DISCOVERY OF NOVEL DRUGS WITH TISSUE AND CELL-TYPE SELECTIVITY FOR CANCER AND INFLAMMATION
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Uncontrolled inflammation is a key factor in multiple disease types, including tissue fibrosis and cancers. The underlying mechanisms and treatment of several of these diseases are still unsolved medical challenges. The studies described in this thesis focused on developing novel cell-type and tissue-selective anti-inflammation and anti-cancer agents that target microinjuries, fibroblast hyperplasia exaggerated extracellular matrix (ECM) deposition and epigenetic dysfunctions. Idiopathic pulmonary fibrosis (IPF) is a life-threatening interstitial lung disease (ILD) of ambiguous cause. IPF is sustained by inflammation caused by chronic injury that promotes inflammatory cytokines release and the accumulation of these cytokines in the bronchial tubes and airways. IPF is a chronic and fatal disease that progressively declines the lung function. Till date, IPF remains untreatable. The FDA approved drugs - pirfenidone (PFD) and nintedanib – are suboptimal in the management of IPF due to their toxic side effects, low potency, cost ineffectiveness and minimal beneficial effect on the patients’ survival rate. In chapter 2 of this thesis, I described four classes of macrolide-based anti-fibrotic agents (28 final compounds) designed to exploit the excellent PK and selective lungs and/or liver tissues distribution activities of the macrolide templates to arrive at novel anti-fibrotic agents that may selectively accumulate within these tissues. I investigated the effects of these compounds on the viability of four cell lines (MRC-5, A549 Hep-G2 and VERO), NF-κB and TGF-β pathways and the levels of fibrosis markers (FN-1, MMP-9, COL1A1, α-SMA). A cohort of these compounds elicit anti-proliferative and anti-inflammatory effects with potency enhancement as high as 1000-fold relative to the standard of care PFD. Based on the data from these experiments, compound 15c was identified as a lead based while the next best compounds are 10c, 11c and 20e. Inspired by the study described in chapter 2, I designed and synthesized macrolide (azithromycin (AZM) and clarithromycin (CLM)) conjugates of three antioxidants – alpha lipoic acid (ALA), fumarate and piperic acid (PIPE) – in chapter 3. After investigation of the cytotoxicity of these macrolide-antioxidant conjugates against cancer cells, normal kidney cell line, and fibroblast cell line, I observed that most of novel compounds showed significant enhancement (more than 100-fold) in cytotoxicity and stronger anti-fibrotic effects relative to their unconjugated antioxidants. Specifically, ALA derivatives showed strong STAT 3 inhibition and extracellular matrix (ECM) components production inhibition effects with attenuation of TGF-β stimulation. Fumarate and PIPE derivatives also demonstrated strong anti-fibrotic effects and Nrf-2 activation. In Chapter 4, I report the discovery that macrolide antibiotic clarithromycin (CLM) undergoes tandem dehydration- cyclization-dehydration reactions, involving C-11 and C-12 hydroxyl groups and the C-9 keto moiety, to furnish a dihydrofuranyl macrolide AO-02-63. I observed that AO-02-63 inhibits the activities of prokaryotic and eukaryotic ribosomes and possibly disrupts the activity of hnRNPs. AO-02-63 also inhibits the proliferation of all cell lines in the NCI-60 panel with low micromolar IC50s and elicits anti-inflammatory activity similar to CLM, although with a 10-fold potency enhancement. The broad anti-cancer activity of AO-02-63 could be due to its inhibition of protein synthesis and mRNA processing, two processes that are vital for the survival of cells. The potential of STAT 3 pathway inhibition as an anticancer and anti-inflammatory strategy is under active investigation in preclinical and clinical settings. Chapter 5 of this thesis focused on validating our hypothesis that simultaneous STAT 3 and histone deacetylase (HDAC) inhibition will lead to more durable anti-proliferative effects in STAT 3-addicted cancer cells. Toward this end, I synthesized 5 pyrimethamine (PYM)-derived compounds and tested them against Hep-G2, A549, VERO, MDA-MB-231, and MCF-7 cell lines. I noticed that these compounds inhibited both HDAC and STAT 3 pathway intracellularly. Interestingly, compounds 12b and 12c showed 6- to 10-fold cell-type selectivity for a STAT 3-dependent, TNBC cell line MDA-MB-231. In Chapter 6, I used an in silico molecular docking tool (Autodock vina) to design three classes of PYM derivatives (total of 12 compounds) as putative STAT 3 inhibitors that function by blocking the DNA binding domain of STAT 3. I synthesized these compounds and profiled their STAT 3 inhibition in a cell free assay. Subsequently, they were analyzed against Hep-G2, A549, VERO, MDA-MB-231, and MCF-7 cell line. I found that class II compounds 11b-d showed 100-fold enhanced cytotoxicity relative to PYM and are also 100-fold better STAT 3 pathway inhibitors. Using a p-STAT 3 DNA binding assay, I found that the STAT 3 inhibition activities of these PYM derivatives are largely due to their direct STAT 3 DNA binding interruption. These PYM-HDAC inhibitors and STAT 3 DNA domain inhibitors could be novel anticancer agents that are selective for STAT 3-addicted cancer cells. In chapter 7, I described results from characterization of the anti-proliferative activities and mechanism of action of 19 glycosylated HDAC inhibitors (HDACi). I found that these compounds are selectively cytotoxic to several HCC cell lines possibly due to GLUT2-mediated uptake with lead compound STR-V-53 significantly more selective for HCC cells. In collaboration with the Petros Lab at Emory University, we found that STR-V-53 is non-toxic to healthy mice (MTD > 100 mg/kg) and effectively suppressed tumor growth in orthotopic murine model of HCC. In addition, we identified STR-V-165 and STR-I-195 as back-up compounds. Collectively, these glycosylated HDACi are promising anti-HCC agents.