Browsing by Author "Hargrove, Amanda E"
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Item Open Access Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures(Science Advances, 2021-11-26) Zafferani, Martina; Haddad, Christina; Luo, Le; Davila-Calderon, Jesse; Chiu, Liang-Yuan; Mugisha, Christian Shema; Monaghan, Adeline G; Kennedy, Andrew A; Yesselman, Joseph D; Gifford, Robert J; Tai, Andrew W; Kutluay, Sebla B; Li, Mei-Ling; Brewer, Gary; Tolbert, Blanton S; Hargrove, Amanda EItem Open Access Deciphering and Leveraging Prominent Features of RNA-Targeting Small Molecules(2021) Wicks, SarahNon-coding RNAs (ncRNAs) are a newly discovered class of biomolecules implicated in biological processes and disease pathogenesis. Small molecules have been proven to be a viable avenue to study biologically relevant ncRNAs; however, the development of RNA-targeting chemical probes has been hindered due in part to a lack of guiding principles for achieving selective RNA:small molecule interactions. Therefore, there is a need to extensively evaluate interactions between chemically diverse small molecules and structurally distinct RNAs in order to comprehensively understand the properties that lead to selective recognition.Recent studies from our laboratory and others have analyzed features of selective RNA-binding compounds and suggested the existence of a privileged chemical space for RNA-targeting small molecules. Consequently, this work aims to examine the potential existence and boundaries of RNA-privileged chemical space. Towards these goals, we have taken three different approaches that generally involve the evaluation of small molecule binding to various RNA targets using indicator displacement assays and computational analysis of molecular features. In the first approach, we explicitly tested and leveraged properties of bioactive, RNA-targeting small molecules through the curation and evaluation of an RNA-biased library. In the second approach, we utilized and evaluated an unbiased library to generally probe and decipher small molecule features that are relevant for targeting RNA. Lastly, in the third approach, we developed and synthesized RNA libraries that consisted of a single secondary structure motif in order to identify favorable features of both RNA and small molecules.
Item Open Access Development of pattern recognition technique for classification of RNA secondary structures by small molecules(2018) Eubanks, ChristopherThree-dimensional RNA structures are notoriously difficult to determine, and the link between secondary structure and RNA conformation is only beginning to be understood. In this dissertation, I will discuss the development of a pattern recognition technique which utilizes differential binding of a receptor to a known analyte to develop patterns of structural motifs, which are then used to evaluate unknown analytes. I applied this method to RNA structures for the first time, revealing guiding principles in RNA:small molecule recognition and supporting our long term goal of classifying unknown RNA conformations and even function. Specifically, an aminoglycoside receptor library was obtained with commercially available aminoglycosides and through simple synthetic approaches. A training set of highly defined RNA secondary structure sequences was identified by computational modeling. I incorporated benzofuranyl uridine, a fluorescent base analogue, into the secondary structure of interest prior to exposing the training set to the small molecule library and the fluorescence changes were used as input for PCA. RNA structures were differentiated based on the five canonical RNA secondary structure motifs. Additionally, unique secondary structures within the Trans-Activation Response element, fluoride riboswitch and pre-queuosine 1 riboswitch could be differentiated based on the position of the fluorophore using the training set clusters. This method provides insight into both the RNA topological and conformational elements and the small molecule ligand properties critical to RNA recognition.
