Design and Evaluation of Hmp Kinase Analogs As Thiamine

Home , Arginine kinase, Ribokinase

DESIGN AND EVALUATION OF HMP KINASE ANALOGS

AS THIAMINE PATHWAY INHIBITORS

DIEGO A. LOPEZ

Presented to the Faculty of the Graduate School of

The University of Texas at Arlington in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

THE UNIVERSITY OF TEXAS AT ARLINGTON

Summer 2016

ii Acknowledgements

This work would not have been possible without the help, support, and motivation of many people. First and foremost, I would like to thank Dr. Frank W. Foss Jr. for his mentorship, guidance, and amazing contribution into my life as a young scientist. I will always look up to him with admiration and follow his academic lessons and life teachings wherever I go.

I would like to express my sincere gratitude to my committee members, Dr. Jeon and Dr. Mandal for their continuous support throughout the years and for their insightful suggestions towards the development of the project. In addition, I would like to thank the

Organic division professors, Dr. Lovely, Dr. Bugarin, and Dr. Pierce for always being cordial and supportive both inside and outside of the chemistry field.

I would also like to thank the numerous people who have contributed into the project, specially Dr. Sumit Bhawal who started the HMP project and developed most of the syntheses present in this study. His hard-work, passion for chemistry, and strive for success will always be remembered by this colleague and friend. I thank all my undergraduate students and members of the Foss’ lab for all the hard work, merit, and joy that have brought into my academic career: Aaron, Andra, Mohammad “Mu”, Pawan,

Shakar, Johny, Twi, Nicky, Kevin and Caitlynn.

Finally, I would like to thank my family and friends who throughout these years, they have not ceased to push me up to this very moment. My most sincere gratitude to my Calasanz community and high five to Andy, Francisco, Jessica, Cynthia, Hakop,

Adam, and Nelli. Lastly, I owe it all to God and my mom for her sacrifice and infinite dedication to me and Alvarito.

July 22nd, 2016

viii

Abstract

DESIGN AND EVALUATION OF HMP KINASE ANALOGS

AS THIAMINE PATHWAY INHIBITORS

Diego A. Lopez, Ph.D. The University of Texas at Arlington, 2016

Supervising Professor: Frank W. Foss Jr.

Current bacterial chemotherapy faces an overwhelming reduction in efficacy due to an increase in bacterial resistance; resulting in a higher clinical demand for novel therapeutics and the investigation for their respective targets. Vitamin pathways exogenous to the human body represent an attractive target for drug development, since the enzymatic machinery is solely and ubiquitously present in many infectious agents.

HMP kinase [E.C 2.7.1.49] is a key enzyme in thiamine metabolism. It catalyzes two subsequent phosphorylations of 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), making it indispensable for microbial survival. Our efforts aimed for a substrate-based approach, where over 50 HMP analogs were synthesized and assayed towards substrate scope and enzyme inhibition. Development of in vitro and in silico analyses were imperative in our search for the needle in a haystack; furthermore, they have aided in the formulation of a structure-activity relationship (SAR) and paved the way for future synthesis. The catalytic activity of HMP was confirmed via a multitude of analytical tools such as UV-Vis, chemiluminescence, liquid chromatography, and mass spectrometry; resulting in the first generation of inhibitors and giving light to a whole new realm of compounds with pro-drug characteristics.

ix Table of Contents

Acknowledgments ...... vii

Abstract ...... ix

List of ilustrations ...... xiii

List of tables ...... xvi

Chapter 1 Introduction to antibiotics ...... 17

1.1 Golden years ...... 17

1.2 Mechanisms of antibiotic action ...... 19

1.3 Mechanisms of antibiotic resistance ...... 22

1.3.1 Prevention of access to target ...... 23

1.3.2 Target modification ...... 23

1.3.3 Molecular bypass ...... 24

1.3.4 Chemical modification ...... 25

Chapter 2 Introduction to thiamine biosynthetic pathway ...... 27

2.1 Vitamin B1: Biosynthesis and Role in Living Systems ...... 27

2.2 The appeal of vitamin pathways as source of novel antibacterials ...... 32

2.3 HMP kinase structure and function in thiamine biosynthesis ...... 34

2.4 Thiamine regulation across species ...... 39

2.5 HMP kinase binding site: substrate & inhibitors ...... 40

2.6 HMPK as drug target ...... 44

Chapter 3 HMP Kinase Overexpression and Purification ...... 47

3.1 Materials and methods ...... 49

3.2 Results ...... 51

3.3 Discussion ...... 53

x Chapter 4 Assay Development and Screening of HMP Analog ...... 56

4.1 Qualitative TLC assay ...... 58

4.1.1 Materials and methods ...... 58

4.1.2 Results ...... 59

4.1.3 Discussion ...... 60

4.2 ADP-Glo chemiluminescence end-point assay ...... 61

4.2.1 Materials and methods ...... 63

4.2.2 Results ...... 64

4.2.3 Discussion ...... 71

4.3 Enzyme coupled kinetic assay ...... 82

4.3.1 Materials and methods ...... 83

4.3.2 Results ...... 85

4.3.3 Discussion ...... 88

4.4 High Performance Liquid Chromatography assay ...... 89

4.4.1 Materials and methods ...... 89

4.4.2 Results ...... 90

4.4.3 Discussion ...... 94

4.5 In vitro whole cell assay and metabolite detection ...... 95

4.5.1 Materials and methods ...... 98

4.5.2 Results ...... 100

4.5.3 Discussion ...... 105

4.5.4 Conclusion and future approach ...... 106

Chapter 5 Experimental ...... 108

5.1 General procedures ...... 106

Appendix A: List of abbreviations ...... 121

xi Appendix B: NMR spectra ...... 124

References ...... 161

Biographical information ...... 168

xii

List of Illustrations

1-1. Top ten leading causes of death as a percentage of all deaths. United States, 1900

...... 17

1-2 Antibiotic deployment by year in the United States...... 18

1-3 Synthetic tailoring of known scaffolds towards successive generation of antibiotics

...... 28

1-4 Targets of the most common and successful antibiotics……………………………….21

1-5 Erm methylates the A2058 position of rRNA disrupting vital interactions with antibiotics; therefore, resulting in resistance. The methyl donor SAM is converted to SAH

(S-adenosylhomoserine)……………………………………………………………………….24

1-6 Hydrogen bond network between the peptidoglycan layer and vancomycin. Acyl-D-

Ala-D-Lac induces resistance to the antibiotic by lowering binding affinity a thousand fold ...... 25

1-7. Accepted mechanisms of serine and metallo-b-lactamases ...... 26

2-1 TPP dependent enzymes (purple) in a mammalian cell and subcellular localization.

TK: transketolase, PDHC: pyruvate dehydrogenase complex, OGDHC: oxoglutarate dehydrogenase complex, BCODC: branched-chain 2-oxo acid dehydrogenase complex, and HACL1: 2-hydroxyacyl-CoA lyase ...... 28

2-2 Decarboxylation mechanism of TPP in pyruvate dehydrogenase. The acetaldehyde formed will become acetyl-CoA before entering the TCA cycle ...... 29

2-3 Thiamine biosynthetic pathway diagram in Plasmodium falciparum. ThiD, ThiM, and

ThiE are the protein-coding genes. Both HMP and THZ have been found to be uptaken from the outside or synthesized de novo in most prokaryotes ...... 31

xiii 2-4 Complete de novo thiamine biosynthetic pathway in bacteria. Glycine and tyrosine are used as starting materials for B. subtilis and E. Coli respectively ...... 32

2-5 A: DHPS catalyzes the coupling between DHPP and pABA in the first steps of the folate biosynthesis. B: DHFR catalyzes the reduction to the piperazine ring by means of

NADPH consumption. C: Sulfamethoxazole and trimethoprim inhibitors for each enzyme respectively ...... 34

2-6 Ribbon diagram of HMP Kinase showing both monomers and the co-crystallized

HMP substrate ...... 35

2-7 A: Phosphorylation reactions catalyzed by HMP Kinase. B: Schematic of the dual catalytic activity of HMP kinase ...... 36

2-8 A: Superposition of ligands in the ATP or ADP binding sites of HMP kinase, T. gondii adenosine kinase, human adenosine kinase, thiazole kinase, and ribokinase ...... 38

2-9 HMP binding site for B. subtillis. Dotted lines reveal the plausible H-bonding with

HMP and the respective distances in Angstroms ...... 41

2-10 Substrates of HMP kinase from B. subtillis ...... 42

2-11 Rugulactone derivatives ...... 43

2-12 Marketed small-molecule drug target by biochemical class ...... 44

2-13 Current HMP analogs incur modifications around C2 (R2), C4 (R4), and C5 (R5) of the heterocycle ...... 46

3-1 Scheme of protein purification through His-tag and affinity chromatography ...... 48

3-2 Gel electrophoresis of recombinant colony PCR ...... 51

3-3 SDS-PAGE of HMP kinase purification ...... 52

3-4 HMP kinase stability upon varying concentrations of glycerol ...... 54

xiv 4-1 General reaction conditions for the in vitro phosphorylation of HMP analogs by HMP kinase ...... 59

4-2 TLC of HMP kinase reaction with natural substrate HMP ...... 60

4-3 Schematic of the ADP-GloTM chemiluminescent assay ...... 62

4-4 Percent phosphorylation of HMP natural substrate in presence of analog inhibitor ... 67

4-5 ATP inhibitors ...... 69

4-6 Activity of commercially available ATP inhibitors ...... 70

4-7 A: Optimization of HMP natural substrate. B: Optimization of HMP kinase enzyme. C:

Tolerance of DMSO solvent in HMPK reaction ...... 71

4-8 Current mechanistic proposal for thiamin phosphate synthase ...... 76

4-9 Molecular docking of substrates ...... 77

4-10 Docking of HMP and inhibitors ...... 78

4-11 Schematic of the pyruvate kinase/lactate dehydrogenase enzyme-coupled assay . 82

4-12 : Phosphorylation of HMP via enzyme coupled assay ...... 84

4-13 Reaction adducts from the HMP kinase ...... 87

4-14 HPLC assay method ...... 90

4-15 Configuration of HPLC-PIESI-MS for detection of negatively charged molecules in positive mode ...... 97

4-16 Charged species detected via PIESI-MS ...... 98

4-17 Minimum inhibitory concentration (MIC) for different bacterial strains ...... 100

4-18 HMP-PP detection from the in vitro enzymatic reaction ...... 101

4-19 Plausible HMP analogs metabolites from in vitro whole-cell assay that are detected via PIESI-MS ...... 102

List of Tables

2-1 TPP-dependent enzymes in Plasmodium falciparum ...... 30

4-1 Preparation of the 1mM series of ATP & ADP standards ...... 63

4-2 Percent phosphorylation of HMP analogs by HMP kinase via chemiluminescence assay ...... 65

4-3 IC50 determination for the most potent inhibitors to date ...... 69

4-4 HMPK inhibitors with milder inhibition potency ...... 81

4-5 Kinetic values obtained for the substrate analogs of HMP ...... 87

4-6 HPLC method for the separation and elution of HMP kinase reaction ...... 91

4-7 HMP analog phosphorylation comparison between ADP Glo and HPLC assay ...... 93

4-8 Percent identity of amino acid sequence for HMP kinase among different bacterial strains ...... 96

4-9 Metabolites detection via PIESI-MS from E. coli K12 strain ...... 103

xvi

Chapter 1

Introduction to Antibiotics: Discovery, Resistance, and Socio-economic Impact

1.1 Golden years

Discovered fortuitously in 1928 and implemented in 1940s, penicillin became the drug of choice to treat previous incurable bacterial illnesses (Figure 1-1); having a wider range of targets and lower side effects than the so-called “sulfa” drugs. This was the start of the golden age for antibiotics. For the next twenty years, an array of compounds with different mechanisms of action took over the chemotherapeutics sector and their success changed the course of mankind; going from a life threatening bacterial infection to a trivial doctor’s visit (Figure 1-2).

12 10 8 6 4

PERCENTAGE 2 0

Figure 1.1-1. Top ten leading causes of death as a percentage of all deaths. United

States, 1900.

Tetracycline Chloramphenicol Ampicillin Gap Streptomycin Vancomycin Cephalosporins Sulfonamides Erythromycin Daptomycin Penicillin Methicillin Linezolid

1930 1935 1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000

Figure 1-2. Antibiotic deployment by year in the United States.

The classes of antibiotics introduced during the period of 1940s through 1960s paved the way for future generations of antibacterials by modification of existing scaffolds

(Figure 1-3); resulting in a handful of well-studied active frameworks but also leading to a nearly thirty-year gap where not a single novel antibacterial was marketed. In fact, since the early 1960s, only four new classes of antibiotics have been introduced yet the $30 billion global antibiotic market is still dominated by antibiotic classes discovered half a century ago. Aside from the introduction of carbapenems in 1985, all antibiotics approved for clinical use between the early 1960s and 2000 were synthetic derivatives of existing scaffolds, accounting up to 73% of the antibacterial New Chemical Entities (NCEs) granted by the U.S. Food and Drug Administration.

1.2 Mechanisms of antibiotic action

A cell inhibited by an antibiotic is in a dynamic state; interaction between an antibacterial agent and its specific binding site will trigger a series of reactions that will ultimately result in inhibition of cell growth and cell division, often leading to apoptosis of the cell.

Antibiotic Class Generation 1 Generation 2 Generation 3 Generation 4

NH HO O H H 2 O O O N N H H Ph R S S N N H S N N N HO S N O O H S N N N S O O O O N N O O O O O O O O HO HO HO HO HO

Penicillins Penicillin G Amoxicillin Ticarcillin Piperacillin

H H O O O N S N S N OH R H N 1 O H S N S N N H S N N N R S O N O N H N 2 O O N N 2 N O NH2 H N N O O 2 N S O O S O O O O O O O HO O HO O HO O HO HO

Cephalosporins Cefalotin Cefuroxime Ceftazidime Cefepime

O O O O O O O O O O F F F OH R1 OH OH OH OH H N N N N N N N N O R2 N HN N O N H R3 H

Quinolones Nalidixic acid Ciprofoxacin Levofloxacin Moxifloxacin

O O O N N R1 N OH O O R3 HO OH HO OH HO N HO N O R2 O O O O N O O O HO N O H O O O O O O O O HO N O O O O O R4 OH O O O O OH

Macrolides Erythromycin Clarithromycin Telithromycin

R2 R3 R4 N HO OH N OH N N N H H OH H H OH H H OH H H OH O H NH2 NH2 NH2 N NH2 R1 OH OH OH N OH H OH O OH O O OH O OH O O OH O OH O O OH O OH O O

Tetracyclines Oxytetracycline Doxycycline Tigecycline

Figure 1-3. Synthetic tailoring of known scaffolds towards successive generation of

antibiotics

There are approximately 200 conserved essential proteins in bacteria, but the number of currently exploited target is very small; current action mechanisms are therefore based on a limited range of bacterial physiology, such as cell wall synthesis

(beta-lactams and glycopeptides), DNA replication (quinolones), DNA transcription

(rifampicin), translation (oxazolidinones, macrolides, chloramphenicol, clindamycin, aminoglycosides, steroids, and tetracyclines), and folic acid metabolism (sulphonamides and trimethoprim (Figure 1-4).

