Graduate Thesis Or Dissertation
 

Pactamycin Inspired Drug Discovery: A Synthetic and Chemoenzymatic Approach

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  • Pactamycin, first reported in 1962, is a potent antitumor antibiotic produced by the soil bacterium Streptomyces pactum. Structurally, it contains a cyclopentitol core unit, a 3-aminoacetophenone (3AAP), a 6-methylsalicylic acid (6-MSA), and a N,N-dimethyl urea. The aminocyclopentitol ring is derived from glucose, possibly via N-acetyl glucosamine (GlcNAc), the 3-aminoacetophenone (3AAP) moiety is derived from 3-aminobenzoic acid (3ABA), and the 6-MSA moiety is produced from acetate by an iterative type I polyketide synthase. Despite some knowledge of its biosynthetic origin, details of the mode of formation of this unique natural product are still elusive. Using genetic, chemical complementation, and biochemical studies we demon-strate that 3ABA is processed by a set of discrete polyketide synthase proteins, i.e. an AMP-forming acyl-CoA synthetase (PtmS), an acyl carrier protein (ACP) (PtmI), and a -ketoacyl-ACP synthase (PtmK), to give 3-[3-aminophenyl]3-oxopropionyl-ACP, which is then glycosylated by a broad spectrum N-glycosyltransferase, PtmJ (Chapter 2). This is the first example of glycosylation of an ACP-bound polyketide intermediate in natural product biosynthesis. Additionally, we demonstrate that PtmO is a hydrolase that is re-sponsible for the release of the glycosylated -ketoacid product from the ACP, and the free -ketoacid product subsequently undergoes non-enzymatic decarboxylation. In addition to the β-ketoacyl-ACP synthase gene ptmK, the pactamycin biosyn-thetic gene cluster also contains a gene (ptmR) that encodes a β-ketoacyl-acyl carrier protein (β-ketoacyl-ACP) synthase (KAS) III. KAS III catalyzes the first step in fatty acid biosynthesis, involving a Claisen condensation of the acetyl-CoA starter unit with the first extender unit, malonyl-ACP, to form acetoacetyl-ACP. KAS III-like proteins have also been reported to catalyze acyltransferase reactions using coenzyme A esters or discrete ACP-bound substrates. Through in vivo and in vitro characterizations of the KAS III-like protein PtmR, we discovered that this enzyme directly transfers a 6-methylsalicylyl moi-ety from an iterative type I polyketide synthase (PtmQ) to the aminocyclopentitol unit in pactamycin biosynthesis (Chapter 3). PtmR is highly promiscuous, recognizing a wide array of S-acyl-N-acetylcysteamines as substrates to produce a suite of pactamycin de-rivatives with diverse alkyl and aromatic features. The results suggest that KAS III-like proteins may be used as versatile tools for modifications of complex natural products for drug discovery. The pronounced biological activity displayed by pactamycin spans across all three phylogenetic domains. Unfortunately the indiscriminate cytotoxicity of pactamycin towards mammalian cells has suppressed its development toward therapeutic appli-cation. Nevertheless, we believe pactamycin is a wellspring of promising biological activity that is waiting to be harnessed. Our previous work demonstrated, through biosynthetic manipulations, production of new pactamycin analogs with pronounced antimalarial activity, lacking significant antibacterial activity, and are about 10–30 times less toxic than pactamycin toward mammalian cells. Furthermore, we have developed a chemoenzymatic process using the promiscuous KAS III-like protein PtmR to produce new pactamycin analogs. Continuing our efforts to draw further on the bountiful activity of the aminocycli-tol core of pactamycin, we have taken a third approach by synthesizing the core amino-cyclopentitol ring which could open up a diverse library of biologically active com-pounds. In Chapter 4, we describe an efficient, modular, and asymmetric synthesis of several aminocyclopentitol compounds resembling the pactamycin pharmacophore be-lieved responsible for its biological activity. The outlined synthesis work has generated four promising biologically active compounds, two of which display modest activity against Gram-positive bacteria, whereas the other two compounds exhibit potent anti-cancer activity against A375 melanoma cells.
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  • Intellectual Property (patent, etc.)
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  • 2017-12-01 to 2019-01-01

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