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

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.

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 macrocyclic 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 falciparum, 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 (manuscript)

IV Synthesis and in vitro biological evaluation of quinolinyl pyrimidines 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 fosmidomycin on Plasmodium falciparum 1-Deoxy-D-xylulose-5-phosphate 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 sammanfattning ...... 69 Acknowledgement ...... 72 References ...... 75

Abbreviations

Acetyl-CoA acetyl coenzyme A AG arabinogalactans AIDS acquired immunodeficiency syndrome ART antiretroviral therapy ATP adenosine triphosphate CB conserved box CNS central neural system COVID-19 coronavirus disease 2019 CPZ chlorpromazine CTD C-terminal domain DDM n-dodecyl-β-D-maltoside DELTA-BLAST domain enhanced lookup time accelerated blast DFD dimer forming domain DMAPP dimethylallyl pyrophosphate DMSO dimethyl sulfoxide DXP 1-Deoxy-D-xylulose 5-phosphate DXR 1-Deoxy-D-xylulose 5-phosphate reductoisomerase EFPIA European federation of pharmaceutical industries and associations ELF European lead factory FI fluorescence intensity FOM fosmidomycin FRET fluorescence resonance energy transfer GAS group A Streptococcus glpT glycerol-3-phosphate transporter HIV human immunodeficiency virus HPLC high performance liquid chromatography JECL joint European compound library MIC minimal inhibitory concentration MRSA methicillin-resistant S. aureus MS mass spectrometry MVA mevalonate IC50 half maximal inhibitory concentration IgG immunoglobulin G IMAC immobilized metal affinity chromatography INH isoniazid

IPP isopentenyl pyrophosphate LAM lipoarabinomannans LepB leader peptidase MDR multiple drug resistant MEP methylerythritol phosphate MOA mode of action mRNA messenger ribonucleic acid NADH nicotinamide adenine dinucleotide NCD non-catalytic domain NDH-2 NADH:quinone oxidoreductase NTD N-terminal domain OG n-Octyl β-D-glucopyranoside PAS para-aminosalicylic acid PDB protein data bank PG peptidoglycans QP quinolinyl pyrimidine SARS severe acute respiratory syndrome S/B signal/background SDS-PAGE sodium dodecyl sulfate–polyacrylamide gel electro- phoresis SEIAS surface-enhanced infrared absorption spectroscopy SP signal peptide SPase I signal peptidase I SPR signal recognition particle TB tuberculosis TPZ trifluoperazine TFPP type IV leader peptidase UQ2 ubiquinone-2 WHO world health organization XDR extensive drug resistant

Introduction

People across the globe have been under the threat of this particular pandemic of COVID-19 for more than a year, which is caused by yet another SARS- like corona virus. With the memory of SARS in 2003 that has not gone too vague, COVID-19 came down more strikingly, and more contagiously. Since the pandemic occurred, 113,498,837 (20210225 11:36pm time zone: GMT+1) people have been infected, while tremendous research funds and effects have been invested on control measures, treatment studies, and research and development of vaccines. The vaccine development for COVID-19 has become the record breaking in human history, which from-development-to-deployment took roughly less than a year. COVID-19 is a little extreme, but it is still merely an infectious disease. Other pathogen-related diseases have been the targets of biological research for decades. Scientists have made great progress since the beginning of pharmacology, and there are much more that modern medicines can do nowadays. The research in our lab mainly focuses on the development of inhibitors as antibiotics that target pathogens causing tuberculosis and malaria. By characterizing the interactions of known inhibitors and target enzymes in vitro, in the whole-cell assay, and structurally at an atom level, inhibitors are purposely chemically modified to improve the potency and/or cell penetration, thus eventually developed to a prodrug. In my thesis, I will give a brief introduction of the history and current situ- ation of some infectious diseases, e.g. tuberculosis, and its corresponding causative agent, Mycobacteria tuberculosis. I will summarize the background and current studies of three selected potential drug targets, signal peptidase type I (SPase I or LepB), 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR or IspC), and NADH dehydrogenase II (NDH-2). LepB has been investigated, because of its critical role in secretion pathway. It has been constructed, produced, and kinetically characterized. A biochemical assay based on a novel FRET substrate was established, and utilized to characterize newly designed inhibitors. DXR is the second enzyme in the MEP pathway which is absent in humans. Four unsaturated fosmidomycin analogues have been co- crystallized with DXR, and the crystal structures of four complexes have been determined by x-ray crystallography. NDH-2 crucial for the viability of bacteria has been characterized kinetically. Several quinolinyl pyrimidine analogues have been tested in the biochemical assay, and exhibited both potency against NDH-2 and antibacterial activity.

11 Infectious diseases An infectious disease is defined as an illness that results from the infection, and more importantly, that can be transmitted from one individual to another. An infection is not only a phenomenon of an invasion of other organisms or body tissues, but also a reaction of the host bodies to the invasion. The reactions may differ, particularly depending on the types of infections and infected body tissues, but in general, fever, fatigue, loss of weight, pains are frequently observed. The causative agents/pathogens consist of bacteria, viruses, fungi, and parasites. Besides the corona virus infected disease 19 (COVID-19) mentioned in the introduction, the other two notorious infectious diseases are also caused by viruses. Influenza is a seasonal respiratory disease caused by influenza A and influenza B viruses, resulting in 3-5 millions severe cases and averagely 0.5 millions deaths annually during 1999-2015 (Lozano et al. 2012). The other one is AIDS. Acquired immunodeficiency syndrome (AIDS) is caused by the infection of human immunodeficiency virus (HIV), accounting for 60 millions incidents and 25 millions deaths since the discovery of the virus in 1981 (World Health Organization 2009). During 40 years of development of antiretroviral therapies (ART), 5 classes of drugs are present in the market, res- cuing millions of lives annually (Dieffenbach and Fauci 2011). It is worth noting that HIV unfortunately cannot be cured completely, but controlled by effectively suppressing the replication of the virus to prolong the life expectancy of patients. The treatments of bacterial infections are relatively convenient. Without interfering with abundant organelles in the host’s cells to proliferate, invaded bacteria are easier to track and target with corresponding antibiotics. Antibi- otics are naturally extracted or artificially synthesized substances that show antibacterial activity. Ever since the discovery of penicillin by Alexander Fleming in 1928, hundreds of types of antibiotics have been applied to treat patients with infections of diverse pathogens. The efficacy and effectiveness of existing antibiotics, however, have become compromised, as more cases of multiple-drug-resistant (MDR) and extensive-drug-resistant (XDR) strains emerged largely and more frequently. The simple reason is the incompatible speed of drug development to bacterial evolutions. The resistance of bacteria to current antibiotics is a ‘widespread serious threat’ that is happening every- where globally (WHO 2017). Tuberculosis is one of the most common bacteria infections in the world.

Tuberculosis Tuberculosis is an infectious disease remaining the leading cause of death from a single infectious agent in adults, caused specifically by Mycobacteria

12 tuberculosis. A quarter of the global population are infected with it. Tubercu- losis initially affects lungs of the hosts, but can occasionally spread to other parts of the body, and if untreated, the death rate can rise up to two thirds of the cases. Tuberculosis can be airborne, resulting in indirect transmission. The symptoms of tuberculosis patients include fever, chronic coughing, chest pain, and weight loss. Generally, patients carrying M. tuberculosis show no symptoms, but when the immune system compromises for other reasons, the tuberculosis emerges to be active (WHO 2020b). TB existed from ancient times. The study of spontaneous mutation fre- quency suggested that TB had evolutionarily emerged first in East Africa 15,000-20,000 years ago (Kapur, Whittam, and Musser 1994; Sreevatsan et al. 1997). Clear evidences found in bone lesions of the mummies in Egypt 5,000 years ago confirmed the long history of TB, such as tubercle bacilli revealed in fresh blood in the trachea and lungs under the microscope (Zimmerman 1979). The first written record of TB was found in Greece, where TB was called as phthisis, and the symptoms were described. More written records were found in Europe, including a clear elucidation of pathogenesis of tuberculosis and a standard concept definition (Daniel 2006). Despite that TB has been present all along the human history, it only became epidemic in the early 1800s. The reasons can be the premise of epidemic, the crowdedness of the population. The industrial revolution dramatically improved the socioeco- nomic conditions, resulting in population increasing more rapidly. Besides, the lack of hygiene, poor nutrition, and poor healthcare synergized the formation of TB epidemics (Bates and Stead 1993). In 1905, Robert Koch became the Nobel Prize laureate in Physiology or Medicine for his discovery of the pathogenesis as tubercle bacillus, Mycobacteria tuberculosis. A tuberculin skin test was invented when Robert Koch was attempting to find a cure for TB (Kaufmann and Schaible 2005). In the mid 1800s, TB rates in the population started to decline, although unfortunately, no cure for TB was found. This probably was due to better living conditions (Lönnroth et al. 2009). The first vaccine for TB and the efficient test promoted the control of TB in the first half of the 1900s in the war time. The first antibiotics discovered to be potent against M. tuberculosis was streptomycin, which became the first drug for the treatment of TB. However, spreptomycin is proven disadvangeously to have neurotoxic side effects, and had lost the efficiency rather fast because of bacteria evolution (Schatz, Bugle, and Waksman 1944). Para-aminosalicylic acid (PAS), was also used as a chemotherapeutic drug for the treatment of TB, but became inefficient quite soon for the same reason as streptomycin (LEHMANN 1946). The treatment of a combination of two drugs was more efficient against TB in clinical trials (DUNNER, BROWN, and WALLACE 1949). The strategy was followed in the future drug development, when a novel antibacterial drug isoniazid was developed by three pharmaceutical companies (Rieder 2009). All three existing drugs were combined as a cocktail treatment, which had been efficiently

13 curing TB patients as a standard procedure for 15 years (Fox, Ellard, and Mitchison 1999). Two more chemotherapeutic drugs, a newly discovered ri- fampicin, and a reinstated drug pyrazinamide were added to the combination (SENSI, MARGALITH, and TIMBAL 1959). And it had been the major treatment for TB since 1960s, and is still in use.

Figure 1. Estimated TB incidence rates, 2019 (WHO 2020b)

WHO has launched the End TB strategy, aiming to completely eliminate TB cases in a long period, with various milestones set at different time points. Thanks to it, The TB incidence rates in the world are declining, but it has not reached the WHO’s first milestone, which is 20 % between 2015 and 2020. 78 countries mostly in Europe and some in Africa, have followed the strategy, and had a reduction of the incidence rates at 20%. However, reductions in the regions were not as satisfactory, while in Brazil the trend is on the contrary rising. All in all, TB, as the leading killer of infectious diseases, accounted for roughly 1.4 millions deaths globally in 2019, including 208, 000 people with HIV (WHO 2020a). Progress to reduce TB incidence rates has been made, and the access for people to TB treatment has increased largely. However, similar to the response of TB to streptomycin and PAS, the multiple-drug-resistant (MDR) and extensively drug-resistance (XDR) strains started to dramatically emerge globally in recent years (Gandhi et al. 2010). The need for novel, effective, antibacterial drugs is urgent, before all existing antibiotics become inefficient to TB.

14 The causative bacterium of tuberculosis, Mycobacteria tuberculosis is a 2- 4 m, rod-shaped bacterium. Its characteristic feature is the thick cell wall and long replication time. M. tuberculosis can replicate in 15-20 hours, compared to 20 min for E. coli. The species from the same genus Mycobacterium are responsible for other diseases, such as leprosy, caused by Mycobacterium leprae, a TB-like disease in AIDS patients, caused by Macobacterium avium (Inderlied, Kemper, and Bermudez 1993). It was also found that livestocks, rarely humans, can be infected with Mycobacterium bovis, to develop TB (Alfred G. Karlson 1970). Mycobacterium smegmatis is asymptomatic when infected, and additionally, it has a shorter replication time than M. tuberculosis, thus has been scientifically useful in research (Gordon and Smith 1955). Unlike other bacteria, which can be distinguished into Gram-positive and Gram-negative bacteria, based on their cell wall features, the Genus Myco- bacteria possess a thick cell wall of peptidoglycans, but show no response in Gram-staining test. This is due to the unique way of constructing of their cellular envelopes. The M. tuberculosis cell envelope consists of four layers: the plasma membrane, the cell wall core, the outer membrane, and the capsule (Kaur et al. 2009). 1), the plasma membrane is similar to conventional biomembrane comprising a phospholipid double layer, except that phosphatidyl- inositol functions as anchoring points for lipoarabinomannans (LAM) cross the cell wall, which has been found to be responsible for bacterial survival from macrophages (Briken et al. 2004). 2), the cell wall core is composed of peptidoglycans (PG) and arabinogalactans (AG). PGs are the construction building blocks to provide the rigidity for the cell shape, and AGs attach PGs to the mycolic acids of the outer membrane (Crick and Brennan 2008). 3), mycolic acids compose the outer cell wall, the thickness of which changes in the different life phases, and in the response to exterior stimuli. This is important for the chemotherapeutic treatments, and the intake of nutrition (Queiroz and Riley 2017). 4), the capsule is the waxy layer on the exocellular surface coating the mycobacterial cells. It consists of the polysaccharide glu- can, which also functions as a protection against macrophages (Lemassu and Daffé 1994). M. tuberculosis, the causative agent of tuberculosis, has been investigated intensively from diverse aspects. The studies of the bacteria have a profound meaning in the development of anti-tubercular treatments.

15 Type I Signal peptidase In 1980, ‘leader peptide hydrolase’ was first discovered by Ziwzinski and Wickner in E. coli, purified ‘6000-fold’ from E. coli membranes, and assayed with an isotopically labelled precursor of bacteriophage M13 coat protein(Schechter 1973a; Zwizinski, Date, and Wickner 1981). Afterwards, the leader peptide hydrolase, annotated as leader (signal) peptidase (Lep) was studied intensively, provided the outer space of cytoplasmic membrane where the enzyme is located, and other features investigated later, consequently, the high potential for druggability. The history of signal peptidase can be traced back to the 1950s when the protein secretion pathway was still a mystery. Until 1971, a signal hypothesis was proposed and presented as a vague description of secretion protein transport, addressing the existence of a functionally conserved signal peptide at amino terminus of secreted proteins. (Blobel and Sabatini 1971). In 1975, Blobel and Dobberstein experimentally confirmed the signal hypothesis, by comparing the size of the translation products from the same mRNA in a protein synthesizing system in vitro, and the authentic secreted protein. He and his group demonstrated the intra-cellular route of non-cytoplasmic proteins starting from synthesis by ribosome, transportation via rough endoplasmic reticulum, and the final localization on the Golgi apparatus prior to secretion (BLOBEL and DOBBERST 1975) A series of experimental observations in immunoglobulin (IgG) and myeloma (MOPC-41) confirmed the signal hypothesis (M. GREEN, P. N. GRAVES, T. ZEHAVI-WILLNER, J. McINNES 1975; Mach, Faust, and Vassalli 1973; MILSTEIN et al. 1972; Schechter 1973b; Schmeckpeper, Cory, and Adams 1974; Swan, Aviv, and Leder 1972; Tonegawa and Baldi 1973)

Signal peptides (SPs) The phenomenon of signal peptide cleavage occurs both in prokaryotic and eukaryotic cells. In most cases, the signal peptides of secreted proteins and transmembrane proteins are removed by signal peptidases, except for integral membrane proteins, whose signal peptides or signal peptides-related sequences are anchored to the membrane (von Heijne 1990). Provided that the function of signal peptide was to partition in cell membranes and be recog- nized by components in the secretion pathway, the structure of signal peptides has shown preferred features for this purpose. Studies of SPs in both prokaryotic and eukaryotic organisms show that they shared a similar structure. Three regions, an amino terminal positively charged region (n-region, 1-5 amino acids long), a hydrophobic region (h-region, 7-15 amino acids long), and a car- boxy-terminal region (c-region, 3-7 amino acids long), have distinct functions in the machinery. The n-region plays an important role in protein translocation. Mutagenesis from a positively-charged amino acid to negatively-charged one

16 in bacterial SPs resulted in much lower rate in translocation, but no evidence showed it would cause a complete silencing (Lehnhardt et al. 1988; Puziss, Fikes, and Bassford 1989; Vlasuk, Inouye, and Inouye 1984) Translocation rate of eukaryotic SPs showed less dependency on the n-region, experimentally confirmed by insertion of amino acids into the n-region in yeast (Brown et al. 1984). The function of the h-region has been studied mostly by mutagenesis. Disruption of the h-region by mutating to charged residues or introducing helix-breaking residues caused severe loss of efficiency in translocation (Fikes et al. 1987; Freudl et al. 1987; Lehnhardt, Pollitt, and Inouye 1987; Michaelis, Hunt, and Beckwith 1986), whereas, increases of hydrophobicity, restoration of the -helical secondary structure to an export defect or extend- ing the length, did the opposite (Bankaitis, Rasmussen, and Bassford 1984; Emr and Silhavy 1983). The c-region determines the specificity of the signal peptidase cleavage site. Sequential analyses around the cleavage sites were carried out both in prokaryotic and eukaryotic organisms. Von Heijne presented the importance of -1,-3 residues before cleavage sites and proposed the (-3, -1)-rule (Von Heijne 1983). More details will be discussed in the peptidase family section. The distribution of three domains, positive-hydrophobic-polar, in the signal sequence, indicated that it could adopt a loop structure to efficiently bind to membranes, which was suggested both experimentally and by biophysical cal- culations. The behavior of SPs was extensively investigated by incubating with detergent micelles, and lipid monolayers and bilayers. With the change of surface pressures, SPs react differently regarding conformational shifts. At physiological surface pressure, SPs possess the secondary structure of an - helix, and insert perpendicularly to the monolayer, while at high surface pressure, they tend to form a monotopic -sheet (Briggs and Gierasch 1984). More evidently, synthetic PhoE SPs binding to the monolayers initially form a helical hairpin with a short connecting loop, which subsequently turns into an - helix, and inserts into the monolayers (Batenburg et al. 1988; von Heijne 1990).

Secretory pathway Signal peptide is not only the recognition site for signal peptidase, but it also is involved in protein transport prior to signal peptide cleavage. The secretion of most secretory proteins in both prokaryotes and eukaryotes employs a similar machinery to conduct protein transport, the complexes of which are both evolutionarily and structurally related (Crowley, Reinhart, and Johnson 1993; Simon and Blobel 1991). The transport machinery, as a trans-membrane complex, is located in the endoplasmic reticulum in eukaryotes, and in the cytoplasmic membrane in prokaryotes. The central component of the machinery, in general, consists of three subunits, SecY/E/G in prokaryotic cells, and their counterparts Sec61// in eukaryotic cells. The heterotrimeric SecYEG and

17 Sec61 are found to have 15 transmembrane -helices and exist in higher oli- gomerization in vivo (Bassilana and Wickner 1993; Breyton et al. 2002). The complete protein translocation machinery recruits a number of proteins in the upstream and downstream portions of the pathway, e.g. SecA, the cytosolic ATPase, an ATP-dependent motor, provides the energy required for the process (Geller 1991). SecY/E are functionally crucial and highly conserved. The SecY is present in all living organisms (Cao and Saier 2003), while SecG is not required for cell viability or evolutionarily close (Matlack, Mothes, and Rapoport 1998; Saier 1994). Two types of pathways have been observed, which can be distinguished by either if signal recognition particle (SRP) receptor is involved or if the translation occurs simultaneously with transport of secretory proteins – co-translational and post-translational pathways. In a co- translational pathway, SRP initially recognizes a signal peptide in a ribosome- nascent protein complex. Consequentially, the complex interacts with SRP and SRP receptor, while the translation is ongoing (Macfarlane and Müller 1995). In the post-translational pathway, instead of SRP, a cytosolic chaper- one is critical for interaction with the signal peptide, keeping the secretory protein unfolded or loosely folded (Barlowe and Miller 2013)(Charles, yeast- book). The two pathways are not mutually exclusive in a particular cell. Co- translational pathway is preferred by signal peptides with highly hydrophobic h-region, whereas the translational pathway is applied more with less hydrophobic peptides.