DNA Gyrase Cell-wall Synthesis

Quinolones Penicillins Cephalosporins Glycopeptides Rifampicin Carbapenems DNA Monobactams

RNA mRNA Polymerase fMet tRNA Folic Acid 30S Sulphonamides Metabolism Trimethoprim

50S 70S initiation complex Oxazolidinones

Protein Synthesis

50S 30S Macrolides Aminoglycosides Chloramphenicol Tetracyclines Clindamycin

Figure 1-4. Targets of the most common and successful antibiotics. Adapted from: The

future challenges facing the development of new antimicrobial drugs

The most successful antibiotic classes have been those with a broad-spectrum and bactericidal properties. These compounds bind to different targets; therefore, diminishing the probability of developing resistance. Ciprofloxacin is a fluoroquinolone antibiotic that targets DNA gyrase and DNA topoisomerase. Ampicillin is semi-synthetic b-lactam that targets several penicillin-binding proteins that are responsible for peptidoglycan synthesis. Streptomycin is an aminoglycoside inhibitor of protein synthesis that bind primarily to 16S ribosomal RNA, which is encoded by several operons.

1.3 Mechanisms of antibiotic resistance

As the “miracle drugs” emerged to treat the very last of infections, bacteria were already embracing a strategy to overcome the new medicaments. In the case of S. aureus, the microbe takes advantage of its high replication rate of about one half hour and a rather low mutation rate of 1 per 1010 base pairs; nevertheless, a resistant phenotype can originate in hours. The problem is not only justified by the rapid accumulation of mutations, but also due to the selective pressures that antibiotics impose. If a drug-resistant phenotype were to evolve and there were no antibiotic present, the same phenotype will likely not flourish. It is only when antibiotics are used that drug-resistant phenotypes gain a selective advantage and survive.

The time period for the resistant strains to arise depends on the type of antibiotic and the mechanism utilized to mitigate its activity; nevertheless, it has been calculated to occur mere months after induction into the market. The major molecular mechanisms of resistance are outlined below.

1.3.1 Prevention of access to target

Compared to Gram-positive bacteria, Gram-negative bacteria are essentially less permeable to foreign substances as their outer membrane forms a permeability barrier.1

Hydrophilic antibiotics cross the outer membrane via channel proteins that were thought to be non-selective to the drug.2 Recent studies, however, show that reduced permeability is achieved via the down-regulation of these porins proteins and the up- regulation of such with higher specificity.3 To make matters worse, bacteria also use multidrug resistance (MDR) efflux pumps in order to transport many of the drugs out of the cell; conferring high levels of resistance to previously clinically useful antibiotics.4 The most important class of efflux pump in Gram negative bacteria are the tripartite resistance-nodulation-cell division (RND) class that encompasses a cell membrane spanning pump, an outer membrane pore, and a periplasmic adapter protein that connects the two.5 This complex machinery represents one of the biggest challenges in drug discovery, urging the search for broad spectrum small molecule inhibitors.6

1.3.2 Target modification

Most antibiotics bind with high affinity to a single target in the cell and thus prevent the normal function of the target. Single point mutations of the target allow for sufficient modification to almost eliminate the binding affinity of the antibiotic without compromising its essential function within the cell. Since the genes that encode specific targets exist in multiple copies; a single point mutation in one of these copies followed by recombination at high frequency between homologous alleles will rapidly result in a cell population with a resistant target, such is the case of linezolid resistance.7 Target

modification can also arise by highly efficient and regioselective modification catalyzed by enzymes. Ribosome methyltransferases are an example of this catalytic resistance strategy (Fig. 1-5). This class of enzymes designated as Erm, methylates the A2058 position of the 23S rRNA automatically conferring resistance to three classes of antibiotics: macrolides, lincosamides, and streptogramins.8

A2058 A2058 N N N N Erm N N N N NH 2SAM 2SAH N 2 H3C CH3

A2058 N N

N N H3C NH O A2058 OH 2 O CH3 N N OH N S OH HO CH O 3 N N O HO OH OH NH2 O O CH3 HN O OH Cl O CH O H3CO 3 Erythromycin N Clindamycin

Figure 1-5. Erm methylates the A2058 position of rRNA disrupting vital interactions with antibiotics; therefore, resulting in resistance. The methyl donor SAM is converted to SAH

(S-adenosylhomoserine)

1.3.3 Molecular bypass

Microbes have evolved in distinct ways to deviate a compounds’ original purpose by lowering its binding affinity and mitigating efficacy. Glycopeptide resistance is a great example of such mechanism; where vancomycin binds to the acyl-D-alanyl-D-alanine terminus of the growing peptidoglycan component of the bacterial cell wall. This dipeptide

serves as a key signaling region for the enzymes in charge of cross-linking adjacent chains of peptidoglycans via a transpeptidation reaction. Vancomycin forms a network of five hydrogen bonds with acyl-D-alanyl-D-alanine, resistance arises when the target is exchanged to the isosteric depsipeptide acyl-D-alanine-D-lactate. The result is an exchange from an amide to an ester linkage; therefore, reducing the hydrogen bonds to four and increasing the electronic repulsion (Figure 1-6).

OR Cl O O

HO OH Cl O O H O H O N N O N N N H N O H R O H HN HO H

HO OH OH

O H O N Acyl-D-Ala-D-Ala R N O H O O O O Acyl-D-Ala-D-Lac R N O H O

Figure 1-6. Hydrogen bond network between the peptidoglycan layer and vancomycin.

Acyl-D-Ala-D-Lac induces resistance to the antibiotic by lowering binding affinity a

thousand fold.

1.3.4 Chemical modification

Enzyme catalyzed inactivation is arguably the pinnacle of bacteria resistance countermeasures.9 Evolution of this complex and efficient catalysis have given bacteria strong and sustained selective resistance towards one of the major antibiotics: b-lactams. The first evidence of b-lactamase activity was reported in the

1940’s, long before penicillin’s widespread clinical use.10 Currently there are two accepted mechanism of drug inactivation: formation of a covalent enzyme intermediate followed by hydrolysis or metal-activation of a nucleophilic water molecule (Fig. 1-7).11

H R H R H N N H S S O O N HN O O O O O Ser-B-lactamases O Ser O H H Ser O Ser O

R R H NH H N H S S O H O R H O N N HN S O O O O O N O O O O 2+ Inactive H Zn O O HO 2+ Zn Zn2+ Zn2+

O O O O Asp Asp

Figure 1-7. Accepted mechanisms of serine and metallo-b-lactamases

Chapter 2

Introduction to Thiamine Biosynthetic Pathway

2.1 Vitamin B1: Biosynthesis and Role in Living Systems

Like other B vitamins, thiamine is an indispensable molecule for all known organisms. This is mainly because, in mammalian cells, its diphosphorylated form, thiamine pyrophosphate (TPP), is the coenzyme for five key metabolic enzymes (Figure

2-1). The most important being mitochondrial pyruvate and oxoglutarate dehydrogenase complexes as well as cytosolic transketolase. Therefore, it is generally believed that thiamine deficiency leads to decreased oxidative metabolism12. In animals, the brain relies heavily on oxidative metabolism to synthesize ATP; therefore, this organ is particularly sensitive to thiamine deficiency. In humans, nutritional thiamine deficiency leads to beriberi, a polyneurotic condition, rapidly reversible after thiamine administration.

In alcoholics, thiamine deficiency can result in typical selective diencephalic brain lesions referred to as Wernicke-Korsakoff syndrome.13

TPP is the active form of vitamin B1. TPP is an important cofactor in the enzyme complexes of pyruvate dehydrogenase (PDH); bridging the glycolysis and tricarboxylic acid (TCA) cycle in cellular respiration via formation of acetyl-CoA. Other TPP-dependent enzymes are transketolase and the branched-chain 2-oxo acid dehydrogenase (BCDOH) where thiamine acts as an acyl-transfer and decarboxylation aid respectively.

Peroxysome Glucose 2-Hydroxy fatty acids (n) Sphingolipids Phytanic acid α-Oxidation HACL1 D-Xylulose-5-phosphate D-Ribose-5-phosphate Glucose-6-phosphate Formyl-CoA Pentose Phosphate TK Glycolysis Shunt Long chain aldehyde (n-1)

Sedoheptulose-7-phosphate D-Glyceraldehyde-3-phosphate

Branched chain amino acids Pyruvate Cytosol

Branched chain 2-oxo acids Pyruvate

β-Oxidation BCODC PDHC

CoA-derivatives Acetyl CoA

Oxaloacetate Citrate

Krebs Cycle

Succinate Oxoglutarate Mitochondrion

OGDHC

Figure 2-1. TPP dependent enzymes (purple) in a mammalian cell and subcellular

localization. TK: transketolase, PDHC: pyruvate dehydrogenase complex, OGDHC:

oxoglutarate dehydrogenase complex, BCODC: branched-chain 2-oxo acid

dehydrogenase complex, and HACL1: 2-hydroxyacyl-CoA lyase 1.

Pyruvate dehydrogenase complex catalyzes the pivotal irreversible reaction that leads to the consumption of glucose in the aerobic energy-generating pathways.14 This intricate enzyme system allows for the transition of pyruvate; the product of the glycolysis cycle into acetyl CoA, the starting material for the TCA or Krebs cycle. The decarboxylation mechanism occurs through the thiamine’s catalytic center at C2 of the thiazolium ring (Figure 2-2).

OR OR

N N S N N N N S

NH2 H NH2 B O - CO2 H O

B H

N S N S O OH H O O

N S CO2

HO H+

Figure 2-2. Decarboxylation mechanism of TPP in pyruvate dehydrogenase. The

acetaldehyde formed will become acetyl-CoA before entering the TCA cycle.

Thiamine pyrophoshate’s catalytic center is located on the thiazole moiety, where deprotonation of 2-hydrogen (pKa ~15) leads to the formation of an ylide or a resonantce stabilized carbene. The next mechanistic steps involve attack of the nucleophilic carbene to a carbonyl-containing carbohydrate, amino acid, etc. resulting in an enamine intermediate where the thiazole serves as an electron sink for the cleavage of the acyl or decarboxylative group according to the enzyme in play15.

Plasmodium falciparum is the pathogenic agent of tropical malaria, a devastating and often deadly disease that caused half a million deaths worldwide out of the 214 million reported cases in 2015 alone. By virtue of its intracellular life cycle, the parasite is highly dependent on the acquisition of host nutrients for its survival, and interference with

the parasite salvage systems for cofactors and other metabolites affords a promising approach for druggability.16

Table 2-1. TPP-dependent enzymes in Plasmodium falciparum.

EC-Number EC-Name Pathway Refs.

1.2.4.1 Pyruvate dehydrogenase Fatty acid biosynthesis and 17 pyruvate metabolism 1.2.4.2 a-ketoglutarate TCA cycle 18 dehydrogenase 1.2.4.4 3-methyl-2-oxobutanoate Branched-chain amino acid 19 dehydrogenase (BCAA) degradation 2.2.1.1 Transketolase Pentose phosphate pathway 20

2.2.1.7 1-Deoxy-D-xylulose- Isoprenoid biosynthesis 21 phosphate synthase

Biosynthesis of TPP in P. falciparum involves two routes: the thiazole and pyrimidine pathways (Figure 2-2). The thiazole pathway uses 5-(2-hydroxyethyl)-4- methylthiazole (THZ), which undergoes phosphorylation by THZ kinase to 5-(2- hydroxyethyl)-4-methylthizole phosphate (THZ-P) with high substrate specificity. The other pathway begins with 4-amino-2-hydroxymethylpyrimidine (HMP). After two consecutive phosphorylations by HMP kinase (ThiD), the HMP diphosphate (HMP-PP) couples with THZ-P via thiamine phosphate synthase (ThiE) to form thiamine monophosphate (TMP). The next steps involve the maintenance and storage of the vitamin by thiamine monophosphate phosphatase (TMPP) and its rapid conversion to the active form via thiamine pyrophosphate kinase (TPK).

Uptake Uptake?

HMP THZ

ThiD

HMP-P ThiM

ThiD

HMP-PP THZ-P

ThiE

TMP

TMPP

Thiamine

TPK

Thiamine Pyrophosphate

Figure 2-3. Thiamine biosynthetic pathway diagram in Plasmodium falciparum. ThiD,

ThiM, and ThiE are the protein-coding genes. Both HMP and THZ have been found to be

uptaken from the outside or synthesized de novo in most prokaryotes.

ThiS PLP Thil-SH Cysteine ThiF OH Thil-SSH HSS-IscS HS-IscS NifS IscS O O O Dxs NH ThiO Pyruvate OH ThiS-COSH H NH2 HO O O (B. subtilis) PO OP O O ThiG Dehydroglycine Glycine HO Deoxy-D-xylulose 5-phosphate ThiH O HO Glyceraldehyde OOC S OP NH2 3-phosphate N COO Thiazole phosphate carboxylate tautomer Tyrosine (E. Coli)

TenI

Thiazole phophate OOC S OP carboxylate N NH NH2 2 ThiE ThiL N N N N S S N N NH2 Hydroxymethyl pyrimidine N OPP2- 2- pyrophosphate OP OPP N TMP TPP

ThiD N

PO O N ThiC NH2

NH2 N OH 5-Aminoimidazole HO Hydroxymethyl OH pyrimidine (HMP) ribotide N

Figure 2-4. Complete de novo thiamine biosynthetic pathway in bacteria. Glycine and

tyrosine are used as starting materials for B. subtilis and E. coli respectively. 22

2.2 The appeal of vitamin pathways as source of novel antibacterials

Due to the intricacy of its role in both bacterial and human metabolism, vitamins offers a valuable source of enzymes available for pathway inhibition. Although vitamin pathways are not a novel concept in the antibacterial realm due to the appearance of the so-called

“sulfa drugs” over fifty years ago and their effect on folic acid or vitamin B9 metabolism; yet “the first miracle drugs” have been a great opponent against disease-causing microbes. Sulfonamides interact with the enzyme dihydropteroate synthase (DHPS) by mimicking the natural substrate p-aminobenzoic acid (pABA) and therefore inhibits the

production of folic acid which yields a bacteriostatic effect. In the late 1960’s, it was found that sulfamethoxaxole, a sulfonamide, and trimethoprim had a synergistic effect in counteracting bacterial growth. Both antibiotics target different enzymes in the folic acid metabolism, and since then, it has been the prescription of choice for urinary tract infections (UTIs) and upper respiratory infections while mitigating the chances for bacteria to develop resistance (Figure 2-4).

A O O O O N DHPS O O 2- HN OPP N HN N H H2N N N H NH H2N N N 2 H

6-Hydroxymethyl-7,8-dihydropterin p-Aminobenzoate 7,8-Dihydropteroate pyrophosphate (DHPPP)

O O R R O N DHFR O N H H H N N HN N HN N H H NADPH NADP+ H2N N N H2N N N H H

Dihydrofolic acid Tetrahydrofolic acid

C O NH2 O S N O O N HN

H2N H2N N O O

Sulfamethoxazole Trimethoprim

Figure 2-5. A: DHPS catalyzes the coupling between DHPP and pABA in the first steps of the folate biosynthesis. B: DHFR catalyzes the reduction to the piperazine ring by means

of NADPH consumption. C: Sulfamethoxazole and trimethoprim inhibitors for each

enzyme respectively

Overall, vitamin pathways offer a myriad of protein machinery that are specific for the biosynthesis of cofactors and the major downstream processes they carry; ranging from cellular respiration to DNA repair. Nevertheless; the most important quality of this enzymatic apparatus is that they are ubiquitous, yet specific to most of the pathogenic microorganisms that healthcare faces worldwide. The result is an enzymatic segregation between prokaryotes and higher animals that depend on their dietary intake for the supply of these important cofactors.