Item Embargo Discovery of RNA-Targeted Small Molecules by Quantitative Structure-Activity Relationship (QSAR) Study and Machine Learning(2023) Cai, ZhengguoRNA is a critical macromolecule in many biological processes by encoding both structural and genetic information. It can serve as the physical template for ribosome read-through during protein synthesis and the intermediary interfering gene expression. For example, messenger RNA encodes specific gene sequence, microRNA regulates expression level of the gene, riboswitch controls translation level and RNA splicing, non-coding RNA provides molecular scaffolding for protein recruitment. Undoubtedly, malfunction of cellular RNAs lead to multiple diseases and targeting disease related RNAs has emerged as the new strategy in many drug development campaigns. Indeed, ribosomal RNA has been utilized as the drug target for a long history and fruitful studies on naturally occurred or synthetic ligands were brought to elucidate the mechanism of translation inhibition. It was the past two decades that witnessed growing research on using small-molecule probes to interrogate non-ribosomal RNAs in various disease pathways.RNA molecules bear distinct chemical properties from proteins that make the design of selective and potent chemical probes challenging. The poor chemical diversity of four building units, immensely charged phosphate backbone, shallow and highly hydrophilic binding pocket, dynamic conformations, all combined render a mysterious ligand space to RNA-targeted small molecules that needs further exploration. A deep understanding of privileged chemotypes or physicochemical properties of RNA-targeting ligands will definitely benefit a broad-scope developing novel chemical entities with desired RNA-interfering outcome. In my thesis work, I first applied the computational approach by building the quantitative structure-activity relationship (QSAR) model to predict the binding profiles of a set of biased ligands scaffolding an amiloride core structure against HIV viral RNA elements. The well-performed model predicted the binding parameters of a set of untested molecules and selected the top-ranked one during lead optimization. The study showed the potential of this computational tool in decision-making during synthesis of RNA-targeted ligands. In the following study, we extended the scope of the QSAR study and leveraged the workflow to cater for the context with diverse structures as substrates. We applied explicit algorithms to build the baseline models to allow easy interpretation of binding behaviors of structurally distinct ligands to HIV-1 TAR. The model first time demonstrated molecular factors that contribute to RNA: small molecule recognition, both kinetically and thermodynamically. The general workflow we described will serve as a powerful computational tool to effectively assess underexplored chemical space and guide decision-making for synthesizing RNA-targeted chemical probes. We then bridged our QSAR approach with the generative deep learning model to pursue de novo ligand design to target SARS-CoV-2 frameshifting pseudoknot. The QSAR model that built on the experimentally validated data provided label annotation of the large training sample for deep learning model. A tree graph-based variational auto-encoder was trained to learn the molecular generation process. Annotated label of each training sample was encoded into the continuous latent space where molecules were reduced their dimensionality and projected. Conditions were applied when sampling new entities from the latent space, leading to the new compounds with desired binding properties. The method mentioned here constitutes the first deep learning practice for automatic chemical design against an RNA target and the first-time application of conditional molecular generation via a junction tree-based variational auto-encoder. Overall, the work presented in this thesis explored possibility of data-driven methods such as QSAR studies and deep learning in accelerating ligand discovery for RNA targets. It is anticipated that these workflows will benefit a wide-range studies in understanding and pursuing RNA-centric drug development, yet slight modifications might be needed for tuning into larger data size.
Item Open Access Insights, Assays, and Strategies for Small Molecule-Based Modulation of the MALAT1 RNA Triple Helix(2020) Donlic, AnitaRecent discoveries of myriad of endogenous RNA transcripts that do not code for proteins has led to a paradigm shift in our understanding of human biology. As the subsequent “RNA revolution” continues to elucidate the disease-related functions and structures of mammalian non-coding RNA molecules, interest in developing small molecule probes and drug leads for these targets is on the rise. For example, the long non-coding RNA MALAT1 (Metastasis-Associated-Lung-Adenocarcinoma-Transcript-1) is overexpressed in and is critical for metastasis in a variety of cancers. A recently characterized triple helix structure at the 3'-end of MALAT1 was shown to be pivotal in stabilizing the transcript, and deletions or a single base substitution - proposed to destabilize this structure - led to a significant decrease in MALAT1 accumulation. While this unique structure represents a novel opportunity for therapeutic intervention, there is a gap in knowledge regarding effective strategies for targeting RNA in general and, in particular, triple helices.
