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Characterizing and Alleviating Androgen Receptor-Mediated Transcriptional Repression of Tumor Suppressor Gene GPER1McDermott, Austin 24 May 2022 (has links)
No description available.
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Transcription factor regulation of amyloid-beta pathway genes by SP1-Modulating compounds : a novel approach in Alzheimer's DiseaseBayon, Baindu L. 07 July 2017 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence of neuritic plaques consisting of extracellular amyloid-beta (Aβ) and neurofibrillary tangles comprised of hyperphosphorylated microtubule associated tau. Aβ is produced following the cleavage of amyloid precursor protein (APP) by the enzyme BACE1. Transcription factors (TFs) are proteins involved in the regulation of gene transcription. Expression levels of some TFs are perturbed in AD. SP1 binding sites on both the APP and BACE1 promoters implicate its potential role in AD. Aβ peptide itself mediates activation of cyclindependent kinase 5 (CDK5), an enzyme which phosphorylates the FOXO (Forkhead Box) TFs. In order to study mechanisms of TF regulation of Aβ production in human models, neuronally differentiated cells as well as a primary human neurosphere culture were used to test the effects of TF-modulating compounds. Our hypothesis is that by targeting relevant TFs via pharmacological inhibitors in human cells, BACE1 activity or APP expression will decrease and Aβ production will be reduced as a result. To test the involvement of TFs in the regulation of APP, we treated several mammalian cells lines and post-mitotic human neuronal cells with roscovitine, mithramycin A (MTM), MTM analogs (MTM-SDK, MTM-SK), and tolfenamic acid (TA). MTM and TA treatment of neurons differentially activated several TFs implicated in AD. Treatment of differentiated neurospheres with MTM led to a significant decrease in APP and SP1 expression along with Aβ40 levels. Epigenetic mechanisms involve alteration of the binding affinity between DNA and transcription factors. We predict that modulation of these TFs may be influenced by epigenetic modifications. To test the effects of drugs on epigenetic markers, histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity was measured. MTM-SDK significantly decreased DNMT activity in differentiated neuroblastoma cells, this may enhance or decrease the ability of SP1 to bind to target DNA and affect transcription of BACE1 or APP. Targeting TF activity is a novel means to manipulate the amyloid pathway. Compounds modifying TF binding to sites on the BACE1 or APP promoters may provide a means to limit the production of amyloid-beta and slow the symptoms of AD.
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INVESTIGATING STRUCTURE AND PROTEIN-PROTEIN INTERACTIONS OF KEY POST-TYPE II PKS TAILORING ENZYMESDowney, Theresa E 01 January 2014 (has links)
Type II polyketide synthase (PKS) produced natural products have proven to be an excellent source of pharmacologically relevant molecules due to their rich biological activities and chemical scaffolds. Type II-PKS manufactured polyketides share similar polycyclic aromatic backbones leaving their diversity to stem from various chemical additions and alterations facilitated by post-PKS tailoring enzymes. Evidence suggests that post-PKS tailoring enzymes form complexes in order to facilitate the highly orchestrated process of biosynthesis. Thus, protein-protein interactions between these enzymes must play crucial roles in their structures and functions. Despite the importance of these interactions little has been done to study them. In the mithramycin (MTM) biosynthetic pathway the Baeyer−Villiger monooxygenase (BVMO) MtmOIV and the ketoreductase MtmW form one such enzyme pair that catalyze the final two steps en route to the final product. MtmOIV oxidatively cleaves the fourth ring of the mithramycin intermediate premithramycin B (PreB) via a Baeyer−Villiger reaction, generating MTM’s characteristic tricyclic aglycone core and highly functionalized pentyl side chain at position 3. This Baeyer−Villiger reaction precedes spontaneous lactone ring opening, decarboxylation, and the final step of MTM biosynthesis, a reduction of the 4′- keto group catalyzed by the ketoreductase MtmW.
