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Dysregulation of the networking of RNA-binding proteins (RBPs) and RNAs drives many human diseases, including cancers, and the targeting of RNA–protein interactions (RPIs) has emerged as an exciting area of RNA-targeted drug discovery. Accordingly, methods that enable the discovery of cell-active small molecule modulators of RPIs are needed to propel this emerging field forward. Herein, we describe the application of live-cell assay technology, RNA interaction with protein-mediated complementation assay (RiPCA), for high-throughput screening to identify small molecule inhibitors of the pre-let-7d–Lin28A RPI. Utilizing a combination of RNA-biased small molecules and virtual screening hits, we discovered an RNA-binding small molecule that can disrupt the pre-let-7–Lin28 interaction demonstrating the potential of RiPCA for advancing RPI-targeted drug discovery.

RNA-binding proteins (RBPs) act as essential regulators of cell fate decisions, through their ability to bind and regulate the activity of cellular RNAs. For protein-coding mRNAs, RBPs control the localization, stability, degradation, and ultimately translation of mRNAs to impact gene expression. Disruption of the vast network of mRNA-protein interactions has been implicated in many human diseases, and accordingly, targeting these interactions has surfaced as a new frontier in RNA-targeted drug discovery. To catalyze this new field, methods are needed to enable the detection and subsequent screening of mRNA-RBP interactions, particularly in live cells. Using our laboratory’s RNA-interaction with Protein-mediated Complementation Assay (RiPCA) technology, herein we describe its application to mRNA-protein interactions and present a guide for the development of future RiPCA assays for structurally diverse classes of mRNA-protein interactions.

Eukaryotic translation initiation factor 4E (eIF4E) is an RNA-binding protein that binds to the m7GpppX-cap at the 5′ terminus of coding mRNAs to initiate cap-dependent translation. While all cells require cap-dependent translation, cancer cells become addicted to enhanced translational capacity, driving the production of oncogenic proteins involved in proliferation, evasion of apoptosis, metastasis, and angiogenesis, among other cancerous phenotypes. eIF4E is the rate-limiting translation factor, and its activation has been shown to drive cancer initiation, progression, metastasis, and drug resistance. These findings have established eIF4E as a translational oncogene and promising, albeit challenging, anti-cancer therapeutic target. Although significant effort has been put forth toward inhibiting eIF4E, the design of cell-permeable, cap-competitive inhibitors remains a challenge. Herein, we describe our work toward solving this long-standing challenge. By employing an acyclic nucleoside phosphonate prodrug strategy, we report the synthesis of cell-permeable inhibitors of eIF4E binding to capped mRNA to inhibit cap-dependent translation.

Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer’s cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome. These patients harbor germline and somatic mutations in Dicer that lead to defective microRNA processing and activity. While most mutations occur within Dicer’s catalytic RNase III domains, alterations within the Platform-PAZ (Piwi-Argonaute-Zwille) domain also cause loss of microRNA production. Using a combination of in vitrobiochemical and cellular studies, we characterized the effect of disease-relevant Platform-PAZ-associated mutations on the processing of a well-studied oncogenic microRNA, pre-microRNA-21. We then compared these results to those of a representative from another Dicer substrate class, the small nucleolar RNA, snord37. From this analysis, we provide evidence that mutations within the Platform-PAZ domain result in differential impacts on RNA binding and processing, adding new insights into the complexities of Dicer processing of small RNA substrates.

On the occasion of the 2023 International Women’s Day on March 8, 2023, we want to celebrate and highlight the contributions of many women volunteers in the American Chemical Society Division of Medicinal Chemistry (ACS MEDI).

As we celebrate International Women’s Day 2024 with the theme “Inspire Inclusion”, the women of the ACS Medicinal Chemistry Division (MEDI) want to foster a sense of belonging, relevance, and empowerment by sharing uplifting stories of what inspired them to become medicinal chemists. In this editorial, we are featuring female medicinal chemistry scientists to provide role models, encouragement, and inspiration to others. We asked women medicinal chemists to contribute a brief paragraph about what inspired them to become medicinal chemists or what inspires them today as medicinal chemists. The responses and contributions highlight their passions and motivations, such as their love of the sciences and their drive to improve human health by contributing to basic research and creating lifesaving drugs.

Protein disorder plays a crucial role in signal transduction and is key for many cellular processes including transcription, translation, and cell cycle. Within the intrinsically disordered protein interactome, the α-helix is commonly used for binding, which is induced via a disorder-to-order transition. Because the targeting of protein–protein interactions (PPIs) remains an important challenge in medicinal chemistry, efforts have been made to mimic this secondary structure for rational inhibitor design through the use of stapled peptides. Cap-dependent mRNA translation is regulated by two disordered proteins, 4E-BP1 and eIF4G, that inhibit or stimulate the activity of the m7G cap-binding translation initiation factor, eIF4E, respectively. Both use an α-helical motif for eIF4E binding, warranting the investigation of stapled peptide mimics for manipulating eIF4E PPIs. Herein, we describe our efforts toward this goal, resulting in the synthesis of a cell-active stapled peptide for further development in manipulating aberrant cap-dependent translation in human diseases.