2.3 HMP kinase structure and function in thiamine biosynthesis

In the current work, the Foss lab has focused on targeting HMP kinase (HMPK,

E.C. number: 2.7.1.49) an important enzyme from the thiamine biosynthetic pathway in charge of carrying two consecutive ATP-dependent phosphorylations within the pyrimidine branch. Although X-ray crystal data has been reported by Ealick et al., there has not been, to the best of our knowledge, any SAR studies nor small molecule analog found to have inhibitory activity. The structure of HMPK was determined at 2.3 Å resolution and the co-crystallized complex with HMP natural substrate was resolved at

2.6 Å from Salmonella typhimurium (Figure 2-5)

Figure 2-6. Ribbon diagram of HMP Kinase showing both monomers and the co-

crystallized HMP substrate

Structurally, HMPK is a homodimer with an active site in each monomer that extends outward along the C-terminal edge of the central b sheet, corroborating a high degree of structural homology to the ribokinase super family; and therefore suggesting an evolutionary progression from the other members of the family. Prospect with HMPK arises from not only being a key step in the thiamine biosynthesis, but also due to the wide range of substrates that ribokinase enzymes accept; ranging from small aromatic compounds to carbohydrates. HMPK also shows a dual functionality as it transfers a phosphate group to both a hydroxymethyl and to a hydroxymethylphosphate moiety; indicating two transition states and the existence of more than one binding mode within the same active site (Figure 2-6)

NH2 NH2 O NH2 O O HMPK HMPK P P P N OH N O O N O O O O O O N ATP ADP N ATP ADP N

NH Mg2+ NH 2 2 O H N O N O P O ADP O O O O O N P O P O P O A N O O O

Anion Hole Anion Hole

O O 2+ NH2 O P O Mg NH2 O P O O O O N N P ADP O O O O O O N P O P O P O A N O O O

Figure 2-7. A: Phosphorylation reactions catalyzed by HMP Kinase. B: Schematic of the

dual catalytic activity of HMP kinase. After the first phosphorylation, a rotation along the

C5-C7 axis (red) occurs in order to stabilize the negative charge within the anion hole.

The most striking difference between HMPK and other member of the ribokinase family is the fact that HMPK carries two consecutive phosphorylations. The structure of

HMPK only suggest one binding site for each the pyrimidine ring and the nucleotide.

Since there is no support that shows any drastic conformational change of the enzyme upon substrate binding, it is reasonable to postulate that the monophosphorylated HMP attains a different binding position in order to facilitate the second phosphorylation.

Phosphorylation of HMP is believed to follow an in-line displacement mechanism where the first transfer of ATP’s g-phosphate to the hydroxyl accepting group is facilitated by ATP itself due to the lack of basic residues around the hydroxyl group. This type of substrate-assisted catalysis has been proposed for other kinase members. 23 After the first phosphorylation, HMP-P undergoes a bond rotation around C5-C7 bond in order to stabilize the negative charge into an “anion hole”; allowing for a second ATP molecule to transfer another g-phosphate onto HMP-P. Even though “anion holes” are structurally conserved across different ribokinases, HMPK is the only enzyme that allows a secondary transfer of phosphate from ATP.

Efforts towards the co-crystallization of HPMK with ATP have been futile. Ealick et al. modeled both ATP and ADP on different ribokinase enzymes and concluded that the binding region is very well conserved due to the overlap among the nucleotide molecules. The highest degree of overlap was found among the a and b phosphate groups, where the g-phosphate shows the highest location variance (Figure 2-7).

A B

Figure 2-8. A: Superposition of ligands in the ATP or ADP binding sites of HMP kinase, T.

gondii adenosine kinase, human adenosine kinase, thiazole kinase, and ribokinase. B:

Computational docking of HMP and HMP-P showing the bond rotation along C5-C7

(AutoDock) C: Binding mode and stabilization of HMP-P by the anion hole

2.4 Thiamine regulation across species

Previous studies suggest that when HMP and hydroxyethylthiazole (HET) are supplied exogeneously, they are taken up by the cell and enter the de novo synthetic pathway; resulting in TPP.24 However, mutations in the thiD and thiM loci results in inactivation of both enzymes responsible to initiate the reaction cascade for thiamine biosynthesis; leading to cell death.25

In a similar fashion, the HMP kinase of S. cerevisiae is encoded by two functionally redundant genes, thi20 and thi21. The predicted products of thi20 and thi21 open reading frames contain 551 amino acids (aa) with 86% amino acid identity. The single deletion strain for thi20 and thi21 reveal thiamine prototrophy, but the simultaneous disruption of both genes lead to thiamine auxotrophy.26 The N-terminal part (1-300 aa) of yeast thi20 and thi21 share about 35% sequence identity with the entire region of the bacterial thiD gene product; raising the possibility that both yeast proteins catalyze two consecutive phosphorylations in the thiamine biosynthetic pathway.

The microorganism Rhizobium leguminosarum has a symbiotic relationship with most leguminous plants in which atmospheric nitrogen is reduced to ammonia in a process called nitrogen fixation. In order to stablish such symbiosis, bacteria must survive in the surrounding soil environment, competing for nutrients. Soluble vitamins such as thiamine, biotin, riboflavin, niacin, and pantothenic acid are released by the legume roots which enhances the ability of bacteria to perform nitrogen fixation. However, microorganisms such as R. leguminosarum doesn’t strive for the uptake of thiamine alone; it has also developed a salvage pathway in order synthesize thiamine from the intermediates HMP and THZ. Mutations of the thiD and thiE genes halt the growth of the microbe and only addition of thiamine is able to restore the growth again.27

2.5 HMP kinase binding site: substrate & inhibitors

Many textbooks and research articles traditionally highlight the remarkable specificity of enzyme action through their lock & key model; nevertheless, enzyme promiscuity has attained a lot of attention lately due to its role in the evolution of new functions.28 Bifunctional or promiscuous proteins reflect ancestral forms from which two different specificities split and originated through a gene duplication event during evolution.29 It is imperative to clarify that catalytic promiscuity is defined as a secondary catalytic activity that has no effect in any known physiological process; where bifunctionality refers to catalytic activities that have physiological relevance and their substrates present significant binding affinities.

The family of ATP-dependent vitamin kinases from the ribokinase superfamily features a single domain with a conserved bab fold (Rossmann), thus structurally diverging from other enzymes of this superfamily. This subfamily includes enzymes such as HMP kinase (HMPK, E.C. 2.7.1.49), hydroxyethyl thiazole kinase (THZK, E.C.

2.7.1.50), and pyridoxal kinase (PLK, E.C. 2.7.1.35). Both HMPK and THZK are part of the de novo biosynthetic vitamin B1 pathway and the latter belongs to the vitamin B6

(PLP) pathway. Although these enzymes have been classified based on either their enzyme activity or structural comparisons, several HMPK/PLK enzymes that are able to phosphorylate both HMP and pyridoxal (PL) have been described, thus conferring a dual function in both the pyridoxal and thiamine biosynthetic pathway.30,31 Moreover, the determinant for specificity towards HMP molecule has been characterized to a single glutamate residue in different bacteria. Glu44 can confer up to eight times higher affinity towards the HMP molecule than its other counterparts (Figure 2-8).28

Met80 H C Gly11 HC 2 HN N H 2.9 3.0 Glu44 Asp23 O H O O H 3.1 2.6 2.6 O O 2.9 H N N O H H N Asp105 H O H 3.4 O 4.5 O 3.0 H S O Cys213 O O S O S O O O CH2 O 2.6 HN 2.9 Gly212 HN CH2 NH3 O O Gly210 Lys176 Glu142

Figure 2-9. HMP binding site for B. subtillis. Dotted lines reveal the plausible H-bonding with HMP and the respective distances in Angstroms (Å). Sulfate ions are present due to the high concentration of salts during the crystallization process. The g-phosphate of ATP

is believed to be positioned where the sulfate close to Gly210 is.23

Known substrates of HMP kinase are highlighted below (Firgure 2-9). The binfunctionality of HMP kinase in both the thiamine and pyridoxal pathway is an attractive characteristic for the development of both PLP or thiamine antimetabolites through a pro- drug approach. One of the main focus of this work highlights the substrate scope of HMP kinase and the bacteriostatic effect that results from catalyzing unnatural HMP analogs.

NH2 NH2 O P N OH N O O O N N

HMP HMP-P

HO H2N O HO HO HO OH OH OH

N N N

PN PM PL

Figure 2-10. Substrates of HMP kinase from B. subtillis. The bottom substrates are

vitamers of the vitamin B6 pathway (PN: pyridoxine, PM: pyridoxamine, and PL: pyridoxal), HMP kinase was not able to carry the second phosphorylation of the pyridoxal

analogs. Competition assays using the above substrates revealed a preference for PL,

followed by HMP, HMP-P, PN, and PM.32

In the other hand, although no HMP-like inhibitors have been studied or revealed,

Sieber et al. assigned rugulactone as the first small molecule inhibitor through a library screening approach33. Rugulactone was known to have antimicrobial properties although no mechanism of action had been validated; nevertheless, the scaffold includes two potential Michael acceptors: an a,b-unsaturated g-lactone, which is known to modify proteins, as in the case of ratjadone34, along with an a,b-unsaturated ketone (Figure 2-

10) The presence of these two electrophilic groups exerts the possibility of enzyme inhibition via covalent modification.

Sieber et al. found that Rugulactone’s activity involves a covalent modification of a cysteine residue via Michael addition to the a,b-unsaturated ketone presented in the molecule. Different analogs were synthesized and screened to properly identify the electrophilic site responsible for the alteration. Inhibitory concentrations (IC50) were found to be in the low micromolar range (14-25 µm) against S. aureus for Ru0 and Ru2 only,

Ru3 and Ru4 exhibited no inhibition of HMP kinase.

O O

O O O O

Ru0 Ru2

O O

O O O O

Ru3 Ru4

Figure 2-11. Rugulactone derivatives. Rugulactone (Ru0) was isolated from the plant

Cryptocarya rugulosa in 2009. Both Ru0 and Ru2 modified Cys213 of the HMP kinase

proving the Michael acceptor site where the covalent modification occurs.

2.6 HMPK as drug target

Biological systems contain only four types of macromolecules with which we can interfere using small molecules therapeutic agents: proteins, polysaccharides, lipids, and nucleic acids. Toxicity, specificity, and inability to obtain potent compounds against the latter three types means that the vast majority of successful drugs achieve their activity by binding to and modifying the activity of a protein. Currently, enzymes still make up to

47% of the marketed small molecule drug target (Figure 2-11); therefore, the insatiable search for novel protein targets should never be disregarded as the efficiency of R&D intensifies.

4 4 4 2 7 1 47 1

Enzymes GPCRs

DNA Integrins

Miscellaneous Other Receptors

Ion Channels Transporters

Nuclear Hormone Receptors

Figure 2-12. Marketed small-molecule drug target by biochemical class. Enzymes and G-

protein coupled receptors (GPCRs) make up to 77% of the commercial targets. 35

Reversible phosphorylation of proteins and lipids is a key component of many cellular signaling pathways including those involved in cell growth, differentiation, proliferation, angiogenesis, apoptosis, cytoskeletal rearrangement, and metabolism 36.

The process of phosphorylation is carried out by protein kinases that catalyze the transfer of a phosphate group from the high energy specie adenosine triphosphate (ATP) or guanosine triphosphate (GTP), to proteins or lipids. In proteins, the hydroxyl acceptor is either a serine, threonine, or tyrosine residue.

They key role of kinases in disease pathophysiology is underscored by the finding that mutations in kinases can lead to dysfunctions in cellular signaling which it can result in diabetes, cancer, cardiovascular, among others 37. For this reason, kinases are an attractive target; however, the current kinase inhibitors only target a small fraction of the 518 protein kinases that comprise the human kinome (~1.7% human genes).

The current work involves the study of the thiamine biosynthetic pathway enzyme, HMP kinase. A unique enzyme with the dual functionality of phosphorylating

HMP and HMP-P along some other substrates from the pyridoxal pathway. HMP kinase poses an attractive target due to several reasons: (a) HMP kinase is part of the ribokinase superfamily. They are known to accept a wide range of substrates and scaffolds and thus arises the possibility of modulation through a pro-drug approach. (b)

The active site is buried deep while the protein undergoes conformational change upon substrate binding; leading to restricted access without solvent effects. (c) HMP kinase is able to phosphorylate HMP-P, leading to the postulation of transition state inhibitors and the stabilization of negatively charged species by the anion hole. (d) HMP kinase is highly conserved across different Gram negative and positive species, including pathogens such as C. difficile categorized by the CDC as urgent threats to humanity (e) Humans and higher mammals lack the complete enzymatic machinery to synthesize thiamine de novo; resulting in a dietary dependency for the cofactor; leading to a plausible higher specificity towards the microscopic organisms.

In the effort to categorize HMP kinase as a target of novel antibacterials, the

Foss group commenced the drug design process through a ligand-based approach.

Knowing the structure of HMP natural substrate along with the amino acid residues that make up the binding pocket of HMPK, my then lab mate and now Dr. Bhawal successfully synthesized a library of HMP analogs with different variations along the C4 and C5 positions of the pyrimidine ring. (Figure 2-11) As the compounds grew in number, the need for expression of HMPK along with a systematic high throughput assay to screen for enzyme inhibition were prioritized. Since then, six different types of assays have been developed and validated in the lab, leveraging our insights and collaborations outside of the organic synthesis realm. The next work involves the over expression and purification of HMP kinase from S. typhimurium and the respective evaluation of HMP analogs via qualitative, spectroscopic, and spectrometric assays.

Het. cycle

R4 N C5 reactive Tail head T R2 N R5 R6 Body

Figure 2-13. Current HMP analogs incur modifications around C2 (R2), C4 (R4), and C5

(R5) of the heterocycle.

Chapter 3

HMP Kinase Overexpression and Purification

Even though the thiazole moiety of thiamine has been extensively studied by

Begley, Chatterjee, and others; the pyrimidine ring has not had the same fate and most of the literature has been published since the late 90’s. Nevertheless, the recent approval of government programs to fight “superbugs” and the incessant struggle to encounter new drug targets; have taken pharmaceutical companies into looking back at old, forgotten vitamin pathways, in a sort of teaching an old dog new tricks.38

HMP kinase expression was initiated by reaching out to Dr. Begley (Texas A&M) in order to request a sample of Thid recombinant DNA for protein expression purposes.

Upon DNA arrival, the Foss’ lab was unable to amplify the gene of interest since the vector could not be identified by the source. At this point, I embarked on the goal of cloning Thid from bacterial stock and express the protein HMPK in-house. In order to facilitate the expression and purification steps, a high expression vector was chosen with three important characteristics available; a T7 promoter region to facilitate DNA transcription, a kanamycin resistant gene for bacterial growth specificity, and an inducible lactose operon (lac) for protein overexpression.

The protein purification steps were carried out by taking advantage of six consecutive hisitidines residues (His-tag) encoded in the vector that will be co-translated with HMP kinase. A nitrilotriacetic acid agarose charged with Ni2+ allows for the histidine residues in the His-tag to bind to the vacant positions in the coordination sphere of the immobilized metal ion with high specificity and affinity (Figure 3-1)

This chapter outlines the cloning of Salmonella’s Thid gene and the overexpression of HMP kinase while optimizing the protein’s purification and stability in non-denaturing conditions with the purpose of assay development later on in the project.

Protein

O O O O O O H H H H H H H H H H H HN C C N C C N C C N C C N C C N C C O

CH2 CH2 CH2 CH2 CH2 CH2

N N N N N N N N NH N N

O Ni O O Ni O O N O O N O O O

O O

Agarose

Figure 3-1. Scheme of protein purification through His-tag and affinity chromatography.

Affinity can be disrupted upon addition of imidazole or low pH buffer.