Towards elucidating these principles, this work takes a three-pronged approach: i) synthesis and evaluation of scaffold-based small molecule libraries to deduce fundamental ligand-triple helix recognition principles, ii) development of robust and high-throughput screening assays to discover functional probes, and iii) structural characterization of the MALAT1 triple helix conformational landscape for small molecule-based regulation. In the first area, we have discovered first small molecule ligands for the MALAT1 triple helix and gained insights into the role of three-dimensional small molecule shape and intramolecular interactions in binding events. Additionally, we established a correlation between ligand-induced thermal stabilization and prevention of exonucleolytic degradation of this target and elucidated binding properties of ligands with triplex-stabilizing functions. In the second area, we developed a high-throughput screening (HTS) technique based on differential scanning fluorimetry and curated an RNA-targeted library to identify novel small molecules with a destabilizing effect on the triple helix. Such thermal melting profiles were predictive of accelerated degradation profiles of the triple helix, thereby providing a robust discovery tool for identifying functional small molecule modulators of this target. In the third area, we conducted chemical probing experiments to characterize secondary structures that may exist in equilibrium with the triple helix and hence represent attractive targets for small molecule stabilization.
Together, this work has and will continue to build a set of small molecule tools, rapid assays, and novel design strategies to enable chemical biology-based interrogation of the MALAT1 triple helix role in cancer biology. This knowledge and tools can be utilized to interrogate the protective mechanism of the MALAT1 triple helix, to identify and develop therapeutic leads, as well as to apply these strategies for the study of other therapeutically relevant RNA.
Item Embargo Interrogating and Elucidating Drivers of Selective RNA-Ligand Interactions(2024) Hay, Emily Grace SwansonRecent efforts to better understand the human genome have uncovered several roles of non-coding RNAs in biology. We have also seen the number of RNA viruses causing epidemics and pandemics rising in recent years, with the most recent being the COVID-19 pandemic. With the growing number of RNA targets came increased efforts to target RNAs for therapeutic purposes. Numerous studies have now been published on RNA-targeting with small molecules, but in the context of all drug-targeting efforts, RNA-targeting is still developing. To date, there is only one FDA-approved drug whose mode of action is known to be caused by binding to non-ribosomal RNA. Despite the limited number of successful drugs, there are growing resources for RNA-small interactions. Many databases have been published detailing RNA-binding small molecules for better understanding the existing ligands that bind RNA. These databases, including the RNA-targeted BIoactive ligaNd Database (R-BIND) curated in the Hargrove lab, have led to a consensus of the bulk properties of small molecules that bind RNA. And while this is a critical first step, there is still much to learn about the types of small molecules that can bind to different RNA structures. RNAs contain a range of structures that fold into varied 3-dimensional shapes that can be targeted by small molecules. We hypothesized that small molecules that bind RNAs with similar structures, containing similar pockets, may have unique properties from molecules that bind to other structures. To date, the concept of small molecule differentiation of RNA structures is underexplored, and thus in this dissertation it will be investigated from a number of approaches. First, we have updated and expanded the scope of the R-BIND database. Specifically, with new ligands being added to the database, we have been able to implement a secondary structure search that identifies lead molecules based on the structures they bind. Further, through linear discriminant analysis we have seen that the small molecules in R-BIND that target unique structural classes have some distinct properties. Additionally, we have shown that small molecules from different libraries (e.g. commercial or synthetic) are enriched in different properties which may also influence the types of targets they are suited for. While understanding the existing literature supported the idea that there are unique properties, we were limited in terms of structural classes to those with several published small molecule binders. Therefore, to continue identifying novel trends in small molecule differentiation of RNA structures, we turned to screening methods to identify small molecules that bind a set RNA secondary and tertiary structures. Initial work was focused on developing a novel peptide-based fluorescent indicator with global RNA binding to use in high throughput screen. In this work nanomolar affinity peptides were identified, however fluorescent-labeling resulted in significant aggregation that could not be overcome for use in high-throughput screens. Ultimately, this study was performed with a small molecule dye as the indicator. A set of >15,000 molecules was screened for binding to nine RNA targets. This screening resulted in a set of 181 hits, 82 of which were unique to one of the nine targets. With these unique hits properties enriched in molecules selective for one RNA structure over another were identified. Of particular interest, we found that molecules that bound to stem loops, which may contain more traditional pockets, were more sphere-like than molecules that bound to g-quadruplexes where stacking is thought to be a binding mechanism. Finally, we investigated the specific recognition of small molecules with an RNA tertiary structure – triple helices. In this work we identified a novel interaction between a small molecule-metal-RNA complex, where small molecule chelation of iron or copper is necessary for binding to RNA. Together this work represents significant efforts to better understand how small molecules recognize unique RNA structures, which can inform future therapeutic design.
