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Title:ISPH fusion proteins, ISPH inhibition, and bisphosphonate mechanism of action
Author(s):O'Dowd, Bing
Director of Research:Oldfield, Eric
Doctoral Committee Chair(s):Oldfield, Eric
Doctoral Committee Member(s):Imlay, James; Gennis, Robert; Chan, Jeff
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):IspH
bisphosphonates
Abstract:In the first part of this work, IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate reductase was studied. In most bacteria, apicomplexan parasites, and plants, the biosynthesis of the five carbon building blocks for terpenes is carried out by IspH. It is an essential enzyme not found in humans and a drug target for infectious diseases as well as herbicides. First, a new class of bacterial IspH we called IspH-RPS1, a fusion protein of IspH with ribosomal protein S1 (RPS1), was characterized. Next, a new class of IspH inhibitors, one without diphosphate groups, was discovered. The characterization of an IspH-RPS1 began with a bioinformatics analysis of the IspH protein family. ~400 sequences out of ~15,000 total sequences in a sequence similarity network of the IspH family were roughly double the length of a typical IspH protein. Most of these sequences contain an IspH domain fused to a RPS1 domain, and most are from obligate anaerobic bacteria. An IspH-RPS1 from Clostridium thermocellum was cloned, expressed, and found to be catalytically active. Mutagenesis studies showed that IspH catalytic activity was independent of the number of RPS1 domains fused to the IspH domain. The sequences being from obligate anaerobes coupled with the oxygen sensitivity of the Fe-S cluster in IspH suggests a possibility of IspH having a secondary function having to do with O2 detection. Next, the way small molecules bind to IspH was studied with the view of gaining insight as to how new inhibitors might be designed. Until the publication of this work, all IspH inhibitors described in the literature contained diphosphate, which make the molecules highly charged and have poor cell penetration. Identifying new inhibitors may lead to the discovery of a cell active IspH inhibitor. Nuclear resonant vibrational spectroscopy (NRVS) and hyperfine sublevel correlation spectroscopy (HYSCORE), along with the examination of numerous IspH and IspG structures revealed the importance of η1 σ-bonding of ligands to the fourth Fe in the 4Fe-4S cluster. Additionally, a virtual screening campaign was carried out by collaborators at the University of California – San Diego, and enzymatic testing of the top hits identified a spirocyclic barbituric acid that exhibited a Ki of ~ 500 nM against Pseudomonas aeruginosa IspH. Synthesis of related molecules identified a benzyl containing analog with a Ki of ~ 3 μM against E. coli IspH. This was the first report of a non-diphosphate containing IspH inhibitor and this finding may help with the development of new IspH inhibitors exhibiting better cell penetration. In the second part of this work, the mechanism of action of bisphosphonates against cancer cell lines were studied. There are two types of bisphosphonates described in the literature: nitrogen containing (NBPs) and non-nitrogen containing bisphosphonates (NNBPs). These drugs are a major class of drugs typically used to treat bone resorption diseases like osteoporosis and Paget’s disease, but also have been found to be effective against cancer and bone metastasis. Bisphosphonates are proposed to target protein prenylation, the epidermal growth factor, or the adenine nucleotide translocase (ANT) via the formation in cells of two ATP-like molecules: an isopentenyl ester of ATP (ApppI) or an ATP analog with the terminal bridging oxygen replaced with CCl2 forming adenosine monophosphate (AMP)-clodronate. Given the high similarity of these metabolites to ATP, it seemed that these molecules might also inhibit kinases. ApppI and AMP-clodronate were screened against a panel of 369 kinases and potent inhibition of some tyrosine kinases by AMP-clodronate was found, while ApppI activity against kinases were less active by ~200x or more. Analogs of AMP-clodronate were synthesized and found to be low nM kinase inhibitors. Bisphosphonate pre-prodrugs that are converted in cells to ATP-analogs were also synthesized and found to inhibit cell-signaling pathways. This work provides clearer explanations behind the clinically observed effects of bisphosphonates, changes our thinking of bisphosphonate mechanism of action, and paves the way for the development of new bisphosphonate drugs for bone resorption, anti-cancer, and anti- inflammatory illnesses.
Issue Date:2018-05-16
Type:Text
URI:http://hdl.handle.net/2142/101638
Rights Information:Copyright 2018 Bing O'Dowd
Date Available in IDEALS:2018-09-27
2020-09-28
Date Deposited:2018-08


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