3.1 Materials and Methods

Cloning. The Thid gene was isolated from Salmonella enterica strain LT2 genomic DNA [ATCC 700720] using primers purchased from integrated DNA technologies (http://www.idtdna.com). A PCR amplification was used to isolate the ORF from genomic DNA and incorporate restriction sites (BamH I/Nhe I) via reaction primers: forward: 5’-(CAT ATG GCT AGC CAA CGA ATT AAC GC)- 3’; reverse: 5’-(TTT TTG

GAT CCC TAC CAC CAC GCG TG) -3’. The DNA isolated by the PCR amplification was ligated into an isopropyl b-D-1-galactopyranoside (IPTG) inducible T7 vector, designated pET-28A(+) (Novagen) that it had been previously treated with both BamH I, Nhe I, and shrimp alkaline phosphatase. Sequence verification of Thid was performed in the life sciences department at UTA by the Genomics Core Facility (http://gcf.uta.edu/). The resulting plasmid pTHID28 was transformed into NovaBlue competent cells (EMD

Millipore), assayed in kanamycin (Kan) media and subject to colony PCR in order to to verify the transformation efficacy.

Media and growth conditions. The pTHID28 recombinant was transformed into chemically competent BL21(DE3) E. coli (Novagen) by heat-shock and grown at 37 oC on a lysogenic broth (LB) agar plate in the presence of 25 µg/ml Kan. After overnight incubation, a single colony was extracted for growth on liquid LB media for inducer optimization (0.1 - 2 mM IPTG). Growth conditions were monitored via UV-Vis at 600 nm

(OD600). Induction was initiated when cells attained a OD600 ~ 0.8 with an IPTG concentration of 0.5 mM and 1 g/l of casamino acids. Additionally, upon addition of the inducer, the temperature was decreased from 37 oC to 25 oC and agitation was set to maintain an O2 concentration of 20% relative to air-saturated media. After 4 hours of incubation, the cells were pelleted by centrifugation (Beckman-Counter Avanti J-E, JA

10.5 rotor) at 8,000 rpm for 20 minutes. The cell paste was stored at -80 oC and the

expression of Thid was confirmed via SDS-PAGE (12% acrylamide) of lysed cells before and after protein induction.

Protein purification. Approximately 10 g of cell paste was suspended in 20 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 25 mM imidazole, 5 mM MgCl2, 1 mM DTT,

1% Triton X-100, pH 8.0), and thawed in an ice bath with 5 mg/mL each of lysozyme, deoxyribonuclease I (DNase I), and ribonuclease I (RNase I) with gentle stirring for 1 hour at room temperature. The cell suspension was sonicated in ice with a 30 seconds on/off pulse cycle for a total of 10 minutes. The resulting cell-free extract was centrifuged

(JA-20 rotor) at 12,000 rpm for 15 minutes at 4 °C. The supernatant was filter-loaded onto a 0.1M NiSO4 charged HiTrap immobilized metal ion affinity chromatography (IMAC)

HP column (GE Healthcare) pre-equilibrated in lysis buffer. The column was washed with five column volumes (25 mL) of IMAC buffer A (100 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The protein was eluted on a linear imidazole gradient (100 mM to 300 mM imidazole in 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The expressed ThiD protein, determined by SDS-PAGE, was dialyzed overnight (Spectra/Por, 6-8 kDa MWCO, 23 mm flat width) with three buffer exchanges and concentrated via VivaSpin Turbo 15 with 10 kDa molecular weight cut-off (Sartorius). Protein concentration was determined via UV-

Vis spectroscopy and standard Coomassie Plus assay (ThermoScientific). Aliquots (1 mL) of HMPK were mixed with 50% glycerol and flash-frozen in liquid nitrogen prior to storage at -80 oC.

Spectroscopy. All UV-Vis measurements were performed on a BMG LabTech

SPECTROstar Omega. All measurements were made in ES Quartz cuvettes (Precision

Cells) or UV-transparent 96-well plate (Corning).

3.2 Results

Cloning. Recombinant Thid from S. typhimurium was obtained as described in section 3.1. As indicated by Figure 3-2, the colony PCR from the transformed NovaBlue competent cells shows the presence of the gene of interest. The band around 750 base pairs (bp) corresponds to the composition of Thid’s total number of amino acids (266 aa) according to previous published results.39

1 2 3 4 5 bp

1000- 750- 500- 300- 150- 50-

Figure 3-2. Gel electrophoresis of recombinant colony PCR (bp: base pairs). Lane 1:

DNA ladder (PCR markers, Promega). Lane 2: PCR amplification of foreign insert into

vector (798 bp). Lane 3: Thid gene amplification directly from S. typhimurium (798 bp).

Lane 4: blank. Lane 5: pET28-A vector uncut (Novagen)

Thid was successfully ligated into the commercially available vector with a kanamycin resistant gene, an N-terminal His-tag used later on for protein purification, a

T7 RNA polymerase promoter region, and an IPTG inducible region for the overexpression of the gene of interest. Gene sequencing also determined the presence of the insert into the high-expression vector and the final transformation was made into

BL21(DE3) E. coli high competent cells.

Protein expression & purification. HMP kinase was first overexpressed and purified in 1996 by Mizote et al. from S. typhimurium for the identification and characterization of an operon involved in thiamine biosynthesis.25 As described in the previous section, the inducible recombinant was optimized to a concentration of 0.5 mM

IPTG and 1 g/L casamino acids in LB broth. The purification of the His-tagged HMP kinase was carried out using an immobilized metal ion affinity chromatography (IMAC) charged with Ni2+. Although the equilibrating/lysis buffer initially contained a low concentration of imidazole (5 mM) to eliminate non-specific binding of unwanted proteins, the buffer was optimized to an imidazole concentration of 25 mM; without any apparent loss of HMP kinase as indicated in Figure 3-3. The elution of HMP kinase occurred after ten column volumes of wash-buffers at a concentration as low as 200 mM of imidazole.

kDa 1 2 3 4 5 6 250

80 60

Figure 3-3. SDS-PAGE of HMP kinase purification. Lane 1: Protein ladder (New England

Biolabs). Lane 2: Whole cell lysate. Lane 3: first wash. Lane 4: second wash. Lane 5: first fraction with elution buffer (200 mM imidazole). Lane 6: second fraction with elution buffer

(250 mM imidazole).

3.3 Discussion

Protein stability. In an effort to minimize the number of protein expression cycles and maximize the life expectancy of HMP kinase, different stability studies were carried out during the early commencing of the project. Initially, the purified HMP kinase was frozen at -20 oC in elution buffer prior transfer to -80 oC for long storage; however, after a single freeze/thaw cycle, a precipitate was visible due to either the long exposure to high concentrations of imidazole or to the lack of a cryogenic preservative. At this point, the need for a rapid buffer exchange was imperative post column purification. Two consecutive methods were executed in order to minimize the effect of imidazole that is well-known in the literature.40 A facile buffer exchange was accomplished first by dialysis at 4 oC over night against a phosphate buffer with no imidazole present. Fresh PBS buffer (pH 7.4) was changed at the second, third, and eight hour of the process. The solution was then concentrated via centrifugation at 4,000 g in an Amicon Ultra-15 centrifugal filter unit with a 10 MWCO (EMD Millipore). The order of steps for the buffer exchange protocol are vital since the reverse order, centrifugation followed by dialysis result in protein precipitation in the dialysis membrane due to the even higher amounts of imidazole present and the slow rate of diffusion at 4 oC. The dialyzed solution was then divided into aliquots and mixed with different amounts of glycerol in order to preserve the integrity of the protein during cryogenic storage and the freeze/thaw process. As indicated in Figure 3-4, the aliquots with at least 25% glycerol were able to sustain at least six freeze/thaw processes before significant reduction of enzyme activity; furthermore, the freezing step was found to be best by using liquid nitrogen instead of the slower process from -20 oC to -80 oC

HMPK Stability

1.2 1

M/min) 0.8 µ 0.6 0.4 0.2 0

Enzyme Activity ( 0* 2.5 5 10 25 Percent Glycerol

Figure 3-4. HMP kinase stability upon varying concentrations of glycerol. Enzyme activity

was measured once a month for a period of 6 months. (*) 0% stock was no longer viable

after the first freeze/thaw cycle

Protein concentration. Proteins usually show absorption maxima between 275 nm and 280 nm, which are caused by the absorbance of the two aromatic amino acids tryptophan (Trp), tyrosine (Tyr), and to a small extent, by the absorbance of cysteine

(Cys, i.e. disulfide linkages).41 Accordingly, the absorption coefficient (e) of a protein can be calculated by counting the number of its Trp, Tyr, and Cys disulfide bonds and using equation [1] as the linear combination of the individual contributions of these amino acids residues.42

� � �� = 5500 � � + 1490 � � + 125 � �

� � �� = 5500 � 5 + 1490 � 2 + 125 � 0

� � �� = 30480

Equation 1. Molar extinction coefficient for S. typhimurium. The amino acids were

computed from the PDB file 1JXH in the RCSB protein data bank website, no disulfide

linkages are present in the X-ray data. Extinction coefficients of HMP kinase were also

calculated for the organisms B. subtilis and E. coli, resulting in 11460 M-1cm-1 31970 M-

1cm-1 respectively.

With the molar absorptivity in hand, the concentration of the protein can be easily calculated by applying Beer-Lambert law. In one of the trials, the theoretical concentration of the protein was computed as 7.06x10-5 M where the experimental value was 7.54x10-5 M via Bradford assay; indicating the accuracy of the mathematical approximation within ±5% margin of error.

Chapter 4

Assay Development and Screening of HMP Analogs

Kinases, enzymes that transfer a phosphate group from one molecule (usually a nucleoside triphosphate) to another, are among the most famous and studied proteins in the life sciences. Although it is difficult to overestimate their diversity or to overstate their function, many of them play a more “blue collar” role in the energy metabolism; while others dictate the regulatory biochemistry of the human body, managing functions of units much larger and numerous than them.

Phosphorylations and dephosphorylations are major post-translational modifications that have a profound effect on protein activity and ultimately the human metabolism. These concepts were integrated into the biochemistry core in the 1950s by the major contributions of Fischer and Krebs; nevertheless, every year new kinases and their activities are still discovered at a high rate. It is these ground breaking discoveries that demand for practical and cost-efficient methods in order to monitor the kinase activity and fast-track the drug discovery pipeline.

The dominant approach to measure kinase activity has long been the direct detection of the phosphorylated product. The main advantage of such approach is specificity which inarguably demonstrates that a specific product is detected and constitutes strong evidence that one has measured the intended activity. However, the disadvantages are not trivial either. The product is usually not able to generate a signal on its own, leading to the addition of other reagents that will generate luminescence, fluorescence, ionizing radiation, or any other measurable readout upon product interaction.

A secondary alternative is the measurement of the consumption of the kinase substrate ATP. The “disappearance” of the nucleotide is easily detected spectroscopically through different commercially available enzyme coupled systems, such as the luciferin/luciferase set-up; resulting in a fast and replicable method without the need for molecular engineering. The disadvantage is the loss of specificity since any degradation of ATP will yield a signal in this system; therefore, ultra-pure and well-characterized components are required.

A similar but different strategy relies on ATP substrate as well. The “reverse” kinase assay is based on the conversion of the ADP byproduct from the phosphorylation step back to ATP. In this method an enzyme is forced to run opposite to the presumed physiological function and with positive DG. The principle of microscopic reversibility holds that any chemical reaction can be run in the reverse sense; therefore, virtually all enzymes are capable of catalyzing forward and reverse reactions. Exceptions occurs when the products are unstable or protein inactivation arises. The solely factor that dictates the flux of a particular product is, therefore, thermodynamics.

The use of large-scale compound screening has become a key component in drug discovery projects in both the pharmaceutical and biotechnology industries.43 High- throughput screening (HTS) processes constitute a major step in the initial drug discovery efforts and involve the use of large quantities of biological reagents, millions of compounds, and the utilization of expensive and innovative equipment; therefore, any optimization to make this process faster, more effective, and less expensive are in continual development.

In an effort to evaluate the activity of the already constructed first generation

HMP library, a rapid, efficient, and replicable method was highly desirable, and along with the recent isolated HMP kinase, it could serve the lab as a starting point for the

development of a structure-activity relationship (SAR) while leveraging our understanding of the enzyme’s catalytic site.

This chapter outlines each of the six assays and new technologies brought into the Foss lab that have advanced our knowledge into the substrate scope of HMP kinase and the plausible inhibitors that have a detrimental effect on bacterial survival. The list of both qualitative and quantitative assays are in chronological order as they were incorporated into the group’s analytical repertoire: (1) Qualitative assay, (2) chemiluminescence assay (3) UV-Vis assay, (4) HPLC assay, (5) in vitro whole-cell assay and metabolite detection assay via paired-ion electrospray ionization (PIESI) mass spectrometry.

4.1. Qualitative TLC assay

The below description relays the first assay carried out after every single batch purification of HMP kinase in order to ratify the non-denaturing conditions and activity of such for further quantitative analysis.

4.1.1 Materials and methods

Adenosine-5’-triphosphate disodium salt hydrate, Tris-HCl and buffer salts were purchased from VWR. All materials were used as obtained, unless otherwise indicated.

Thin layer chromatography (TLC) assay was performed on EMD Merck TLC silica gel 60

F254, 250 µm thickness. Purified HMP natural substrate and HMP analogs reactions were set up according to Figure 4-1. Reaction buffer included 40 mM Tris-HCl and 20 mM MgCl2 at pH 7.4. At selected time points, 10 µL sample aliquots were removed, heat- denatured at 85 oC for 30 seconds, and cooled down to 0 oC. The reaction mixture (1 µL) was spotted onto a silica TLC plate alongside the starting material and a reaction without

HMPK enzyme as negative control. After spotting the sample on the TLC plate, a heat gun was utilized to completely dry the plate prior to elution. The TLC plate was eluted in isopropanol, water, ammonium hydroxide system in a ratio 7:1:1 respectively. After elution, the TLC was dried once again and the reaction components were visualized under UV light and/or stained with ninhydrin for assay analysis.

HMPK, ATP (2.5 eq.) O O R4 Tris-HCl Buffer pH 7.4 R4 4-12 h, 37 oC P O P O N R5 N R5 O O

R2 N R6 R2 N R6

Figure 4-1. General reaction conditions for the in vitro phosphorylation of HMP analogs

by HMP kinase. Excess ATP was added assuming the double catalytic activity of the

enzyme was taking place.

4.1.2 Results

The enzymatic reaction was performed in a water bath at 37 oC for at least 4 hours before any product was noticeable. Figure 4-2 reveals a sample of the developed

TLC.

A) B)

1 2 3 4 5 HMP Analogs

Figure 4-2. A: TLC of HMP kinase reaction with natural substrate HMP. Line 1: control,

line 2: t = 1 h, line 3: t = 2 h, line 4: t = o/n, line 5: HMP-P standard. B: TLC of HMP

kinase reaction with different analogs, the red circle represents the new formed product.

Reagents are visible under UV light and no staining was required.

4.1.3 Discussion

Qualitative substrate screening was performed via thin layer chromatography to analyze product formation. Although this method does not corroborate the phosphorylated product, it is a good first-time assay to confirm the activity of the enzyme and to screen for the substrate scope of HMPK. The change in retention factor (Rf) for the alcohol substrate vs. the phosphorylated product is about 0.8 and no differentiation from the mono or diphosphorylated products can be validated after TLC elution. Ninhydrin staining was not needed in order to visualize the product formed.

The substrate scope of HMPK will be discussed in the next sections that include quantitative assays.

4.2 ADP-Glo chemiluminescence end-point assay

In the search for a more quantitative method where we could ratify the druggability of HMPK and the efficacy of the analog library, we focused our attention into commercially available assays that could give us a reliable, rapid, and replicable data.