Item Embargo Investigating the Structure:Dynamics:Function Relationship of the MALAT1 Triple Helix(2023) Kassam, Kamillah JenaThe “noncodingRNA (ncRNA) revolution” in the late 20th and early 21st century triggered the transition from scientists viewing ncRNA as cellular junk to realizing that ncRNA plays a variety of roles in biological functions in both healthy and disease related processes. With this discovery came the desire to drug the transcriptome and develop therapies to ameliorate diseases that had previously been thought of as undruggable. Among the potential RNA targets discovered was the ncRNA MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) a long non-coding RNA that is expressed at relatively high levels in cells. MALAT1 was first identified as a marker for lung cancer, then as an oncogenic transcript shown to be over accumulated in several different cancer phenotypes. Previous work has shown the therapeutic potential of knocking down this transcript, making it an attractive potential target. In addition, the 3′-end of the mature MALAT1 transcript forms a U•A-U rich triple helix that evades normal cellular degradation pathways through the sequestration of an A-rich tail between two U-rich regions. Targeting this region with small molecules was shown to decrease metastasis in an organoid model, indicating the promise of the region as a drug target. However, the most effective targeting of an RNA must begin with understanding the underlying structure:dynamics:function relationship. Towards that end, this work aims to increase the understanding of the structure:dynamics:function relationship of the 3′-end of MALAT1 by: 1) probing the conformational landscape of the triple helix in different sequence contexts, 2) examining the protective function of the triple helix in different sequence contexts, and 3) investigating binding-competent structures found in the MALAT1 triple helix ensemble. In the first aim, we show that the 3′-end of MALAT1 is predicted to form modular, independently folding secondary structures. In addition, we report evidence of non-triplex contacts forming within the triple helix, supporting the presence of alternate, non-triple helix structures in the ensemble of the MALAT1 3′-end. In the second aim, we probe the change in protective function of the triple helix within different native sequence contexts and report the development of an enzymatic assay that we believe will be of use in probing the protective function of RNA triple helices and other RNA motifs in general. In the third aim, we investigate binding-competent structures within the triple helix ensemble through use of a mutation construct.
Item Open Access RNA-Targeted Small Molecule Ligand Discovery via Imine-Based Dynamic Combinatorial Chemistry(2021) Umuhire Juru, AlineIn addition to coding for proteins, RNA molecules play direct functional roles in cellular metabolism, including the regulation of chromatin architecture and the regulation of gene expression at the transcriptional and translational level. Due to their many roles, RNA molecules have been implicated in the progression of cancers, neurological and infectious diseases. As a result, RNA is being pursued as a potential drug target for many indications. The better pharmacokinetic properties and cell delivery profiles of small molecules, as well as their non-immunogenicity, makes them a more attractive option for targeting RNA compared to antisense- and peptide-based technologies. However, despite several emerging examples of small molecules that target RNA selectively in the cell, RNA drug discovery remains challenging. For example, although the discovery of small molecule properties that allow selective targeting of certain RNA topologies would expedite RNA drug and probe discovery, such investigations may suffer from the lack of novel small molecule libraries. Towards the expansion of small molecule libraries available for RNA ligand discovery, in this work we developed an approach that allows rapid tandem synthesis and screening of a large number of novel small molecules against an RNA of interest. Specifically, we used imine-based dynamic combinatorial chemistry, which allows template-guided assembly of ligands from aldehydes and amine building blocks. In a proof-of-concept study, we demonstrated the feasibility of imine-based dynamic combinatorial chemistry approach on a known RNA-binding scaffold. Additionally, we discovered guiding principles for increased reactivity of aromatic amines towards reductive amination in buffer. This information is critical in ensuring that a high percentage of the expected library members can indeed be accessed in aqueous conditions. Finally, we observed that bicyclic amines led to increased RNA-binding of the amiloride-based library, supporting the potential of this method to identify preferred chemotypes for different RNA topologies. In subsequent studies, we designed three 120-member libraries utilizing relatively simple commercial aldehydes and amine building blocks. To test the applicability of this method to RNAs with complex 3D architecture, we tested the libraries against a three-way junction RNA from the HIV-1 genome packaging signal. Hit compounds identified in this screen were independently synthesized and shown to have higher RNA-binding activity compared to non-hits. Our results show that imine-based dynamic combinatorial chemistry is a promising tool that can enable rapid discovery of ligands for RNAs with a range of size and 3D complexity. This capability will in turn expedite our understanding of small molecule properties that lead to preferential recognition of various RNA structural motifs.
