Charting the sequence-activity landscape of peptide inhibitors of translation termination.

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

Recent structural advances in constrained helical peptides.

Given the ubiquity of the ⍺-helix in the proteome, there has been much research in developing mimics of ⍺-helices, and most of this study has been toward developing protein-protein interaction inhibitors. A common strategy for mimicking ⍺-helices has been through the use of constrained, helical peptides. The addition of a constraint typically provides for conformational and proteolytic stability and, in some cases, cell permeability. Some of the most well-known strategies included are lactam formation and hydrocarbon "stapling." Beyond those strategies, there have been many recent advances in developing constrained peptides. The purpose of this review is to highlight recent advances in the development of new helix-stabilizing technologies, constraint diversification strategies, tether diversification strategies, and combination strategies that create new bicyclic helical peptides.

Non-electrophilic NRF2 activators promote wound healing in human keratinocytes and diabetic mice and demonstrate selective downstream gene targeting.

The transcription factor NRF2 plays an important role in many biological processes and is a promising therapeutic target for many disease states. NRF2 is highly expressed in the skin and is known to play a critical role in diabetic wound healing, a serious disease process for which treatment options are limited. However, many existing NRF2 activators display off-target effects due to their electrophilic mechanism, underscoring the need for alternative approaches. In this work, we investigated two recently described non-electrophilic NRF2 activators, ADJ-310 and PRL-295, and demonstrated their efficacy in vitro and in vivo in human keratinocytes and Leprdb/db diabetic mice. We also compared the downstream targets of PRL-295 to those of the widely used electrophilic NRF2 activator CDDO-Me by RNA sequencing. Both ADJ-310 and PRL-295 maintained human keratinocyte cell viability at increasing concentrations and maintained or improved cell proliferation over time. Both compounds also increased cell migration, improving in vitro wound closure. ADJ-310 and PRL-295 enhanced the oxidative stress response in vitro, and RNA-sequencing data showed that PRL-295 activated NRF2 with a narrower transcriptomic effect than CDDO-Me. In vivo, both ADJ-310 and PRL-295 improved wound healing in Leprdb/db diabetic mice and upregulated known downstream NRF2 target genes in treated tissue. These results highlight the non-electrophilic compounds ADJ-310 and PRL-295 as effective, innovative tools for investigating the function of NRF2. These compounds directly address the need for alternative NRF2 activators and offer a new approach to studying the role of NRF2 in human disease and its potential as a therapeutic across multiple disease states.

Elucidating Sequence-Assembly Relationships for Bilingual PNA Biopolymers.

Nucleic acids and proteins possess encoded "languages" that can be used for information storage or to direct function. However, each biopolymer is limited to encoding its respective "language." Using a peptide nucleic acid (PNA) scaffold, nucleobase and amino acid residues can be installed on a singular backbone, enabling a single biopolymer to encode both languages. Our laboratory previously reported the development of a "bilingual" PNA biopolymer that incorporates a sequence-specific nucleic acid code interspersed with hydrophobic (alanine) and hydrophilic (lysine) amino acid residues at defined positions to produce amphiphilic character. We observed the amphiphilic amino acid residues directing the biopolymer to undergo self-assembly into micelle-like structures, while the nucleic acid recognition was harnessed for disassembly. Herein, we report a series of bilingual PNA sequences having amino acid residues with varying lengths, functional group charges, hydrophobicities, and spacings to elucidate the effect of these parameters on micelle assembly and nucleic acid recognition. Negative charges in the hydrophilic block or increased bulkiness of the hydrophobic side chains led to assembly into similarly sized micelles; however, the negative charge additionally led to increased critical micelle concentration. Upon PNA sequence truncation to decrease the spacing between side chains, the biopolymers remained capable of self-assembling but formed smaller structures. Characterization of disassembly revealed that each variant retained sequence recognition capabilities and stimuli-responsive disassembly. Together, these data show that the amino acid and nucleic acid sequences of amphiphilic bilingual biopolymers can be customized to finely tune the assembly and disassembly properties, which has implications for applications such as the encapsulation and delivery of cargo for therapeutics.

Structure-Activity Relationships of the Antimicrobial Peptide Natural Product Apidaecin.

