Browsing by Subject "Chemical biology"

Heat Shock transcription Factor 1 (HSF1) has long been recognized as the master regulator and signal integrator in the eukaryotic proteotoxic stress response. Revealed by recent discoveries in cancer, the functions of HSF1 have extended far beyond its canonical role in protein folding, further encompassing critical functions in anti-apoptosis, invasion and metastasis, energy metabolism, DNA damage repair, and evasion of host immune surveillance. Meanwhile, both our understanding of the molecular basis of HSF1 regulation as well as available biochemical tools to investigate such details are lacking. Based on an in vitro ligand binding approach, the studies presented in this thesis were dedicated to the identification, validation, and characterization of a direct, first-in-class, small-molecule HSF1 inhibitor. The pharmacological inhibition of HSF1 occurs through small-molecule stimulation of nuclear, but not cytoplasmic HSF1 degradation, which attenuated prostate cancer cell proliferation, inhibited the HSF1 cancer gene signature and arrested tumor progression in multiple therapy-resistant animal models of prostate cancer. The identification of a direct small-molecule HSF1 inhibitor provides a unique pharmacological tool for future HSF1 research and serves as a significant proof-of-concept for pharmacologically targeting HSF1 for anti-cancer treatment approaches.

In 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.

O-linked β-N-acetylglucosamine (O-GlcNAc) exerts myriad effects on protein localization, activation, inhibition, stability, conformational changes, or degradation. However, the biochemical effects of O-GlcNAc on the vast majority of substrates is unknown. Recently, we and others have shown that several coat protein complex II (COPII) components including SEC23A, SEC24C, and SEC31A are O-GlcNAcylated. The COPII coat complex consists of protein coated carriers that mediate secretory trafficking from the endoplasmic reticulum. To determine the effects of O-GlcNAc on COPII we used a combination of chemical, biochemical, cellular and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. We demonstrate that individual O-GlcNAcylation sites of SEC23A are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of cranio-lenticulo-sutural dysplasia (CLSD).

Next, we developed a proteomic workflow to address the challenges of identifying and quantifying novel changes in substrate O-GlcNAcylation in response to a stimulus. Current methods of O-GlcNAcome enrichment suffer from issues with specificity, reproducibility, time-resolution, or require specialized hardware. We developed a novel, unbiased glycoproteomics workflow to survey global changes in O-GlcNAc in response to stimuli. Our approach utilizes both stable isotope labeling with amino acids in cell culture (SILAC) for quantitation and metabolic labeling of O-GlcNAc for enrichment. Using our glycoproteomics workflow we examined the effects of brefeldin A (BFA), a fungal metabolite that disrupts vesicle trafficking, and cytokine deprivation on a pro-B cell line. We identified changes in the O-GlcNAcylation of Coatomer subunit gamma-1 (COPG) a coat protein complex I (COPI) component in response to BFA. Interestingly, COPI mediates traffic from the Golgi to the ER, as well as within the Golgi, and is the specific target of BFA. O-GlcNAcylation of COPI components may have effects similar to O-GlcNAc on COPII, possibly altering membrane binding or the trafficking of specific cargo.

Finally, we identified a candidate O-GlcNAc-mediated binding part of SEC23A using a combination chemical biology tools and mass spectrometry (MS). We identified ankycorbin, a vertebrate specific protein with no known function, as the candidate SEC23A O-GlcNAc-mediated binding partner. However, our attempts to validate this interaction were inconclusive.

Overall, this work examines the role of O-GlcNAc in the vertebrate secretory pathway. We demonstrate the effects of O-GlcNAc on SEC23A in the COPII pathway and identify a potentially novel method of COPI protein trafficking regulation via the O-GlcNAcylation of COPG.

RNA 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.