Item Open Access Small Molecule Targeting of Disease Relevant RNA Secondary and Tertiary Structures(2022) Zafferani, MartinaThe discovery of the many active roles of RNA in diseased pathways propelled the current ‘RNA Renaissance’. As a result, RNA is no longer regarded as a passive intermediate between DNA and proteins, but as a key player in human disease and an attractive therapeutic target. Small molecules are uniquely poised for RNA-targeting due to their potential for favorable bioavailability, cell permeability, and spatio-temporal control, overcoming the current limitations of gene editing and sequence-based technologies. Nevertheless, questions regarding favorable properties that render small molecules specific for RNA and, ideally, selective for one RNA target over others are far from being answered. Thus, to elucidate possible trends that can expedite future targeting efforts we leveraged both synthetic and commercially available approaches in targeting disease relevant structures in mammalian and viral RNAs. We designed and synthesized a focused library and evaluated it using a holistic approach against the RNA triple helix motif present at the 3’-end of the mammalian oncogenic long non-coding RNA MALAT1. The development of triple helix-focused assays enabled us to assess small molecules’ affinity and their effect on the triplex structural stability and enzymatic digestion. This study resulted in the first example of synthetically tuned small molecules that can bi-directionally modulate the susceptibility of the triplex to enzymatic degradation. In a subsequent study we leveraged the well-established relationship between the structural stability of the MALAT1 triple helix and its refractivity to degradation as a case-study to develop a high-throughput platform that directly reports on the effects of mutations or small molecules on the stability of the triple helix. Optimization and application of this platform to RNA G-quadruplexes and pseudoknots corroborates the potential of this assay to bridge the current gap between affinity-based assays and cell-based activity. With the emergence of SARS-CoV-2, we focused our efforts in identifying potential RNA targets within the viral genome that would be amenable to small molecule targeting with the goal of developing new antiviral agents. Specifically, we first focused on conserved RNA structures at the 5’-end of the viral genome and utilized an in-house focused library based on the known RNA-binding scaffold amiloride. Through evaluation of small molecules leads using a plethora of in vitro and in cellulo assays we identified three small molecule leads that inhibited viral replication by targeting structures at the 5’-end of the genome. This study represented the first example of RNA-targeted SARS-CoV-2 antivirals. We then focused on an RNA structure present at the interface of two overlapping open reading frames and responsible for programmed ribosomal frameshifting, and essential process in coronavirus lifecycle. Screening of an RNA-biased commercially available library curated by our laboratory resulted in the identification of a small molecule selective for the CoV-2 pseudoknot over other disease-relevant triple helices. The work presented herein provides essential insights into RNA:small molecules molecular recognition events and provides tools and workflows that will significantly expedite future targeting efforts against the RNA structures presented in this work and others yet to be identified or explored.
