of host defense peptides (HDPs) to serve as new antibiotics. Informed by our prior structure-activity work, we demonstrate that transposition of an N-terminal biphenyl motif from full-length LL-37 onto the previously-described central activity region (residues 18-29) improves activity against gram-negative bacteria by >16-fold. We further improve upon this lead derivative, termed FF-14, using stabilizing modifications such as D-amino acids and C-terminal amidation as well as selected N-lipidation moieties, and demonstrate the transferability of biphenyl motif activity to longer cathelicidin scaffolds. Mechanistic interrogation reveals that the biphenyl pharmacophore increases both inner and outer membrane permeabilization while promoting helical structure in the 14-mer without directly impacting peptide stability. Inclusion of the LL-37 biphenyl motif is thus a portable strategy for augmenting short cathelicidin activity and better approximating the activity profile of LL-37 itself in short sequences.

microbiology↗

bioRxiv2025

Peptaibiotic-inspired antimicrobials based on human cathelicidin

The prevailing model of cathelicidin function holds that peptide helicity leads to membrane permeabilization, which in turn leads to killing of gram-negative bacteria. Using a paired Ala-Aib mutagenesis approach to isolate sidechain and structure-dependent functions, we demonstrate here that the effect of helicity on gram-negative killing in a model cathelicidin-derived scaffold is species-dependent. We then leverage this insight to derive lead compounds with up to 32-fold improvements in selectivity for bacterial over mammalian cells. Further interrogation of the mechanistic basis for selectivity demonstrates that, while helicity may predict permeabilization, permeabilization does not predict killing of gram-negative bacteria. Thus, neither helicity nor permeabilization is a universal requirement for cathelicidin-mediated killing.

microbiology↗

bioRxiv2025

Protective antigen-mediated delivery of an anti-CRISPR proteinfor precision genome editing

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size (6-54 kDa) and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and non-viral vectors) or are not applicable in vivo (e.g., nucleofection), limiting therapeutic applications. To address these problems, we developed the first protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that LFN-Acr binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

biochemistry↗

bioRxiv2024

ProCyon: A multimodal foundation model for protein phenotypes

Characterizing human proteins remains a major challenge: approximately 29% of human proteins lack experimentally validated functions and even well-annotated proteins often lack context-specific phenotypic insights. To enable universal modeling of protein phenotypes, we present PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOW, a multimodal foundation model that utilizes protein sequence, structure, and natural language for generating and predicting protein phenotypes across diverse knowledge domains. PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOW is trained on our novel dataset, PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOW-IO_SCPLOWNSTRUCTC_SCPLOW, with 33 million protein phenotype instructions. On dozens of benchmarking tasks, PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOW performs competitively against single-modal and multimodal models. Further, PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOW conditionally retrieves proteins via mechanisms of action of small molecule drugs and disease contexts, and it generates candidate phenotypic descriptions for poorly characterized proteins, including those implicated in Parkinsons disease that were identified after PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOWs knowledge cutoff date. We experimentally confirm PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOWs predictions in multiple sclerosis using post-mortem brain RNA-seq, identifying novel MS genes and elucidating associated pathway mechanisms consistent with cortical pathology. PO_SCPLOWROC_SCPLOWCO_SCPLOWYONC_SCPLOW paves the way toward a general approach to generate functional insights into the human proteome.

bioinformatics↗

bioRxiv2024

In vivo Antibody Painting for Next Generation Weight Loss Drugs

Peptide-based therapeutics are currently in great demand but often suffer from rapid clearance and accumulation in off-target tissues which continue to present barriers in their clinical translation. Here, we developed an electrophilic peptide for the attachment of therapeutics to native immunoglobulin (IgG) in vivo, enabling the bioorthogonal covalent linkage, or painting, of peptide drugs of choice to circulating IgGs directly in live animals. Native IgG painting with glucagon-like peptide-1 (GLP-1) results in sustained body weight loss and prolonged blood glucose management after one dose. Such technology might revolutionize the next generation of long-acting peptide-based medicines.

bioengineering↗

bioRxiv2024

TDC-2: Multimodal Foundation for Therapeutic Science

Drug discovery AI datasets and benchmarks have not traditionally included single-cell analysis biomarkers. While benchmarking efforts in single-cell analysis have recently released collections of single-cell tasks, they have yet to comprehensively release datasets, models, and benchmarks that integrate a broad range of therapeutic discovery tasks with cell-type-specific biomarkers. Therapeutics Commons (TDC-2) presents datasets, tools, models, and benchmarks integrating cell-type-specific contextual features with ML tasks across therapeutics. We present four tasks for contextual learning at single-cell resolution: drug-target nomination, genetic perturbation response prediction, chemical perturbation response prediction, and protein-peptide interaction prediction. We introduce datasets, models, and benchmarks for these four tasks. Finally, we detail the advancements and challenges in machine learning and biology that drove the implementation of TDC-2 and how they are reflected in its architecture, datasets and benchmarks, and foundation model tooling.

bioinformatics↗

bioRxiv2023

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons

Antigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LFN). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings.

