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Biosynthetic Access to the GEX1A Scaffold: A Novel Therapeutic Approach for Niemann-Pick Type C Disease

thesis
posted on 2018-04-02, 00:00 authored by Eve A. Granatosky

GEX1A, alternatively known as herboxidiene, is a polyketide natural product isolated from Streptomyces chromofuscus that is known to target the spliceosome. Besides its reported anticancer and herbicidal activity, our group has discovered that GEX1A is able to reverse the cellular phenotype associated with Niemann-Pick Type C, or NPC, disease. NPC is a rare lysosomal storage disorder, caused by mutations to the npc1 or npc2 genes. We have shown that GEX1A is able to disperse the characteristic intracellular cholesterol blockade in NPC1 mutant cells similar to other therapeutic agents under investigation for this disease, at notably low doses.

We developed a platform for the sustainable production of GEX1A from S. chromofuscus, by optimizing the media composition and growth conditions for this microorganism. We also determined that fermentation of S. chromofuscus in the presence of small molecules that trigger the native autoregulatory system of Streptomyces has a positive impact on the production of GEX1A. We sought to inhibit specific stages of biosynthesis catalyzed by polyketide tailoring enzymes, by incubating S. chromofuscus with an exogenous inhibitor of cytochrome P450 monooxygenases. Treatment with one specific inhibitor effectively resulted in the production of two putative deoxygenated GEX1A analogues by S. chromofuscus. We also attempted to manipulate GEX1A biosynthesis in S. chromofuscus through genetic manipulation, by inactivating one or more of the genes involved in tailoring the GEX1A scaffold.

We finally examined the effects of GEX1A on in vitro models of NPC disease at the cellular and molecular levels. We determined that several semisynthetic analogues of GEX1A could have a comparable impact on the NPC phenotype as the natural product and highlighted the effects of GEX1A on the levels and isoforms of the NPC1 protein produced in response to treatment. Lastly we performed RNA sequencing to study the influence of GEX1A on the NPC transcriptome. A complete analysis of this expression data, which includes isoform-specific information relevant to GEX1A’s role in splicing modulation, is underway, and will be a valuable contribution to understanding how this natural product, and potentially other structurally related scaffolds, has a beneficial therapeutic effect in the context of NPC disease.

History

Date Created

2018-04-02

Date Modified

2019-10-23

Defense Date

2018-03-21

Research Director(s)

Richard E. Taylor

Committee Members

Shahriar Mobashery Holly Goodson

Degree

  • Doctor of Philosophy

Degree Level

  • Doctoral Dissertation

Program Name

  • Chemistry and Biochemistry

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