The ADP-Glo kinase assay is a luminescence assay that provides a universal, homogeneous, and high-throughput method to measure the kinase activity by quantifying the amount of ADP produced in any given reaction. The ADP-Glo system can be used to monitor the activity of any ADP-generating enzyme (Kinase or ATPase). The end-point assay scheme is described in Figure 4-3 and it works in two steps; first, after the kinase reaction takes place, addition of ADP-Glo reagent will deplete the unreactive or remaining

ATP. Second, the kinase detection reagent is added in order to convert the generated

ADP back into ATP and this one will be substrate for the luciferin/luciferase reaction where the light output can be traced back to the activity of the original enzyme. The assay is sensitive enough to detect very low amounts of ADP (20 nM) and can detect

ADP in a reaction containing up to 1 mM ATP in a linear fashion which is beneficial for systems with low enzymatic turnover.

O COOH Luciferase N N N N OAMP

O S S O S S Luciferin Luciferyl adenylate ATP Pi

AMP

N N O hv (560 nm) O S S Oxyluciferin

Figure 4-3. Top: Schematic of the ADP-GloTM chemiluminescent assay (Promega)

involving the kinase reaction, ATP depletion step, ADP depletion step, and light readout.

Bottom: Reaction of luciferin/luciferase system contained in the kinase detection reagent.

Reaction proceeds once ADP is converted back to ATP and the light output is stable for

up to three hours.

4.2.1 Materials and methods

ADP-GloTM Kinase Assay can detect small changes in ATP to ADP conversion.

Whether assaying low activity kinases whose ATP turnover rate is low or using small concentrations of enzymes that produce minimal amounts of ADP, a high signal-to- background ratio must be obtained. In order to estimate the amount of ADP produced in the kinase reaction, a calibration curve was produced in order to correlate luminescence to the conversion of ATP to ADP. These conversion curves represent the amounts of

ATP and ADP available in a reaction at the specified conversion ratio (Table 4-1)

Table 4-1. Preparation of the 1mM series of ATP & ADP standards

Well Number 1 2 3 4 5 6 7 8 9 10 11 12

1 mM ADP 100 80 60 40 20 10 5 4 3 2 1 0

(mL)

1 mM ATP 0 20 40 60 80 90 95 94 97 98 99 100

(mL)

All the calibrations and enzymatic reactions were performed in 50 mM Tris-HCl buffer, 25 mM NaCl, 20 mM MgCl2, 0.5 mM DTT, and pH 7.4. The substrate scope of

HMP kinase was carried out by screening HMP analogs at a concentration 100 mM, 200 mM ATP, and 40 nM HMPK. All reactions were left running for at least 4 hours and at this point an equal volume (25 µL) of ADP-GloTM reagent was added to stop the kinase reaction and deplete the unconsumed ATP, leaving only ADP and a small background

ATP. The ADP-GloTM reagent was stirred at room temperature for an hour according to manufacture recommendations; which, at this point, a two equivalent volume of Kinase

Detection Reagent was added (50 µL) in order to convert the ADP back into ATP and introduce luficerin and luciferase to yield the light output. This reaction was let stir efficiently for at least forty minutes at room temperature before measuring the chemiluminescence in a BMG LabTech multiwall plate reader equipped with a 560 nm monochromator.

The screening of HMP inhibitors were carried out in the same fashion as above with HMP natural substrate concentration at the Km value (25 µM) in order to not oversaturate the enzyme and inhibitor concentrations at 200 µM to observe any mild activity present. ATP inhibitors were screened at concentrations 5x higher than its counterpart ATP.

4.2.2 Results

Substrate SAR. All HMP alcohol analogs were screened for catalytic activity and the results are summarized in Table 4-2 below. Every enzymatic reaction was done in triplicates and the same HMPK batch was utilized during the assays.

Table 4-2. Percent phosphorylation of HMP analogs by HMP kinase via chemiluminescence assay

Activity study for analogs with modifications at C5 position

NH2 R1 R2 N OH % phosphorylation

R1=H ; R2=H (HMP) 100±1

R1=CH3 ; R2=CH3 <1

R1=H ; R2=CH3 <1

R1=H ; R2=Ethyl <1

R1=H ; R2=Vinyl <1

R1=H ; R2=Pentyl <1

R1=H ; R2=Phenyl <1

Activity study for analogs with modification at C4 position

R4 N OH % phosphorylation

R4=NH2 (HMP) 100±1

R4=NHCH3 30±2

R4=N(CH3)2 <1

R4=OCH3 <1

R4=H <1

R4=Morpholine <1

Activity study for analogs with modification at C2 position

NH2 N OH % phosphorylation

R N 0

R0=CH3 (HMP) 100±1

R0=Ethyl 22±4

R0=OCH3 63±4

R0=SCH3 58±2

R0=NH2 130±16

R0=Phenyl <1

R0=H 10±6

Activity study for analogs with homologation at C5 position

NH2 % phosphorylation N nOH

n=1 (HMP) 100±1

n=2 <1

n=3 <1

Activity study for conformational restriction at C5

NH2 % Phosphorylation

N OH <1

Activity study for non-alcohol analogs

NH2

N X % Phosphorylation

x=OH (HMP) 100±1

x=NH2 35±1

x=NHCHO <1

2- x=PO3 (HMP-P) 50±5

Analog Inhibition SAR. HMP analogs including key synthetic intermediates were screened for inhibition at a concentration 10x higher than the concentration of HMP natural substrate ([HMP] = Km = 25 µM). Every enzymatic reaction was carried out in triplicates and with the same purified HMPK batch. Analogs that show catalytic activity were not able to be assayed for inhibition through the chemiluminescence method; therefore, a HPLC assay was developed in order to validate the current data (see section

4.4). Results are summarized in the following Figure 4-4.

HMP Analogs Inhibition Screening 120

100

Percent Phosphorylation Percent 40

111106105102102101100 98 98 98 98 98 98 96 96 96 95 95 95 94 91 91 86 85 85 83 75 73 71 71 62 56 55 42 10 0 B8 D8 A4 A6 B0 E9 A5 E0 C2 C7 B1 A3 D6 D9 B2 F1 F5 F2 F3 F4 C4 B9 B7 A7 F0 D3 B5 A0 C8 E6 E8 E2 D5 C3 E3 Analogs [200 µM]

NH2 NH2 O NH2 O NH2 O NH2

2- NH2 N OPO3 N N N O N N OH H N N N N N

E8 E2 D5 C3 E3

Figure 4-4. Percent phosphorylation of HMP natural substrate in presence of analog inhibitor. Compounds that show significant decrease in catalytic activity (60% and below)

were further analyzed for IC50.

IC50 calculation. Compounds E8, E2, D5, C3, and E3 were investigated further to determine IC50 values. HMP concentration was the same as before, at its Km value (25

µM). A log scale was utilized for the inhibitors ranging from concentrations of 1000 -

0.001 µM. The data is summarized in the table below.

Table 4-3. IC50 determination for the most potent inhibitors to date. All reactions were done in triplicates and value determined using OriginPro graphing program.

Analog Structure IC50 (µM)

NH2

2- E8 N OPO3 >200

NH2 O NH E2 N N 2 150 H N

NH2 O D5 N O 75

NH2 O C3 N 44

NH2 E2 N OH 22

ATP Inhibitors. Since HMP kinase belongs to the ribokinase superfamily with highly conserved ATP binding sites, a series of known ATP inhibitors were screened in order to evaluate the efficiency of such and to study the possible synergistic effect with the already active HMP analogs. The ATP inhibitors are shown in Figure 4-5:

O O O O O O O O EtO O O O O P O P P P HO O P P O EtO O O O O HO Cl Cl

Phosphonoacetic Diethylphosphonoacetic Etidronate Chlodronate acid (PAA) acid (DEPAA)

Figure 4-5. ATP inhibitors. Phosphonoacetic acid (PAA) and diethylphosphonoacetic acid

(DEPAA) are pyrophosphate analogs commercialized under the trade name Foscavir and

show antiviral activity via selective viral DNA polymerase inhibition. Etidronate and

Chlodronate are anti-osteoporotic drugs that inhibit specific mitochrondiral ATP-

dependent enzymes.44

Results of the effect of the aforementioned compounds are depicted in Figure 4-6 for both HMP, HMP-P, and E3 analog.

ATP Inhibitors in HMP Enzymatic Reaction

Control 100 DEPAA 95 PAA 91 Etidronate 104 Clodronate 101

80 85 90 95 100 105 110 Percent Phosphorylation

ATP Inhibitors in HMP-P Enzymatic Reaction

HMP-P Control 100

DEPAA 96

PAA 89

Etidronate 95

Clodronate 91 80 85 90 95 100 105 Percent Phosphorylation

Figure 4-6. Activity of commercially available ATP inhibitors. A: ATP inhibitors (100 µM)

were screened in the HMP natural substrate reaction along with ATP (50 µM) B:

Inhibition of the second catalytic step of HMPK by ATP inhibitors. The same

concentrations were used as above.

4.2.3 Discussion

Assay optimization. Before embarking in the high throughput spectroscopy assay, a series of optimizations were carried out in order to maximize the utility of the assay itself and the activity of HMP kinase. Substrate, enzyme, and DMSO concentrations were screened during the enzymatic reaction in order to minimize interference and variations between trials. The results are depicted in the figure below:

HMP Optimization

3.50E+06

3.00E+06

2.50E+06

2.00E+06 y = 4980.4x + 511981 1.50E+06 R² = 0.99314 1.00E+06

5.00E+05

Relative Light Units (RLU) Units Light Relative 0.00E+00 -100 0 100 200 300 400 500 600 [HMP] (µM)

HMPK Optimization

4000000 3500000 3000000 2500000 2000000 RLU 1500000 1000000 500000 0 0 0.5 1 1.5 2 2.5 3 [HMPK] (µM)

DMSO Tolerance

140000

120000

100000

80000

RLU 60000

40000

20000

Percent DMSO

Figure 4-7. A: Optimization of HMP natural substrate. B: Optimization of HMP kinase

enzyme. C: Tolerance of DMSO solvent in HMPK reaction

Given the results above, the optimal concentration of HMP was below 2 mM before any substrate inhibition was observed. The concentration of HMPK that fits within the linear range of the relative light units was found to be between 2.5 nM-40 nM at a 1 mM concentration of HMP. DMSO, interestingly, was found to be tolerant up to 7% of the reaction mixture volume before any interference with the light output was recorded; nevertheless, manufacturing recommendations state no more than 3% per well working volume.

Substrate scope. The results summarized in Table 4-2 demonstrate a specific yet interesting characteristic about HMP kinase. Several modification along the pyrimidine ring were included in a library of analogs; starting with the fifth position, different alkyl groups such as methyl, ethyl, pentyl, and dimethyl were installed in order to test the stearic factor at the catalytic site (C5-reactive head) of the heterocyclic ring. Vinyl and phenyl groups were added under the suspicion that they would have p-interactions with hydrophobic residues within the active site and should any interaction allow them to adopt the correct conformation for catalysis. However, HMP kinase proves to maintain a stearic requirement for its substrates at the fifth position and no significant phosphorylation was recorded for these modifications.

Elongation of the reactive head of HMP analogs; in other words, carbon homologation and conformational restriction did not result in any phosphorylation activity.

The C5 position exposes a very stringent requirement for catalysis, the hydroxyl group has to be only one carbon away from the pyrimidine ring and no other structural variance allows it to undergo phosphorylation.

The fourth position of the pyrimidine ring was screened based on stereoelectronic factors. Methylation of the exocylic amine reduces its phosphorylation capability significantly (32%) but dimethylation appears to disrupts one of the major hydrogen bonding networks with Glu44; therefore, rendering it unable to undergo catalysis. Substitution of the primary amine for a hydrogen atom resulted in no phosphorylation activity, leading to the conclusion that this functional group is of highly importance as a source of hydrogen bonding in the HMP kinase substrates. Increasing the bulk further yet creating a new source for hydrogen-boding, resulted in no appreciable catalysis.

In order to test the dual activity of HMPK, the monophosphorylated HMP substrate was screened. Keeping in mind that a conformational bond rotation is required in order to stabilized the substrate in the anion hole, our hope for HMP-P to bind into the binding site was proven right, and about half of the phosphorylated HMP underwent phosphorylation to HMP-PP as compared to HMP substrate. Given that HMP requires two equivalents of ATP in order to carry out the consecutive phosphorylations, it is not surprising that HMP-P only consumed half the equivalents of ATP, though this required slower time frame. Substitution of the 5-hydroxyl group to its isostere amine, surprisingly, resulted in 35% phosphorylation; amplifying, by definition, its catalytic function. The enzyme commission number is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. As a system of enzyme nomenclature, every EC number is associated with a recommended name for the respective enzyme. HMP kinase is classified as follows:

EC 2.-.-.-: Transferases (20,783 PDB entries)

EC 2.7.-.-: Transferring phosphorous-containing groups (12,237 PDB entries)

EC 2.7.1.-: Phosphotransferases with alcohol groups as acceptors (1,360 PDBs)

EC 2.7.1.49: Hydroxymethylpyrimidine kinase (4 PDBs)

Even though, there are phosphotransferases with amine groups as acceptors known in the literature such as histidine and arginine kinases, 45,46 this is the first evidence of a ribokinase member being able to phosphorylate a primary amine, further amplifying the range of substrates accepted by such members and also uncovering hidden pathways for which HMPK may be part of, within the microbial and/or plant metabolism.

Finally, the vast majority of compounds able to be phosphorylated by HMPK had modifications further away from the reactive center. The tail or C2 position of HMP analogs were modified with specific groups once the elongation of the methyl to an ethyl group and the presence of a phenyl moiety resulted in low to no phosphorylation.

Methoxy (bacimethrin) and thiomethyl groups at the C2 position were phosphorylated by about the same amount, 63% and 58% respectively. Furthermore, having an amino group increased the phosphorylation output by 30% higher than the natural substrate

(130% total). The need for a small and electron donating grouP at this carbon center increases the efficiency of phosphorylation, it may as well increase the binding affinity due to a new source of hydrogen bonding possibly with Met80 within the active site. The effect of having an electron donating vs. electron withdrawing group at this center is crucial not only for this catalytic step but also for the subsequent thiamine phosphate synthase step of thiamine’s biosynthetic pathway, as described by Begley et al.47 Where the current mechanism supports a SN1 step in the thiazole coupling process with HMP.

O O P O H H Mg+2 N O P O N O O S O N OH N P O O

O H H O P O Thiamine N H H Mg+2 phosphate synthase N O N N S P O N O N O S O O N OH N P O P OH O O O

Figure 4-8. Current mechanistic proposal for thiamin phosphate synthase. Electron

donating groups will stabilize the carbocation intermediate, leading to higher kcat values.

Substitution of the methyl group by a hydrogen at this position results in significant decrease in phosphorylation (10%) due to the electronic factors that are relevant to the alkyl group.

Computational docking studies were carried out in order to visualize the binding pose of such. The overlap of the substrate analogs indicates a similar binding attachment with the active site of the enzyme (average binding affinity: -4.5 kcal/mol). On the other hand, non-substrate analogs show a dysfunctional binding pose (Figure 4-9, B) where the alcohol group is significantly far away from g-phosphate of ATP.

In silico docking was performed using Autodock Vina run through Pyrx and the results visualized through PyMol and Discovery Studio. Ligands were prepared by generating a 3D structure in ChemBioDraw 3D, followed by an MM2 minimization and transferred into AutoDock Tools in order to assign Gasteiger charges, nonpolar

hydrogens, and setting up any torsional restrictions. HMPK crystal structure (PDB: 1JXH) was prepared using Discovery Studio in order to remove the co-crystallized natural substrate, sulfate ions, and water molecules. A 12-20 Å cubic search space was placed on the HMP’s binding pocket and the calculations ran with an exhaustiveness of 16 iterations.

Figure 4-9. A. Molecular docking of substrates HMP, HMP-P, B5, F5, and G4 analogs. B.

Molecular docking of HMP, 2-phenyl HMP, and 4-deasmino HMP. Drastic changes in the

binding pose are visible for all non-substrate analogs.