Item Embargo Structural characterization of the long noncoding RNA SChLAP1 reveals therapeutically tractable interfaces of RNA:protein recognition(2024) Falese, James PaulLong noncoding RNAs (lncRNAs) are non-proteinogenic RNAs greater than 200 nucleotides in length and can play roles in both housekeeping processes and disease conditions. Prostate cancer is one of the most commonly occurring forms of cancer, and it constitutes the second-leading cause of cancer-related death among American men despite the availability of treatment options. Thus, there is an urgent and unmet need to characterize the molecular drivers of aggressive prostate cancer ultimately to facilitate the development of next-generation prostate cancer therapies. The lncRNA Second Chromosome Locus Associated with Prostate 1 (SChLAP1) has been identified in multiple studies as prognostic of poor patient outcomes and a driver of aggressive prostate cancer in in vivo and cellular models. Despite its association with aggressive prostate cancer, its mechanism of action has remained elusive. We hypothesized that structure-function characterization of SChLAP1 would enable identification of functionally important regions worthy of follow up biological study and therapeutic targeting. Herein, we report our efforts toward this goal. Specifically, we performed SHAPE-MaP on SChLAP1, which allowed us to generate an experimentally informed structure model and identify protein binding regions. Subsequent native gel electrophoresis of a retroviral-derived element within SChLAP1 revealed a heterogenous structure significantly sensitive to metal concentration and RNA preparation. We also provide evidence that SChLAP1 binds chromatin as part of its biochemical mechanism. Lastly, we report our initial efforts to identify SChLAP1- targeted small molecules targeting likely functional substructures based on our data. This work provides a comprehensive analysis of SChLAP1 structure-function relationships, which we hope will inspire future efforts to therapeutically target SChLAP1 and favor better outcomes for prostate cancer patients.
Item Open Access Structure-Function Relationships of Long Non-coding RNA in Prostate Cancer(2020) McFadden, Emily JosephineThe noncoding RNA (ncRNA) revolution has revealed myriad RNA species that play critical roles at all stages of life, including embryogenesis and disease progression. For example, three long ncRNA (lncRNA), Hox Transcript Antisense Intergenic RNA (HOTAIR; ~2.5 kb), Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT1; ~6.7 kb) and Second Chromosome Locus Associated with Prostate-1 (SChLAP1; ~1.5 kb), are basally expressed in normal prostate tissue but are dysregulated in prostate cancer. HOTAIR scaffolds the PRC2 and LSD1 protein complexes to selectively methylate and demethylate, respectively, histone proteins, thereby regulating downstream gene expression. MALAT1 acts in trans at nuclear speckles during mRNA post-transcriptional processing while SChLAP1 acts in cis to influence oncogenic gene expression. Initial work on HOTAIR developed technical skills that were then applied to the lncRNAs MALAT and SChLAP1 to learn more about their role in prostate cancer. There is compelling evidence that the 3′–end of MALAT1 is a triple helix structure that acts as a molecular knot, driving transcript accumulation in cancer cells and furthering their metastatic potential, but we currently lack any biophysical data detailing the relationship between SChLAP1 structure and function. In general, the relationship among lncRNA structure, dynamics, and function is not well understood; for example, even with high-resolution structures of the MALAT1 triple helix, questions remain regarding the role of intrinsic dynamics in transcript stability or protein binding. As lncRNA represent an underexplored therapeutic avenue, this work aims to investigate the role of lncRNA structure and dynamics in driving prostate cancer metastasis. This work uses biophysical and biochemical methods including chemical probing, NMR, SAXS, and native gels to study the two lncRNA MALAT1 and SChLAP1 and learn more about their respective structure-function relationships. From this work, we have found a discrete structure within the lncRNA SChLAP1 that is highly structured and implicated in driving metastasis via protein recognition. Additionally, our preliminary studies regarding MALAT1 support the presence of non-triplex states that require further characterization. Overall, this work supports a deeper understanding of lncRNA structure as it relates to their function in cancer and provides examples for the biophysical analysis of large and/or structurally complex RNA.
Item Open Access Sulfamate Esters Guide Remote C–H (Hetero)arylation Reactions and Target Disease-Relevant Non-coding RNAs(2022) Kwon, KitaeAlcohols are ubiquitous motifs in the constellation of organic molecules. Given their prevalence in the world around us, new synthetic methods for forging new bonds on alcohols and preparing biologically active alcohol derivatives provide toolkits to solve challenging problems in organic chemistry. First, new bond-forming reactions with alcohols streamline access to complex alcoholic scaffolds commonly found in natural products and pharmaceuticals. Second, commercial and synthetic availabilities of alcohols offer rapid entry to new chemical space or expansion of pre-existing chemical space for biologically active molecules. My dissertation meets these motivations by masking alcohols as sulfamate esters, which are useful traceless linkers for C–H functionalizations and heteroatom-rich bioactive molecules.