Signal peptidase families Peptidases were categorized into families based on the amino acids in the catalytic site, including the serine, threonine, cysteine, aspartic, metallo-, and un- known types (summarized by Nail D. Rawlings, with assistance of bioinformatics). This taxonomy offers direct information on the active site, and the enzymes within the same family share similar structures in the core domain and modes of action (MOA). Generally, signal peptidase (EC No. 3.4.X.X) is an enzyme that catalyzes the cleavage of N-terminal signal peptide from the protein precursors, the nascent products of which are secreted proteins and membrane proteins, and that is involved in transports of the mature proteins to their destinations. According to the peptidase taxonomy, signal peptidases were sub-grouped into three families. Type I, type II and Type IV signal peptidases act with serine, aspartic acid and aspartic acid, respectively, in the active site. The work mainly focused on signal peptidase I, therefore, it will be discussed further below. Signal peptidase II (EC No. 3.4.23.36, SPase II), known as lipoprotein- specific signal peptidase, cleaves not only signal peptides of bacterial lipoproteins, but also -amylase, a non-lipoprotein in Bacillus subtilis (Tjalsma, Zanen, et al. 1999). SPase II mainly resides in the inner cytoplasmic cell membrane with four transmembrane domains, two in the periplasmic space, and

18 both N- and C- termini facing the cytoplasm (Innis et al. 1984). The cleavage of a signal peptide by SPase II occurs simultaneously with the translocation of nascent lipoproteins through the cytoplasmic membrane (Hussain, Ichihara, and Mizushima 1982). The active site of SPase II consists of two aspartic acids residues catalyzing the cleavage (Tjalsma, Zanen, et al. 1999). Lipoproteins constituting 2-3% genomes in bacteria, are essential for bacterial viability, and pathogenicity (Vogeley et al. 2016). All signal peptides from prolipoproteins possess a consensus peptide named the lipobox ([LV][ASTVI] [GAS] C), with a universally conserved cysteine residue at position +1, and a highly conserved leucine residue at position -3 (Tjalsma, Zanen, et al. 1999). SPase II shares neither sequential nor structural similarity with SPase I. Moreover, given the different active sites from SPases, SPase II clearly has a different MOA than SPase I (Date and Wickner 1981), and lipoproteins are essential for bacteria viability, suggesting its irreplaceable role in the protein secretory pathway in Gram-negative bacteria (Yamada, Yamagata, and Mizushima 1984). However, depletion of SPase II in B. subtilis does not entirely inhibit the growth of cells (Tjalsma, Kontinen, et al. 1999), possibly indicating that an alternative lipoprotein secretion pathway exist in Gram-positive bacteria. Type IV leader peptidase (EC No. 3.4.23.43, TFPP), which has been found in both Gram-negative and Gram-positive bacteria, was named prepilin peptidase after its functionality. TFPP catalyzes the cleavage and N-terminally methylated type four leader peptide from type IV prepilins (Strom, Nunn, and Lory 1993). Both signal peptide from substrate preproteins, and the active site of TFPP are highly conserved, evidently different from other substrate – enzyme pairs (Strom and Lory 1987). Studies show that the major TFPP domains and aspartic acid residues in the active site are exposed in the cytoplasmic space with six transmembrane helices (Szabó, Albers, and Driessen 2006). Provided with the evidences mentioned above and the location of methyl source, S-adenosyl, it is most likely that the cleavage of TFPP occurs in the cytosol (Strom and Lory 1987). Three signal peptidases were discovered in 1980s, but due to the cruciality for the growth, and virulence of abundant pathogens, and druggability, Both type I and II SPases have been studied intensively in inhibition and antibiotics. Type I SPases exist in a broader spectrum of pathogens in the research to date, and the main domain structure of type I SPases from species are available, thus type I SPase was selected as one of fascinating targets in my study.

Signal peptidase I Signal peptidase I (3.4.21.89) was discovered very early to cleave leader signal peptide from secretory protein precursors in various species. The topology study shows that SPase I, an integral transmembrane protein, has two transmembrane segments at N-terminus, and the main domain and both N- and C-

19 termini are facing periplasmic space, where the cleavage of signal peptide occurs (Moore and Miura 1987; Wolfe, Wickner, and Goodman 1983). Moreo- ver, it was experimentally confirmed that the cleavage of signal peptide from preproteins occurs post-translocationally (Kaderbhai, Harding, and Kaderbhai 2008). Site-directed mutagenesis studies suggest Ser90 and Lys145 in E. coli SPase I, forming a catalytic dyad, are critical for proteolytical activity (Tschantz et al. 1995). Although His-Ser-Asp triads are dominantly more common in the protease family, SPase I with the active dyad exhibits similar catalytical efficiency (Suciu, Chatterjee, and Inouye 1997). The membrane spanning domains of SPase I from Bacillus subtilis was found to be involved in recognition of cleavage site of substrate, together with the assistance of hydrophobic h-region in the substrate, to remain high fidelity of cleavage (Carlos et al. 2000).

SPase I Sequences SPase I (leader peptidase, LepB, UniProtKB - P00803 (LEP_ECOLI)) from E. coli, comprises of 324 amino acid residues, with a molecular weight of 35960 Da. The sequence is shown below.

>sp|P00803|LEP_ECOLI Signal peptidase I OS=Escherichia coli (strain K12) OX=83333 GN=lepB PE=1 SV=2 MANMFALILVIATLVTGILWCVDKFFFAPKRRERQAAAQAAAGDSLDKATLKKVAPKPGW LETGASVFPVLAIVLIVRSFIYEPFQIPSGSMMPTLLIGDFILVEKFAYGIKDPIYQKTL IETGHPKRGDIVVFKYPEDPKLDYIKRAVGLPGDKVTYDPVSKELTIQPGCSSGQACENA LPVTYSNVEPSDFVQTFSRRNGGEATSGFFEVPKNETKENGIRLSERKETLGDVTHRILT VPIAQDQVGMYYQQPGQQLATWIVPPGQYFMMGDNRDNSADSRYWGFVPEANLVGRATAI WMSFDKQEGEWPTGLRLSRIGGIH

The sequence was searched by Domain Enhanced Lookup Time Accelerated blast (DELTA-BLAST) against swissprot Database (Boratyn et al. 2012). The feature of DELTA-BLAST searching a requested sequence against a collection of short protein profiles, helps detect homologies with high partial similarity. The searching organisms were filtered to Mycobacteria (taxid:85007), Enterococcus (taxid:1350), Staphylococcus (taxid:1279), Klebsiella (taxid:570), Acinetobacter (taxid:469), Pseudomonas (taxid:286), enterobac- teria (taxid:543), Plasmodium (taxid:5820), Homo (taxid:9605), which are of our interest, and/or infectious disease-related. The multiple sequence alignment showed SPase I from Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter sp. have ~ 18-40 % identity, with overall 25-75 % sequential coverage, except for SPase I from Salmonella enterica showing 93% identity with 100% coverage. Overall in the sequence alignment, ten conserved boxes are observed, at least five of which have been annotated to date. Conserved box (CB) 1-3 are

Figure 2. Sequence alignment of SPases I from various pathogens. CB – conserved box. transmembrane helical domain 1 (from E. coli residue 4-28), cytoplasmic domain, and transmembrane helical domain 2 (from E. coli residue 58-76), respectively, except for S. aureus SPase I that is missing first two conserved domains. CB 4 contains one of the catalytic residue, S91(from E. coli) and the

21 residues surrounding it, supporting the active site in the right conformation. The other residue of the catalytic dyad, K145 and the following residue Arg146 that are strictly conserved among 8 species, compose CB 7. CB 9 has conserved Gly272 and Ser278 interacting together, and Asp280 and Arg282 forming a salt bridge, which are essential for SPase I structural stability (Paetzel, Dalbey, and Strynadka 2000). CD 5, 6, 8, 10 have not been annotated to have any impact on stability or catalytic activity of SPase I.Surprisingly, no SPase I from Plasmodium sp. was shown in the DELTA-BLAST alignment or Psi-BLAST (Data not shown), due to low similarity. The sequence comparison of E. coli SPase I and the big signal peptidase (UniProtKB - Q6PSM6 (Q6PSM6_PLAFA)) by ClustalOmega (Sievers et al. 2011), however, showed high similarity in CD4, CD7 mentioned above, strongly suggesting PfLepB recruit the same catalytic dyad. The type I signal peptidase is experimentally confirmed to be present in Plasmodium falciparum, yet the catalytic mechanism of PfLepB have not been understood completely, except that the self- cleavage was observed in in vitro experiment (Sharma et al. 2005). The mis- alignment in the amino terminal region where the transmembrane domains of E. coli SPase I reside, indicates P. falciparum SPase I utilizes a distinguished topology for membrane docking. Only Mitochondrial inner membrane protease subunit 1 from Homo Sapi- ens (UniprotKB Number: Q96LU5.1), which has 166 amino acids compared to 324 aa in EcLepB, possesses the highest sequential similarity to signal peptidase I, with 38% and 60% identity to two inconsecutive fragments. The sequence analysis of SPase I addressed the catalytic mechanism employed universally by filtered pathogens and the large diversion between pathogens and humans, strongly suggesting a promising drug target. In our research, a full-length lepb sequence (as found in Gene ID: 947040) plus a His6-tag were inserted to A pEXP5-CT/TOPO vector (Life Technolo- gies), amplified, and verified by Sanger sequencing (NGI, Uppsala). The ac- tual sequence, with residues differing from the wild type shown in lowercase, is:

MANMFALILVIATLVTGILWCVDKFFFAPKRRrerhhhhhhArDSLDKATL KKVAPKPGWLETGASVFPVLAIVLIVRSFIYEPFQIPSGSMMPTLLIGDFI LVEKFAYGIKDPIYQKTLIETGHPKRGDIVVFKYPEDPKLDYIKRAVGLP GDKVTYDPVSKELTIQPGCSSGQACENALPVTYSNVEPSDFVQTFSRRNG GEATSGFFEVPKNETKENGIRLSERKETLGDVTHRILTVPIAQDQVGMYY QQPGQQLATWIVPPGQYFMMGDNRDNSADSRYWGFVPEANLVGRATAIWM SFDKQEGEWPTGLRLSRIGGIH

The verification of catalytic dyad was done by mutagenesis from Ser91 to Al- anine.

22 Structure and activity relationship Long prior to 3D structure of SPase I available, the membrane topology of SPase I from different species was both predicted in silico based on sequences, and verified experimentally (Figure 3). The first membrane topology of E. coli SPase I was proposed to possess two consecutive N-terminal -helices spanning cross the inner cytoplasmic membrane, with the major catalytic domain facing the periplasm (Whitley, Nilsson, and von Heijne 1993). SPase I from typical Gram-negative bacteria, e.g. Legionella pneumophila (Lammertyn et al. 2004), Rhodobacter capsulatus (Klug et al. 1997), have been proven to have a similar domain arrangement, with two hydrophobic helices, while in typical Gram-positive bacteria, only one N-terminal helix is present (Paetzel, Dalbey, and Strynadka 2000). The existence of one helix predicted by HMMTOP (Tusnády and Simon 2001), is in agreement with the sequence alignment, e. g. Staphylococcus aureus.

Figure 3. The membrane topology of EcLepB (PDB code: 1KN9, chain A). EcLepB have two transmembrane segments, with both ends pointing towards the periplasm, and a loop located in the cytosol. The orientation of the main domain of EcLepB relative to inner membrane was determined based on the location of the catalytic dyad and an exposed hydrophobic surface (Paetzel, Dalbey, and Strynadka 1998)

Structural alignment of experimentally determined homologues from different species is considered more accurate, thus more informative, as high structural similarity indicates a similar mode of actions, e.g. the substrate binding site, the catalytic site, and moreover, profound understanding of evolutionary trace

23 to preliminarily guide the research on the new homologues from other species. However, the limiting premise of this approach is the lack of sufficient solved structures. Due to the difficulty of crystallization in this particular case, where transmembrane helices of membrane proteins form a bundle outside the catalytic domain, lacking constraints for a right orientation of the main domain, A truncated SPase I from E. coli was constructed to as a soluble, catalytically active protein with 2-75 residues (Paetzel et al. 1995). Paetzel and his group have solved complex structures of the same 2-76 construct with a -lactam inhibitor (PDB identity: 1B12, (Paetzel, Dalbey, and Strynadka 1998)), with a lipopeptide based inhibitor(1T7D, (Paetzel et al. 2004)), with an arylomycin A2, and a  sultam inhibitor (3IIQ, (Luo et al. 2009)), with a lipoglycopeptide arylomycin variants (3S04, (Liu et al. 2011)), together with an apo-structure (1KN9, (Paetzel, Dalbey, and Strynadka 2002)). A complex of E. coli SPase I with a new chemically modified arylomycin derivative bound was structurally characterized (6B88, (P. A. Smith et al. 2018)). The first SPase I from Gram- positive bacterium Staphylococcus aureus was solved as an apo-enzyme (4wvg), and in a complex with a cleavable signal peptide (4wvh, 4wvi), and an inhibitor peptide(4wvj), separately (Ting et al. 2016). Therefore, SPase I only from two species, E. coli and S. aureus, have been structurally determined, and they shared high similarity in the conserved domain, especially in the substrate binding pocket. The arrangement of secondary structure is nearly identical, except that two  strands forming a -hairpin and a C-terminal helix in E.coli are missing in S. aureus. While large differences are observed in non- conserved domain, where only a -sheet consisting of three -strands and one helix are found in common. A candidate worth mentioning is SipA from Strep- tococcus pyogenes that is critical for group A Streptococcus (GAS) pili polymerization. SipA with a different Asp-Lys dyad exhibiting no SPase activity, shares the similar -strand enriched fold in the catalytic domain, yet the exact function is unclear (4K8W, 4N31, 2013, (Young et al. 2014)). Another two SPases I (4NV4, Bacillus anthracis. 4ME8, Enterococcus faecalis) are largely truncated, and no descriptions are available from PDB (https://www.rcsb.org). SPase I is a mainly -strand protein, comprising two antiparallel -sheet domains, and five helices on the surface. The loop, with the catalytic residue Ser91, between the first and second  strand, resides in parallel with the sixth -strand with the other catalytic residue Lys146, forming a substrate binding groove and catalytic center. When the peptide-like inhibitors are bound in the substrate binding groove, they form an intensive network of hydrogen bonds with the residues flanking catalytic dyad. The groove appeared more exposed, and significant conformational changes are observed in S1 and S3 subsites. The terminology of S1 and S3 was derived from a study of papain active site mapping (Berger 1968), in which, S X are a collection of residues or more specifically, atoms that are relevant to the P X residue in the substrate, e. g.

24 S1 subsite of E. coli SPase I consists of non-polar atoms from residues 86, 87, 88, 90, 91, 95, which are responsible for P1 recognition, and S3 subsite for P3 are residues 84, 86, 101, 132, 142, 144. Essentially, binding of the inhibitor triggered the rotation of Phe84 and Ser88, by ~80 and ~180 respectively, and also expanded the S1 pocket resulting from the different positioning of Leu95, Ile144 and Tyr143 side chains. however, surprisingly, the positioning of two residues from catalytic dyad does not change spatially (Paetzel, Dalbey, and Strynadka 2002). Of three water molecules (WAT1, WAT2, WAT3) that are located in the catalytic groove in the apo-enzyme structure, two were absent in the inhibitor-enzyme complex, suggesting that perhaps these two water molecules or one of them is involved in deacrylating. Functionally, Ser91 as a nucleophilic residue, attacks the scissile bond from the si-face rather than re-face at the cleavage site, which is unique from other

Figure 4. Structure of SPase I A) schematic presentation of SPase I secondary structure arrangement. B) overall structure ((E. coli, PDB code: 1B12). C) detailed interactions between residues and ligand Penem

25 proteases utilizing Ser-His-Asp catalytic triad (Black and Bruton 1998). The general base residue Lys146 places mostly likely WAT3, which is closest to Ser91 and Lys146 in space, in the right orientation, and activates its role as a deacrylating water. and Ser88 hydroxyl group serves to form the SPase oxyanion hole, and to stabilize the tetrahedral oxyanion intermediate. However, the mechanism of SPase I cleavage process has not been fully understood (Paetzel 2014). Non-catalytic domain (NCD) structurally differs from species to species, and the function was unclear. Recent computational studies on modelling of a signal-peptide-included peptide and SPase I complex suggested a possible role of NCD in recognizing the substrate after the cleavage site Ala-X-Ala, and more importantly, adapting a proper conformation to increase the specificity (Choo, Tong, and Ranganathan 2007). We have been dedicated to establish a platform investigating both the structure of full-length EcLepB including the transmembrane segments, and a biochemical assay capable of evaluating compounds with potential inhibitory activity. The full length lepb gene from E. coli was constructed, inserted to a pEXP5-CT/TOPO vector (Life Technologies), and transformed to E. coli BL21 C43(DE3) strains, which is popular for a high-level overexpression of membrane proteins (2008, Wagner). EcLepB with two transmembrane segments was extracted from the membrane fraction with detergents Triton X- 100, n-dodecyl-β-D-maltoside (DDM), n-Octyl β-D-glucopyranoside (OG), after the lysis of cell pellets. The final yield of EcLepB with three detergents was of no difference, neither was catalytic activity at later stage. However empirically, DDM and OG statistically increase the success rate of crystallization of membrane proteins (2013, Oliver). The his-tagged EcLepB was purified with immobilized metal affinity chromatography (IMAC), to roughly 95% purity, followed by buffer exchange with a PD-10 column (GE Healthcare) to remove high concentration imidazole. The purified EcLepB in the final buffer (10 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM -mercapto- ethanol, 0.5% reduced Triton X-100), was ready to be tested in the enzymatic activity assay. EcLepB solution was concentrated to approximately 20 mg/ml, and ready for crystallization. The purification of E. coli full-length S91A LepB was successful, and produced a similar amount of protein as wild-type. Vapor-diffusion crystallization was initially performed for EcLepB with com- mercially available crystallization screens, at protein-resovior volume ratio 1:1, with the assistance of crystallization robot mosquito® crystal (TTP labtech, UK). Commercial screens as, Morpheus, JCSG+, JBS bbuffer screen, structure, memstart&memsys were purchased from Molecular Dimen- sions (https://www.moleculardimensions.com), the last of which was specifically designed for membrane protein crystallization. EcLepB was cocrystal- lized with inhibitors synthesized by our colaborators, e.g. decanoyl-PTANA-

26 aldehyde, JEDI975, JEDI976, at divert molar ratios. No ordered crystal formation has been observed. The focus of our work has been shifted to development of activity assay and inhibitor exploration.

