Browsing by Subject "Chemical probes"

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    Deciphering Cryptic Stereochemistry in Polyketide Biosynthesis and Functional Characterization of β-processing Domains in Pikromycin Synthase
    (2015-07) Li, Yang
    Combinatorial biosynthesis of polyketides through engineering the respective biosynthetic pathways represents a promising approach to natural products discovery. However, a lack of information on substrate and stereochemical specificity of polyketide synthases (PKS) currently hinders these efforts. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase (KR) and dehydratase (DH) domains of PKS. PKS KRs stereoselectively reduce the β-keto chain intermediate while dictating the orientation of the α-substituent. The DHs of PKS, which generate an α,β-unsaturated bond through dehydration of a β-alcohol, have extremely high stereospecficity towards substrates and can prematurely terminate the nascent polyketide when presented with unnatural substrates. We have developed a chemical tool-based approach which was verified using KR and DH of pikromycin PKS module 2 (PikKR2) as a model system. Triketide substrate mimics were designed to increase stability (incorporating a non-hydrolyzable thioether linkage) and minimize non-essential functionality (truncating the phosphopantetheinyl arm). The identities of reduction and dehydration products as well as steady-state kinetic parameters were revealed by a LC-MS/MS analysis of synthetic standards. Additionally, the substrate specificity was interrogated with a systematic series of synthetic triketides containing altered stereogenic centers. Furthermore, the mechanism of PikDH2 catalyzed dehydration was investigated by site-directed mutagenesis, evaluation of the pH dependence of catalytic efficiency (Vmax/KM), and through kinetic characterization of a mechanism-based inhibitor.
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    Synthesis and Evaluation of Transvalencin Analogues and Adenylation Inhibitors as Antitubercular Agents and Chemical Probes
    (2013-06) Nelson, Kathryn Marie
    >Mycobacterium tuberculosis (Mtb), the etiological agent of pulmonary tuberculosis, is the leading cause of death due to an infectious disease worldwide. Due to a lack of new drug development, poor fidelity to currently available therapeutics, and repeated exposure to therapeutics, Mtb has become multidrug resistant, extensively drug resistant, and even totally drug resistant in some patients. With nearly 9 million deaths and 1.4 million new cases reported by the World Health Organization (WHO) in 2011, new therapeutics that act by novel mechanisms of action are desperately needed to fight this global health threat. Herein we describe our efforts to develop new antitubercular agents by attacking the bacteria's need for iron. This approach involves the inhibition of the biosynthetic pathway to produce siderophores, small molecule iron chelators responsible for acquiring iron in limiting conditions, such as a human host. A prototypical inhibitor of the initiating enzyme in this pathway, MbtA, had previously been developed by our lab, and was used to develop a small set of analogues for in vivo evaluation. We employed Sprague-Dawley rats to evaluate the oral bioavailability of our compounds, revealing that the pKa of the linker nitrogen of the scaffold had a large effect on compound permeability.In addition, we studied the mechanism of action of our parent inhibitor, Sal-AMS, through the development of a photoaffinity probe to label and pull down proteins for target identification. A probe containing a benzophenone moiety for photo-crosslinking and a small alkyne handle for attachment of an imaging or enrichment tag was successfully synthesized. This probe was successful in identifying the intended enzyme of interest (MbtA) as a binding partner, but did not yield any additional hits, suggesting Sal-AMS is a highly specific inhibitor. We also studied a natural product, transvalencin Z, that had been reported as selective against mycobacteria. This compound was very similar in structure to the mycobactins from Mtb, suggesting that it might interfere with iron acquisition or homeostasis. We successfully synthesized the 4 possible diastereomers of the reported structure in an attempt to define the absolute stereochemistry of the natural product, but were unable to match spectroscopic data to the literature report. We attempted to confirm the true stereochemistry through activity testing, but again found our negative results to be in stark contrast to those reported by the discovery group. Finally, we aided a collaborator in the development and synthesis of a probe against DhbE in Bacillus subtilis. Dr. Jun Yin of the University of Chicago was studying the substrate specificity of adenylation domains, and chose an enzyme highly homologous to our target MbtA. We designed a probe that incorporated a similar inhibitor, DHB-AMS, and a long flexible linker with a biotin attached for Dr. Yin's unique yeast cell display assay. Dr. Yin was able to utilize these probes to successfully identify mutant adenylation enzymes with altered specificity towards nonnative substrates. This technique is an exciting new way to potentially access analogues of natural products through manipulation of the biosynthetic machinery, instead of through the organic chemist. These studies have continued to advance our understanding of a new mechanism of action against Mtb, and have brought us one step closer to a preclinical candidate.