Adaptation of RiPCA for the Live-Cell Detection of mRNA-Protein Interactions.

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.

Structural Insights into the Advances and Mechanistic Understanding of Human Dicer.

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.

Comparative Biochemical Studies of Disease-Associated Human Dicer Mutations on Processing of a Pre-microRNA and snoRNA.

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 vitro biochemical 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.

An ECD and NMR/DP4+ Computational Pipeline for Structure Revision and Elucidation of Diphenazine-Based Natural Products.

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.

Optimization of RiPCA for the Live-Cell Detection of Pre-MicroRNA-Protein Interactions.

Advancements in methods for identifying RNA-protein interactions (RPIs) on a large scale has necessitated the development of assays for validation of these interactions, particularly in living cells. We previously reported the development of RiPCA (RNA interaction with protein-mediated complementation assay) to enable the cellular detection of the well-characterized interaction between the pre-microRNA, pre-let-7, and its RNA-binding protein (RBP) partner Lin28. In this study, the applicability of RiPCA for the detection of putative pre-miRNA-protein interactions was explored using an improved RiPCA protocol, termed RiPCA 2.0. RiPCA 2.0 was adapted to detect the sequence specificity of the RBPs hnRNP A1, Msi1, and Msi2 for reported pre-microRNA binding partners. Additionally, the ability of RiPCA 2.0 to detect site-specific binding was explored. Collectively, this work highlights the versatility of RiPCA 2.0 in detecting cellular RPIs.

The RNA-binding protein SART3 promotes miR-34a biogenesis and G1 cell cycle arrest in lung cancer cells.

MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of most biological processes. Global miRNA biogenesis is altered in many cancers, and RNA-binding proteins play a role in miRNA biogenesis, presenting a promising avenue for targeting miRNA dysregulation in diseases. miR-34a exhibits tumor-suppressive activities by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor BCL-2, among other regulatory pathways such as Wnt, TGF-ß, and Notch signaling. Many cancers exhibit down-regulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo However, the post-transcriptional mechanisms that cause miR-34a loss in cancer are not entirely understood. Here, using a proteomics-mediated approach in non-small-cell lung cancer (NSCLC) cells, we identified squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RNA-binding protein with no previously reported role in miRNA regulation. We found that SART3 binds pre-miR-34a with higher specificity than pre-let-7d (used as a negative control) and elucidated a new functional role for SART3 in NSCLC cells. SART3 overexpression increased miR-34a levels, down-regulated the miR-34a target genes CDK4/6, and caused a cell cycle arrest in the G1 phase. In vitro binding experiments revealed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Our results provide evidence for a significant role of SART3 in miR-34a biogenesis and cell cycle progression in NSCLC cells.

A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation.

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein that forms an α-helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding-induced structural changes are low-throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.

The role of olefin geometry in the activity of hydrocarbon stapled peptides targeting eukaryotic translation initiation factor 4E (eIF4E).

Hydrocarbon stapled (HCS) peptides are a class of cross-linked α-helix mimetics. The technology relies on the use of α,α'-disubstituted alkenyl amino acids, which fully contrain the helical region to typically yield peptides with enhanced structural ordering and biological activity. Recently, monosubstituted alkenyl amino acids were disclosed for peptide stapling; however, the impact that this tether has on HCS peptide structure and activity has not yet been fully explored. By applying this HCS to the disordered peptide eIF4E-binding protein 1 (4E-BP1), we discovered that this type of tethering has a dramatic effect on olefin geometry and activity of the resultant stapled peptides, where the putative trans isomer was found to exhibit enhanced in vitro and cellular inhibitory activity against eIF4E protein-protein interactions. We further demonstrated that the metathesis catalyst used for ring-closing metathesis can influence monosubstituted HCS peptide activity, presumably through alteration of the cis/trans olefin ratio. This study represents one of the first in-depth analyses of olefin isomers of a stapled peptide and highlights an additional feature for medicinal chemistry optimization of this class of peptide-based probes.

Inhibitor of MYC identified in a Kröhnke pyridine library.

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.

High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes.

MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

Second-Generation Cap Analogue Prodrugs for Targeting Aberrant Eukaryotic Translation Initiation Factor 4E (eIF4E) Activity in Drug-Resistant Melanoma.