Biochemical assay (paper I and II) Decades ago, when Blobel and his group first experiementally confirmed signal hypothesis, an assay to essentially measure the cleavage activity was developed (W. P. Smith et al. 1977). known secretory proteins with signal peptide, e.g. f1 pre-coat protein, were labeled with radioactive atoms, and subsequently analyzed by SDS-PAGE and fluorography (W. P. Smith et al. 1977; Zwizinski and Wickner 1980) The first assay to decide the kinetics of cleavage activity of SPase I, was developed, by separating the peptide products by re- verse phase liquid chromatography, followed by identification and quantifica- tion in amino acid analysis (Dev, Ray, and Novak 1990). The calculated kcat and Km for a series of chemically synthesized peptides with different lengths were ranging from 0.1 to 119 h-1 and from 0.8 to 3.7 mM, respectively. it had been observed that kcat for short peptides were tremendously lower than ones for secreted preproteins, while Km much higher, indicating that SPase I exhibits significantly higher catalytic activity as well as greater binding affinity for preproteins (Chatterjee et al. 1995), before a suitable activity assay for short peptides was developed. A continuous assay theoretically based on Fluores- cence Resonance Energy Transfer (FRET), was widely popular in the research of protease activity. A known SPase I substrate or a signal-mimicking peptide was synthesized with a pair of FRET donor and quencher attached. The overlapping of absorbance wavelength of quencher and the emission wavelength of fluorophore in a specific substrate, provides an approach to measure the concentration of cleaved substrate quantitively, thus to kinetically characterize the proteolytic activity of SPase I (Stein, Barbosa, and Bruckner 2000; Zhong and Benkovic 1998). Multiple potential substrates with FRET pairs have been used in the in vitro biochemical assay to date.

Y(NO2)–FSASALA↓KIK(Abz) (MBP) K5-L10-Y(NO2)FSASALA↓KIKAbz-NH2 (MBP) KLTFGTVK(Abz)PVQAIA↓GY(NO2)EWL (Streptokinase 8) (Dabcyl)-AGHDAHA↓SET-(EDANS) (SceD, (Bockstael et al. 2009)) Decanoyl-LTPTAKA↓ASKIDD-OH

In our study, a peptide substrate (dabcyl-VGGTATA↓GAFSRPGLE- (EDANS)) was designed and purchased (Genecust, Europe), based on a sequence from Mycobacteria tuberculosis antigen 85A, with dabcyl and EDANS, attached to amino terminus and carboxyl terminus, respectively. EDANS (2-aminoethyl)aminonaphthalene-1-sulfonic acid, as the fluorophore known for a long fluorescent lifetime, when excited at 340nm, fluoresces at

27 478nm, where DABCYL(4-(4-dimethyl- aminophenylazo) benzoic acid) ab- sorbs efficiently. This pair were selected for detecting HIV protease activity, due to its high quenching efficiency and complete spectral overlap of EDANS emission and DABCYL absorbance (Zhong and Benkovic 1998). To chemically couple EDANS to the peptide substrate, an extra glutamic acid residue was added to the C-terminus. Three additional substrates based on streptokinase 8 sequence were synthesized, and two of them were mutated to Phe and Ile at position Trp residue. EcLepB activity assay was conducted mostly with Mtb antigen 85A with a FRET pair, as the native and F-mutated substrate derived from Streptokinase 8 appeared not completely soluble in the assay, and with I-mutated substrate, EcLepB has been proven to have significantly lower affinity and turnover rates. 3 µl protein at a final concentration of 50 nM was mixed with 3 µl 10% DMSO, considering the feasibility of the assay for following inhibition study. Simultaneously, 3 µl 0.5 % tween-20 was mixed with 21 µl 10 µM substrate solution. 6 µl protein mix was pipetted into an OptiPlate-384F plate (Perkin Elmer), followed by the addition of 24 µl substrate mix to start the reaction. Triplicates were performed in each sample. The fluorescence intensity was monitored continuously with an Envision 2104 multilabel plate reader (Perki- nElmer). A pair of an excitation filter (340nm) and an emission filter (535nm) were used, essentially compatible with the FRET pair. To accurately convert the velocity of Fluorescence intensity (FI) change to velocity of product concentration increase, the formula 1 below was applied. vnM/s is the catalytic velocity in units of nM/s. vFI/s, reaction rates in units of FI/s, was determined from a time period with a linearly increasing FI. FIt=0 is the fluorescence intensity at time 0, while FIt= represents the maximum FI at the endpoint when the cleavage reaction has been ongoing for an infinite time till theoretically all substrate was hydrolyzed.

Enzymatic kinetics of EcLepB proteolytic activity was characterized by titrat- ing the substrate concentration from 100 to 0.78 µM. Subsequently, kinetic parameters were calculated by applying non-linear curving fitting to the Mich- aelis-Menten equation in GraphPad Prism®. In our assay, the kcat of EcLepB -1 for M. tb antigen 85A was 135.18 s , and the Km was 20 µM. For I-mutated -1 Streptokinase 8, the Km and kcat of EcLepB were 60 µM and 90.1 s . EcLepB showed higher specificity to MBP derivative substrate (K5-L10- Y(NO2)FSASALAKIKAbz-NH2 (MBP), with a Km of 0.6 µM, and a kcat of -1 1.5 s , while for the substrate without chemical modification (Y(NO2)– 2 3 -2 - FSASALAKIK(Abz) (MBP), the Km and kcat were 10 ~10 µM, and 10 ~10 3 s-1. Bruton and his group also presented a novel substrate (Decanoyl-LTP-

28 -1 TAKAASKIDD-OH) with a Km of 29 M and a kcat of 67s designed based on the in silico sequential analysis of 17 signal peptides from S. aureus (Bruton et al. 2003). It has been reported that for a preprotein substrate other than a FRET-labelled peptide substrate, pro-ompA-nuclease A, the Km of -1 EcLepB was 16.5 µM, and the kcat was 8.7 s (Chatterjee et al. 1995). Another full length protein precursor The SPase I from Streptococcus pneumoniae has been kinetically characterized with substrate Streptokinase 8, as Km 118.1 µM -1 and kcat 0.032 s (Peng et al. 2001). Kinetic parameters for other substrates, obtained via other analytical methods, e.g. HPLC, HPLC-MS, exhibit significantly lower affinity and turnover rates (Bruton et al. 2003; Dev, Ray, and Novak 1990; Kuo et al. 1994). Introduction of S91A mutation in EcLepB almost completely silenced the enzymatic activity, with 1.3 % residual activity, compared to the wild-type, confirming that S91 is essential for LepB proteolytical activity. Studies of LepB from Mycobacteria smegmatis and Mycobac- teria tuberculosis were performed similarly to EcLepB, as well as similar conclusions were drawn, except that MsLepB and MtLepB showed 5 folds and 10 folds decreased enzymatic activity. Furthermore, instability of MtLepB was observed, as the activity of MtLepB has lost in the freeze-thaw cycle, so we mainly performed our research only on EcLepB.

Inhibitor development (paper I and II) Due to the essentiality in bacteria growth (Cregg, Wilding, and Black 1996; Date and Wickner 1981), non-effect of known protease inhibitors, and locat- ing in the extra-cytoplasmic spase, SPase I has been considered as a potentially good antibiotic target. The available known protease inhibitors showed no inhibitory effect on SPase I (Black 1993; Kuo et al. 1994), to a certain extent, indicating that SPase I employs a unique catalytic mechanism, compared to other proteases. One natural signal peptide of M13 procoat protein was first reported to be inhibitory on SPase I in in vitro experiment, besides high concentration of salts. A small molecule, 5-S penem, was first found to be inhibitory to SPase I, the complex structure of which was solved (1B12, (Kuo et al. 1994)). Hence, to date, four distinct groups of inhibitors have been confirmed to target LepB from divert species. Class 1, penem derivatives. Since the discovery of the inhibitory effect on SPase I, the penem has been chemically modified largely, creating a library of penem derivatives. Quite a few have been reported extraordinarily potent on SPase I, e.g. 6-(hydroxyethyl) 5S enantiomer has an IC50 of 0.92 µM at the protein concentration 25 nM (Allsop et al. 1996); by intracellular cyclization, (5S)-tricyclic penem showed strong potency to SPase I, with an IC50 of 0.6 µM (Hu et al. 2003); C2-ethylamine-(5S)-penem showed 0.63 µg/ml, more importantly, increased the potency against Y. pestis and S. epidermidis by 8 folds (Yeh et al. 2018). Studies of penem-derived inhibitors also provoked a

29 better understanding of the catalytic mechanism, that Ser91 nuleophilically breaks the peptide bond of the substrate on the si-face (Black and Bruton 1998). Class 2,  - aminoketone (MD3) was discovered to be potent against M. tuberculosis (Ollinger et al. 2012), S. aureus and E. coli under-expressing LepB (Barbosa et al. 2002), and multidrug-resistant Gram-negative bacteria (Personne et al. 2014), showing inhibition on the bacterial growth, and bactericidal activity. Interestingly, MD3 only inhibited the growth of E. coli under- expressing LepB temporarily, with rescued growth after a short period of in- cubation, not mentioning that wild-type E. coli is completely resistant to MD3 alone. MD3 showed an IC50 of 5.0 µM on EcLepB in a biochemical assay, while an MIC of 4.5 µg/ml on S. aureus ATCC 13709 in vivo (Barbosa et al. 2002). By the regulation of lepb expression level in M. tuberculosis, the sus- ceptibility of M. tuberculosis to MD3 has changed accordingly, indicating LepB is the essential target. MD3 exhibited inhibition on M. tuberculosis H37Rv, LepB-overexpressed M.tb, LepB-underexpressed M. tuberculosis, with MIC values of 17.7 µM, 35.4 µM, and 8.8 µM, respectively. MD3 has a major disadvantage that it is stable neither in Tris buffer nor in 100% DMSO (Barbosa et al. 2002). Class 3, naturally produced antibiotics. Krisynomycin and actinocarbasin are known natural antibiotics from Streptomyces fradiae and Actinoplanes fer- rugineus, respectively, individually with no potency against methicillin-resistant S. aureus (MRSA). However, They synergize with imipenem, and highly restore the potency against MRSA and LepB enzymatic activity, hypo- thetically due to the prevention of the expression of secreted proteins that are required for penem resistance. They deceased the MICs for a panel of clinical isolates by 16 folds, ranging from 0.06 ~ 8 µg/ml . In an enzymatic assay, Krisynomycin and actinocarbasin showed extensively strong inhibition to LepB with IC50 values of 120 and 50 nM, respectively (Therien et al. 2012). Class 4, peptide-like inhibitors or peptide derivatives. The strategy for peptide-like inhibitors was to produce a compound that mimics the recognition site and promotes the competitive binding to LepB, without cleaving the secreted preproteins that are vital for bacteria growth. The early approach was to synthesize the known peptide substrates carrying mutations that increase the affinity to LepB. The assignment of proline residue at P1’ position showed a competitive inhibition on SPase I (Barkocy-Gallagher and Bassford 1992; Nilsson and Heijne 1992). Based on the substrate (Decanoyl-LTPTAKAAS- KIDD-OH) demonstrated above, -ketoamide incorporated substrate (Deca- noyl-LTPTA--ketoamide-SKIDD) has been proven to be inhibitory on SPase I from S. aureus, with an IC50 of 0.6 M (Bruton et al. 2003). A series of Bruton’s substrates modified at different lengths have been tested against S. aureus SPase I, and the results have been analyzed. A composition of TAKAPS residues was the minimum requirement for inhibition, and used as a starting scaffold for inhibitor development. It is worth noting that none of

30 these compounds were inhibitory to EcLepB. Based on this interesting discovery, A compound (decanoyl-PTANA-aldehyde) was synthesized, and tested in the same system as mentioned above. It has largely elevated inhibitory activity against both SaSpsB and EcLepB, with respective IC50 values of 0.09 µM and 13.4 µM. (Buzder-Lantos et al. 2009). Arylomycins originally produced by Streptomyces sp. Tü 6075 are lipohex- apeptides, which have been found to show antibacterial activities against E. coli, S. aureus, Yersinia pestis (Höltzel et al. 2002; Schimana et al. 2002; Steed et al. 2015), and confirmed to specifically target SPase I, instead of blocking secretion channel or polarizing the environmental membrane (P. A. Smith and Romesberg 2012). Arylomycin A and B have a similar peptidyl sequence, as D-Met1-Ser2-D-Ala3-Gly4-N-methyl-4-hydroxyphenylgly5(MeHpg5)-L- Ala6, with only a differentiated residue at postion 7, a tyrosine in Arylomycin A, and 3-NO2 substituted tyrosine in B. Both arylomycin compounds were cyclized by a [3,3]-biaryl bridge between MeHpg and Tyr. Saturated C11 ~C15 with n, iso, and anteiso fatty acid tails were added to N-terminus by acylation. Both exhibited potency against Gram-positive bacteria, especially Rhodococcus erythropolis and Brevidacillus brevis. By comparison, the substitution of 3-nitro tyrosine in arylomycin Bs significantly increased the antibiotic activities. A complex structure comprising catalytic domain of SPase I from E. coli, arylomycin A2 and morpholino--sultam derivative was solved by crystallography, and dose-response analysis further confirmed the competitive binding mode of SPase I to arylomycin analogues (Luo et al. 2009). More interestingly, lack of observation of synergy from arylomycin A2 and morpholino--sultam on inhibitor binding, and the complex structure (3IIQ) confirmed two independent binding sites, although they are physically close to each other (Liu et al. 2011). The optimization of Arylomycin families are ongoing, yet so far Arylomycins are still considered as latent, but promising broad-spectrum antibiotics (Tan and Romesberg 2012).

Figure 5. Known LepB inhibitors

31 Our investigation of chemical modifications on decanoyl-PTANA-aldehyde has been aiming to synthesize compounds more potent to bacteria, and more inhibitory to EcLepB in particular (Higuchi, Robert, I; Roberts, Tucker, Curran; Smith, Peter, Andrew; Campbell, David, Paraselli 2013). Essentially, compounds designed are expected to be more chemically stable, but equally reactive, therefore, modifications at both termini were performed. The decanoyl tail was removed or replaced by three diverted groups. Simultaneously aldehyde was substituted with different warhead, such as boronic acid, -ketoamide, ester (Figure 6). All synthesized compounds were evaluated in the biochemical assay, and antibacterial assay against a panel of various bacterial strains. The very best compound comprising bulkier 4-(4-hexylphenyl)benzoyl group and boronic ester warhead with an IC50 of 12 nM was selected as a lead for future studies.

Figure 6. The PTANA analogues. Red square: various fatty acid tails; Blue square: various warheads. (De Rosa et al. 2017). 50j: red3, blue3; 50k: red4, blue3; 50m: red4, blue3; 50n: red4, blue4.

From the analysis in the biochemical assay, surprisingly, chemically modified decanoyl-PTANA-aldehyde based on the structures of other known inhibitors did not show increased inhibitory activity, e.g. replacement of aldehyde to - ketoamide, introduction of proline at p1’ position, removal of fatty acid tail, a

32 truncation of peptide PTANA sequence. The replacement of aldehyde to boronic ester and/or acid (2015, Patent, Smith, 2013, Patent, Higuchi) eventually helped dramatically increase the potency by 25 folds against EcLepB, and the compound was chosen as a starting model. The fatty acid was designed to corporate with membranes in vivo or to detergent-formed micelles in vitro, thus is related to substrate placement in activity dyad, and as a consequence, is related to binding affinity and catalytic activity. We replaced the decanoyl with bulkier aromatic fatty acid, and found that 4-(4-hexylphenyl)benzoyl group elevated the inhibitory activity by 4 folds compared to decanoyl- PTANA-boronic ester (50j, 50k). The potency of active lipopeptide inhibitors was tested against a panel of strains, including, Gram-negative bacteria, E. coli, and SKAP out of ESKAPE pathogens. Despite of the high inhibitory activity against EcLepB, these inhibitors did not show remarkable potency against any strains. Introduction of a positively charged residue at P5 proline position has been seen to highly improve the potency against E. coli (D22, lpxC mutant, drug-hypersensitive) and S. aureus wild-type (50m, 50n). Our hypothesis is that the positively charged residue could improve the permeability through the membrane. Cytotoxicity and hemolysis were measured to evaluate the potential druggability. Two pairs of inhibitors showing interesting inhibitory activity and/or potency against Gram-negative strains unfortunately act poorly in both assays. 50j and 50k that are PTANA-boronic ester differing with fatty acid tails showed high cytotoxicity and relatively mild hemolytic activity. 50m and 50n differing only in the warhead, with either boronic ester or boronic acid behaved poorly in both assays (Paper I). The cyclization of MeHpg and tyrosine in arylomycin has hinted us as a possible scheme to synthesize compounds that improve the antibacterial activity, and have lessened cytotoxicity and hemolysis. To date, no macrocyclic peptide mimics have been reported as protease inhibitors. Hence, three unique differentiated macrocyclic scaffolds were synthesized, linking P2 and boronic ester. It should be noted that to keep the original residue an asparagine in P2, the amide was replaced by a triazole. Besides, the replacement of positively charged residue at P5 was kept, as it has been confirmed to have superior potency to inhibitors including neutral residues at P5. In the biochemical assay, all macrocyclic inhibitors exhibited slightly decreased inhibitory activity, compared to linear reference analogues, with IC50s ranging from 40 – 100 nM. The same cytotoxicity and hemolysis assays were conducted, the results of which are scientifically more informative, suggesting more promising hits as a lead in future study. Unfortunately, the overall results indicate that antibacterial activity of inhibitors are significantly co-related to cytotoxicity and hemolysis. To sum up, breaking the correlation remained as a huge challenge for expanding a potential drug library targeting SPase I (Paper II). An inhibition assay was developed to evaluate the biochemical effects of compounds specifically on EcLepB, based on the well-established activity assay. Compared to the activity assay, prior to start of the reaction, an additional

33 step, incubating the protein with the compounds for 10 min, was found nec- essary for the stability of the assay, and observably a better inhibitory effect. The inhibitors were synthesized by collaborators from department of medicinal chemistry, and stored in 10 mM 100 % DMSO stock. The single-concentration inhibition assay was conducted at 10 µM, 20 µM, 100 µM, depending on the solubility estimated by chemists. The compounds with inhibition higher than 50% were selectively proceeded for the dose-response assay, at a series of concentrations, ranging from 10 µM to 0.00192 µM. By non-linear regression analysis of dose-response relation fitting into a four-parameter sigmodal curve, IC50 values were calculated in GraphPad Prism®. The analysis showed that compounds inhibit the proteolytic reaction in a significantly concentration-dependent manner, indicating no compounds bind to EcLepB covalently. The EcLepB inhibition assay was investigated by the European Lead Fac- tory (ELF, https://www.europeanleadfactory.eu) as a feasible enzymatic assay for high throughput screening. ELF has constructed a compound library (Joint European Compound Library, JECL), containing a large number of diverse high quality compounds that could potentially good starting points for the development of inhibitors with novel modes of action or chemotypes, if found potent in the assay. The collection has roughly over 500,000 compounds with various physicochemical features and high estimation of drug-likeness, and is expected to cover a wide range of biological targets (Besnard et al. 2015)(Karawajczyk et al. 2015). Projects in ELF are strictly required to meet such criteria as, signal/background (S/B) > 3, and Z’ > 0.6, stability of the assay, etc. Prior to official acceptance by ELF, the EcLepB assay was therefore optimized for feasibility for high throughput screening, pursuing prefer- ably higher S/B, and larger Z’. In the optimization process, the assay was adjusted in terms of time duration, plate selection, and different combinations of enzyme-substrate concentrations. The assay eventually reached all criteria in the initial feasibility testing, and continued as an accepted project. Upon acceptance, a primary assay was run with approximately 450,000 compounds, followed by a confirmation assay. A number of compounds were thus selected for dose-response curves. After two rounds of dose-response assays, besides potency and selectivity tests, a qualified hit list of compounds with non-peptide scaffolds was confirmed. There will be investigated further, but the compounds are currently protected by agreements within European Federation of Pharmaceutical Industries and Associations (EFPIA, https://www.efpia.eu).