Another example of co-dependent post-PKS tailoring enzymes from the gilvocarcin biosynthetic pathway is composed of GilM and GilR. These two enzymes form an unusual synergistic tailoring enzyme pair that does not function sequentially. GilM exhibits dual functionality by catalyzing the reduction of a quinone intermediate to a hydroquinone and stabilizes O-methylation and hemiacetal formation. GilM mediates its reductive catalysis through the aid of GilR that provides its covalently bound FADH(2) for the GilM reaction, through which FAD is regenerated for the next catalytic cycle. A few steps later, following glycosylation related events unique to each gilvocarcin derivative, GilR dehydrogenates the hemiacetal moiety created by GilM to establish the formation of a lactone and the final gilvocarcin chromophore. To achieve a better understanding of post-type II PKS tailoring enzymes and their protein-proteininteractions for the benefit of future combinatorial biosynthetic efforts two specific aims were devised.
Specific aim 1 was to investigate the structure of MtmOIV and the role of active site residues in its catalytic mechanism.
Specific aim 2 was to integrate the function of GilM and its protein-protein interactionswith GilR that lead to their synergistic activity and sharing of GilR’s bicovalently bound FAD moiety.
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DEVELOPMENT OF MITHRAMYCIN ANALOGUES WITH IMPROVED EFFICACY AND REDUCED TOXICITY FOR TREATMENT OF ETS-DEPENDENT TUMORS IN EWING SARCOMA AND PROSTATE CANCEREckenrode, Joseph Michael 01 January 2019 (has links)
Introduction: Genetic rearrangements in Ewing sarcoma, prostate, and leukemia cells result in activation of oncogenic ETS transcription factor fusions. Mithramycin (MTM) has been identified as an inhibitor of EWS-FLI1 transcription factor, a gene fusion product responsible for oncogenesis in Ewing sarcoma. Despite preclinical success, a phase I/II clinical trial testing MTM therapy in refractory Ewing sarcoma was terminated. Liver and blood toxicities resulted in dose de-escalation and sub-therapeutic exposures. However, the promise of selectively targeting oncogenic ETS transcription factors like EWS-FLI1 prompted us to undertake the discovery of more selective, less toxic analogues of MTM. MTM is a potent inhibitor of ubiquitous SP1 transcription factor, likely inducing non-specific toxicity. In collaboration with two medicinal chemistry groups, two semi-synthetic efforts were implemented to develop novel analogues of MTM. The first effort utilized the biosynthetic product mithramycin SA (MTMSA) to modify C3-side chain. The second effort utilized an oxime linker directly formed on MTM’s C3-side chain (MTM-oxime; MTMox). Here I present the pharmacological assessment of over 75 novel MTM analogues towards selectively targeting oncogenic ETS transcription factors, like EWS-FLI1, over ubiquitous transcription factors, like SP1.
Methods: Novel MTM analogues were evaluated for selective cytotoxicity against ETS fusion-dependent cell lines. Selectively cytotoxic analogues were evaluated for inhibitory effects on several gene promoters in TC-32 reporter cell lines, a Ewing sarcoma cell line dependent on EWS-FLI1, transfected with luciferase reporter vector. Cloned reporter vectors incorporated NR0B1 (EWS-FLI1 binding), β-actin (SP1 binding) and CMV (non-specific) gene promoters. Furthermore, gene (mRNA) and protein expression changes of EWS-FLI1 and SP1, as well as regulated target genes, namely NR0B1, VEGFA and BCL-2 were evaluated with MTM analogue treatments. The MTM analogues with most selective activity in vitro were administered to mice by intravenous bolus dose for pharmacokinetic analysis. The MTM analogues with highest systemic exposure from each semi-synthetic effort, namely MTMSA-Trp-A10 and MTMox-24, were further evaluated. Metabolic stabilities in whole blood, plasma, and tumor cell matrices, and across multiple species were compared with MTM. Moreover, intrinsic hepatic clearances were estimated using mouse liver microsomes. Tumor and liver distributions were estimated in tumor bearing mice. Additionally, the effect of organic anionic transporter polypeptides (OATP) on distribution of MTM was investigated. Maximum tolerated doses were evaluated for lead MTM analogues, having both selective activities in vitro and high systemic exposure, compared to MTM. Complete blood cell counts and plasma alanine aminotransferase activity were measured to evaluate dose-dependent blood and liver toxicities, respectively. ETS fusion-dependent and non-dependent cell lines were implanted subcutaneously into immunocompromised mice for efficacy studies. Average tumor volumes and survival were tracked for mice receiving treatment, compared to MTM and vehicle treatment.
