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Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2020 Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens LU LU ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-1148-7 UPPSALA urn:nbn:se:uu:diva-435815 2021 Dissertation presented at Uppsala University to be publicly examined in Room A1:111a, BMC, Husargatan 3, Uppsala, Friday, 16 April 2021 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Christian Cambillau. Abstract Lu, L. 2021. Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2020. 91 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1148-7. As antibiotic resistance of various pathogens emerged globally, the need for new effective drugs with novel modes of action became urgent. In this thesis, we focus on infectious diseases, e.g. tuberculosis, malaria, and nosocomial infections, and the corresponding causative pathogens, Mycobacterium tuberculosis, Plasmodium falciparum, and the Gram-negative ESKAPE pathogens that underlie so many healthcare-acquired diseases. Following the same- target-other-pathogen (STOP) strategy, we attempted to comprehensively explore the properties of three promising drug targets. Signal peptidase I (SPase I), existing both in Gram-negative and Gram-positive bacteria, as well as in parasites, is vital for cell viability, due to its critical role in signal peptide cleavage, thus, protein maturation, and secreted protein transport. Three factors, comprising essentiality, a unique mode of action, and easy accessibility, make it an attractive drug target. We have established a platform, investigating the protein purification, enzymatic kinetics, and inhibition. A full-length SPase I from E. coli, including two transmembrane segments, was produced and purified in the presence of 0.5 % Triton X-100. In the in vitro biochemical assay, it exhibits proteolytic activity on antigen 85A from M. tuberculosis, with a Km of 20 µM and a kcat of 135 s-1. A series of macrocyclic oligopeptides that have been proven inhibitory to E. coli SPase I also showed potency against a panel of Gram-negative bacteria. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is responsible for the production of methylerythritol phosphate (MEP) in the non-mevalonate pathway of isoprenoid biosynthesis, and is thus essential for cell growth. DXRs from M. tuberculosis and P. falciparum have been under investigation in our lab for years. I addressed structural and biochemical characterizations of PfDXR with analogs of 3-(N-formyl-N-hydroxyamino)propyl- phosphonate (fosmidomycin) and 3-(N-acetyl-N- hydroxyamino)propyl- phosphonate (FR-9000098), two natural products showing potency against P. falciparum. Chemical modifications, methylation at Cg, and double bond formation between Ca and Cb, were investigated to increase the pathogenicidal activity. Crystallographic complex structures of PfDXR and four novel compounds inhibitory to PfDXR in a dose- dependent manner were solved, and ligand binding will be discussed in detail. Type II NADH dehydrogenase (NDH-2) is an essential component in the respiratory chain, playing an important role in electron transfer. Biomembrane-bound NDH-2 from M. tuberculosis was over-expressed in E. coli, as well as the homolog from M. smegmatis. The purified NDH-2s were kinetically characterized, and showed a similar affinity to previously reported NDH-2s expressed M. smegmatis. A collection of novel inhibitors in the scaffold of quinolinyl pyrimidines were synthesized and tested for inhibition in a biochemical assay. Keywords: LepB, DXR, NDH-2, fosmidomycin, quinolinyl pyrimidine Lu Lu, Department of Cell and Molecular Biology, Structural Biology, Box 596, Uppsala University, SE-751 24 Uppsala, Sweden. © Lu Lu 2021 ISSN 1651-6214 ISBN 978-91-513-1148-7 urn:nbn:se:uu:diva-435815 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-435815) To Yani List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I De Rosa M, Lu L, Zamaratski E, et al. Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB). Bioorganic Med Chem. 2017;25(3): 897-911. doi:10.1016/j.bmc.2016.12. Lu L and De Rosa M. contributed equally II załaj N, Lu L, Benediktsdottir A, et al. Boronic ester-linked mac- rocyclic lipopeptides as serine protease inhibitors targeting Esch- erichia coli type I signal peptidase. Eur J Med Chem. 2018; 157:1346-1360. doi:https://doi.org/10.1016/j.ejmech.2018.08. 086 Lu L and załaj N contributed equally III MEPicide: ,-Unsaturated Fosmidomycin N-acyl Analogs as inhibitors that selectively target DXR from Plasmodium falcipa- rum, the deadliest causative parasite of human Malaria. Lu Lu, Xu Wang, Rachel L. Edwards, Amanda Haymond, Rober C. Brothers, Helena Boshoff, Robin D. Couch, Audrey R. Odom, Cynthia S. Dowd, Sherry L. Mowbray, Alwyn. T. Jones (manu- script) IV Synthesis and in vitro biological evaluation of quinolinyl pyrim- idines targeting type II NADH-dehydrogenase (NDH-2) Lu Lu, Linda Åkerbladh, Shabbir Ahmad, Vivek Konda, Sha Cao, An- thony Vocat, Louis Maes, Stewart T. Cole, Diarmaid Hughes, Mats Larhed, Peter Brandt, Anders Karlén, Sherry L. Mowbray (manuscript) Reprints were made with permission from the respective publishers. Additional Publications I Sulfonimidamide Linked Antibacterial Oligopeptides as Type I Signal Peptidase Inhibitors: Synthesis and Biological Evaluation Andrea Benediktsdottir, Lu Lu, Sha Cao, Edouard Zamaratski, Sherry L. Mowbray, Diarmaid Hughes, Anders Karlén and Anja Sandström (manuscript) II Inhibition of gamma-carbon modified analogues of fosmidomy- cin on Plasmodium falciparum 1-Deoxy-D-xylulose-5-phos- phate reductoisomerase. Lu Lu, Alwyn T. Jones, Sherry Mow- bray (manuscript) Contents Introduction ................................................................................................... 11 Infectious diseases .................................................................................... 12 Tuberculosis ......................................................................................... 12 Type I Signal peptidase ............................................................................ 16 Signal peptides (SPs) ........................................................................... 16 Secretory pathway ............................................................................... 17 Signal peptidase families ..................................................................... 18 Signal peptidase I ................................................................................. 19 SPase I Sequences................................................................................ 20 Structure and activity relationship ....................................................... 23 Biochemical assay (paper I and II) ...................................................... 27 Inhibitor development (paper I and II) ................................................ 29 Conclusions and future perspectives.................................................... 34 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/IspC) ........... 36 Isoprenoids ........................................................................................... 36 MVA and MEP pathway ..................................................................... 37 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/IspC) ...... 40 DXR sequences .................................................................................... 40 DXR-targeted drug development ......................................................... 43 The PfDXR structure ........................................................................... 46 PfDXR complexes with unsaturated FOM analogues (Paper III) ....... 48 Conclusions and future perspectives.................................................... 51 NADH dehydrogenase II (Paper IV) ........................................................ 53 Sequences and structures ..................................................................... 54 In vitro biology .................................................................................... 59 evaluation of quinolinyl pyrimidines ................................................... 61 conclusions and future perspectives .................................................... 65 Summary of papers ................................................................................... 66 Paper I: Design, Synthesis and In Vitro Biological Evaluation of Oligopeptides Targeting E. coli Type I Signal Peptidase (LepB) ....... 66 Paper II: Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase. ............................................................................................. 66 Paper III: C-delta analogues of unsaturated Fosmidomycin targeting Plasmodium falciparum 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase (manuscript) ........................................................... 67 Paper IV: Synthesis and in vitro biological evaluation of quinolinyl pyrimidines targeting type II NADH-dehydrogenase (NDH-2) (manuscript) ......................................................................................... 68 Populärvetenskaplig
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