Conclusions and future perspectives Bacterial Type I signal peptidase has been considered as a promising target for the development of novel potential antibiotics against pathogenic bacteria, for several reasons: the essentiality for the viability of the bacteria, a Ser/Lys activity dyad that is insensitive to inhibitors for other proteases, and easy accessibility due to its location in the outer space of cytoplasmic membrane.

34 Furthermore, SPase I is conserved among both Gram-negative and Gram-positive bacteria, especially the architecture in the catalytic domain. On the contrary, eukaryotic signal peptidases commonly possess a conserved histidine to form a Ser/His dyad. All evidences suggest that inhibitors for SPase I can be potentially developed as promising broad-spectrum antibiotics. We have managed to construct a full-length EcLepB, including two transmembrane segments; to express EcLepB in E.coli with a relatively high yield; to purify soluble and functional EcLepB with the triton X-100 to a high purity; to develop a biochemical assay using a novel substrate, feasible for high throughput screening; to explore a collection of inhibitors that are highly inhibitory to EcLepB. Given the well-established platform, SPases I from various pathogens can be investigated, such as Mycobacteria species, Plasmodium falcifarum, and ESKAPE pathogens. Novel designs of inhibitors that could break the co-relation between potency against bacteria and cytotoxicity/hemolysis should be considered, including recruiting a novel mode of action.

35 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/IspC) Isoprenoids are the primary or secondary metabolites, such as heme, steroids, and cholesterol, that are crucial for all living organisms (Spurgeon, S. L. & Porter 1981). Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are, two building blocks needed to make all of them (Sacchettini and Poulter 1997). It had long been considered that all living organisms produce IPP from Acetyl-CoA through mevalonate pathway (MVA pathway, (Spurgeon, S. L. & Porter 1981)). However, in the 1990s, an alternative pathway was found in bacteria, algae, and the plastids of higher plants, which begins with pyruvate (Arigoni et al. 1997; Rohmer et al. 1993). This methylerythritol phosphate (MEP, or non-mevalonate) pathway was also found in the apicoplasts of Plasmodium falciparum, the causative parasite of malaria (Snow et al. 2005). Malaria, as one of the most widespread diseases in the world, accounts for approximately 1 million deaths per year (Jomaa et al. 1999)(2005, Snow). As the most deadly parasite in the Plasmodium genus, P. falciparum has developed extensive resistance to drugs currently available on the market. Fosmidomycin (3-(N-formyl-N-hydroxyamino)propyl- phos- phonate) and its derivative FR-9000098 (3-(N-acetyl-N-hydroxyamino)propyl- phosphonate) were originally found in nature, and have potency against Plasmodium falciparum. In the MEP pathway, DXR catalyzes the isomerization of 1-deoxy-D-xylulose 5-phosphate (DXP), which is followed by the reduction to produce the 2-C-methyl-D-erythritol 4-phosphate (MEP). In our lab, we have targeted various enzymes that are involved in the MEP pathway, pro- ducing isoprenoids, for example, 1-deoxy-D-xylulose 5-phosphate synthase (DXP synthase, DXS), condensing pyruvate and glyceraldehyde to produce DXP; DXP reductoisomerase (DXR), converting DXP to MEP; MEP cytidylyltransferase (IspD), and others of the eight enzymes involved. My studies specifically focused on the second enzyme DXR in the MEP pathway, DXR, especially the complex structures with fosmidomycin or FR-9000098 analogues.

Isoprenoids In living organisms other than viruses, isoprenoid biosynthesis is vital for cell growth. More than 65,000 isoprenoids, a diverse class of natural organic products, have been found in all three domains of life. The presence of the oldest isoprenoids, 2-methylhopane hydrocarbon derivatives, was found in the organic-rich sediments from 2.5 billion years ago (Summons et al. 1999). They have been identified as primary and secondary metabolic intermediates and dominantly involved in various biological functions, including lipids in membranes, hormones, signal transduction pathway, regulators of gene expression, electron transfer chain, and so forth (Gershenzon and Dudareva 2007;

36 Hoshino and Gaucher 2018; Lange et al. 2000; Santner, Calderon-Villalobos, and Estelle 2009). Plants produce the largest class of isoprenoids, which are the key components in the secretory structure to attract pollinators, as well as to defend against predators and pathogens. A large proportion of commercial products are the secondary metabolites of isoprenoids from plants, such as rubber, organic acids, and pigments (Gershenzon and Dudareva 2007). Sev- eral commercial drugs are isoprenoids, as the natural products of plants. Camptothecin and taxol, found in the stembark of Camptotheca acwninata and Taxus brevifolia, respectively, were used in the cancer chemotherapeuti- cal treatments (Wall 1998). Interestingly, artemisinin, for the discovery of which Tu Youyou shared the 2015 Nobel Prize in Physiology or Medicine (Tu 2016), was combined with other organic chemicals to show high efficacy against uncomplicated P. falciparum malaria and non-falciparum malaria (Borrmann et al. 2004). Isoprenoids are exclusively synthesized by condensation of two universal building blocks, isoprenoids precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP)(Sacchettini and Poulter 1997). Diversification of isoprenoids is generated by chemically modifying the pol- ymerized IPP and DMAPP, for example, through polymerization catalyzed by prenyltransferase to create triterpenes (C30) and tetraterpenes(C40), and even longer linear intermediates, and through cyclization to form more commer- cially popular terpenes and terpenoids(Greenhagen 2003). All isoprenoids are naturally synthesized in two independent pathways – mevalonate (MVA) pathway and methylerythritol phosphoate (MEP) pathway. The elaborate details will be discussed in the following section.

MVA and MEP pathway The isoprenoid precursor biosynthesis pathway starting from the condensation of acetyl-CoA (mevalonate (MVA) pathway) had long been considered ubiq- uitous among all living organisms (Bloch 1992; Spurgeon, S. L. & Porter 1981) However, recently an alternative pathway named after an intermediate in the pathway, methylerythritol phosphate (MEP), was discovered in bacteria, and algae, as well as the plastids in higher plants. (Arigoni et al. 1997; Rohmer et al. 1993). It is now known the malaria causative parasite, Plasmodium falciparum also synthesizes isoprenoids in its apicoplasts through the latter pathway(Jomaa et al. 1999). The use of two distinct essential pathways in parasites and humans provides a potentially promising drug targets for malaria treatment. The MVA pathway comprises five enzymatic reactions converting acetyl- CoA to the final products IPP and DMAPP in the last two steps. 1) condensation of acetyl-CoA and acetoacetyl-CoA occurs to form 3-hydroxy-3-methyl- glutaryl-CoA (HMG-CoA, in the presence of HMG-CoA synthase (HMGS). 2) HMG-CoA is reductively deacylated to form MVA, by enzyme HMG-CoA

37 reductase (HMGR). The HMG-CoA reductase is critical and rate-limiting in the pathway. 3) MVA is subsequently dual phosphorylated to form pyrophospho-MVA, by enzymes MVA kinase (MVK) and phospho-MVA kinase (PMK). 4) Pyrophospho-MVA is ATP-dependently decarboxylated to form IPP, catalyzed by the enzyme pyrophospho-MVA decarboxylase (MPD). 5) In the final step, the carbon-carbon double bond of IPP is isomerized catalytically by enzyme IPP isomerase. The reaction is reversible with an equilibrium that favors DMAPP formation.

Figure 7. The MVA pathway

The novel isoprenoid biosynthesis pathway (MEP pathway) was discovered, via the research on incorporation of isotopic C13-labeled precursors, attempting to determine the origin of the carbon atoms of isoprenoids in several bacteria (Rohmer et al. 1993). Isoprenoid precursors, IPP and DMAPP were found to be formed by condensation of pyruvate-derived acetaldehyde instead of HMG-CoA and MVA, which confirmed the existence of a novel pathway. The essentiality of the MEP pathway in many human pathogens and the absence in humans lead to an extensive investigation of the component enzymes with the final goal of developing potential drugs targeting them. All the enzymes have been biochemically and/or structurally characterized in the pathway since the discovery (Gräwert et al. 2011; Hale et al. 2012; Obiol-Pardo, Rubio-Martinez, and Imperial 2011; Wiemer and Wiemer 2010)(Wiesner and Jomaa 2006). The enzymes all have conventionally scientific names, but several of them are otherwise named in letters with the prefix Isp, indicating their order in the pathway. Seven steps of enzymatic reactions compose the MEP pathway, in addition to the isomerization of IPP to DMAPP. 1) Distinct from the MVA pathway, the biosynthesis of isoprenoid precursors originally begins with the condensation of pyruvate and glyceraldehyde 3-phosphate (GAP) to produce 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by DXP synthase (DXS). 2) DXP is then rearranged to give 2-C-methyl-erythrose-4-phosphate, and subsequently reduced to produce 2-C-methyl-D-erythritol 4-phosphate (MEP), by the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR/IspC), with the energy provided by the conversion of NADPH to NADP+. We will explain DXR and the DXR-catalyzed reaction in details in the following section. 3) MEP and cytidine 5’-triphosphate (CTP) are coupled to form 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME), catalyzed

38 by 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). 4) An ATP-dependent reaction to convert CDP-ME to 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phophate (CDP-ME2P) is catalyzed by CDP-ME kinase (IspE). 5) In the release of CMP, CDP-ME2P is internally cyclized to form 2- C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP), catalyzed by 2-C- methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). 6) MECDP is subsequently converted to 1-hydroxy-2-methyl-2(E)-butenyl-4-diphosphate (HMBPP) by HMBPP synthase (IspG). 7) Finally the IPP and DMAPP are produced from HMBPP by the catalysis of HMBPP reductase (IspH). The in- terconversion of IPP and DMAPP is identical to the final step in the MVA pathway, involving the same IPP isomerase, to produce DMAPP. However, the HMBPP synthase catalyzes the biosynthesis of two isoprenoid precursors, thus IPP isomerase is not essential in the MEP pathway. Our lab has been involved in the investigation of enzymes along the MEP pathway for decades. Our work has included the structural and biochemical characterization of DXRs from M. tuberculosis (Andaloussi et al. 2011; Björkelid et al. 2012; Henriksson et al. 2006, 2007), and P. falciparum (Chofor et al. 2015; Jansson et al. 2013; Sooriyaarachchi et al. 2016), and IspD from M. tuberculosis(Björkelid et al. 2011). FOM analogues showing high potency in the biochemical assay against Plasmodium falciparum are of more interest, than those aimed at the M. tuberculosis enzyme (for which compounds active against the bacteria are lacking), hence, I have concentrated on the structural study of PfDXR complexes.

Figure 8. The MEP pathway

39 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/IspC) DXR was first confirmed to be involved in the MEP pathway in an E. coli mutagenesis study (Takahashi et al. 1998). EcDXR coded by a single gene yaeM (later named dxr and ispC) was overexpressed and identified as DXR/IspC, responsible for MEP biosynthesis. It catalyzes rearrangement from DXP to 2-C-methyl-erythrose-4-phosphate and subsequent reduction of 2-C-methyl-erythrose-4-phosphate to produce MEP in the second reaction in the MEP pathway. This hypothesis of a two-step reaction was experimentally proven by kinetic competency of synthesized 2-C-methyl-erythrose-4-phosphate for the reduction step (Hoeffler et al. 2002). It was found to require NADPH as the preferred cofactor rather than NADH, and a divalent metal ion to coordinate the interactions in the binding site, most commonly as Mg2+ and Mn2+(Lange and Croteau 1999). Crystallographic structures solved previously show DXR is a homodimer, with a subunit molecular weight of roughly 45 kDa.

Figure 9. The DXR catalytic reaction

DXR sequences Since the first discovery of DXR in E. coli, quite a few DXRs from different organisms have been identified, and structurally characterized, e.g. E. coli (1JVS, 1T1R, 1T1S (Yajima et al. 2002, 2004), 1Q0L, 1Q0H, 1Q0Q (Mac Sweeney et al. 2005)), Zymomonas mobilis (1R0L, 1R0K (Ricagno et al. 2004)), Mycobacteria tuberculosis (2C82, (Henriksson et al. 2006), 2JD2, 2JD1, 2JD0, 2JCY, 2JCX, 2JCV, 4AIC (Henriksson et al. 2007), 4A03 (Björkelid et al. 2012)), Yersinia pestis (3IIE, Osipiuk, to be published), Ther- motoga maritima (3A06, 3A14 (Takenoya et al. 2010)), Plasmodium falciparum (3AU8, 3AU9, 3AUA (Umeda et al. 2011), 5JBI, 5JAZ, 5JC1, 5JMP, 5JMW, 5JNL, 5JO0 (Sooriyaarachchi et al. 2016)), Acinetobacter baumannii (4ZN6, Fairman, to be published), Vibrio vulnificus (5KQO, 5KRV, 5KRY, 5KS1, 5KRR (Ussin et al. 2018)), Staphylococcus schleifer (6MH4, 6MH5

40 (Edwards et al. 2020)), Moraxella catarrhalis (4ZQE, 4ZQF, 4ZQG, 4ZQH, Birkinshaw, to be published). PfDXR (UniProtKB ID: Q8IKG4) from Plasmodium falciparum (isolate 3D7) has 488 amino acids including a hypothetical signal peptide. Its molecular weight as a monomer is 55,757 Da. The amino-acid sequence is shown below.

>sp|Q8IKG4|DXR_PLAF7 1-deoxy-D-xylulose 5-phosphate reductoisomerase, apicoplastic OS=Plasmodium falciparum (isolate 3D7) OX=36329 GN=DXR PE=1 SV=1 MKKYIYIYFFFITITINDLVINNTSKCVSIERRKNNAYINYGIGYNGPDNKITKSRRCKR IKLCKKDLIDIGAIKKPINVAIFGSTGSIGTNALNIIRECNKIENVFNVKALYVNKSVNE LYEQAREFLPEYLCIHDKSVYEELKELVKNIKDYKPIILCGDEGMKEICSSNSIDKIVIG IDSFQGLYSTMYAIMNNKIVALANKESIVSAGFFLKKLLNIHKNAKIIPVDSEHSAIFQC LDNNKVLKTKCLQDNFSKINNINKIFLCSSGGPFQNLTMDELKNVTSENALKHPKWKMGK KITIDSATMMNKGLEVIETHFLFDVDYNDIEVIVHKECIIHSCVEFIDKSVISQMYYPDM QIPILYSLTWPDRIKTNLKPLDLAQVSTLTFHKPSLEHFPCIKLAYQAGIKGNFYPTVLN ASNEIANNLFLNNKIKYFDISSIISQVLESFNSQKVSENSEDLMKQILQIHSWAKDKATD IYNKHNSS

The multiple sequence alignment (CLUSTAL Omega) included DXR amino- acid sequences from Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, (SKAP out of ESKAPE pathogens), Plasmodium falciparum, Mycobacterium tuberculosis, Yersinia pestis, Zymomonas mobilis. No DXRs from Enterococcus faecalis, Entero- bacter sp. are found in the Unitprot database. Generally, DXRs among all organisms listed are highly conserved with the identities ranging from 36% to 71%, except that PfDXR possesses an extended N-terminal signal peptide and apicoplast-targeting sequence. KpDXR has the highest similarity to YpDXR, with the identity at 71%, while the lowest identity is between PfDXR and MtDXR. A tryptophan-containing loop (His293-Gly299 in PfDXR) identified to be critical for enzymatic activity (Kholodar et al. 2014) is identical in nine orgranisms excluding P. falciparum and M. tuberculosis, which have Lys295 & Lys297, and Thr202 & Ser204 flanking Trp296 and Trp203, respectively. The NADPH-binding site, based on the MtDXR-NADPH complex, includes Tyr21-Ile24, Gly47-Ala49, and Gly206. The first cluster and Gly206 from nine species are identical, while NADPH interacts with the backbone nitro- gens of residue 47-49, showing that conservation of the second cluster is not strictly required (Henriksson et al. 2007). Asp231, Glu233, Glu315 in PfDXR in the catalytic site are identical among nine species. In summary, the overall structures of DXRs may vary, however, all potentially crucial amino acid residues are highly conserved, e.g. in the NADPH binding site and the DXP- binding site.