The first part of my dissertation describes photochemically mediated, nickel-catalyzed γ-C(sp3)–H (hetero)arylation reaction between sulfamate esters and (hetero)aryl bromides to affect traditionally challenging net C(sp2)–C(sp3) cross-coupling reactions. Hitherto, there were no general methods that convert γ-C(sp3)–H bonds of aliphatic alcohols into γ-C(sp3)–C(sp2) bonds under mild photochemical conditions. Fortunately, this transformation was realized by photochemically generating nitrogen-centered free radical intermediate and introducing nickel catalyst to orchestrate radical relay/C(sp2)–C(sp3) cross-coupling cascade reaction. The second part of my dissertation applies sulfamate esters in the arenas of medicinal chemistry and biochemistry. Herein, these heteroatom-enriched masked alcohols were surveyed as a novel class of small molecule ligands for targeting disease-relevant non-coding RNAs. Informed by machine learning and rational molecular design, I developed a new generation of sulfamate esters that should herald a new chemical space for RNA therapeutics.
Item Open Access Towards Guiding Principles for Targeting RNA: Rational Approaches to Design and Synthesize RNA-Biased Small Molecule Libraries(2018) Morgan, Brittany SuzanneRNA is increasingly recognized as a therapeutic target in many diseases, including viral and bacterial infections, neurodegenerative disease, and cancer. Despite this, no FDA-approved drugs target RNA, aside from select antimicrobials that recognize highly abundant ribosomal RNA. Currently, most RNA-targeted, small molecule screens utilize commercially available libraries; however, these libraries are presumably biased to protein-binding chemotypes, leading to low hit rates for RNA and/or the identification of promiscuous ligands with limited efficacy in biological systems. Additionally, previous attempts to characterize distinct guiding principles for targeting RNA were unsuccessful, potentially due to the choice of criteria (e.g. in vitro binding) or the limited selection of parameters (e.g. Lipinski’s rules). As a result, few rationally designed, RNA-focused libraries have been developed and screened against non-ribosomal RNAs, hindering the identification of chemical probes and the study of disease-causing RNAs. Therefore, the goal of my dissertation work is to elucidate guiding principles for selectively targeting RNA and utilize the rules to rationally design and synthesize novel RNA-biased libraries.
Toward this goal, 2-oxazolidinone was chosen as the core for small molecule synthesis, as the scaffold is present in FDA-approved antimicrobials that target ribosomal RNA as well as small molecules that inhibit the T-box riboswitch in vitro. In addition to being diversity-oriented, the developed synthetic scheme utilized commercially available, chiral amino acid derivatives, which allowed for stereochemical control and therefore, the study of stereocenters in RNA binding and selectivity. Five scaffolds and fifteen bis-substituted small molecules were synthesized, and a preliminary screen identified a secondary structure binding preference for RNA bulge motifs. In parallel, the RNA-targeted BIoactive ligaNd Database (R-BIND) was curated, which was comprised of RNA-binding ligands with activity in cell culture and animal models. The library was compared to in vitro RNA binders and FDA-approved drugs from which several guiding principles were identified for bioactive RNA-binding ligands: i) compliance to common medicinal chemistry rules; ii) a statistically significant shift in rod-like character; and iii) a statistically significant increase in nitrogen atom count and ligand rigidity. These rules were utilized to develop a biology-oriented synthesis based on the 2-oxazolidinone core and bioactive RNA chemical space. The strategy included a novel scaffold design, a subunit library inspired by the building blocks in R-BIND, and a method to select small molecules for synthesis based on similarity to known RNA bioactives. Future work will include the diversification and expansion of the 2-oxazolidinone-based libraries, screening of the small molecules against secondary structure libraries as well as therapeutically relevant RNAs, and validation and exploration of bioactive RNA-privileged chemical space. It is expected that the design strategies and guiding principles identified in this and future work will establish an avenue to create and/or select additional RNA-focused libraries, facilitating the discovery of novel RNA-targeted chemical probes and therapeutics.