Melanoma is the deadliest form of skin cancer with a 5-year survival rate of less than 20%. While significant strides have been made in the field of kinase-targeted and immune-based therapies for melanoma, the development of resistance to these therapeutic agents has hindered the success of treatment. Drug-resistant melanoma is particularly reliant on enhanced cap-dependent translation to drive the production of oncoproteins that promote growth and survival. The m7GpppX cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting factor of cap-dependent translation initiation, and its overexpression in melanoma tumors has been shown to drive resistance to BRAFV600E kinase-targeted inhibitors. These findings point to eIF4E-targeted therapies as a promising strategy to overcome drug resistance in melanoma. Herein, we build upon our previous work of developing cell-permeable cap analogue inhibitors to design second-generation cap analogues that inhibit eIF4E-mediated cap-dependent translation in drug-resistant melanoma cells.

Live cell screening to identify RNA-binding small molecule inhibitors of the pre-let-7-Lin28 RNA-protein interaction.

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.

MNK-driven eIF4E phosphorylation regulates the fibrogenic transformation of mesenchymal cells and chronic lung allograft dysfunction.

Tissue fibrosis remains unamenable to meaningful therapeutic interventions and is the primary cause of chronic graft failure after organ transplantation. Eukaryotic translation initiation factor (eIF4E), a key translational regulator, serves as convergent target of multiple upstream profibrotic signaling pathways that contribute to mesenchymal cell (MC) activation. Here, we investigate the role of MAP kinase-interacting serine/threonine kinase-induced (MNK-induced) direct phosphorylation of eIF4E at serine 209 (Ser209) in maintaining fibrotic transformation of MCs and determine the contribution of the MNK/eIF4E pathway to the pathogenesis of chronic lung allograft dysfunction (CLAD). MCs from patients with CLAD demonstrated constitutively higher eIF4E phosphorylation at Ser209, and eIF4E phospho-Ser209 was found to be critical in regulating key fibrogenic protein autotaxin, leading to sustained ß-catenin activation and profibrotic functions of CLAD MCs. MNK1 signaling was upregulated in CLAD MCs, and genetic or pharmacologic targeting of MNK1 activity inhibited eIF4E phospho-Ser209 and profibrotic functions of CLAD MCs in vitro. Treatment with an MNK1/2 inhibitor (eFT-508) abrogated allograft fibrosis in an orthotopic murine lung-transplant model. Together these studies identify what we believe is a previously unrecognized MNK/eIF4E/ATX/ß-catenin signaling pathway of fibrotic transformation of MCs and present the first evidence, to our knowledge, for the utility of MNK inhibitors in fibrosis.

Empowering Voices: Inspiring Women in Medicinal Chemistry.

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.

Empowering Voices: Inspiring Women in Medicinal Chemistry.

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.

Development of a high-throughput screen and its use in the discovery of Streptococcus pneumoniae immunoglobulin A1 protease inhibitors.

Streptococcus pneumoniae relies on a number of virulence factors, including immunoglobulin A1 protease (IgA1P), a Zn(2+) metalloprotease produced on the extracellular surface of the bacteria, to promote pathogenic colonization. IgA1P exhibits a unique function, in that it catalyzes the proteolysis of human IgA1 at its hinge region to leave the bacterial cell surface masked by IgA1 Fab, enabling the bacteria to evade the host's immune system and adhere to host epithelial cells to promote colonization. Thus, S. pneumoniae IgA1P has emerged as a promising antibacterial target; however, the lack of an appropriate screening assay has limited the investigation of this metalloprotease virulence factor. Relying on electrostatics-mediated AuNP aggregation, we have designed a promising high-throughput colorimetric assay for IgA1P. By using this assay, we have uncovered inhibitors of the enzyme that should be useful in deciphering its role in pneumococcal colonization and virulence.

Repositioning of an existing drug for the neglected tropical disease Onchocerciasis.

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness.

Contemporary Progress and Opportunities in RNA-Targeted Drug Discovery.

The surprising discovery that RNAs are the predominant gene products to emerge from the human genome catalyzed a renaissance in RNA biology. It is now well-understood that RNAs act as more than just a messenger and comprise a large and diverse family of ribonucleic acids of differing sizes, structures, and functions. RNAs play expansive roles in the cell, contributing to the regulation and fine-tuning of nearly all aspects of gene expression and genome architecture. In line with the significance of these functions, we have witnessed an explosion in discoveries connecting RNAs with a variety of human diseases. Consequently, the targeting of RNAs, and more broadly RNA biology, has emerged as an untapped area of drug discovery, making the search for RNA-targeted therapeutics of great interest. In this Microperspective, I highlight contemporary learnings in the field and present my views on how to catapult us toward the systematic discovery of RNA-targeted medicines.

Design of Cell-Permeable Inhibitors of Eukaryotic Translation Initiation Factor 4E (eIF4E) for Inhibiting Aberrant Cap-Dependent Translation in Cancer.

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.