M Sequence Logo G M S K P K D L I D D A M A D Q R M KKY I Y I YFFFI T I T I N D LV I NNTSK C V S I E RRKNNA Y I N Y GIGY N GPD N K I T K S RRC KR I K L C K M T N S T I G R I K R T

Ruler 2 110203040506070

Ruler 1 80 90 100 110 120 130 140 150 Consensus k q v t I L G S T G S I GxS T L D VxRxN P- - - d r FxV v A L v A G k N V- xe L v e Q C l E FxP r Y A V vxD exs A exL kx l l-x-xG sp|Q8IKG4|DXR_PLAF7 I N V A I F G S T G S I G T N A L N I I R E C N K I E N V F N V K A L Y V N K S V- N E L Y E Q A R E F L P E Y L C I H D K S V Y E E L K E L V K N I K D sp|P9WNS1|DXR_MYC... L R V V V L G S T G S I G T Q A L Q V I A D N P- - - D R F E V V G L A A G G A H L D T L L R Q R A Q T G V T N I A V A D E H A A Q R V------sp|P45568|DXR_ECOLI K Q L T I L G S T G S I G C S T L D V V R H N P- - - E H F R V V A L V A G K N V- T R M V E Q C L E F S P R Y A V M D D E A S A K L L K T M L Q Q- Q G tr|A0A033V6T8|A0A0... K N I A I L G A S G S V G Q Q A L D V I R E H A- - - S Q F N L V A F S V G K N I-A Y A Q N I I E E F H P E L I S V Q N E E D I D K L K------H tr|A0A378ABW1|A0A3... K Q L T V L G S T G S I G C S T L D V V R H N P- - - G R F S V A A L V A G K N V- D R M V E Q C L E F T P R Y A V M D D A Q S A E R L R T R L H E- H G tr|A0A2P1ASG3|A0A2... Q S V C I L G V T G S I G R S T L K I L G Q H P- - - D K Y S V F A V S A H S R I-S E L V E I C K Q F R P K V V V V P E Q K I A E L K T L F A Q Q- N I sp|Q9KGU6|DXR_PSEAE Q R I S V L G A T G S I G L S T L D V V Q R H P- - - D R Y E A F A L T G F S R L- A E L E A L C L R H R P V Y A V V P E Q A A A I A L Q G S L A A- A G sp|Q8ZH62|DXR_YERPE K Q L T I L G S T G S I G N S T L S V V R A N P- - - E L F K V T A L V A G R N V- R E M A Q Q C L E F S P R Y A A M S D E H S A K S L R L L L A E- Q G sp|Q9X5F2|DXR_ZYM... R T V T V L G A T G S I G H S T L D L I E R N L- - -D R Y Q V I A L T A N R N V- K D L A D A A K R T N A K R A V I A D P S L Y N D L K E A- - L-A G

C A R L E E V V A A S E Q T R V D E S R R L K T D I R H D N K E L Y D E H E A E A V H H F K A A G V A K A A Sequence Logo N S K A N S G N V L Q C E E L A V K L N H D G I K A E D D D F Q A R P E S R R K P Q S L Q Q S N V E A A N R I I A F N I A D A R Q D I C N T A G L F K I R D L N A L H A E I I E M A H R L V L T Q P K M L L Q G L A F F A H A I A R L C K Q T A H H Q S L S S A Q I V D H N Q Q K A V S T M Q Q E T N T Q E K K L Y N K R L K R T I V V LF G V TS G S VI G R N A L S L L Q Q C N K I E S V Y R L T GA V Y G H G S L L T Y A Y R L R R R H T V V V V S I S N P Q V I Q S V T G S V K N I N I Ruler 2 80 90 100 110 120 130 140 150

Ruler 1 160 170 180 190 200 210 220 230 Consensus s r t E V L s G q q Axc E v A a lxxV D q V M A A I V G A A G LxP T L A A I r A G KxV L L A N K E S L V txGx L f M d A V r-xs g A q L L P V sp|Q8IKG4|DXR_PLAF7 Y K P I I L C G D E G M K E I C S S N S I D K I V I G I D S F Q G L Y S T M Y A I M N N K I V A L A N K E S I V S A G F F L K K L L N I H K N A K I I P V sp|P9WNS1|DXR_MYC... - G D I P Y H G S D A A T R L V E Q T E A D V V L N A L V G A L G L R P T L A A L K T G A R L A L A N K E S L V A G G S L V L R A A R- P- - G Q I V P V sp|P45568|DXR_ECOLI S R T E V L S G Q Q A A C D M A A L E D V D Q V M A A I V G A A G L L P T L A A I R A G K T I L L A N K E S L V T C G R L F M D A V K-Q S K A Q L L P V tr|A0A033V6T8|A0A0... L N I E I V H G D A G L L A V A T Y H N T D I L L N S I V G S I G L R P T I A A I E A G I D I G L A N K E T L V A A G E L V M S K A R- E H N V N I L P V tr|A0A378ABW1|A0A3... C R T E V L S G Q Q A A A E V A A L D E V D Q V M A A I V G A A G L V P T L A A I R A G K T V L L A N K E S L V T C G R L F M E A V Q- Q S G A R L L P V tr|A0A2P1ASG3|A0A2... S D I D V L A G Q K G L V D I A S H T D V D I V M A A I V G A A G L L P T L A A V K A G K R V L L A N K E A L V M S G E I M M Q A A R- D H Q A L L L P V sp|Q9KGU6|DXR_PSEAE I R T R V L F G E Q A L C E V A S A P E V D M V M A A I V G A A G L P S T L A A V E A G K R V L L A N K E A L V M S G A L F M Q A V K-R S G A V L L P I sp|Q8ZH62|DXR_YERPE S D T E V Y S G E T A A C E L A A L D D V D Q V M A A I V G I A G L P S T L A A I R A G K Q V L L A N K E S L I T C G K L F M D E V K-R S R A Q L L P I sp|Q9X5F2|DXR_ZYM... S S V E A A A G A D A L V E A- A M M G A D W T M A A I I G C A G L K A T L A A I R K G K T V A L A N K E S L V S A G G L M I D A V R- E H G T T L L P V

L D E Q R Q C Q L D E S Q V A T D A R T A R K T A P R F R Q G A H E A L Sequence Logo S D E V L A D D V E I V I C P L A Q V S C G I A A M H E K V A R E A T C F M M E A R N A N I I Y D S A I M A F I I K V S I E K G I K D E A K Q M G M K D G K D K R L H E L A A V L K M L L D A A A C E I L G D I L I K N A S F V K K N T I L N P C A K L L A S N N V L N V S V I I A A K R A H H Q T V Y S V R P V F G S T G M T R M V T Y P S T DW T V I S LI I GS S Q G L Y A T M YA A L M T GN I Q L G L A N K E T LI VI S G G S I L L S L L Q I P K R V V I V P I Ruler 2 160 170 180 190 200 210 220 230

Ruler 1 240 250 260 270 280 290 300 Consensus D S E H N A I F Q S L Pxn i q------l--n G V s k I l L T A S G G P F R e TxL a e L axV T PxQ A C a H P N W S M G r K I S V D S A sp|Q8IKG4|DXR_PLAF7 D S E H S A I F Q C L D N N K V L K T K C L Q D N F S K I N N I N K I F L C S S G G P F Q N L T M D E L K N V T S E N A L K H P K W K M G K K I T I D S A sp|P9WNS1|DXR_MYC... D S E H S A L A Q C L R G G T P------D E V A K L V L T A S G G P F R G W S A A D L E H V T P E Q A G A H P T W S M G P M N T L N S A sp|P45568|DXR_ECOLI D S E H N A I F Q S L P Q P I Q H N L- - -G Y A D L E Q N G V V S I L L T G S G G P F R E T P L R D L A T M T P D Q A C R H P N W S M G R K I S V D S A tr|A0A033V6T8|A0A0... D S E H S A I F Q C L N G E K K------E Q V N K L I I T A S G G S F R D L T R T E L K S V T R D E A L N H P N W S M G Q K I T I D S A tr|A0A378ABW1|A0A3... D S E H N A I F Q S M P E T I Q Q H L- - -G Y A D L A R N G V S S I L L T G S G G P F R E T A V A E L A A M T P D Q A C R H P N W S M G R K I S V D S A tr|A0A2P1ASG3|A0A2... D S E H N A I F Q S L P H N Y L Q A- - - - D R T G Q P Q L G V S K I L L T A S G G P F L N H S L E Q L T H V T P Q Q A C K H P N W S M G Q K I S V D S A sp|Q9KGU6|DXR_PSEAE D S E H N A I F Q S L P R N Y A D------G L E R V G V R R I L L T A S G G P F R E T P L E Q L A S V T P E Q A C A H P N W S M G R K I S V D S A sp|Q8ZH62|DXR_YERPE D S E H N A I F Q S L P E R I Q R Q L- - -G Y S S L N E N G V S R I I L T G S G G P F R E T P L S Q F S D V T P D Q A C A H P N W S M G R K I S V D S A sp|Q9X5F2|DXR_ZYM... D S E H N A I F Q C F P H H N R------D Y V R R I I I T A S G G P F R T T S L A E M A T V T P E R A V Q H P N W S M G A K I S I D S A

N Q E A H E I A S P A G D Q N G L E L A S R G K D Q A E K E E Sequence Logo K A E N A T A C K V S H H L D H A D T P D N S K Q Q D G Y N G R N I L M E L N N P P H K K E D R E A A S L Y L R R R Q L P R N F N D D S F Q E Q F L H T R F R N G K Q R N R L N L N L A I L G G S S D V E N K I D S E H S A LI AF Q C M R R T T V R Q T K C L Q R T S S P I V Y VI V S L V I TC S S GGPS F Q T W A V T D M T N M T S Q R A V Q H P T WSK M G P MK NI T L DN S A Ruler 2 240 250 260 270 280 290 300

Ruler 1 310 320 330 340 350 360 370 380 Consensus T M M N K G L Ex I E A r W L Fxa s axQ i E V V v H P Q S V I H S M VxYxD G S V L A Q l GxP D M R T P I a H AxA W P e R vxs g V k P L D F c sp|Q8IKG4|DXR_PLAF7 T M M N K G L E V I E T H F L F D V D Y N D I E V I V H K E C I I H S C V E F I D K S V I S Q M Y Y P D M Q I P I L Y S L T W P D R I K T N L K P L D L A sp|P9WNS1|DXR_MYC... S L V N K G L E V I E T H L L F G I P Y D R I D V V V H P Q S I I H S M V T F I D G S T I A Q A S P P D M K L P I S L A L G W P R R V S G A A A A C D F H sp|P45568|DXR_ECOLI T M M N K G L E Y I E A R W L F N A S A S Q M E V L I H P Q S V I H S M V R Y Q D G S V L A Q L G E P D M R T P I A H T M A W P N R V N S G V K P L D F C tr|A0A033V6T8|A0A0... T M M N K G L E V I E A K W L F D L D I D Q I E T V L H K E S I I H S M V E F K D T S V I A Q L G T P D M R T P I Q Y A F T Y P E R Y T R D A E K L N L A tr|A0A378ABW1|A0A3... T M M N K G L E Y I E A R W L F N A S A Q Q M E V L I H P Q S V I H S M V R Y Q D G S V L A Q L G E P D M R T P I A H T M G W P Q R L N S G V K P L D F C tr|A0A2P1ASG3|A0A2... T L M N K G L E L I E A C H L F S I S E H F V T V V V H P Q S I I H S M V Q Y V D G S T L A Q M G N P D M C T P I A H A L A W P E R L Q T N V P A L D L F sp|Q9KGU6|DXR_PSEAE S M M N K G L E L I E A C W L F D A Q P S Q V E V V I H P Q S V I H S M V D Y V D G S V I A Q L G N P D M R T P I S Y A M A W P E R I D S G V S P L D M F sp|Q8ZH62|DXR_YERPE T M M N K G L E Y I E A R W L F N A S A E Q I E V V L H P Q S V I H S M V R Y H D G S I L A Q M G T P D M R T P I A H A M A Y P M R V S S G V A P L D F C sp|Q9X5F2|DXR_ZYM... T M M N K G L E L I E A Y H L F Q I P L E K F E I L V H P Q S V I H S M V E Y L D G S I L A Q I G S P D M R T P I G H T L A W P K R M E T P A E S L D F T

E N A D I E R D E D V S K C L A S E Q I Q L N A S G Y V L K D Sequence Logo C W D V V H A A I V A P F A E S V V L T M E N D M R R S N V H I F M C T A G I H D Y H P G N K A H E V I G I G T G L E F D I K S I F L P K V A K Q G K T M A K Q L N L P Q Q K S L Y T M W Q M D A P L H S L MV N K G L E Y I E T Y L L F S V Q P Q R F T T I L H K E SC I I H S MC V T F L D T S T I SA Q I Y Y P DM Q L P I Q L S F T Y P R R Y T R P L S S CL DN M T Ruler 2 310 320 330 340 350 360 370 380

Ruler 1 390 400 410 420 430 440 450 460 Consensus x x S q L t Fxa P D yxR F P C L k L A m q Axxa G G a A P T v L N A A N E I A V A A F Lxxk I r F t D I ax I i e d V L e kxd--xxE---P sp|Q8IKG4|DXR_PLAF7 Q V S T L T F H K P S L E H F P C I K L A Y Q A G I K G N F Y P T V L N A S N E I A N N L F L N N K I K Y F D I S S I I S Q V L E S F N S Q K V S E N S E sp|P9WNS1|DXR_MYC... T A S S W E F E P L D T D V F P A V E L A R Q A G V A G G C M T A V Y N A A N E E A A A A F L A G R I G F P A I V G I I A D V L H A A D Q W A V E---P sp|P45568|DXR_ECOLI K L S A L T F A A P D Y D R Y P C L K L A M E A F E Q G Q A A T T A L N A A N E I T V A A F L A Q Q I R F T D I A A L N L S V L E K M D- -M R E---P tr|A0A033V6T8|A0A0... Q I G Q L N F K E M D F D R F K C I Q Y A Y E A I K I G G T M P V V L N A V N E V A V N K F L N N E I T F L E I E D M I G E A M K N H E- - V I E---H tr|A0A378ABW1|A0A3... Q L S N L S F S A P D Y T R Y P C L K L A M D A F D V G Q A A T T T L N A A N E E S V A A F L H G D I R F T D I A A V N L A V L D K M D- - L Q E---P tr|A0A2P1ASG3|A0A2... E Y S Q L N F Q A P D T Q K F P A L N L A R Q A M R A G G L A P T I L N A A N E I A V E A F L M E R I G F T S I P Q V V E H T L E K L E- - N A A---A sp|Q9KGU6|DXR_PSEAE A V G R L D F Q R P D E Q R F P C L R L A S Q A A E T G G S A P A M L N A A N E V A V A A F L E R H I R F S D I A V I I E D V L N R E A- - V T A---V sp|Q8ZH62|DXR_YERPE K V G A L T F T T P D Y Q R Y P C L K L A I D A C N A G Q A A T T A L N A A N E I S V M A F L D S K I R F T D I E V I N R T V V E G L L- - L S E---P sp|Q9X5F2|DXR_ZYM... K L R Q M D F E A P D Y E R F P A L T L A M E S I K S G G A R P A V M N A A N E I A V A A F L D K K I G F L D I A K I V E K T L D H Y T- -P A T---P

E E A G A K L L A Q F A K E D D K L T Q A D K M K I A N V A M V V Y G V D R T A A E P A A E E Q D K C E R D I L G A E A I Sequence Logo S D R G A T I A I E E A N H K N I I Q F P N K D G A H H E A K Q T R A L D E D Q V Q H A E M N G V K A L A Q V A A R K P E F C Q A N S L I E E F P K G H N F L M R N Y E L P E L A V A N Q L M L K R M S A E K K E L M Q Q S H E I S E S R F E I R I C R T Q S M S H R H P S Q R T K R H N N S G A S T Y R T W S F T T M DS L T V Y PK A V T YL A S D SA M V V G N T Y T V T Y N A V N E V T N M L F L M S Q I T YF S S I V S M V S T A V N S Y T S W P T T E NSV Ruler 2 390 400 410 420 430 440 450 460

Ruler 1 470 480 490 500 Consensus x Sxxd V Lx I Dx xA R s v Axq vxs r l a s------sp|Q8IKG4|DXR_PLAF7 D L M K Q I L Q I H S W A K D K A T D I Y N K H N S S------sp|P9WNS1|DXR_MYC... A T V D D V L D A Q R W A R E R A Q R A V S G M A S V A I A S T A K P G A A G R H A S T L E R S sp|P45568|DXR_ECOLI Q C V D D V L S V D A N A R E V A R K E V M R L A S------tr|A0A033V6T8|A0A0... P D L E T I L K I D A T Y K S K D Y G V------tr|A0A378ABW1|A0A3... Q S I D D V L V I D A E A R A I A H Q Q L Q R L V A Q A------tr|A0A2P1ASG3|A0A2... E S I E C I L D K D K V A R S V A Q Q Y I S S I G G------sp|Q9KGU6|DXR_PSEAE E S L D Q V L A A D R R A R S V A G Q W L T R H A G------sp|Q8ZH62|DXR_YERPE T S V E E V L V I D R K A R D V A A Q V I A K L N N------sp|Q9X5F2|DXR_ZYM... S S L E D V F A I D N E A R I Q A A A L M E S L P A------

E E A S A V Q L A W S Q Q I A S D D V A R A S A D K G E A Sequence Logo C I D E L L D Q V N K H D I M N D V R K A A L H Q P C V K K R I R G N G E V D Q L K H S I G S S I E Q N T A Q T K M Q P E R W T T M K T I LF S V Q S V YA K I R DA Y R Y Y T G M V N V A IASTA K PGAAGRHA ST L E R S Ruler 2 470 480 490 500

Figure 10. The sequence alignment of DXRs from diverse species

42 DXR-targeted drug development The prevalence of the MEP pathway in pathogenic bacteria and parasites, and its absence in humans, highlight DXR as a potential drug target, with inhibitors expected to have limited toxicity to humans. Due to the fact that multiple drug-resistant (MDR) and extensively drug-resistant (XDR) strains are occur- ring more frequently, effective novel antibiotics with a broader spectrum are urgently needed (Michael, Dominey-Howes, and Labbate 2014; Prestinaci, Pezzotti, and Pantosti 2015). DXRs are vital for the survival of both M. tuberculosis and P. falciparum, and correlations between DXR inhibition and antibacterial and antimalarial activities have been observed. Hence, DXR inhibitor design attracted medicinal chemists’ attention. DXR is known as a homo- dimeric enzyme, where each monomer consists of three domains, the N-terminal NADPH binding domain (NTD), the dimer-forming domain (DFD), the C-terminal domain (CTD). The structural details will be discussed in structure session. Based on two important domains, the NADPH binding domain and the DXP binding domain, all designed inhibitors can be categorized into two groups. 1) NADPH-mimicking inhibitors. A series of novel 3’,N6-disubstituted adenosines sharing the same scaffold as NADPH, exhibited potency against P. falciparum strain Dd2 and PfDXR enzymatic activity in the biochemical assay. Unfortunately, the 3’, N6-disubstituted adenosines inhibited only 25% of PfDXR activity, yet it can be considered as an alternative lead for the future drug design (Herforth et al. 2004). 2) Inhibitors bound to the DXP binding pocket need to further be sub-categorized into two groups. One applies the strategy of imitating the DXP scaffold in the inhibitor design. (3R,4S)-3,4-Dihydroxy-5-oxohexylphosphonic acid, a DXP isosteric analogue, was designed in a similar DXP scaffold to bind in the catalytic site with no production of MEP. It showed neither significant inhibition of EcDXR, nor of E. coli growth. Surprisingly, it was converted by DXR into (3R,4R)-3,4,5-trihydroxy-3-methylpentylphosphonic acid, isomeric MEP analogue. Because of the phosphonate group replacing the phosphate group, both affinity of DXR to the inhibitor and turnover rate have been compromised (Meyer et al. 2003). The second sub-class of inhibitors includes derivatives of fosmidomycin (FOM), a natural product, and its acetyl analogue FR-900098 (Kuzuyama et al. 1998; Zeidler et al. 1998). FOM was first identified as a potent inhibitor for EcDXR with a Ki of 38 nM, and for Z. mobilis DXR with a Ki of 600 nM. The inhibitory mechanism of FOM consists of a combination of a slow tight- binding competitive inhibition against DXP, and an uncompetitive inhibition against NADPH (Koppisch et al. 2002). FOM was subsequently evaluated in the biochemical assay, and found to have IC50s of 8 nM against EcDXR (Kuzuyama et al. 1998), 80 nM against MtDXR (Henriksson et al. 2007), and 36 nM against PfDXR (Chofor et al. 2015). FR900098 showed equally high

43 inhibitory activity against EcDXR with an IC50 of 30 nM, 160 nM against MtDXR, and 18 nM against PfDXR. FOM and FR900098 were identified to be potent against E. coli and P. falciparum in the whole-cell assays (Jomaa et al. 1999). On the contrary, these two compounds showed insufficient inhibition against M. tuberculosis, due to the lack of the glycerol-3-phosphate transporter (glpT) in Gram-positive bacteria and Mycobacteria, which is specifically responsible for a facilitated transport of FOM (Sakamoto et al. 2003). Hence, medicinal chemistry efforts of FOM have been put to increase the permeation through the Gram-positive cell walls (Dhiman et al. 2005; Nair et al. 2011; Uh et al. 2011). Besides, fosmidomycin treatment has a significantly high cure rate for uncomplicated P. falciparum in humans (Lell et al. 2003; Missinou et al. 2002) and P. vinckei in mice (Jomaa et al. 1999). However, side effects, as headache, weakness, appeared in the patients, more concern- ingly, the treatment would cause unneglectable increase in gametocyte carrier (Lell et al. 2003; Missinou et al. 2002). Due to its highly-charged nature, fosmidomycin has stability issues in plasma, with a half-life time of 2.5h, and poor pharmacokenitics (Kuemmerle et al. 1985). It also has been proven to have lower efficacy in children under 3 years of age (Lanaspa et al. 2012). FR900098 exhibits highly increased antimalarial activity intraperitoneally in P. vinckei-infected mice, compared to FOM (Jomaa et al. 1999). Moreover, it presents low cytotoxicity in a mouse model, and no significant evidence of genotoxicity in humans (Wiesner et al. 2016). However, high dose requirement of FR900098 (Missinou et al. 2002) limits the clinical use, due to the poor oral bioavailability. This could be explained as high lipophilicity in physiological environment. To date, an extensively large collection of chemical modifications have been introduced into FOM and FR900098 to increase the lipophilicity, permeation, and thus, antibacterial and antimalarial activity, e.g. 1) introduction of an ester group to the phosphonate moiety ((Uh et al. 2011), 2) extension of the N-acyl or N-hydroxyl moieties with aromatic groups (San Jose et al. 2016), and 3) substitution on the -, or -carbon atoms, relative to the phosphorus (Kunfermann et al. 2013). Strategy 1 was intended to increase the lipophilicity so that the FOM analogues would be capable of penetrating the greasy cells wall. However, the most potent FOM analogue showed only weak inhibition with MIC values of 25-500 g/mL against M. tuberculosis. Strategy 2 namely bi-substrate strategy, intended to occupy both DXP and NADPH binding sites with a single inhibitor, has been proven to be less potent against M. tuberculosis than FOM. Strategy 3 successfully produced FOM analogues with lower IC50s against DXRs in the biochemical assay. In summary, structure-activity relationship (SAR) data indicated that novel FOM analogues must maintain the hydroxamate group contributing to stabilize the metal ion- coordinated network, phosphonate fragments, and three-carbon linker chain, to be potent (Gadakh et al. 2015).