microRNAs (miRNAs) are small gene regulatory RNAs, and their expression has been found to be dysregulated in a number of human diseases. To facilitate the discovery of small molecules capable of selectively modulating the activity of a specific miRNA, we have utilized new high-throughput screening technology targeting Dicer-mediated pre-miRNA maturation. Pilot screening of ~50,000 small molecules and ~33,000 natural product extract libraries against pre-miR-21 processing indicated the potential of our assay for this goal, yielding a campaign Z′ factor of 0.52 and an average plate signal-to-background (S/B) ratio of 13. Using two-dimensional screening against a second pre-miRNA, pre-let-7d, we evaluated the selectivity of confirmed hits. The results presented demonstrate how high-throughput screening can be used to identify selective small molecules for a target RNA.

microRNAs (miRNAs) are small gene regulatory RNAs, and their expression has been found to be dysregulated in a number of human diseases. To facilitate the discovery of small molecules capable of selectively modulating the activity of a specific miRNA, we have utilized new high-throughput screening technology targeting Dicer-mediated pre-miRNA maturation. Pilot screening of ~50,000 small molecules and ~33,000 natural product extract libraries against pre-miR-21 processing indicated the potential of our assay for this goal, yielding a campaign Z′ factor of 0.52 and an average plate signal-to-background (S/B) ratio of 13. Using two-dimensional screening against a second pre-miRNA, pre-let-7d, we evaluated the selectivity of confirmed hits. The results presented demonstrate how high-throughput screening can be used to identify selective small molecules for a target RNA.

Discovery and structure elucidation of natural products available in infinitesimally small quantities are recognized challenge. This challenge is epitomized by the diphenazine class of molecules that contain three bridged stereocenters, several conformations, ring fusions, and multiple spatially isolated phenols. Because empirical NMR and spatial analyses using ROESY/NOESY were unsuccessful in tackling these challenges, we developed a computational pipeline to determine the relative and absolute configurations and phenol positions of diphenazines as inhibitors of eukaryotic translation initiation factor 4E (eIF4E) protein–protein interactions. In this pipeline, we incorporated ECD and GIAO NMR calculations coupled with a DP4+ probability measure, enabling the structure revision of phenazinolin D (4), izumiphenazine A (5), and baraphenazine G (7) and the structure characterization of two new diphenazines, baraphenazine H (3) and izumiphenazine E (6). Importantly, through these efforts, we demonstrate the feasibility of NMR/DP4+ analysis for the determination of phenol positions in phenazine-based molecules, further expanding the limits of computational methods for the structure elucidation of complex natural products.

The RNase III endoribonuclease Dicer was discovered to be associated with cleavage of double-stranded RNA in 2001. Since then, many advances in our understanding of Dicer function have revealed that the enzyme plays a major role not only in microRNA biology but also in multiple RNA interference-related pathways. Yet, there is still much to be learned regarding Dicer structure–function in relation to how Dicer and Dicer-like enzymes initiate their cleavage reaction and release the desired RNA product. This Perspective describes the latest advances in Dicer structural studies, expands on what we have learned from this data, and outlines key gaps in knowledge that remain to be addressed. More specifically, we focus on human Dicer and highlight the intermediate processing steps where there is a lack of structural data to understand how the enzyme traverses from pre-cleavage to cleavage-competent states. Understanding these details is necessary to model Dicer’s function as well as develop more specific microRNA-targeted therapeutics for the treatment of human diseases.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells over normal cells; however, tumor cells may develop TRAIL resistance. Here, we demonstrate that this resistance can be overcome in the presence of bacterial acylhomoserine lactones (AHLs) or AHL-producing bacteria through the combined effect of TRAIL-induced apoptosis and AHL-mediated inhibition of inflammation regulated by NF-κB signaling. This discovery unveils a previously unrecognized symbiotic link between bacteria and host immunosurveillance.

Assays armed with catalytic signal amplificationhave arisen as superior systems for ultrasensitive detection of analytes. A conceptually new enzyme assay called cat‐ELCCA (catalytic assay using enzyme‐linked click‐chemistry) is described, in which an enzyme‐linked azide is utilized to arm the assay with catalytic fluorescence signal amplification. By using this assay technology, the first high‐throughput screen for recently disclosed ghrelin O‐acyltransferase (GOAT) was developed.

Assays mit katalytischer Signalverstärkungsind überlegene Systeme für hoch empfindliche Nachweisverfahren. Ein neuartiger Enzym‐Assay namens cat‐ELCCA (catalytic assay using enzyme‐linked click‐chemistry) wird beschrieben, bei dem ein enzymgebundenes Azid die katalytische Fluoreszenzverstärkung bewirkt. Diese Technik kam beim ersten Hochdurchsatz‐Screennig auf die kürzlich entdeckte Ghrelin‐O‐Acyltransferase (GOAT) zur Anwendung.

The compounds (I) as antagonists of GOAT are promising anti‐obesity and anti‐diabetes drug targets.