Results: Evaluation of MTM analogues from both semi-synthetic efforts revealed that conjugation of MTM C3-side chain with tryptophan (Trp) and/or phenylalanine (Phe) improved selective cytotoxicity against ETS fusion-dependent cell lines. This was highlighted by MTMSA-Trp-A2 (also refer to as MTMSA-Phe-Trp) and MTMSA-Trp-A10 (also refer to as MTMSA-Trp-Trp), with selective indices of 19.1 and 15.6, respectively, compared to MTM (1.5). Similarly, MTMox-23 (also refer to as MTMox-Phe-Trp) and MTMox-20 (also refer to as MTMox-Trp) had selectivity indices of 4.6 and 4.5, respectively. These selectively cytotoxic MTM analogues inhibited EWS-FLI1-mediated transcription 10-fold more effectively than both non-specific CMV-mediated and SP1-mediated (via β-actin promoter) transcription in TC-32 reporter cell lines. Moreover, gene (mRNA) and protein expression of EWS-FLI1 and regulated gene, NR0B1, were inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Similarly, SP1 and target genes, VEGFA and BCL-2, gene (mRNA) and protein expressions were also inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells.
Conjugation of Trp and/or Phe to C3-side chain of MTM increased systemic exposure in vivo. Most impressively, the addition of two Trp residues, namely MTMSA-Trp-A10 and MTMox-24 (also refer to as MTMox-Trp-Trp), resulted in systemic exposure increases of 218- and 42-fold, respectively, after intravenous (IV) bolus dose. Metabolically, tryptophan/phenylalanine conjugated MTM analogues are liable to esterase activity on carboxy-methyl functional group. Very rapid de-methylation in biological matrix was observed with MTMox-24, compared to MTMSA-Trp-A10, suggesting a regiospecific effect. However, esterase activity was limited to rodent matrices and demethylation occurred at significantly diminished rates in non-human primate and human plasma. MTM analogues were not susceptible to p450-mediated metabolism, with negligible loss in mouse liver microsome assay compared to verapamil control. MTM (1mg/kg) and MTMox-24 (6mg/kg) were detected in subcutaneously implanted (flank) LL2 tumors and liver homogenates after IV bolus dose. Interestingly, MTMSA-Trp-A10 (2mg/kg) was not. Despite a 3-fold increase in systemic exposure with rifampin oral pretreatment, an OATP inhibitor, exposure of MTM was unaffected in Oatp knockout mouse model. Exposure of MTM in liver tissue was 8.4-fold higher compared to tumor tissue with low tissue clearance. This agrees with the lack of metabolism observed in liver microsomes and may provide a mechanism for clinically observed liver toxicity.
MTMSATrp-A10 had a single maximum tolerated dose (MTD) of 0.75mg/kg, compared to 1mg/kg for MTM, administered by IV bolus. In contrast, MTM-oxime analogues (MTMox-20, -23, -24 and -25) had single maximum tolerated doses of 20 – 25mg/kg. These increased tolerances are the result of additive differences in whole blood stability, cytotoxicity and systemic exposure. At a dose of 0.75mg/kg, administered every 3 days, MTMSA-Trp-A10 did not result in an efficacious result in tumor xenograft studies. These studies remain under further investigation, but the result may indicate high plasma protein binding of MTMSA-Trp-A10 and lack of free fraction available within tumor. The most selective MTM-oxime analogue in vitro, MTMox-23, significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0025, day 16, one-way ANOVA multiple comparisons test) compared to MTM (p=0.1174, day 16) and extending survival for 17 days out of 48 days on study (p=0.0003, Log Rank (Mantel-Cox) single comparison test) with treatment at MTD every 3 days, compared to vehicle. Additionally, the MTM-oxime analogue with highest systemic exposure, MTMox-24, also significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0003, day 21, one-way ANOVA multiple comparisons test) compared to MTM (p=0.032, day 21) and extending survival for 12 days out of 37 days on study (p=0.0004, Log Rank (Mantel-Cox) single comparison test) with treatment, compared to vehicle.