Figure 11. FOM, FR900098, synthesized FOM analogues from three strategies.

An inhibitor with double-bonded (-carbon and -carbon, and CH2OCOtBu binding to phosphonate moiety displayed the most potent inhibition against M.tuberculosis cell growth, with an MIC of 9.4 g/mL (Jackson et al. 2014). A double-bonded FOM analogue with two pivaloyloxymethyl (POM) phosphonate was tested in a whole-cell assay, and showed highly potent inhibition against P. falciparum with an IC50 of 13 nM, as well as high in vivo efficacy in the Plasmodium berghei-infected mice. Moreover, it displayed no significant cytotoxicity to HepG2 cells with an IC50 larger than 50 M (Wang X, Edwards R, Ball H et al. 2019). In the collaboration with medicinal chemists,

45 a series of FOM analogues with an unsaturated propyl chain (-carbon and - carbon double-bonded) possessing an phenyl ring with linkers of different carbon atoms to acetyl group were designed, and synthesized. This novel approach was designated as a combination of strategy 2 and an increased planarity on the inhibitors. Four double-bonded inhibitors 1, 2, 3, 4 were synthesized with H, a phenyl ring, a phenyl ring with a linker of two carbon atoms, a phenyl ring with a linker of three carbon atoms at the N-acyl position, respectively.

Figure 12. Four unsaturated FOM analogues

The PfDXR structure The first crystal structures of PfDXR were determined to guide the refinement of fosmidomycin performance in anti-malarial activity (Umeda et al. 2011). Three PfDXR structures were solved, as a PfDXR-NADPH-Mn2+ complex, a PfDXR-FOM or -FR900098 complex with NADPH and Mg2+ present. The initial phases of PfDXR-NADPH complex were determined by molecular replacement with E. coli DXR dimer (PDB code: 1ONN), considering the high sequence similarity between two DXRs, followed by the refinements at 1.86Å (PDB code: 3AU8). Subsequently, PfDXR-FOM and PfDXR-FR900098 complexes (PDB code: 3AU9 and 3AUA) were solved by molecular replacement with PfDXR-NADPH complex, at resolution 1.9Å and 2.15Å. PfDXR is a homodimer with the N-terminal peptide cleaved off (residues 75-488), in which each molecule consists of three domains, the N-terminnal NADPH binding domain (residues 77-230), the central catalytic domain (residues 231-369), a linker region (370-395), and the C-terminal domain (396- 486). Three domains are forming V-shaped tertiary complex with the central catalytic domain at the vertex. In each subunit, one NADPH molecule binds 2+ 2+ in the binding cleft, and one Mg or Mn coordinates the intensive network with NADPH, inhibitors and amino acids from PfDXR. The NADPH binding domain (residues 77-230) possesses a seven-stranded -sheet, and two sets of three -helices, conforming a sandwich fold with two sets of three -helices flanking the -sheet, similar to the characteristic

46 Rossmann fold. The tertiary arrangement of secondary structures in Rossmann fold are generally two  motifs, and the NADPH binding domain dis- plays differently by one pair of additional  motif inserted after 3. The NADPH molecule is placed in the fashion similar to EcDXR-NADPH complex. The NADPH is binding in the binding domain with the nicotinamide pointing perpendicular to the catalytic domain, making interactions with the metal ion.

Figure 13. The overall structure of PfDXR in complex with NADPH, Mn2+, and FOM is presented in cartoon mode. Yellow, NADPH binding domain; blue, DXP binding domain; green, C-terminal domain.

47 The catalytic domain (residues 231-395) is an / structure interacting with the other subunit to form a dimer. It is composed of four -strands and five - helices, slightly smaller than the NADPH binding domain. No complex structures with DXP have been determined, but FOM analogues known to compet- itively bind in the catalytic have been co-crystallized. PfDXR-FOM complex showed that FOM is located in the center of the four helices and at the end of two parallel -strands in the -sheet. The loop (residues 291-299) functioning as a lid covering the catalytic site has been observed in DXR-inhibitor complex structures from other species. the details of the network in the catalytic domain will be discussed later. The catalytic domain contributes dominantly for homodimer formation, creating an eight-stranded -sheet, with four from each subunit, and a two-stranded -sheet in the linker that connects the catalytic domain and the C-terminal domain, from each individual subunit. The function of C-terminal domain is not clear, but it mainly seems to have a structural role.

PfDXR complexes with unsaturated FOM analogues (Paper III) As described previously in the inhibitor session, four unsaturated FOM analogues and PfDXR complexes were characterized structurally by x-ray crystallography. Four complexes of 1, 2, 3, 4 have been refined to resolutions in the range 2.4Å, 1.86Å, 2.13Å and 2.05 Å. A complex structure with PfDXR and FR900098 has been included, and refined to 1.9Å, for comparison. In general, PfDXR preparation was conducted as described previously (2015, Chofor; 2019, Sooriyaarachchi). The Pfdxr gene (generously contributed by Sanjeewani Sooriyaarachichi) with a truncation of the first 74 amino-acid residues and N-terminal His-tag was inserted into the plasmid pEXP-5-CT/TOPO, and subsequently transformed into E.coli TOP10 cells (Invitrogen, US) for am- plification. Plasmids with the truncated pfdxr were selected, and transformed into E.coli C43(DE3) for expression. Cell pellets with expressed PfDXR were thawed and lysed with a cell disruptor in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 5% (v/v) glycerol, 0.01 mg/mL RNase A and 0.02 mg/mL DNase, 2 mM DTT, and a tablet of cOmplete, EDTA-free protease inhibitor cocktail (Roche)). N-terminally His-tagged PfDXR was purified by nickel immobilized metal affinity chromatography (IMAC), followed by size exclusion chromatography (SEC) on a HiLoad 16/60 Superdex 200 preparative grade column (Amersham Biosciences) pre-equilibrated with 20 mM Tris-HCl pH 7.8, 5% glycerol, 300 mM NaCl, 2 mM DTT. The protein purity was analyzed by SDS-PAGE. The concentrated protein was mixed at a 1:1 molar ratio with each ligand (1, 2, 3, 4, FR900098) immediately before crystallization screening trials. Drop volumes were 100 nL of the protein:ligand complex and 100 or 200 nL of Morpheus screen (Molecular Dimensions, (Gorrec 2009)) set up with a Mosquito robot (TTP Labtech, UK). Reservoir volumes were 50 L. Crystals appeared within 1-3 days at 22C in wells D12b, F12a, C12, and A12a.

48 Diffraction data of the PfDXR complexes with 1, 3 and 4 were collected at ID29 at the European Synchrotron Radiation Facility Grenoble, France (ESRF), and complex with 2 was collected at the MAX II BioMAX beamline at Lund, Swe- den. All crystals were in triclinic space group P1, with similar dimensions of the unit cell (near a=51Å, b=56Å, c=86Å, =103°, =103°, =101°). Diffraction frames were integrated and scaled with XDS (Kabsch 2010) and Scala (P. Evans 2006). Datasets showed minor anisotropy, hence, the final resolution limits for different ligand complexes were determined by CC(1/2) along different Eigen vectors in Aimless (P. R. Evans and Murshudov 2013). The data anisotropy and resolution limits were visually and statistically verified in O (Jones et al. 1991). Given the same triclinic P1 space group, and high similarity of cell dimensions, the initial phases for the PfDXR-1 PfDXR-2, PfDXR-3, and PfDXR-4 complexes were determined by rigid-body refinement with REFMAC5 (Murshudov, Vagin, and Dodson 1997) and the coordinates of PfDXR- FOM- analogue complex (Sooriyaarachchi, unpublished data). They were then followed by restrained refinement in REFMAC5, to generate the initial structures and difference density maps. Thereafter, the structures were manually rebuilt in O (Jones et al. 1991) and refined in several rounds with REFMAC5. Solvent molecules initially were placed based on the respective model structures, followed by manual rebuilding and editing in O. In building of the ligands with the qds tools in O, the C-C bonds of the four ligands were altered from a single to double bond based on the structure of FR900098. 1 is the FR900098 analogue with a double-bonded C-C. In addition, upon the removal of the methyl ketone group in FR900098, 2, 3, and 4 were manually extended at the acetyl-carbon with a phenyl group with 0, 2, or 3 carbon linkers. Simultaneously, the stereochemical restraints were defined for the ligands. All four ligands were fitted well in the 2Fo-Fc maps (Figure X). All four complexes with unsaturated FR900098 analogues have similar overall structures to the FR900098 complex, with RMSDs in the range 0.5-0.8 Å after the least-square alignment of C atoms. Each catalytic site contains a metal ion binding site and a phosphate binding site, both of which are occupied by the FOM analogues. In all active sites of five complexes, the FOM analogues have essentially the same metal coordination with their hydroxamate group, and make the same interactions with the PfDXR and some crucial water molecules. The side chains of several polar residues, Asn311 and His293, and the backbone nitrogen of Ser269 interact with the phosphonate moiety in the FOM analogues within distances in the range 2.7 – 3.0Å, A dis- torted octahedral network comprises hydrogen bonds between the metal ion and two oxygens from the hydroxamate group in the ligands, carboxyl oxygens from Asp231, Glu233, and Glu315, which are generally highly conserved. However, minor variations exist in the flexible flap (291-299) crucial for the ligand placement. In line with DXR complexes from other organisms, the flap is parallel to the backbone of FOM analogues, with the Trp296 forming a stacking interaction to the backbone and the salt bridge mentioned earlier

49 (phosphonate and H293). The catalytic pocket is observed to have an open and a closed form, depending on the binding of DXP or DXP mimics, which involves a significant movement of the flap. This results in a rigid-body movement of the NADPH-binding domain and the C-terminal domain rotating with respect to each other (Björkelid et al. 2012)

Figure 14. Ligand 1, 2, 3, 4 in respective 2Fo-Fc map.

The hope for this series of ligands was to evaluate the fitting regarding the introduction of increased planarity in the backbone, and to efficiently occupy both the DXP site and the NADPH-binding site. The complex with ligands 3, 4 and the PfDXR complexes with both FOM and NADPH (PDB code: 3AU9) were superimposed (Figure X) to compare the arrangements. The backbone of ligands 3 and 4 are essentially overlapped, but slightly different from FOM. The rigidity resulting from the carbon-carbon double bond restrained the movement of atoms within the two new inhibitors. However, the spatial location of catalytic residues showed no significant difference. The phenyl ring of ligand 4, with a linker of three carbon atoms was pointing towards the NADPH-binding site, overlapping the position of the amide in the nicotinamide, while ligand 3 was not. This confirmed that a linker with three carbon atoms is a minimum requirement for the design of bi-substrate inhibitors.

Figure 15. The spatial arrangement of ligand 4 (coloured in element) in the catalytic site. FOM and NADPH (yellow) from 3AU9; ligand 3 (pink).

Conclusions and future perspectives P. falciparum, the causative parasite of malaria, accounts for roughly half a million deaths per year. The pivotal role of DXR in the organism’s isoprenoid precursor biosynthetic pathway has led to it being considered as a promising drug target for antimalarial drugs. A natural product fosmidomycin and its analogue FR900098 displayed extraordinary inhibitions in the biochemical assay against DXRs from diverse pathogens, in addition to antimalarial activity. Such compounds have, however, showed only limited activity against other important pathogens such as M. tuberculosis. A series of unsaturated FOM analogues with linkers of various lengths were designed and synthesized, in order to combine two strategies that have been proven to improve the druggability of FOM and FR900098. Unsaturation in the backbone of the inhibitors was introduced to increase the planarity, thus hopefully enhancing the membrane permeation. The extension at the acetyl group with a phenyl ring via linkers followed the scheme of a bi-substrate strategy, i.e. expecting an individual inhibitor to occupy both the NADPH-binding site and the DXP site. Four PfDXR-inhibitor complexes have been structurally characterized, providing atomic details needed for guidance. Our expectation was fulfilled with ligand 4, which has a C-C double bond and a phenyl ring with a linker of three carbon atoms. It extended the phenyl ring into the nicotinamide position in the NADPH-binding site, based on the comparison to the PfDXR- NADPH-FOM complex structure. Other inhibitors with shorter linkers pointed the phenyl ring out in different directions, indicating that a linker of three carbon atoms is the minimum requirement for bi-substrate design. This series of compounds has been adjusted in hydrophobicity by introducing the

51 planarities of the double bond; they now can be tested in the whole-cell assay against Gram-positive bacteria, which were found to be completely resistant to FOM and FR900098. Besides, based on the discovery of the minimum requirement of a three-carbon linker, a large collection of chemical groups mimicking nicotinamide can be introduced to replace the phenyl ring in ligand 4.

52 NADH dehydrogenase II (Paper IV) Type II NADH:quinone oxidoreductase (NADH dehydrogenase II, NDH-2, EC NO.: 1.6.5.3) plays a critical role in respiratory chains of organisms throughout the three life domains, Archaea, Bacteria, and Eukarya (Marreiros et al. 2017). It is a monotopic membrane protein, catalyzing the reduction of menaquinone derivatives to establish, and maintain the electrochemical potential between inner and outer cellular spaces. This protonmotive is important in regards of ATP synthesis, nutrient transport, and so forth. NDH-2s are the only dehydrogenases in some pathogens, while in other pathogens, two other different NDHs were found. In the case of M. tuberculosis, three different dehydrogenases in the respiratory chain were found in the genome, but surprisingly, only NDH-2 was identified to be essential for the viability and persis- tence of hypoxic, non-replicating M. tuberculosis (Rao et al. 2008). Hence, the essentiality of NDH-2s in pathogens and the absence of homologues in humans make it a potential drug target. The order and location of the substrate binding and the mechanism of NDH-2 catalysis are still not fully understood. In the recent years, a few structures from different organisms have been determined at atomic resolutions. Thus, scientists attempted to grasp a better understanding of NDH-2, based on the structures observed. Feng suggested two binding pocket separately for NADH and quinone (PDB code: 4G73 (Feng et al. 2012)). The placement of quinone-like inhibitor in C. thermarum also indicated two separated binding sites (Petri et al. 2018). while Momi Iwata presented two individual complexes with respective NADH and quinone, but superimposition showed overlapping of two substrates (Iwata et al. 2012). Two distinct substrate binding sites were confirmed with EcNDH-2 by surface-enhanced infrared absorption spectroscopy (SEIAS) (Salewski et al. 2016). Two hypotheses of NDH-2 catalytic mechanisms have been proposed accordingly. Approximately two decades ago, when no high-resolution structures were available, the mechanism was deducted by the study of enzymatic kinetics. A ping-pong mechanism was proposed to have the substrate binding in a timely order in the same site (Eschemann et al. 2005; Velázquez and Pardo 2001). A study of MtNDH-2 proposed two-site ping-pong mechanism, in which quinone binds to a distinguished site than NADH (Yano et al. 2014). Recently structure and inhibition studies guided scientists to propose a novel hypothesis of NDH-2 catalytic mechanism, that a ternary complex is established with NADH and quinone bound to the NDH-2 simultaneously during the catalysis (Sena et al. 2015; Yu Yang et al. 2011) In our study, we have investigated both NDH-2s from both M. tuberculosis and Mycobacterium smegmatis. The ndh genes coding NDH-2s were constructed into pEXP5-CT vector, and then subsequently transformed to E. coli BL21(DE3) C43 cell strains for expression. The membrane-associated NDH- 2s were purified with 8 mM CHAPS, after the solubilization of the membrane

53 fraction with 15 mM CHAPS. The enzymatic activity of NDH-2s was continuously monitored by spectrophotometrically measuring the consumption of NADH at 340 nm in a biochemical assay. With minor adjustments, the biochemical assay was feasible for evaluation of inhibitors targeting NDH-2s in large-scale. A series of quinolinyl pyrimidines (QP) analogues were designed, synthesized, and subsequently analyzed in the biochemical assay, in whole- cell assay against a panel of pathogenic cell strains, and in the cytotoxicity test. A clear correlation between the IC50 values and the calculated MICs was observed in M. tuberculosis, while no correlation between MICs and cytotoxicity was not evident, strongly suggesting that this series of compounds can be considered as antibacterial drug lead for further optimization.

Sequences and structures

Figure 16. The pairwise sequence alignment of MtNDH-2 and MsNDH-2.

