Browsing by Subject "Drug discovery"

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.

As non-coding RNAs are increasingly implicated in cellular regulatory functions and disease states, there is a need to deepen our understanding of RNA structure-function relationships as well as to develop methods targeting RNA with small molecules. The transactivation response element (TAR) RNA from human immunodeficiency virus type 1 (HIV-1) is an established drug target for the development of anti-HIV therapeutics and has served as a model system for understanding RNA dynamics and RNA:ligand interactions. Like many RNAs, HIV-1 TAR is a highly flexible molecule that experiences dynamics ranging from local fluctuations in base orientation and interhelical angles to higher-order dynamics that transiently alter base pairing away from the ground state (GS) secondary structure. The work presented in this thesis is aimed at developing approaches targeting TAR with small molecules that integrate its broad range of structural dynamics.

First, nuclear magnetic resonance (NMR) chemical shift mapping is applied in concert with fluorescence binding assays and computational docking to efficiently characterize the TAR-binding modes of a focused library of amiloride derivatives. Through this work, amiloride is established as a novel RNA binding scaffold with interesting structure-activity relationships. Ultimately, this approach yielded ten novel TAR binders with demonstrated selectivity for TAR over tRNA and with up to a 100-fold increase in activity over the parent dimethyl amiloride compound.

Next, we demonstrate that ensemble-based virtual screening (EBVS) is a powerful approach to predict ligand binding for flexible RNA targets. Here, we generate a library to evaluate EBVS enrichment by subjecting HIV-1 TAR to experimental high-throughput screening against ~100,000 drug-like small molecules. EBVS against a dynamic ensemble of the TAR GS determined previously by combining NMR spectroscopy data and molecular dynamics (MD) simulations scores hits and non-hits with an area under the receiver operator characteristic curve of ~0.85-0.94 and with ~40-75% of all hits falling within the top 2% of scored molecules. Importantly, the enrichment was shown to depend on the accuracy of the ensemble.

Finally, we explore the novel strategy of specifically targeting non-native RNA excited state conformations inspired by the fact that their altered secondary structures are likely functionally inactive and highly unique. We use a mutational stabilize-and-rescue approach to demonstrate that TAR ES2 dramatically inhibits TAR activity in cells, suggesting that stabilizing the ES conformation with small molecules would similarly inhibit activity. To pursue TAR ES2 as a potential target, we have determined the first-ever dynamic ensemble of an RNA ES using a combination of MD and NMR residual dipolar couplings (RDCs) measured on a highly accurate ES2-stabilizing mutant. This dynamic ensemble was subjected to our validated EBVS approach to identify small molecules that bind and stabilize TAR ES2. Using NMR chemical shift fingerprinting, we have identified molecules that bind the TAR ES2 structure, including two that induce significant broadening in wtTAR consistent with chemical exchange and two that show a preference for TAR ES2 over the GS.

Together, this work explores multiple novel strategies for structure-specific RNA targeting.

Dengue viruses (DENV) are mosquito‐borne flaviviruses that pose a continued and growing threat to global health. There are estimated to be 390 million DENV infections each year, and because there is no vaccine or approved therapeutic treatment, developing a small‐molecule treatment is imperative. Possible small‐molecule drug therapies for DENV could be immune system modulators, inhibitors of DENV‐required host factor, or inhibitors of a viral gene product. In this study, we chose to take the latter approach and focused our drug discovery efforts on the most highly conserved flaviviral protein, non‐structural protein 5 (NS5). NS5 contains two major domains, each with different enzymatic activities. The N‐terminus has methyltransferase activity, and the C terminus, an RNA‐dependent RNA polymerase (RdRp). The activities of both domains are purine‐dependent, and therefore both domains contribute to the purine‐binding properties of NS5. Inhibition of either of these domains in NS5 results in inadequate propagation of DENV, and the purine‐binding domains present ideal drug targets for disrupting these activities. These factors make NS5 protein an ideal candidate target for our small‐molecule library screen.

A high‐throughput fluorescence‐based screen was employed to identify anti‐DENV compounds based on their ability to competitively bind NS5. The screen was performed by binding green fluorescent protein NS5 fusion protein (GFP‐NS5) to immobilized ATP resin, and then performing parallel elutions using over 3,000 distinct compounds. One compound in particular, HS‐205020, was able to competitively elute GFP‐NS5 from the ATP resin and also exhibited antiviral activity in both the U937+DCSIGN human monocyte cell line and BHK‐21 cells. Additionally, HS‐205020 was able to inhibit DENV NS5 RNA polymerase activity in vitro. HS‐205020 is chemically distinct from the majority of previously reported NS5 inhibitors, which are nucleoside analogs that can cause severe toxicity in animal studies. In contrast, over the concentration range that produced anti‐DENV effects, HS‐205020 showed comparable viabilities to ribavirin, an FDA approved hepatitis C virus (HCV) therapeutic. These findings support HS‐205020 as a potential dengue antiviral candidate, and its chemical scaffold represents as an ideal starting compound for future structure‐activity relationship studies.