Conclusion: These studies in whole highlight the importance of exposure (pharmacokinetics; PK), toxicity and efficacy (pharmacodynamics; PD) relationships. The cytotoxicity and high systemic exposure of MTMSA-Trp-A10 directly contributes to its lower tolerated dose. However, despite a similar tolerated dose to MTM, systemic exposure remains 163-fold higher at the MTD. High systemic exposure may be attributed to high plasma protein binding, but also reduces the exposure of free MTMSA-Trp-A10 within the tumor tissue, which drives the efficacious response. In contrast, the less cytotoxic and rapidly de-methylated MTM-oxime analogues allow for 25-fold higher tolerances in mice. This unique metabolism and clearance may prevent exposures required to induced systemic blood and liver toxicities induced by MTM. Moreover, at these highly tolerated doses, the initial systemic exposure at MTD is highest among analogues tested, which resulted in an efficacious response with MTMox-23 and MTMox-24 treatment in tumor xenograft models. It remains to be determined if these PK/PD relationships can be reproduced in additional animal models, including human, without inducing toxicity. Nonetheless, these initial studies in mice demonstrate that a more selective, more tolerated analogue of MTM has potential for clinical success in treating ETS fusion-dependent tumors.
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Chemoenzymatic Studies to Enhance the Chemical Space of Natural ProductsChen, Jhong-Min 01 January 2015 (has links)
Natural products provide some of the most potent anticancer agents and offer a template for new drug design or improvement with the advantage of an enormous chemical space. The overall goal of this thesis research is to enhance the chemical space of two natural products in order to generate novel drugs with better in vivo bioactivities than the original natural products.
Polycarcin V (PV) is a gilvocarcin-type antitumor agent with similar structure and comparable bioactivity with the principle compound of this group, gilvocarcin V (GV). Modest modifications of the polyketide-derived tetracyclic core of GV had been accomplished, but the most challenging part was to modify the sugar moiety. In order to solve this problem, PV was used as an alternative lead-structure for modification because its sugar moiety offered the possibility of enzymatic O-methylation. We produced four PV derivatives with different methylation patterns for cytotoxicity assays and provided important structure-activity-relationship information.
Mithramycin (MTM) is the most prominent member of the aureolic acid type anticancer agents. Previous work in our laboratory generated three MTM analogues, MTM SA, MTM SK, and MTM SDK by inactivating the mtmW gene. We developed new MTM analogues by coupling many natural and unnatural amino acids to the C-3 side chain of MTM SA via chemical semi-synthesis and successfully made some compounds with both improved bioactivity and in vivo tolerance than MTM. Some of them were consequently identified as promising lead-structures against Ewing’s sarcoma.
The potential of selectively generating novel MTM analogues led us to focus on a key enzyme in the biosynthetic pathway of mithramycin, MtmC. This protein is a bifunctional enzyme involved in the biosynthesis of TDP-D-olivose and TDP-D-mycarose. We clarified its enzymatic mechanisms by X-ray diffraction of several crystal complexes of MtmC with its biologically relevant ligands. Two more important post-PKS tailoring enzymes involved in the biosynthesis of the MTM side chains, MtmW and MtmGIV, are currently under investigation. This would not only give us insight into this biosynthetic pathway but also pave the way to develop potentially useful MTM analogues by engineered enzymes.
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