First generation inhibitors. Our HMP analog library was screened towards enzyme inhibition by the ADP Glo system were a decrease in the relative light units will yield the first generation inhibitors of HMPK. Compound E8, HMP-P, exhibits substrate inhibition since it competes with the binding site in presence of HMP (IC50 >200 µM)

Compounds E2, D5, and C3 show a specific acyl group at the fifth position where the most electrophilic carbonyl group exhibits the highest level of inhibition (IC50 = 150 µM,

75 µM, and 44 µM respectively). Finally, the best inhibitor to date is E3, where the conformational restricted allylic alcohol shows an IC50 of 22 µM.

This inhibition capability significantly decreases upon reduction of the double bond. Furthermore, if we look at all the analogs that comprise a substantial inhibition of the phosphorylation of HMP, the results are much clearer (See Table 4-4)

Figure 4-10. A: Docking of HMP and inhibitors E3, C3, D5, and E8. Overlap is consistent

with the substrate analogs. B: Ligand binding interaction is conserved with major

hydrogen bonding interactions with Glu44, Gly11, Met80, and Gly210 residues.

Table 4-4. HMPK inhibitors with milder inhibition potency.

Analog Structure Percent Inhibition (PI)

NH2 OH 29 E6 N

N NH2 O

C8 N O 29

N NH2 O

A0 N OH 27

N NH2

B5 N NH2 25 N NH2

D3 N OH 14

Homologated analog E6 shows the next highest inhibition, followed by allylic alcohol derivatives (C8 and A0). In these cases, the presence of the conformational restricted feature allows for higher inhibition vs. its reduced counterpart (D3). Primary amine analog B5 is the only substrate analog that also prevails in the inhibition screening by competing with HMP during the catalytic process. The one carbon homologation with the same unsaturated feature as E3 (vinylic vs allylic alcohol) has not been yet synthesized by the group and it must be screened in order to prove the correlation between homologation and unsaturation. The second generation inhibitors are under way

along with some functionalities from the substrate SAR. More of this topic will be covered at the end of this chapter.

ATP inhibitors. The idea of screening ATP inhibitors came from the dual catalytic function of HMP kinase. Taking advantage of two complete different binding conformations and the possible formation of an intermediate with a tight binding could result in a suicidal inhibitor. The addition of an ATP inhibitor would either have an agonistic or antagonistic effect in either case.

Etidronate, chlodronate, phosphonoacetic acid, and diethylphosphonoacetic acid were screened in the HMP kinase reaction. From Figure 4-6, it is clear that these ATP analogs have no difference in the RLU output over the control reaction; however, they have a significant effect over the secondary phosphorylation of HMP-P, specially chlodronate and phosphonoacetic acid. A possible explanation to this occurrence can be found in the mechanism for which HMPK carries its catalytic activity. HMP kinase is believed to follow an ordered sequential mechanism, where HMP substrate binds first followed by an ATP molecule. Once the first phosphorylation occurs, ADP leaves, HMP-P rearranges inside the active site and a second molecule of ATP arrives to generate the final HMP-PP product. Since the known ATP analogs have only a small effect on the second step within the overall reaction, it is likely that they inhibit the binding of the monophosphorylated intermediate by destabilizing the attachment into the anion hole of the protein.

The same ATP inhibitors were screened along with the first generation HMPK inhibitors; but to our dismay, they did not generate a synergistic effect. Compound E3, allylic alcohol, extends further away towards the C-5 center; therefore, disrupting the binding of the phosphate analogs, rendering them inoperable.

4.3 Enzyme coupled kinetic assay

In an effort to look deeper into the chemical steps of the active site, the Foss group focused its attention in kinetic assays for the purpose of complementing the ADP

Glo assay along with kinetic data for the enzyme substrates. Figure 4-11 shows the schematic of pyruvate kinase and lactate dehydrogenase enzyme coupled assay. The

ATP-dependent assay consists in three steps. The ATP depletion by HMPK catalysis will activate the dephosphorylation activity of pyruvate kinase by means of ATP regeneration.

Although this step is non-pontaneous, the lactate dehydrogenase step is; therefore, the overall coupled system has a DG < 0. The reduction of pyruvate occurs rapidly by means of oxidation of nicotinamide adenine dinucleotide (NADH) and therefore the absorption maximum at 340 nm reduces over time since the newly formed NAD+ is aromatic in nature.

H R H R N 4 N 4 HMPk 2- N OH N OPO3

R2 N R2 N ATP ADP

O OPO 2- O O O 3 PK O NADH

NAD+

O OH

Figure 4-11. Schematic of the pyruvate kinase/lactate dehydrogenase enzyme-coupled

assay and HMP kinase. NADH depletion is monitored at lmax: 340 nm

4.3.1 Materials and methods

Enzyme activity was measured spectrophotometrically at 25 oC by the pyruvate kinase/lactate dehydrogenase enzymes from rabbit muscle (Sigma Aldrich). The ATP- dependent consumption of NADH was monitored continuously at 340 nm using a

SPECTROstar Nano plate reader (BMG Labtech). Catalytic activity was determined in 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 5 mM MgCl2, 0.5 mM NADH, 0.5 mM phosphoenolpyruvate, 0.5 mM ATP, 6 U pyruvate kinase, 10 U lactate dehydrogenase,

0.5 µM purified HMPK, and various concentrations of HMP and other substrates.

Substrate scope. All of the plausible substrates were dissolved in DMSO and saturating concentrations (1mM) were used to study the catalytic activity of HMP kinase.

Final concentration of DMSO was kept under 10% of overall working volume (200µL).

The reaction mixture was set up as above, HMP was replaced with each individual analog and ran in duplicates. The reaction was incubated for ten minutes at 25 oC and the cascade initiated by the addition of ATP

4.3.2 Results

In order to obtain kinetic information regarding the substrates, a range of substrate concentrations were screened. Substrates were studied from concentrations of 2 mM and serial diluted to 8 µM. Figure 4-12 displays a typical plot from obtained from initial rates of reactions and Table 4.5 shows its corresponding Michaelis-Menten approximation derived from it.

HMP Saturation Curve

1400

1200 y = -1.6183x + 1151.5 R² = 0.96821 1000 y = -2.2231x + 1152.6 800 R² = 0.97457 y = -3.2095x + 1198.5 600 R² = 0.98077 [NADH] y = -4.3905x + 1185.9 400 R² = 0.99126 y = -5.7138x + 1239 200 R² = 0.99784 y = -6.1388x + 1252.2 0 R² = 0.9899 0 50 100 150 200 Time (m)

Figure 4-12. Top: Phosphorylation of HMP via enzyme coupled assay and monitored

through NADH oxidation at 340 nm. [HMP] 2 mM through 8 µM. Bottom: Michaelis-

Menten fitting to the initial rates of reaction

Table 4-5. Kinetic values obtained for the substrate analogs of HMP

-1 HMP analog Km (µM) Vmax kcat (s )

(µM/minute)

NH2 20 6.4 0.19 N OH

NH2 O P N O O 62 1.3 0.22 O N

NH2 N OH 121 4.5 0.39

MeO N

NH2 N OH 152 4.0 0.37

MeS N

NH2 N OH 280 7.6 0.97

H2N N

NH2 30 2.5 0.21 N NH2

4.3.3 Discussion

The PK/LDH enzyme coupled assay served useful in defining the kinetic parameters for the substrate scope of HMPK. The analogs were tested for pyruvate kinase and lactate dehydrogenase inhibition before the start of the study. None of the analogs were found to be disruptive for the consumption of NADH up to concentrations of

5 mM. Concentrations of ATP, PK, and LDH were optimized previously in triplicates in order to obtain a linear range towards the consumption of NADH.

The lowest Km values correspond to the HMP natural substrate and the only other analog with modification in the fifth position, the aminomethyl-HMP. The monophosphorylated HMP exhibits higher Km and slower rate, presumably due to the stereoelectronic incumbency that it must overcome to access the binding site while having a negatively charged group. Methoxy-HMP and thiomethyl-HMP show similar parameters with high Km values and yet almost identical rates (kcat).

Amino-HMP showed the biggest surprise, since it catalyzes almost 30% percent more than the natural substrate (ADP Glo assay), it also shows the largest Km value, yet the highest Vmax and turnover number. The theory that a non-chemical step involved in the slow release of the product may be taking place; therefore, it is affecting the enzyme affinity for such compound.

4.4 High performance liquid chromatography assay

One of the major disadvantages of the enzyme-coupled assay is the indirect measurement of HMP kinase substrates and products. As long as ATP is consumed, the reaction cascade initiates, leading to oxidation of NADH and an ambiguous read out. In order to validate the previous spectroscopy assays, our group embarked in developing a high performance liquid chromatography (HPLC) assay, which could directly measure intermediates and products. Hydrophilic interaction liquid chromatography (HILIC) has been shown successful in the separation and quantification of nucleotides; therefore, we focused our attention in attempting to separate and quantify the consumption of ATP in our kinase reaction via this method.48,49

Figure 4-13 shows the reaction adducts from the HMP kinase reaction with its natural substrate. The best baseline separation was achieved via FRULIC-N column

(AZYP, 4.6x150 mm, 5 µm) with a mobile phase consisting of acetonitrile/25mM ammonium acetate gradient (up to 75/25) and a flow rate of 1.0 mL/min. However, after several trials with HMP analogs, no baseline separation was achieved, rendering errors in the quantifications of the product.

Figure 4-13. Reaction adducts from the HMP kinase and HMP natural substrate after 10

hours of reaction time. Reagent standards controls were recorded at the given retention

times. Column: FRULIC-N 4.6x150 mm, flow rate 1.0 mL/min

Given the fact that we were unable to attain baseline separation with the rest of the enzymatic reactions and that the reaction products are negatively charged, we attempted the separation via paired-ion liquid chromatography. In this type of chromatography, an ion-pair reagent such as tetrabutylammonium hydrogen sulfate is added to the mobile phase and allowed to come to equilibrium with the reverse phase column. The alkyl groups of the ion-pair reagent will be held strongly by the nonpolar stationary phase (C18), leaving the charged functional ammonium ion protruding into the mobile phase. At this point, the negatively charged species can be attracted to the immobilized ion-pair reagent, providing chromatographic retention.

4.4.1 Material and methods

An enzymatic reaction consisting of 200 µM HMP and/or analog, 400 µM ATP, and 500 nM HMPK were prepared in 50 mM Tris-HCl buffer containing 20 mM MgCl2 and

1 mM DTT (pH 7.4) and run overnight at 37 °C. The reaction was terminated via heat denaturation (90 oC for 60 seconds) followed by ice bath (5 minutes) and the protein precipitated by centrifugation (12,000 rpm for 10 minutes). The supernatant was filtered using a Spin-X 0.22 µm centrifuge cellulose tube filter (Costar) and analyzed via ion- paired chromatography. Samples (2 µl) were injected on a C18 reverse phase column

(Phenomenex, Luna 5u C18(2) 100 A 250 mm x 4.60 mm) at a flow rate of 1 ml/min. ATP depletion was monitored at 242 nm. Retention times were confirmed by NMR verified standards. The HPLC method is illustrated in the table below:

Table 4-6. HPLC gradient protocol for the separation and elution of HMP kinase reaction

Minutes % buffer(b) % methanol % 4mM TBAHS(a) 0 75 15 10

14 25 25 50

16 50 40 10

18 75 15 10

30 75 15 10 (Reconditioning) (a) TBAHS: tetrabutylammonium hydrogen sulfate. (b) buffer = 100 mM phosphate buffer (pH 6.6), containing 4 mM TBAHS.

4.4.2 Results

The order of elution from a reverse phase C18 column under ion-pairing conditions is identical to that observed under normal phase conditions; HMP analogs are eluted first followed by their phosphorylated forms, then ADP, and finally ATP. As seen in

Figure 4-14, the reaction adducts were separated accordingly and the level of phosphorylation quantified based on the amount of ATP consumed from the calibration curve below:

ATP calibration curve

600 y = 1.5286x R² = 0.99513 400 200

% Rel. area 0 0 50 100 150 200 250 300 [ATP] (µM)

Figure 4-14. HPLC assay. Top: ATP calibration curve monitored at 242 nm wavelength.

Bottom: HPLC elution at time = 0 and time = 10 hours for the enzymatic reaction of HMP.

ATP calibration curve and monitoring determined the amount of phosphorylation taking

place in the reaction mixture. All reactions were run in triplicate.

The rest of the substrate analogs were screened in the same fashion and the results compared to the ADP Glo assay in order to confirm the validity of the chemiluminescence one. The comparison is listed in table 4-7 below:

Table 4-7. HMP analog phosphorylation comparison between ADP Glo and HPLC assay

Substrate ADP Glo (Percent HPLC (Percent

phosphorylation) phosphorylation)

NH2 100±2 100±10 N OH

NH2 N OH 58±2 65±14

MeO N

NH2 N OH 63±4 56±11

MeS N

NH2 N OH 130±16 155±20

H N N 2

NH2 35±10 59±10 N NH2

NH2 O P N O O 47±5 58±18 O N

4.4.3 Discussion

Ion-pair liquid chromatography successfully resulted in the separation of the HMP kinase adducts. Tetrabutylammonium hydrogen sulfate is a good ion-pair reagent that enables the separation of negatively charged species, conferring them a sort of non-polar behavior. The measurement of the degree of phosphorylation was chosen to be monitored via ATP degradation since some of the phosphorylated analogs will co-elute at the same retention time as ADP, leading to miscalculations.

Overall, the results from HPLC corroborated the phosphorylation data from the chemiluminescence assay. Methoxy-HMP and thiomethyl-HMP phosphorylate about the same amount in relationship to HMP natural substrate. HMP-P also was able to enter into the active site and undergo phosphorylation about half the amount as HMP. 5- methylamino-HMP will phosphorylate the least compared to all the analogs, and 2-amino-

HMP will phosphorylate the highest. 2-amino-HMP shows again the rapid efficiency for phosphorylation. The presence of the monophosphorylated specie was not detected through the HPLC method and the assumption that the intermediate never leaves the active site persists, likely because of the electrostatic interaction with the anion hole is very high; -5.8 kcal/mol for HMP-P vs. -4.5 kcal/mol for HMP according to docking studies.

Although the present assay is based on ATP depletion; once again, it corroborates the formation of a phosphorylated specie by the appearance of a “new” peak and the increase of ADP concentration. It was our interest to get direct confirmation and to be able to detect this newly formed species my mass spectrometry. Unfortunately, the HPLC method was incompatible with MS due to the suppression of ionization by the phosphate buffer and ion-pair regent in the mobile phase.

4.5 In vitro whole cell assay and metabolite detection

Having developed a SAR within the substrate scope of HMPK, we focused our attention into screening such compounds in whole-cell systems. Bacimethrin, a known prodrug that is able to incorporate itself into the thiamine biosynthetic pathway at the

HMPK stage, undergoes all subsequent de novo reactions, yielding the non-natural methoxy-thiamine. Therefore, it was our belief that the rest of HMP analogs with modifications at the C-2 position would be able to integrate themselves into the cell and go through the reaction cascades that lead to the nonfunctional thiamine analog, disrupting the thiamine dependent enzyme functions.

A series of bacterial strains were chosen in order to screen the antibiotic susceptibility of HMP analogs. A rapid BLAST amino acid sequence yielded the percent identity in table 4-8. Identity is defined as the extent to which two nucleotide or amino acid sequences have the same residues at the same positions in an alignment. The bacterial candidates were both Gram-positive and Gram-negative in nature: E. coli, P. aeuriginosa, K. pneumoniae, S. aureus, and M. smegmatis; a fast-growing, non- pathogenic homologous of M. tuberculosis.