54 MtNDH-2 (UniProtKB - P95160 (P95160_MYCTU) from Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) is composed of 463 amino acid residues, with a molecular weight of 49,619 Da, while MsNDH-2 (UniProtKB - A0QYD6 (A0QYD6_MYCS2) from Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (Mycobacterium smegmatis) has 457 amino acid residues, and the molecular weight is 48,980 Da. The pairwise sequence alignment is shown in figure 15. The sequence alignment showed that MtNDH-2 and MsNDH-2 are highly conserved throughout the whole sequence, with the identity of 81.9 % and the similarity of 90.3 %. Due to the instability of MtNDH-2 occasionally observed in purification and the freeze-and-thaw cycle during storage, the experimental analysis with MsNDH-2 is more informative, and of more accurate reflection of its true characteristics. Therefore, in my study, most of the experiments were conducted with MsNDH-2. Up to date, structures of NDH-2 have been determined by x-ray crystallography, from the yeast Saccharomyces cerevisiae (PDB code: 4G6H, 4G9K (Feng et al. 2012; Iwata et al. 2012)), from Caldalkalibacillus thermarum (PDB code:4NWZ, 5KMS, 5WED, 6BDO (Blaza et al. 2017; Heikal et al. 2014; Nakatani et al. 2017; Petri et al. 2018)), from Staphylococcus aureus (PDB code: 5NA1 (Sousa et al. 2017)), and from Plasmodium falciparum (PDB code: 5JWA (Yiqing Yang et al. 2017). The sequences of NDH-2s were extracted from the published structures, except for MtNDH-2. Subsequently, the sequence alignment was conducted via the MUSCLE algorithm (Edgar 2004). Five conserved boxes of amino acid residues were found, CB1-CB5. elucidation CB1 contains the residue Ser61 in the FAD binding site that is highly conserved among three species, and within the same CB1, the nitrogen in the backbone of Trp63 and Gly64, are somewhat conserved among five species. Although they are not identical, but they are in the same category of amino acids, for example, Ser61 in ScNDH- 2 was substituted with two aromatic residues Tyr21 in SaNDH-2, Phe24 in MtNDH-2. Arg85 was proposed to involve in FAD binding, but it is not conserved in these species. CB2 has been nominated with any functions. CB3 showed significantly high similarity among five species, with 237Gly-242Glu almost identical to each other. This CB3 was found to interact with the flavin moiety in the FAD in CtNDH-2, and possibly get involved in the quinone protonation in both SaNDH-2 and PfNDH-2 (Iwata et al. 2012; Nakatani et al. 2017; Sousa et al. 2017). CB4 includes two identical residues Trp326 and Gly329 in a connecting loop between the NBD and the CTD, which have not been found any functional information to date. CB5 comprises seven amino acid residues, three of which are identical among five species. No functions have been reported for this highly conserved loop in any species, but by ana- lyzing the structures, this loop is perpendicularly pointing to pyrophosphate moiety of the FAD, possibly involved in the FAD placement. The Gln391-

Figure 17. The sequence alignment by MUSCLE, of ScNDH-2, PfNDH-2, SaNDH- 2, CtNDH-2, and MtNDH-2.

Gln394 in CB6, which are highly conserved among five species, have been identified to be involved in quinone binding in SaNDH-2, CtNDH-2.Struc- ture-wise, all four structures are highly similar in conformation. The Chain As of CtNDH-2, SaNDH-2, and PfNDH-2 were superimposed to ScNDH-2 with the RMSDs of C at 1.26 Å, 0.76 Å, and 0.77 Å, respectively.

Figure 18. The overall structure of ScNDH-2. The FBD, green; the NBD, tint; the CTD, yellow, Subunit b, grey. The FAD is coloured by atoms.

In general, NDH-2 is a monotopic membrane homodimer with a FAD molecule and a metal ion bound in each subunit. As no MtNDH-2 structures have been solved, the numbering of residues in the following are according to ScNDH-2 (PDB code: 4G9K, (Iwata et al. 2012). In each subunit, NDH-2 consists of three domains: 1) the FAD binding domain (FBD, residues 43-178 and 342-442), 2) the NADH-binding domain (NBD, residues 179-341) and C- terminal membrane anchor domain (CTD, residues 443-513). The former two domains are adapted to typical Rossmann folds. Besides the interaction to the membrane, the CTD is also involved in dimerization with intensive interactions between two helices (residues 488-506). Other interactions involving both FBD and NBD contribute largely for dimerization. The spatial arrangement of substrates was elucidated in ScNDH-2 and CtNDH-2. Two groups proposed contradicting results on the overlapping of NADH and quinone in ScNDH-2. In UQ5-NADH-NDH-2 complex, the alkenyl group of UQ5 is located in the cleft, pointing outwards to the solvent, while the six-carboned ring is half exposed, physically near isoalloxazine ring of FAD. UQ2 adapted a different conformation, with the six-carboned ring pointing to NADH, overlapped with nicotinamide group. The placement of HQNO is similar to UQ5, with a tail extended to the solvent, and the double ring close to FAD. Two types of arrangement represent two hypotheses of NDH-2 catalytic mechanisms.

Figure 19. The spatial arrangement of NADH, quinone/quinone-like inhibitor and FAD in ScNDH-2 (upper left, 4G73), ScNDH-2 (upper right, 4GAV and 4GAP), CtNDH-2 (below, 5KMS and 6BDO), NDH-2 three domains presented as mesh in green, tint and yellow.

In vitro biology We mainly established a workflow, including the expression, and the purification of NDH-2s from M. smegmatis and M. tuberculosis, an in vitro assay to quantitively characterize the enzymatic activity, and evaluate the potent inhibition against both NDH-2s. Inhibitors were designed, and synthesized by chemists with whom we collaborate. Full lengths of ndh genes from M. smegmatis (MsNDH-2, MSMEG_3621, Gene ID: 4531957 from str. MC2 155) and M. tuberculosis (MtNDH-2, M. tuberculosis H37Rv|Rv1854c|Ndh: 469 aa - probable NADH dehydrogenase, NDH-2) with his-tags were amplified using the genomic DNA as the template, and subsequently inserted into the pEXP5-NT vector with TOPO ligation. The correctly constructed vectors with insertions of NDH-2s were transformed to E. coli BL21(DE3) C43 for expression (Wagner et al. 2008). The transformed cells were cultured in Luria-Bertani medium, followed by the induction of expression with IPTG when the cultures reached an OD600 of 0.6-1. The cells were incubated for 21 h at 22 C with gentle shaking. Expression with other E.coli strains did not produce stable, active NDH-2s. The pelleted cells were resuspended, and disrupted in lysis buffer (0.1 M potassium phosphate pH 7.0, 1 mM PMSF, plus a complete protease inhibitor cocktail pill (Roche)). The lysed cells were centrifuged to remove the cell debris. Then the ultracentrifu- gation was applied to the supernatant from the previous step, so that the membrane fractions were obtained. The pellets of membrane fractions were resuspended in the solubilizing buffer (0.1 M phosphate buffer, pH 7.0, containing

59 15 mM CHAPS), and the protein concentration was adjusted to 4 mg/mL. Dif- ferent detergents were tested, but CHAPS gave the most satisfactory results. The mixture was incubated at 4 C with gentle stirring, followed by ultracen- trifugation to remove the insolubilized membranes. The supernatants were mixed with TALON Cobalt resin, preequilibrated with IMAC buffer 0.1 M potassium phosphate buffer, pH 7.0, containing 8 mM CHAPS. The resin was washed with IMAC buffer plus 20 mM imidazole to get rid of unspecific binding, then subsequently eluted with IMAC buffer with 250 mM imidazole. The eluted protein was analyzed in concentration and purity by SDS-PAGE. The final yields for MsNDH-2 and MtNDH-2 were 3.8 mg/ L and 0.48 mg/L cell culture, respectively. The MsNDH-2 and MtNDH-2 protein sequences are shown below with different amino acids from the wild-type indicated in lower case.

>MSMEG_3621 MSHPGATASDRHKVVIIGSGFGGLTAAKTLKRADVDVKLIARTHHLF QPLLYQVATGIISEGEIAPATRVILRKQKNAQVLLGDVTHIDLENKT VDSVLLGHTYSTPYDSLIIAAGAGQSYFGNDHFAEFAPGMKSIDDAL ELRGRILGAFEQAERSSDPVRRAKLLTFTVVGAGPTGVEMAGQIAEL ADQTLRGSFRHIDPTEARVILLDAAPAVLPPMGEKLGKKARARLEKM GVEVQLGAMVTDVDRNGITVKDSDGTIRRIESACKVWSAGVSASPLG KDLAEQSGVELDRAGRVKVQPDLTLPGHPNVFVVGDMAAVEGVPGVA QGAIQGRYAAKIIKREVSGTSPKIRTPFEYFDKGSMATVSRFSAVAK VGPVEFAGFFAWLCWLVLHLVYLVGFKTKIVTLLSWGVTFLSTKRGQ LTITEQQAYARTRIEELEEIAAAVQDTEKAAShhhhhh >M. tuberculosis H37Rv|Rv1854c| - PROBABLE NADH DEHYDROGENASE NDH MgSPQQEPTAQPPRRHRVVIIGSGFGGLNAAKKLKRADVDIKLIART HHLFQPLLYQVATGIISEGEIAPPTRVVLRKQRNVQVLLGNVTHIDL AGQCVVSELLGHTYQTPYDSLIVAAGAGQSYFGNDHFAEFAPGMKSI DDALELRGRILSAFEQAERSSDPERRAKLLTFTVVGAGPTGVEMAGQ IAELAEHTLKGAFRHIDSTKARVILLDAAPAVLPPMGAKLGQRAAAR LQKLGVEIQLGAMVTDVDRNGITVKDSDGTVRRIESACKVWSAGVSA SRLGRDLAEQSRVELDRAGRVQVLPDLSIPGYPNVFVVGDMAAVEGV PGVAQGAIQGAKYVASTIKAELAGANPAEREPFQYFDKGSMATVSRF SAVAKIGPVEFSGFIAWLIWLVLHLAYLIGFKTKITTLLSWTVTFLS TRRGQLTITDQQAFARTRLEQLAELAAEAQGSAASAKVAShhhhhh

the in vitro biochemical assay have been developed in a plate format. The de- crease of NADH concentration was spectrophotometrically monitored at 340 nm continuously for 2 h, using an NADH extinction coefficient of 6220 cm- 1M-1. The enzyme and inhibitors were pre-mixed and incubated with the assay buffer 0.1 M HEPES, pH 7.0, 5%(v/v) DMSO, 3 mM CHAPS ,5 M FAD and 100 M NADH for 10 min. the substrate mix contains 0.1 M HEPES, pH 7.0, 5 % (v/v)DMSO, 3 mM CHAPS, 5 M FAD, and 50 M menadione.

60 Different electron acceptors, such as UQ1, UQ5, UQ10 were tested, while menadione provided a stable assay, so was selected for further research. The de- crease of absorbance at 340 nm was measured for 2 h, and the rates were determined from the linear portions of each reaction curve. NDH-2s were incubated with 0.2 mM NADH, and then the reaction was started by adding the electron acceptor at serial concentrations. Kinetic parameters of each individual electron acceptor were calculated by non-linear curve fitting of the rates at different concentrations to Michaelis-Menten equation.

Table 1. the kinetic parameters of MsNDH-2 and MtNDH-2

MsNDH-2 MtNDH-2

Electron ac- -1 kcat/Km -1 kcat/Km Km (M) kcat ( s ) Km (M) kcat ( s ) ceptor (s-1*M-1) (s-1*M-1)

Menadione 34 4.0 1.1*105 37.5 2.0 9.5*104 6 UQ2 0.78 1.4 1.7*10 menadione 1001 4231 4.2*106 1. reference value (Yano et al. 2014)

Km conventionally indicates the affinity of the enzyme to the substrate. MsNDH-2 showed higher affinity to UQ2 than menadione, and slightly lower turnover rates as well. Compared to MsNDH-2, MtNDH-2 showed similar affinity to menadione, but the catalytic constant is 270 folds lower. Kcat/Km is the catalytic efficiency, reflecting both binding affinity and catalytic rate, thus representing the overall ability of the enzymatic catalysis. MsNDH-2 exhibited roughly 10 folds higher efficiency to UQ2 than to menadione, but to keep track of the behaviour of the NDH-2 from different batches, and to process the data comparably to previous ones, menadione was still used in the assay. Com- parison with the kinetics of the MtNDH-2 expressed in M. smegmatis (Yano et al. 2014) and MsNDH-2 suggested further that MtNDH-2 expressed in E. coli needs optimization in expression and purification to be more representative and accurate in the biochemical assay. MsNDH-2 were proceeded in the inhibitor evaluation. evaluation of quinolinyl pyrimidines The isoniazid (INH) had been discovered to be potent against M. tuberculosis, and used as a member in a triple-drug cocktail treatment (Rieder 2009). The resistance to INH was possibly linked to the decreased NDH-2 activity in the mutant. The reduction in the NDH-2 activity resulted in an intracellular im- balance of NADH/NAD+, thus the compromised antitubercular potency of INH in cells (Lee, Teo, and Wang 2001; Miesel et al. 1998; Vilchèze et al.

61 2005). Phenothiazines are a group of compounds sharing the same heterocyclic structure, and have anti-mycobacterial activity. Early in 1970s, four phenothiazine derivatives, chlorpromazine, levomepromazine and diethazine and promethazine effectively and synergistically inhibited the growth of M. tuberculosis, when two compounds were combined (Molnár, Béládi, and Földes 1977). Trifluoperazine (TPZ), belonging to phenothiazines, has been found to completely inhibit the growth of M. tuberculosis H37Rv and INH-resistant M. tuberculosis strains (Ratnakar and Murthy 1992). 50 g/mL TPZ can arrest th the growth of M. tuberculosis H37Rv on the 10 day. Chlorpromazine and thioridazine inhibit the respiration of the multi-drug-resistant M. tuberculosis strains (L. Amaral et al. 1996). CPZ was also found to specifically inhibit MtDNH-2 in vitro, with an IC50 of 10 M (Weinstein et al. 2005). Till 2006, phenothiazines were confirmed to target NDH-2 in the respiration chain, and inhibit the activity of MtNDH-2 (Yano et al. 2006). Phhenothiazines were not considered as potential drug leads because of effectively required doses higher than potent CNS activities (Leonard Amaral and Kristiansen 2000). A large group of quinolones have been characterized intensively on NDH-2s from S. aureus, S. cerevisiae, Yarrowia lipolytica, P. falciparum, M. tuberculosis (Eschemann et al. 2005; Griffin et al. 2011; Hong et al. 2017; Lin, Groß, and Bohne 2009; Pidathala et al. 2012; Sena et al. 2015; Yamashita et al. 2007) Yamashita and his group has reported a quinolone derivative displayed the lowest IC50 of 0.2 M on the ScNDH-2(Ndi1) (Mogi and Kita 2009). Recently a ScNDH-2 complex structure has been solved with 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). It showed an IC50 of 10.5 M on the wild-type NDH- 2. However, because of the nature of competitive binding, and excess of menadione in the assay, the residual activity of NDH-2 remained 40% (Petri et al. 2018). A quinolone analogues from a quinolone screen have displayed ex- tremely high potency against MDR M. tuberculosis in whole-cell assay, with an IC50 of 140 nM (Hong et al. 2017). Two compounds in the thioquinazoline scaffold and tetrahydroindazole scaffold inhibit the growth of M. tuberculosis H37Ra with IC50 values of 0.43 M and 6.6 M respectively (Harbut et al. 2018). All quinolone analogues do not display high cytotoxicity against mammalian cells. Polymyxin B has been identified as NDH-2 inhibitors, with IC50 of 1.6 g/mL to MsNDH-2, and in whole-cell assay against E. coli, K. pneumoniae, A. baumannii with IC50 in the 100 M scale (Deris et al. 2014; Mogi and Kita 2009). A compound in the quinone-pyrimidine scaffold was chemically modified at 2-position of the quinone ring, and the pyrimidine ring with a phenyl ring, and has been proven to show largely elevated potency against MtNDH-2 and M. tuberculosis (Shirude et al. 2012). The best compound 4-F- phenyl-quinolynnyl pyrimidine- 4-Cl-phenyl exhibited an IC50 of 43 nM to MtNDH-2, and MIC of 1.91 M against M. tuberculosis. The structure-activity relationship analysis suggested that two hydrophobic groups at both ends were required for strong potency of the compounds in this particular scaffold.

Figure 20. Known inhibitors. Chlorpromazine, CPZ; Trifluoperazine, TPZ; 2-heptyl-4-hydroxyquinoline-N-oxide, HQNO.

63 All inhibitors were designed, synthesized, based on the QP as the starting lead, in collaboration with chemists from Department of medicinal chemistry and organic pharmaceutical chemistry, Uppsala university. They are pre-incubated with MsDNH-2, as the enzyme mix, at 22 C for 10 min, and then the reaction was started by adding the substrate mix to the enzyme mix. When calculating the IC50s for inhibitors, the compounds were added to the assay in a two-fold sequential dilution in the range 10 to 0.002 M. the IC50 values were calculated by applying non-linear curve fitting to sigmoidal four-parameter dose- response regression curve in GraphPad Prism®. Inhibitors have been applied to other assays, such as a whole-cell assay against a panel of stains, MIC assays on parasites, to explore the potent inhibition more thoroughly. These experiments were conducted in collaborations with other parties. We have investigated 25 QP analogues with the major focus on the substitution at 4-position in pyrimidine. 25 compounds were evaluated in the biochemical assay and in the MIC assay.

Figure 21. the chemical modification on quinolinyl pyrimidine. 1) Reference com- Mt pound 13a, IC50 = 1.6 M, MIC = 2.6 M; 2) 4-((tert-butyl carbamate)aminome- Mt thyl)piperidin-1-yl, IC50 = 0.35 M, MIC = 6.8 M; 3) 4-((tert-butyl carba- Mt mate)amino)piperidin-1-yl, IC50 = 0.95 M, MIC = 6.1 M.

Compared with the IC50 reported previously (Shirude et al. 2012), the same reference compound (13b) showed highly elevated IC50, possibly due to the natural characteristics of the purified NDH-2. Shirude et al. tested the potency on MtDNH-2, and all inhibition studies in our lab have been carried out with MsNDH-2. The MICMt from two separate experiments are relatively diverse as well, from <0.81 M to 2.6 M. Data analysis deducted that the IC50 values calculated for MsNDH-2 and MtNDH-2 exhibit a clear correlation, suggesting that the IC50 values are clearly representative of the potency against MsNDH- 2. Two QP analogues with the similar bulky head showed comparable inhibitory potency on MsNDH2 and M. tuberculosis, indicating that a large chemical group may be needed to fit into the binding site to gain higher affinity. Besides, correlations between the IC50 and potency against efflux defective

64 mutant E. coli, but not wild-type, indicate that lack of the inhibition on wild- type bacteria might be due to the membrane permeation of the compounds. No correlation of IC50 and cytotoxicity for QP analogues was observed, suggesting these two properties can be optimized separately in this series. Recently several NDH-2 structures have been solved at high resolutions, and docking could help rationalize our SAR, and guide for future optimization. As in CtNDH-2 complex structure with HQNO, the quinolone moiety is bur- ied in the hydrophobic substrate-binding groove, while the tail is extended, and exposed in solvent. If QP analogues would bind to the groove, probably the bulky head would be accommodated in the hydrophobic groove. conclusions and future perspectives we have been dedicated to constructing a system to express NDH-2s from M. tuberculosis and M. smegmatis in E. coli. Perhaps, due to the lack of post- translational modifications, MtNDH-2 does not present its best quality in stability and activity, compared to MtNDH-2 expressed in M. smegmatis. Both NDH-2s were purified with IMAC, and kinetically characterized in an in vitro biochemical assay. The assay was designed to continuously monitor the de- crease of NADH concentration at 340 nm in a spectrophotometer when catalysis occurs. The assay is also feasible to evaluate the potency of inhibitors targeting NDH-2s. A series of quinolinyl pyrimidine analogues were synthesized, and two of them showed comparable inhibition against both MsNDH-2 and M. tuberculosis. More work needs to be done to improve the stability and enzymatic activity of MtNDH-2 and MsNDH-2, such as change of expression hosts, optimization of the purification procedures. With more stable mycobacterial NDH-2s, a crystal structure complexed with substrates or ligands would dramatically broaden our understanding of the catalytic mechanism, and the mode of action. So would mutagenesis based on the sequence alignment.