immunology↗

bioRxiv2023

Discovery of reactive peptide inhibitors of human papillomavirus oncoprotein E6

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.

molecular biology↗

bioRxiv2023

A transferrin receptor 1-targeted PNA-peptide conjugate inhibits microRNA-21 expression in cardiac and other mouse tissues

MicroRNAs (miRNAs) are implicated in the onset and progression of a variety of diseases. Modulating the expression of specific miRNAs is a possible option for therapeutic intervention. A promising strategy is the use of antisense oligonucleotides (ASOs) to inhibit miRNAs. Targeting ASOs to specific tissues can potentially lower the dosage and improve clinical outcomes by alleviating systemic toxicity. We leverage here automated peptide nucleic acid (PNA) synthesis technology to manufacture an anti-miRNA oligonucleotide (antagomir) covalently attached to a 12-mer peptide that binds to transferrin receptor 1. Our PNA-peptide conjugate is active in cells and animals, effectively inhibiting the expression of miRNA-21 both in cultured mouse cardiomyocytes and different mouse organs (heart, liver, kidney, lung, and spleen), while remaining well-tolerated in animals up to the highest tested dose of 30 mg/kg. Conjugating the targeting ligand to the PNA antagomir significantly improved inhibition of miRNA-21 in the heart by over 50% relative to the PNA alone. Given the modulation of biodistribution observed with our PNA-peptide conjugate, we anticipate this antagomir platform to serve as a starting point for pre-clinical development studies. Table of Contents Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/536802v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@645857org.highwire.dtl.DTLVardef@1e3be1corg.highwire.dtl.DTLVardef@d6286aorg.highwire.dtl.DTLVardef@1f76032_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisConjugating T12, a peptide targeting transferrin receptor 1 (TfR1), to a peptide nucleic acid (PNA) oligonucleotide targeting microRNA-21 increases delivery of the PNA-T12 conjugate to cardiac tissue relative to PNA alone.

molecular biology↗

bioRxiv2023

Branched multimeric peptides as affinity reagents for detection of α-Klotho protein

-Klotho is a protein associated with aging that is expressed in the kidney, parathyroid gland, and choroid plexus. As a transmembrane protein, it acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. -Klotho has an extracellular domain that can be cleaved, released and circulated in the blood stream as a soluble form. Decreased levels of -Klotho are an indication of chronic kidney disease and other age-associated diseases. Detecting or labeling transmembrane and soluble -Klotho is a longstanding challenge that has impeded the in-depth understanding of its role. Here we describe branched multimeric peptides that recognize -Klotho with high affinity and selectivity in the biological milieu. The branched peptides are prepared in a single-shot synthesis by parallel automated fast-flow synthesis in under one hour. The branched -Klotho-binding peptides show improvement in affinity relative to the monomeric versions and can be used to label Klotho for live imaging in kidney cells. Our results demonstrate the potential of automated flow technology to deliver peptide-based reagents with complex architecture and improved affinity for the selective binding of target proteins in physiological settings.

molecular biology↗

bioRxiv2022

Rapid Single-Shot Synthesis of the 217 Amino Acid-Long N-Terminal Domain of Pyocin S2

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remains challenging. To address this limitation, we employed automated fast-flow peptide synthesis (AFPS) for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 hours and purified to yield milligrams quantities of the desired product. To our knowledge, the 217 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor (FpvA) and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching (FRAP). These modified, synthetic S-type pyocins domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

synthetic biology↗

bioRxiv2022

In-cell penetration selection-mass spectrometry produces noncanonical peptides for antisense delivery

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) has reached the market after 30 years of investigation due to challenges in the discovery of new, more efficient sequences. We developed a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) to discover peptides from a synthetic library capable of delivering macromolecule cargo to the cytosol. This method was inspired by recent in vivo selection approaches for cell-surface screening, with an added spatial dimension resulting from subcellular fractionation. A representative peptide discovered in the cytosolic extract, Pep1a, is nearly 100-fold more active toward antisense phosphorodiamidate morpholino oligomer (PMO) delivery compared to a sequence identified from a whole cell extract, which includes endosomes. Pep1a is composed of D-amino acids and two non--amino acids. Pulse-chase and microscopy experiments revealed that while the PMO-Pep1a conjugate is likely taken up by endosomes, it can escape to localize to the nucleus. In-cell PS-MS introduces a means to empirically discover unnatural synthetic peptides for subcellular delivery of therapeutically relevant cargo.

bioengineering↗

bioRxiv2021

Cell-Penetrating D-Peptides Retain Antisense Morpholino Oligomer Delivery Activity

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise non-penetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMO) resulted in higher muscle concentration, increased exon-skipping and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical L-peptides. We hypothesized that mirror-image D-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities, while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency which may be useful for determining which peptide sequence to pursue in further pre-clinical studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/462617v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@887d8borg.highwire.dtl.DTLVardef@ea664aorg.highwire.dtl.DTLVardef@e1b1e1org.highwire.dtl.DTLVardef@1652d47_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