With the growing crisis of antimicrobial resistance, it is critical to continue to seek out new sources of novel antibiotics. This need has led to renewed interest in natural product antimicrobials, specifically antimicrobial peptides. Nonlytic antimicrobial peptides are highly promising due to their unique mechanisms of action. One such peptide is apidaecin (Api), which inhibits translation termination through stabilization of the quaternary complex of the ribosome-apidaecin-tRNA-release factor. Synthetic derivatives of apidaecin have been developed, but structure-guided modifications have yet to be considered. In this work, we have focused on modifying key residues in the Api sequence that are responsible for the interactions that stabilize the quaternary complex. We present one of the first examples of a highly modified Api peptide that maintains its antimicrobial activity and interaction with the translation complex. These findings establish a starting point for further structure-guided optimization of Api peptides.

Synthesis and Evaluation of Noncovalent Naphthalene-Based KEAP1-NRF2 Inhibitors.

The oxidative stress response, gated by the protein-protein interaction of KEAP1 and NRF2, has garnered significant interest in the past decade. Misregulation in this pathway has been implicated in disease states such as multiple sclerosis, rheumatoid arthritis, and diabetic chronic wounds. Many of the known activators of NRF2 are electrophilic in nature and may operate through several biological pathways rather than solely through the activation of the oxidative stress response. Recently, our lab has reported a nonelectrophilic, monoacidic, naphthalene-based NRF2 activator which exhibited good potency in vitro. Herein, we report a detailed structure-activity relationship of naphthalene-based NRF2 activators, an X-ray crystal structure of our monoacidic KEAP1 inhibitor, and identification of an underexplored area of the NRF2 binding pocket of KEAP1.

SAR Study on Estrogen Receptor α/ß Activity of (Iso)flavonoids: Importance of Prenylation, C-Ring (Un)Saturation, and Hydroxyl Substituents.

Many botanicals used for women's health contain estrogenic (iso)flavonoids. The literature suggests that estrogen receptor beta (ERß) activity can counterbalance estrogen receptor alpha (ERα)-mediated proliferation, thus providing a better safety profile. A structure-activity relationship study of (iso)flavonoids was conducted to identify ERß-preferential structures, overall estrogenic activity, and ER subtype estrogenic activity of botanicals containing these (iso)flavonoids. Results showed that flavonoids with prenylation on C8 position increased estrogenic activity. C8-prenylated flavonoids with C2-C3 unsaturation resulted in increased ERß potency and selectivity [e.g., 8-prenylapigenin (8-PA), EC50 (ERß): 0.0035 ± 0.00040 µM], whereas 4'-methoxy or C3 hydroxy groups reduced activity [e.g., icaritin, EC50 (ERß): 1.7 ± 0.70 µM]. However, nonprenylated and C2-C3 unsaturated isoflavonoids showed increased ERß estrogenic activity [e.g., genistein, EC50 (ERß): 0.0022 ± 0.0004 µM]. Licorice (Glycyrrhiza inflata, [EC50 (ERα): 1.1 ± 0.20; (ERß): 0.60 ± 0.20 µg/mL], containing 8-PA, and red clover [EC50 (ERα): 1.8 ± 0.20; (ERß): 0.45 ± 0.10 µg/mL], with genistein, showed ERß-preferential activity as opposed to hops [EC50 (ERα): 0.030 ± 0.010; (ERß): 0.50 ± 0.050 µg/mL] and Epimedium sagittatum [EC50 (ERα): 3.2 ± 0.20; (ERß): 2.5 ± 0.090 µg/mL], containing 8-prenylnaringenin and icaritin, respectively. Botanicals with ERß-preferential flavonoids could plausibly contribute to ERß-protective benefits in menopausal women.

Steroid receptor/coactivator binding inhibitors: An update.

The purpose of this review is to highlight recent developments in small molecules and peptides that block the binding of coactivators to steroid receptors. These coactivator binding inhibitors bind at the coregulator binding groove, also known as Activation Function-2, rather than at the ligand-binding site of steroid receptors. Steroid receptors that have been targeted with coactivator binding inhibitors include the androgen receptor, estrogen receptor and progesterone receptor. Coactivator binding inhibitors may be useful in some cases of resistance to currently prescribed therapeutics. The scope of the review includes small-molecule and peptide coactivator binding inhibitors for steroid receptors, with a particular focus on recent compounds that have been assayed in cell-based models.