65 Summary of papers Paper I: Design, Synthesis and In Vitro Biological Evaluation of Oligopeptides Targeting E. coli Type I Signal Peptidase (LepB) In this work, I have successfully established a platform, including LepB construct design, protein purification, a biochemical assay, enzymatic kinetics, and inhibitor hit identification. 1) A new construct of full-length EcLepB was expressed in E.coli BL21(DE3) C43 cell strains with a yield of roughly 4.5-5 mg/L cell culture. 2) EcLepB was purified with IMAC to 95% purity, and a round of buffer exchange to remove high concentration of imidazole, and to a buffer that is suitable for the future purposes. 3) A biochemical assay was developed to characterize kinetical parameters with a novel substrate derived from Mycobacteria tuberculosis signal peptide of antigen 85 A with a FRET pair attached. The Km and kcat of EcLepB on this substrate are 20 M and 139s-1. 4) The assay was further optimized to confer the feasibility to quantitively identify the inhibitory activity of several lipopeptide-based inhibitors derived from a known inhibitor. Data confirmed that the inhibitor with the sequence of PTANA with 4-(4-hexylphenyl)benzoyl group at N-terminus and a boronic acid at C-terminus exhibit the highest inhibition with an IC50 of 6 nM. Inhib- itors with a similar fatty acid tail and similar warheads of boronic bases showed similar inhibitions, however, unfortunately, also high cytotoxicity and high activity in hemolysis test.

Paper II: Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase. This paper was a continued investigation of Paper I, expecting to explore more scaffolds in LepB inhibitors. The PTANA-based inhibitor was cyclized, hinted by cyclized arylomycins and 5S-penem that exhibit strong potency against bacteria. The macrocyclic lipopeptides with the link between P2 and boronic bases have been confirmed to inhibit the LepB activity with IC50s ranging from 40-100 nM. Simultaneously, cytotoxicity and hemolysis tests suggest they are not satisfactory candidates as antibacterial drugs, but a promising lead. Further modifications need to be done to disturb the correlation of inhibitory activity and cytotoxicity.

66 Paper III: C-delta analogues of unsaturated Fosmidomycin targeting Plasmodium falciparum 1-Deoxy-D-xylulose-5- phosphate Reductoisomerase (manuscript) Fosmidomycin and FR-9000098 have been confirmed to show parasiticidal activity against Plasmodium falciparum, targeting DXR involved in the MEP pathway. We designed a construct of PfDXR that has successfully been overexpressed in E. coli BL21(DE3) C43, and purified by IMAC and SEC, with the final yield of 1.2 mg/ 8 L culture. PfDXR was concentrated to 20 mg/ml, and co-crystallized with previously tested inhibitors in the FR-9000098 scaffold in the presence of Mn2+. Three FR-9000098 analogues with double- bonded C-C and/or a phenyl ring with various lengths to N1, showed inhibitory activities with IC50s roughly 50 nM. Three crystals were in triclinic P1 space group, with similar dimensions in the unit cell (51Å, 56Å, 86Å, 103°, 103°, 101°). All four complex structures have been crystallographically determined at resolutions in the range 1.86 Å, 2.45 Å, 2.13 Å, 2.05 Å. Given the high similarity in structures, the initial phases were determined by rigid body refinement with search model PfDXR-FN3 complex, followed by restrained refinement in refmac5. Subsequently, the ligands and surrounding amino acid residues were manually rebuilt with the qds tools in O. the C-C bonds of the three ligands were altered from a single to double bond based on the structure of FR9000098. In addition, two ligands were extended at the C with a phenyl group, and with the benzyl group connected by two carbons. N-terminal NADPH binding domains from four complexes undergo minor rigid body movement, and more details of conformational changes in the flap region are discussed.

67 Paper IV: Synthesis and in vitro biological evaluation of quinolinyl pyrimidines targeting type II NADH-dehydrogenase (NDH-2) (manuscript) Type II NADH dehydrogenase (NDH-2) is identified as a crucial opponent of electron transfer in different microbial pathogens. We have conducted investigations, in order to biochemically characterize a series of quinolinyl pyrimidine (QP) analogues targeting NDH-2 from Mycobacteria tuberculosis, and Mycobacteria smegmatis. MtNDH-2 and MsNDH-2 were constructed, overexpressed in E. coli BL21(DE3) C43, and successfully purified in the presence of 8 mM CHAPS with the yield of approximately 1.5 mg/L cell culture. The enzymatic kinetics of both NDH-2s were analyzed in a biochemical assay by monitoring spectrophotometrically the consumption of NADH at 340 nm. 5 M cofactor FAD was proven to be required in the assay for the catalytic activity of NDH-2. The Km and kcat of MsNDH-2 for the electron receptor, men- -1 adione, were 34 M and 4.0 s , and the Km and kcat of MtNDH-2 for menadione were 37.5 M and 2.0 s-1. Due to the instability of MtNDH-2 and similar catalytic activity, more analyses were performed with MsNDH-2. A collection of QP derivatives were synthesized with the chemical modifications at the 4- position on the pyrimidine ring, aiming to increase the solubility of the known inhibitor. 25 compounds showed inhibition to MsNDH-2, with IC50 values ranging from 0.35 M to >100 uM. Simultaneously, the antibacterial activity of these compounds were analyzed against a spectrum of Gram-negative bacteria, the ESKAPE pathogens, and M. tuberculosis, as well as cytotoxicity and hemolytic activity. The best two compounds with the IC50s of 0.35 M and 0.95 M exhibited correlations between potencies against NDH-2 enzymes and against efflux defective mutant E. coli but not wild-type E. coli, suggesting optimization should be focused on membrane permeability. Cytotoxicity analysis showed that all compounds were cytotoxic, yet that no correlations to enzymatic inhibition were observed, thus, two properties can be optimized separately. In conclusion, QP analogues tested in a functioning biochemical assay targeting MsNDH-2 did not prove a solid extensive capability of antibacterial activity, however, can be considered as a drug lead requiring optimi- zations.

68 Populärvetenskaplig sammanfattning

Smittsamma sjukdomar har blivit en stor fråga inom folkhälsan, t.ex. den ökända COVID-19, tuberkulos, malaria, för att inte tala om att patogenerna, som orsakar dessa sjukdomar har utvecklat multipel eller omfattande läkeme- delsresistens mot de läkemedel som finns på marknaden. Detta gör att behovet av nya, effektiva läkemedel är angeläget globalt.

Ungefär en fjärdedel av världens befolkning är smittad med tuberkulos och sjukdomen står för 1 miljon dödsfall per år. Patienter som är infekterade med tuberkulos har vanligtvis milda symtom, men när immunsystemet inte funge- rar, t.ex. som vid infektion av andra sjukdomar, kan tuberkulos vara en mycket aktiv sjukdom och ha stor påverkan på patientens lungor eller någon annan vävnad. Tuberkulos orsakas av en patogen som heter M. tuberculosis, som är en stavformad bakterie med ett mycket tjockt hölje. Det tjocka höljet är en av dess försvarsmekanismer, så att vissa antibiotika, som kan döda andra bakterier, inte är effektiva mot M. tuberculosis.

Malaria är en annan smittsam sjukdom och överförs genom myggor. Malaria- infektion gör så att röda blodkroppar sprängs, ger därmed anemi, och var an- svarig för ungefär 500 000 dödsfall under 2019. Parasiterna, som orsakar malaria finns i samma släkte, kallat Plasmodium, men majoriteten av inciden- terna med svåra symtom orsakas av Plasmodium falciparum. P. falciparum är en encellig eukaryot parasit, som har utvecklat läkemedelsresistens under evo- lutionen.

På vårt lab har vi ägnat oss åt forskning om proteiner som skulle kunna vara potentiella läkemedelsmål från olika patogener, såsom M. tuberculosis, P. falciparum och ESKAPE patogenerna, som orsakar vissa sjukhusförvärvade sjukdomar. Vi har följt en strategi som heter Same Target Other Pathogens (STOP) i vår forskning. STOP-strategin innebär att genom att studera samma enzym från olika arter har vi utvidgat vår kunskap om målets egenskaper så att bättre hämmare kan designas och syntetiseras. Vi har försökt hitta enzymerna som 1) endast finns i patogener, 2) är signifikant olika hos människor, 3) är väsentliga för patogenernas livskraft. En bonuspoäng skulle vara en lätt- tillgänglighet för hämmare, till exempel är cellväggen hos M. tuberculosis tjock, men kanske är enzymerna i cellväggen eller utanför membranet mer exponerade, så lättare att slå på. Vi har valt tre enzymer, Signalpeptidas I från

69 E. coli (EcLepB), 1-deoxy-D-xylulos 5-fosfat reduktoisomeras från P. falciparum (PfDXR) och NADH: kinon-oxidoreduktas från M. smegmatis (MsNDH-2).

Signalpeptidas I är ett membranprotein som ligger i den yttre delen av det inre membranet, med en eller två -helixar som dockar in i membranet. Det katalyserar avlägsnandet av en viss peptidsekvens, nämligen signalpeptiden från utsöndrade proteiner, och blir därmed involverad i proteinmognaden och den extracellulära transporten. Proteinet är därför avgörande för bakterietillväxt. Då det inte finns någon homolog hos människor, har det ansetts vara ett bra läkemedelsmål. Ett fullängds EcLepB konstruerades, uttrycktes i E. coli och renades med hjälp av detergenten Triton X-100. En serie oligopeptidliknande hämmare och makrocykliska hämmare designades och syntetiserades av våra samarbetspartners i kemi. Dessa hämmare har visat sig binda konkurrenskraf- tigt till EcLepB och hämmar enzymets aktivitet. Tyvärr har det alltid funnits ett samband mellan hämning och cytotoxicitet för dessa hämmare, så fler op- timeringar måste göras.

1-deoxy-D-xylulose 5-fosfat reduktoisomeras (DXR) är det andra enzymet i MEP-vägen, en metabol process som producerar föregångarna till isoprenoider. Isoprenoider är en stor samling av mer än 65 000 organiska föreningar som är primära eller sekundära metaboliska mellanprodukter, involverade i många biologiska processer, såsom uppbyggnad av cellmembranet, signal- transduktion och reglering av genuttryck. MEP-vägen upptäcktes för ungefär trettio år sedan och finns endast i bakterier, alger, plastider från högre växter och i plastiden hos Plasmodium. För ett läkemedel som kallas fosmidomycin (FOM) har det upptäckts att det inte bara riktar sig mot DXR-enzym in vitro utan också visar hämmande effekt mot P. falciparum. Men på grund av de biverkningar patienter får om FOM tas oralt och dess dålig stabilitet i plasma måste föreningen optimeras kemiskt. Ett funktionellt system för uttryck av PfDXR i E. coli har utvecklats, och kristallstrukturer har lösts med fyra olika FOM-analoger som har en dubbelbindning mellan C och C, och en fenyl- ring med länkar av olika kolatomer. Elektrontätheten hos hämmarna var väl- definierad efter flera förfiningsrundor, vilket tyder på att hämmarnas placering var tillförlitlig. De strukturella karakteriseringarna av målenzymet DXR med hämmare hjälper oss att förstå mekanismen och bindningen bättre och leder till optimering av hämmare för framtida läkemedel.

Typ II NADH: kinon-oxidoreduktas (NADH-dehydrogenas, NDH-2) spelar en viktig roll i andningskedjorna som upprätthåller balansen mellan elektro- kemisk potential mellan inre och yttre cellulära utrymmen. Enzymet är avgö- rande för ATP-syntes, näringstransport med mera. NDH-2 är ett monotopiskt membranprotein (binder till en sida av membranet) som katalyserar redukt- ionen av kinonderivat. Det har tre fördelar som gör att det kan vara ett bra

70 antimycobakteriellt läkemedelsmål, 1) det är viktigt för tillväxt hos M. tuberculosis; 2) det är lättillgängligt; 3) det finns inga homologer hos människor. Men på grund av instabiliteten hos MtNDH-2 har det mesta arbetet utförts på NDH-2 från M. smegmatis, som har 81,9% sekvensidentitet till MtNDH-2. En hämmare, nämligen kinolinylpyrimidin (QP), uppvisade mycket god hämning av både MtNDH-2 och M. tuberculosis, men tyvärr var det cytotoxiskt för humana celler. Vårt mål var att behålla QPs förmåga att hämma enzymaktivi- teten samtidigt som vi ville reducera dess cytotoxiska egenskaper. För detta skapades ett arbetsflöde som inkluderar uttryck av MsNDH-2 och MtNDH-2, bestämning av enzymatisk aktivitet i en in vitro biokemisk analys, och karak- terisering av hämmare. 25 analoger har syntetiserats och testats i vår analys med QP som referensförening för metoden. QP visade en sämre hämmande aktivitet mot MsNDH-2 i våra händer, men två liknande QP-analoger visade jämförbar hämmande styrka mot MsNDH-2.

Sammanfattningsvis har forskning om tre potentiella läkemedelsmål genom- förts. Våra resultat har gett oss en bättre förståelse för enzymmekanismerna och har väglett oss i hur de hämmare som riktar sig mot dessa tre enzymer ska förbättras.

71 Acknowledgement

It has been quite a journey, a long one. I would like to take the chance to thank the people who have been with me in this fantastic journey. I just want to show my deepest gratitude to my supervisors, Sherry L. Mowbray, and Alwyn T. Jones. Thank you, Sherry, for believing in me, for being always positive on what I have done, for being so kind and considerate when it came to family issues. Thank you, Mr., Sir, for being there for me, for all the crystallographic workshops, for the humors of all these years, and for the clicking sounds from your wooden slippers in the corridor. It is always good to have you around. Thank you, Torsten Unge, for bringing me into this lab, and for all the nights together at the beams. Thank you, Terese Bergfors, for all the crystals you have made in our projects, for the discussions about the languages, and the most importantly, for showing me how organized a person can be. I also want to thank all the people who contributed to this thesis. A special thanks to Sanjeewani Sooriyaarachchi, for the wonderful work on DXR that made mine go smoothly, for tolerating me smoking, for being a big sister in our shared office. To Annette Roos, for all the helps in the LepB project, for the popular science, for the clothes you brought to my kids. To Shabbir Ah- med and Adrian Suarez Covarrubias, for the weekly meetings on NDH-2, for the tips in protein purification and assays, for all the discussions about politics. In the group of Anders Karlén: Peter brandt, Linda Åkerbladh, Maria De Rosa, Natalia Zalaj, Andrea Benediktsdottir, Edouard Za- maratski, and Vivek Konda, for the excellent collaborations, and the compounds you provided us. Also thanks to Diarmaid Hughes, for the beautiful science in whole-cell assays in parallel, to Sha Cao, for MIC evaluation of compounds, and for the phone calls made for our research. To Henrik Wadensten and Per Andrén, for the assistance in operating the MS instru- ments. To Xueliang Ge, for the materials you offered in the NDH-2 project, and tips on the growing and lysis of M. smegmatis. I still remember when I first came to Sweden as a master student, I barely could speak a full sentence in English. Lars Lijas and Maria Selmer were the teachers of my first course – structures and functions of macromolecules. Thank you for opening a door to a new world for me, and for helping me blend into this new society. The experience would not have been the same if there weren’t good colleagues and friends. Thank you, Stefan Knight, for providing ideas in science,

72 and for discussions of everything in the lunch room. Thank you, Sandesh Kanchugal for being a boss, for caring everyone and everything in the lab, and for the Pokemon you traded with me. Thanks to Wangshu Jiang for going on the specific trip together with me where I collected the datasets used in my thesis. Thank you Soneya Majumdar for insightful ideas in science and in life. It was always inspiring to talk to you. To Li Zhang, for sharing a lot experiences about experiments and taking care of the kids, and for the useful medical advices. You are more than a true doctor. Thank you, Annika Söder- holm, Julia Griese, Daniel Larsson, and Silvia Trigüis, for creating a warm and caring environment in the lab. Thank you all who have been on the data collection trips with me all these years, it has always been pleasant, and full of surprises. Thanks to everyone in the administration: Åsa Hammarborg, Mathilda Olsén, Akiko Cerenius, Frida Österdahl, Lena Jansson, Anders Eriksson, Erling Wikman, Ali elfadil, and Jessica Lindberg, for your pa- tience and help all these years. I also want to take the opportunity to express my thanks again to Terese Bergfors, Sanjeewani Sooriyaarachchi, and Sandesh Kanchugal for going to China, and experiencing one of the most memorable moments in my life with me. Thanks also goes out to colleagues and friends in the present and in the past: Lena Henriksson, Magnus Persson, Evalina Andersson, Christopher Bjökelid, Ana Jansson, Henrik Ingvarsson, Mark Harris, Nina Bäckbro, Ana Laura Stern, Avenash Punekar, Alena Stsiapanva, Ulrich Eckhard, Dirk Mauer, Cisco Marcos Torres, Wimal Ubhayasekera, Karin Valegård, Dirk Hasse, Ana Larsson. You have made the lab a great place to be. Friends from my master program, thank you, Mubin Mohammed Rah- man, for inviting me over for dinners now and then, and for a wonderful trip in Helsinki. To Hannes Beyer, Roy Prasad, for the fun we had together, and the cricket. Special thanks to Ziquan Yu, Jiazhong Guo, Cheng Xu, Song Sun, for the good old days, when we sweat on the basketball court, when we drove all the way to the countryside, when we lay down on the grass doing nothing, when we kept on talking about nothing with a bottle in hand. To Wei Zhang, for always understanding me, sometimes even without a word being said, for encouraging me when I was lost. Good to have you all by my side. Thanks to all my Chinese friends, you have made my life brighter, in the endless darkness. Lei Chen, Hua Huang, Xiaohu Guo, Zhoujie Ding, Hui Xu, Shiying Wu, Zhibin Zhang, Lu Zhang, Jin Zhao, Junhong Yan, Yue Cui, Jingji Zhang, Zhen Liao, Jingyi Liu, Yang Chen, Shun Yu, Miao Wu, Wei Xia, Zhirong Fu, Di Yu, Chuan Jin, Yanhong Pang, Gang Pan, Hao Li, Wei Sun, Xiaomei Chen. Last but not least, thank you, my beloved other half, Yani Zhao, for com- pleting me, for deciding to spend the rest of your life with me, for all the efforts

73 and sacrifices you have made for the family, for bringing two little angels to us, for everything. I love you. 我爱你！ To Pengyu Lu and Pengchen Lu, for being not sick during the last moment when dad was writing his thesis, for growing so fast, for occasionally wanting to hold dad while asleep, for everything. You three are the best things that ever happened to me. I love you! 感谢我的父母，你们永远是我最坚强的后盾，感谢你们信任我，放开手让我实现自己的梦想，感谢你们对我的体谅和宽容。感谢雅妮的父母，把你们心爱的女儿交给我，一起生活在遥远的北欧，感谢你们理解和包容。感谢你们四位父母轮班千里迢迢从中国来到瑞典，不辞辛苦帮我们照顾大圣和小仙儿，帮助我完成学业。

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91 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2020 Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)

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