Table 4-8. Percent identity of amino acid sequence for HMP kinase among different bacterial strains. BLAST (protein-protein) for HMPK amino acid sequence

Species Gram (+) / Gram (-) Percent Identity S. aureus NCTC 8325 (+) 44

M. smegmatis (+) 45

M. bovis BCG (+) 39

E. faecalis (+) 42

S. pneumoniae (+) 46

S. epidermidis (+) 44

C. difficile (+) 42

K. pneumoniae 13883 (-) 90 ATCC

P. aeuriginosa PA01 (-) 37

E. coli K12 (-) 91

P. falciparum n/a 27

Salmonella enterica (-) 100

In order to confirm the phosphorylation events from all the previous studies, including the whole cell assay; mass spectrometry was highly needed in our pharmacophore pursuit. Due to ionization suppression, the method developed in the

HPLC assay could not be applied to the liquid chromatography-mass spectrometry (LC-

MS) instrument. At this moment, we turned our attention into a similar ion-pair chromatography techinque. Paired Ion Electrospray Ionization-mass spectrometry

(PIESI-MS) involves introducing low concentrations of structurally optimized ion-pairing reagents (IPRs) into the sample flow, thereby allowing the anions to be measured with

high sensitivity in the positive ion mode as the anion/IPR associated complexes.50 Figure

4-15 shows instrumental configuration of HPLC-PIESI-MS

Figure 4-15. Configuration of HPLC-PIESI-MS for detection of negatively charged

molecules in positive mode.51

With a new analytical technique in hand, the detection of the phosphorylation activity in the high throughput assays along with the metabolites from the whole cell assays would be facilitated.

Figure 4-16. Charged species detected via PIESI-MS. The overall charge of the complex

must be positive in order to be detected in the positive mode of the mass spectrometer.

4.5.1 Materials and methods

Antibiotic susceptibility. Cultures of E.coli K12, P. aeuriginosa PA01, K. pneumonia ATCC 13882, S. aureus NCTC 8325, and M. smegmatis MC2 155 were inoculated in Mueller Hinton Broth (Becton Dickingson, Franklin Lakes, NJ) and grown overnight at 37 oC with medium agitation (200 rpm). The overnight cultures were then diluted (1:1000) into DifcoTM M9 minimal media (BD, Franklin Lakes, NJ) prepared according to manufacturing recommendations with 20% glucose as the only carbon source. The inocula were transferred into a 96-well plate containing the compounds to be tested. Bacterial growth was monitored spectrophotometrically (OD600) with a

SPECTROstar Nano plate reader (BMG Labtech, Cary, NC). A ten-fold serial dilution of each compound was tested in duplicate. Negative controls were run without the presence of any analog for each bacterial strain. Sulfamethoxazole and isoniazid were used as positive controls.

PIESI. The solvents used for the analysis were MS-grade and were purchased from

Honeywell Burdick and Jackson (Morristown, NJ). The cationic reagent 1,3-Propanediyl- bis(tripropylphosphonium) difluoride (BTPP) was purchased from Sigma-Aldrich (St.

Louis, MO). Solutions of anions were prepared at a concentration of 10 µg/mL. The concentration of the ion-pairing reagent was 10 µM. The ion-pairing reagent was introduced to the Shimadzu LCMS-8050 from a Shimadzu LC-6A pump (Shimadzu,

Columbia, MD) at a flow rate of 100 µL/min. The ESI-MS conditions were set as follows:

Spray voltage of 3 kV, capillary temperature of 350 oC, capillary voltage of 11 kV, sheat gas flow at 37 AU, and the auxiliary gas flow at 6 AU. Methanol/water solution (1:1) and the ion-pairing reagent were introduced into a mixing tee before entering the mass spectrometer at a total flow rate of 0.4 mL/min. The samples were injected through a sample loop of 5 µL and SIM mode was utilized for the respective mass to charge ratios.52

Metabolite extraction. 1 mL culture of E. coli K12 was grown as described above until it reached an OD600 = 0.6 in M9 minimal media. At this point, HMP analogues were introduced for a final highest concentration of 1 mM. The cultures were incubated for an additional 10 hours at 37 oC. At this point, the bacteria were pelleted by centrifugation for

5 minutes at 5,000 x g, the supernatant was removed, and the cells suspended in 150 µL of 40:10 methanol:water. The suspension was immersed in dry ice-acetone bath for 15 minutes followed by centrifugation at 12,000 rpm for 10 minutes at 4 oC. The clear extract was removed and the process repeated two more times. The final extraction included sonication in an ice bath for 15 minutes at 42 kHz and all the fractions combined.

The collected extracts were filtered in a Spin-X 0.22 µm centrifuge cellulose tube filter in order to remove all the remaining cell debris. Positive culture controls contained HMP

and commercial available thiamine hydrochloride. All trials were run in triplicates and the metabolites measured as described above in PIESI.

4.5.2 Results

Antibiotic susceptibility. The substrate analogs of HMP resulted successful towards the growth inhibition of the bacterial strains. It is worthy to mention that the compounds of interest were inactive in rich media such as LB or Mueller Hinton broths.

However, when the bacteria strains are grown in minimal media, the effect of HMP analogs are detrimental for the survival of the cell. The results are outlined in Figure 4-17.

Figure 4-17. Minimum inhibitory concentration (MIC) for different bacterial strains.

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Analogs thiomethyl-HMP, methoxy-HMP, and amino-HMP show activity towards growth inhibition in both gram positive and negative species. The minimal effective concentrations that inhibits the growth in each species ranges from low micromolar (1

µM) up to 1 mM concentration. The efficiency trend is as follows: thiomethyl-HMP < methoxy-HMP < amino-HMP. The other substrate analogs of HMPK and/or in vitro inhibitors showed no growth inhibition in the whole-cell system. The 2-amino-HMP analog was the only candidate effective against M. smegmatis.

Having proof of concept that the HMP analogs were effective towards bacterial growth inhibition, the number of action mechanisms are immeasurable. Nevertheless,

PIESI allowed us to determine the possible fate of such analogs by means of detection of the metabolites from the thiamine biosynthetic pathway.

Figure 4-18. HMP-PP detection from the in vitro enzymatic reaction. The HMP-PP adduct

and ion pair form a complex with m/z of 660.300

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Figure 4-19. Plausible HMP analogs metabolites from in vitro whole-cell assay that are detected via PIESI-MS. Neutral (HMP) and overall positively charged (thiamine) species

do not require pair ion for their detection.

102

Table 4-9. Metabolites detection via PIESI-MS from E. coli K12 strain.

R Intermediate Formula MW (g/mol) Detected m/z

in PIESI1

mode

HMP C6H9N3O 139.0 140.1

HMP-P C6H9N3O4P 218.0 580.3

HMP-PP C H N O P 298.0 660.3 -Me 6 10 3 7 2

Thiamine C12H17N4OS 265.1 265.1

TMP C12H16N4O4PS 343.0 705.4

TPP C12H17N4O7P2S 423.0 785.3

SMe-HMP C6H9N3OS 171.0 172

SMe-HMP-P C6H10N3O4PS 250.0 612.3

SMe-HMP-PP C H N O P S 329.9 691.3 -SMe 6 10 3 7 2

SMe-Thiamine C12H17N4OS2 297.0 297.1

SMe-TMP C12H16N4O4PS2 375.0 737.4

SMe-TPP C12H17N4O7P2S2 455.0 817.3

OMe-HMP C6H9N3O2 155.0 156.1

OMe-HMP-P C6H9N3O5P 234.0 596.3

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OMe-HMP-PP C6H10N3O8P2 313.9 675.3

-OMe OMe-Thiamine C12H17N4O2S 281.1 281.1

OMe-TMP C12H16N4O5PS 359.0 721.4

OMe-TPP C12H17N4O8P2S 439.0 801.3

NH2-HMP C5H8N4O 140.0 141.1

NH2-HMP-P C5H8N4O4P 219.0 581.3

NH2-HMP-PP C5H9N4O7P2 298.9 660.3 -NH2

NH2-Thiamine C11H16N5OS 266.1 266.1

NH2-TMP C11H15N5O4PS 344.0 706.4

NH2-TPP C11H16N5O7P2S 424.0 786.3

1. Ion pair used: BTPP, molecular weight = 362 g/mol

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4.5.3 Discussion

HMP analogs were unable to assert any biological activity in Mueller Hinton broth possibly due to the myriad of components found in the beef infusion and casein acid hydrolysate from where it is made. To further isolate de novo synthesis targets, M9 minimal salts were chosen and tested as antibiotic susceptibility media. HMP analogs showed a bacteriostatic activity when tested against different bacterial strains as seen from the growth curves and no evidence of bactericidal activity was found. At this point, we aimed to prove the theoretical pro-drug mechanism inherent by these analogs via mass spectrometry.

Paired-ion electrospray ionization mass spectrometry (PIESI) resulted successful in the detection of the negatively charged species from the HMP kinase reaction. These adducts were unable to be detected via electrospray ionization mass spectrometry (ESI-

MS) and it is known that the limits of detection (LODs) for this technique are sometimes inadequate for such measurements53. When operating in the negative ion mode with standard chromatographic solvents (primarily water, methanol and acetonitrile), the corona discharge is more prevalent, which leads to an unstable Taylor cone and ultimately provokes signal instability and poor limits of detection54. PIESI introduces an ion pair reagent that allows the species to be detected in the more sensitive positive mode. The anionic analyte is also brought into a much higher mass range (analyte-ion pair complex), where the chemical noise is greatly reduced.

The detection of metabolites from each and every HMP analog proves the admission of such compounds into the cells, possibly via a transporter protein,55 and integration within the thiamine biosynthetic pathway. The amino-HMP analog obtained the highest efficacy towards growth inhibition and it is not surprising since such analog is

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also able to phosphorylate at a higher rate than its natural substrate counterpart in the

HMPK system; thereby, validating the proposed dissociative mechanism upon attachment with the thiazole complement (see Figure 4.8). Amino-HMP was also the solely agent effective against M. smegmatis, possibly due to the likely higher specificity of such compound towards the transporter protein.

4.5.4 Conclusion and future approach

The course of the HMP project commenced with the synthesis of small molecule analogs; however, with the need to screen the multitude of compounds, I focused my attention into developing quick, high-throughput, and reliable techniques for the monitoring of the enzyme activity/inhibition. The substrate scope of the enzyme, although stringent against steric modifications in the fourth and fifth position of the pyrimidine ring, it is amenable to small isostere changes in the second and fifth position. Furthermore, these non-natural substrates are able to infringe a crucial metabolic disruption via formation of thiamine analogs according to LC-PIESI-MS data. These pro-drug active compounds were found serendipitously and have yielded a new area of study towards the targeting of riboswitches and vitamin pathways as sources of novel antibacterials.

The first generation HMPK inhibitors, although they were not fully studied in terms of inhibitory mechanisms, reveal a fundamental conformational restriction and a conserved p system in the fifth position that yields moderate micromolar inhibition. To our dismay, these compounds failed to disrupt any bacterial growth in the whole-cell assays.

For the next generation analogs, I would like to see validated the capabilities of the long forgotten Michael acceptors, as in the case of rugulactone, in order to covalently modify HMP kinase but also taking advantage of the 2-amino feature in the pyrimidine ring, due to its high rate of reaction and enzyme affinity.

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Since HMPK exhibits the dual phosphorylating function, all the alcohol analogs synthesized in our lab should be phosphorylated and assayed towards a transition state approach. Preliminary data shows the efficacy of some ATP analogs, especially in the second phosphorylating step of the enzyme; therefore, synthetic tailoring of such compounds with known ATP analogs may give light to a whole new class of ligand-based family of compounds.

Lastly, the exploration of thiamine analogs or compounds that are able to incorporate themselves into thiamine’s biosynthetic pathway should never cease. Due to the promiscuity of the ribokinase family members, more compounds not limited to pyrimidine systems should be assayed towards substrate analysis and their corresponding metabolites detected via PIESI-MS.

“THE FUTURE OF HUMANITY AND MICROBES WILL LIKELY EVOLVE…AS

EPISODES OF OUR WITS VERSUS THEIR GENES”

DR. JOSHUA LEDERBERG

1958 Nobel Prize in Physiology or medicine

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Chapter 5. Experimental

5.1 General procedures

Reagents were purchased in the purest commercial form from Sigma Aldrich or Alfa

Aesar. Anhydrous solvents were purchased from EMD drisolv. Line of solvents through

VWR. All the reagents were used as obtained from the commercial vendors unless otherwise specified. 300 MHz 1H NMR and 75 MHz 13C NMR analysis were performed in a JEOL ECX 300 instrument. 500 MHz 1H NMR and 125 MHz 13C NMR analysis were performed in a JEOL eclipse Plus 500 instrument. Chemical shifts were recorded in reference to residual solvent peaks (DMSO-d6 residue = 2.50 ppm, CDCl3 residue = 7.26 ppm, D2O = 4.79 ppm, and CD3OD = 3.31 ppm). The following abbreviations indicate the peak multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, qu = quintet, and bs = broad singlet. Chromatography was performed on P60 silica gel (mesh

230-400) and TLC plates were purchased from Merck, F254, and 250 µm thickness. High resolution mass spectrometry was analyzed in the Shimadzu Center for Advanced

Analytical Chemistry (SCAAC) at UTA via the Shimadzu LCMS-IT-TOF mass spectrometer. IR analysis was recorded with neat samples in a Shimadzu FT-IR.

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4-Amino-2-methylpyrimidine-5-carbonitrile (1): Acetamidine hydrochloride (9.45 g, 100 mmol) was slowly added to a (~2.3 M) solution of sodium ethoxide, prepared by adding sodium (3 g) to dry ethanol (60 mL) at 0 °C. The solution was filtered through Celite and the filtrate mixed with ethoxymethylenemalononitrile (6.10 g, 50 mmol). The dense yellow precipitate which formed immediately, (slightly exothermic) was swirled for 20 min to produce a paste, which was filtered and washed with cold methanol followed by ether to obtain a yellow solid (4.76 g, 71%) after drying.

The material was recrystallized from hot ethanol; m.p. 247-248 °C (lit 63 250-251 °C); IR

(neat, cm-1) 3381, 3334, 3017, 2978, 2849, 2757, 2666, 2224, 1674, 1281, 1240, 958;

1H NMR (300 MHz, DMSO-d6) δ 8.50 (s, 1H), 7.78 (bs, 2H), 2.38 (s, 3H); 13C NMR (75

MHz, DMSO-d6) δ 169.9, 162.2, 160.9, 115.6, 86.5, 25.8. HRMS (ESI-TOF) m/z calculated for C6H7N4 [M+H] + 135.0665, found 135.0659.

4-Amino-5-carboxy-2-methylpyrimidine (2): 4-Amino-2- methylpyrimidine-5-carbonitrile (1) (2.5 g, 19 mmol) was refluxed for 2 h in 20 mL of 10%

KOH (aq.) and the solution filtered hot and acidified with glacial acetic acid resulted into a precipitate on cooling. The precipitate was filtered and washed with cold water and dried under vacuum to afford the acid as light yellow needles (2.6 g, 90%). Recrystallized from water to give fine needle-like crystals; m.p. 272-273 °C (lit 64 270-270.5 °C); IR (neat, cm-1) 3224, 3079, 2351, 1960, 1661,1567, 1316; 1H NMR (500 MHz, DMSO-d6) δ 8.61

(s, 1H), 7.79 (bs, 2 H), 2.38 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 165.8, 163.8,

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162.9, 148.3, 103.8, 21.2; HRMS (ESI-TOF) m/z calcd for C6H8N3O2 [M+H] + 152.0466, found 152.0467.

Methyl-5-carbomethyoxy-6-aminopyrimidine (3): The amino acid (2)

(5.0 g, 33 mmol) was dissolved in 15 g of concentrated sulfuric acid and was warmed gently at 50 °C. A mixture of methanol (20 mL) and sulfuric acid (4 g) was added. The resulting solution was refluxed at around 110°C until it turned clear (~ 30-45 min).

Methanol (20 mL) was again added and refluxed for an additional 45 min. The resulting dark yellow/ brownish mixture was cooled, poured into ice-water and neutralized with

Na2CO3 until the pH~7-8. The precipitated ester was filtered and washed with cold water

(120 mL) and dried to afford (3.8 g, 73%) of faint yellow solid; m. p. 175-177 °C; IR (neat, cm -1) 3434, 3264, 3022, 2951, 2851, 1688, 1630, 1439, 1234, 1102; 1H NMR (500

MHz, CDCl3) δ 8.80 (s, 1H), 7.79 (bs, 1H), 6.05 (bs, 1H), 3.89 (s, 3H), 2.53 (s, 3H); 13C

(125 MHz, DMSO-d6) δ 170.5, 166.1, 162.0, 158.9, 101.4, 51.9, 25.7; HRMS (ESI-TOF) m/z calculated for C7H10N3O2 [M+H] + 168.0768, found 168.0756.

Amino-5-hydroxymethyl-2-methylpyrimidine (4, HMP): Diethyl ether

(200 mL) was added to (1.5 g, 40 mmol) of lithium aluminum hydride at 0 °C. Then (5.0 g,

30 mmol) of 2-methyl-4-amino- pyrimidinecarboxylate (3) was dissolved in THF (100 mL) was added slowly. The reaction was allowed to attain room temperature and stirring was

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continued for 12 h. The reaction was quenched with 2-3 mL of NH4Cl at 0 °C and stirred for 30 min, following which the slurry was filtered using Celite. After washing the filter bed with 50 mL of a mixture of ethyl acetate and methanol (2:1), the solvents were removed under reduced pressure. The crude material was purified by flash silica gel chromatography with hexane/ethyl acetate/methanol (50:40:10), Rf = 0.35, to obtain the product as a faint yellow semisolid mass. The product then crystallized out as a white powder, (2.6 g, 62%) from a mixture of methanol and ethyl acetate (~1:9); m.p 191-193

°C (lit 63 193-194 °C); IR (neat, cm-1) 3427, 3384, 3286, 3198, 3005, 2971, 2872, 2729,

2682, 1687, 1599, 1000; 1H NMR (300 MHz, DMSO-d6) δ 7.92 (s, 1H), 6.52 (bs, 2H),

5.10 (t, J = 5.1 Hz, 1H), 4.30 (d, J = 5.5 Hz, 2H), 2.29 (s, 3H); 13C NMR (75 MHz,

DMSO-d6) δ 165.8, 161.9, 153.3, 114.1, 58.2, 25.6; HR-MS (ESI-TOF) m/z calcd for

C6H10N3O [M+H] + 140.0818, found 140.0814.

Ethyl 4-amino-2-(methylthio)pyrimidine-5-carboxylate (5): To a stirred solution of ethyl-4-chloro-2-methylsulfanyl-5-pyrimidinecarboxylate (4.50 g, 19.4 mmol) in methanol was added ~7N NH3 (14 mL) in methanol at 0 °C and the mixture was stirred for 2 h at room temperature in a sealed glass vessel. The reaction mixture was then diluted with ethyl acetate (75 mL) and washed with saturatedNaHCO3 solution (1 x

40 mL). The organic layer was dried over MgSO4, filtered and concentrated. The crude product crystallized out from the mixed solvent of ethyl acetate/hexanes (~40:60) to give

(3.76 g, 90%) of ethyl 4-amino-2-methylsulfanyl-5-pyrimidinecarboxylate 5 as a white solid; m.p. 125-127 °C(lit80 128-129 °C.); IR (neat, cm-1) 3410, 3266, 3130, 2973, 2931,

1687, 1622, 1563, 1532; 1H NMR 500MHz (DMSO-d6) δ 8.69 (s, 1H), 7.84 (bs, 1H), 5.76

111

(bs, 1H), 4.34 (q, J = 6.9 Hz, 2H), 2.51 (s, 3H), 1.37 (t,J = 6.9 Hz, 3H); 13C NMR 125

MHz (DMSO-d6) δ 176.0, 166.3, 161.7, 158.9, 101.1, 61.0, 14.2. HR-MS (ESI-TOF) m/z calcd for C8H12N3O2S [M+H]+ 214.0645, found214.0636.

(4-Amino-2-(methylthio) pyrimidin-5-yl) methanol (6): To a solution of ethyl-4-amino-2-methylthiopyrimidine-5-carboxylate (5) (2.0 g, 9.4 mmol) in dry THF (20 mL) was added LiAlH4, 2M in THF (9.4 mL, 18.8 mMol) dropwise at 0 °C and the reaction mixture was stirred at room temperature for 4h. After 4 h, the mixture was quenched at 0 °C with 30% aqueous Na2SO4 (1.5 mL) and stirred for84another 1 h.

The slurry was filtered through Celite. After washing the filter bed with (2 x 100 mL) mixture of ethyl acetate and methanol (2:1), the solvents were removed under reduced pressure. The crude material was purified by flash silica gel chromatography with hexane/ethyl acetate/methanol (50:40:10), Rf= 0.4, to obtain the product as a faint yellow mass. The product then crystallized out as a white powder, (0.72 g, 45%) from a mixture of methanol and ethyl acetate (~10:90); m.p.101-103 °C (lit80 101-104 °C); IR (neat, cm-

1) 3322, 3161, 2713, 1653, 1588, 1545, 1477, 1343, 1255, 1166; 1H NMR (500 MHz,

DMSO-d6) δ 7.84 (s, 1H), 4.45 (s, 2H), 2.47 (s, 3H); 13C NMR 125 MHz (DMSO-d6) δ

170.4, 162.3, 152.7,111.9, 58.5, 12.6; HRMS (ESI-TOF) m/z calcd for C6H10N3OS

[M+H]+ 172.0547, found 172.0544.

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2-(2,4-Diaminopyrimidin-5-yl) acetonitrile81 (7): To a freshly prepared solution of sodiumethoxide (1g sodium in 25 mL absolute ethanol) guanidine hydrochloride (3.10 g, 33 mmol) was added and the reaction mixture was refluxed for 30 min. The turbid white solution was filtered through Celite and washed with ethanol (15 mL). To the filtrate (4.0 g, 33 mmol) ethoxymethylenemalononitrile was added in portions and the reaction mixture was allowed to stir at room temperature for 10 h. The reaction mixture was concentrated to dryness and the residue was dissolved in hot glacial acetic acid followed by a reflux to dissolve the crude material. Following a quick filtration, and on cooling yellow crystals started to precipitate out. The product was filtered, washed with ether (3 x 40 mL) and then evaporated underreduced pressure to remove any remaining acetic acid.to obtain (3.1 g, 63%) as a yellow solid; m.p.~312°C (decomp.) (lit81 315 °C);

IR (neat, cm-1) 3452, 3431, 3338, 3091, 2811, 2711, 2202, 1650, 1584, 1547,1476,

1282; 1H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.09 (bs, 2H), 6.95 (bs, 2H); 13C

NMR. (125MHz, DMSO-d6) δ 164.1, 163.8, 162.8, 118.0, 78.9. HRMS (ESI-TOF) m/z calcd for C5H6N5 [M+H]+136.0618, found 136.0621.

4-Amino-2-methylpyrimidine-5-carbaldehyde81 (8): To a solution of diaminonitrile (7) (2.0.g, 14.8 mmol) in 97% formic acid (20 mL) was added (~2.5 g

Raney nickel 50% slurry in water) and the mixture refluxed for 4 h. TLC in ethyl acetate

(Rf = 0.3) indicated completion of reaction. The mixture was filtered through Celite and washed with formic acid (30 mL). The filtrate was concentrated under reduced pressure

113

and stirred with ~28% ammonia to pH ~7 to obtain a free flowing solid which was washed with cold water and dried to obtain (1.5 g, 73%) aldehyde as an off white solid. The crude material was used as such for the reduction step m.p. > 250 °C (decomp.) (lit81 273-275

°C); IR (neat,cm) 3379, 3314, 3117, 2854, 1627, 1588, 1498, 1234, 1058; 1H NMR (500

MHz, DMSO) δ 9.44 (s,1H), 8.30 (s, 1H), 7.79 (s, 1H), 7.54 (s, 1H), 7.16-7.10 (bs, 2H);

13C NMR (125 MHz, DMSO) δ 189.6,167.2, 164.6, 162.9, 106.4. HRMS (ESI-TOF) m/z calcd for C5H7N4O [M+H]+ 139.0614, found 139.0609.

(2,4-Diaminopyrimidin-5-yl) methanol (9): To a methanolic solution of 2,4-diaminopyrimidine-5-carbaldehyde 8 (0.65 g, 4.7 mmol) was added sodium borohydride (0.27 g, 7.05mmol) and the mixture was stirred at room temperature for 8 h. Excess methanol was added to the reaction to quench it after TLC in ethyl acetate/methanol 80:20 indicated the disappearance of startingmaterial. The crude mixture was purified by silica gel chromatography, ethyl acetate/methanol (90:10) to

(60:40), Rf = 0.25 to obtain (0.3 g, 31%) of the alcohol as a faint yellow solid; m.p. 216-

218 °C. (lit82 218-223 °C); IR (neat, cm-1) 3400, 3330, 3132, 3014, 2929, 2912, 2866,

1556, 1064; 1H NMR (500 MHz,DMSO) δ 7.58 (s, 1H), 6.16 (bs, 2H), 5.88 (bs, 2H), 4.9

(bs, 1H), 4.2 (s, 2H); 13C NMR (125 MHz,DMSO-d6) δ 163.3, 163.2, 155.3, 107.4, 58.6;

HRMS (ESI-TOF) m/z calcd for C5H9N4O [M+H]+ 141.0771,found 141.0777.86

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4-Amino-2-phenylpyrimidine-5carbonitrile (10): Sodium (0.8 g) was dissolved in ethanol (15mL). To this was added (6.0 g, 38 mmol) of benzamidine hydrochloride. The resulting solution was stirred for 10 min followed by addition of ethoxymethylene malononitrile (4.42 g, 36 mmol). The mixture was heated at 40 °C for 1 h, followed by Celite filtration, washed with methanol and dried overnight to obtain the nitrile as a white solid with salt; m.p. 230-231 °C; IR (neat, cm-1) 3407, 3332, 3232,

3065,3054, 3026, 2226, 1642, 1540, 1477, 1409; 1H NMR (500 MHz, DMSO-d6) δ 8.72

(s, 1H), 8.33-8.31 (m,2H), 7.96 (bs, 2H), 7.54-7.49 (m, 3H); 13C NMR (125 MHz, DMSO- d6) δ 165.3, 163.1, 162.1, 137.0,132.1, 129.3, 128.8, 116.3, 88.1. HRMS (ESI-TOF) m/z calcd for C11H9N4 [M+H]+ 195.0676, found195.0685.

4-Amino-2-phenylpyrimidine-5-carboxylic acid (11): The crude nitrile 10 (5 g) was subjected to hydrolysis with 15% KOH in 1:1 THF/water mixture (250 mL) at 100 °C for 16 h. TLC in hexane/ethyl acetate/methanol (40:45:15), Rf ~ 0.3 indicated the disappearance of starting material. The acid was extracted with THF (1 x 50 mL). The crude acid was then subjected to silica gel chromatography under the same condition as the TLC to obtain 2.5 g of the acid as an off white solid;260-262 °C; IR (neat, cm-1) 3481, 3346, 3309, 3171, 3059, 2758, 1675, 1623, 1571, 1392; 1H NMR(500 MHz,

DMSO-d6) δ 8.82 (s, 1H), 8.35-8.32 (d, 2H), 8.12 (bs, 1H), 7.51-7.49 (m, 3H); 13C NMR

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(125 MHz, DMSO-d6) δ 169.0, 165.4, 164.6, 163.1, 162.2, 156.2, 137.7, 137.0, 132.0,

131.4, 128.9, 128.5,116.3, 106.0, 88.1; HRMS (ESI-TOF) m/z calcd for C11H8N3O2 [M-

H]- 214.0622, found 214.0612.

Methyl-4-amino-2-phenylpyrimidine-5-carboxylate (12): The acid 11 (1.0 g, 4.6 mmol)was esterified with methanol (20 mL) in the presence of excess sulfuric acid (5mL) in a modified Fisher esterification procedure. The mixture then refluxed for 5 h to obtain the crude product. Further purification was done by triturating with 50% mixture of ethyl acetate/hexane. The desired material was then obtained (0.67 g, 64%) as a faint yellow powder; m.p. 55-57 °C; 1R (neat, cm-1) 3413, 3334, 3272,3225,

3175, 3118, 3072, 3029, 2993, 2952, 2845, 1698, 1570, 1414, 1303, 1237, 1189; 1H

NMR (500MHz, DMSO-d6) δ 8.99-8.97 (s, 1H), 8.41-8.38 (d, 2H), 7.82 (bs, 1H), 7.49-

7.46 (m, 3H), 5.8 (bs, 1H),3.91-3.87 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 166.9,

162.8, 159.9, 137.1, 131.5, 128.8, 128.6,103.0, 52.2. HRMS (ESI-TOF) m/z calcd for

C12H12N3O2 [M+H]+ 230.0924, found 230.0915.

4-Amino-2-phenylpyrimidine-5-yl) methanol (13): The ester 12

(0.62 g, 2.6 mmol) wasdissolved in dry THF (50 mL) maintained at 0 °C. to the solution

116

(0.29 g, 7.85 mmol) of LiAlH4 powder was slowly added. The reaction mixture was stirred at room temperature for 4 h, when TLC in hexane/ethyl acetate/methanol (50:5:5), Rf =

0.45 indicated disappearance of starting material. The reaction mixture was then quenched with saturated NH4Cl (1 mL), extracted with ethyl acetate/THF, 1:2(75 mL) in the presence of saturated NaCl (20 mL), concentrated and purified by silica gel chromatography with eluent composition same as TLC to obtain the desired alcohol (0.29 g, 55%) as a white solid; m.p. 132-134 °C; IR (cm-1) 3437, 3297, 3170, 2922, 2854,

1626, 1402; 1H NMR (500 MHz,DMSO-d6) δ 8.31-8.30 (m, 2H), 8.18 (s, 1H), 7.45-7.44

(m, 3H), 6.7 (bs, 2H), 5.18 (t, J1 = 5.7 Hz, J2 = 5.2Hz, 1H), 4.41 (d, J = 5.2 Hz, 2H); 13C

NMR (125 MHz, DMSO-d6) δ 162.4, 162.2, 153.7, 138.7, 130.4,128.7, 127.9, 115.6,

58.4; HRMS (ESI-TOF) m/z calcd for C11H12N3O [M-H]- 200.0829, found 200.0831.

4-amino-2-bromopyrimidine-5-carbonitrile (21): To a solution of sodium (5.5 g, 238 mmol) in methanol (160 mL) was added cyanamide (10 g, 238 mmol) over 5 min. The suspension stirred for another 10 min followed by addition of ethoxymethylenemalononitrile (29 g, 238 mmol) in portions. The resulting yellowish solution was evaporated under reduced pressure. The residue was treated with chloroform and recrystallized from methanol to obtain the salt. To (10 g, 70 mmol) of this compound, was added ~110 mL of HBr (48% in water) to effect cyclization. The mixture was diluted with water (200 mL) filtered by suction and washed with water to afford the desired compound 21 in 71% yield. The compound was found to be rather unstable and

1 the crude material was used as such to the next step. H NMR 500 MHz (DMSO-d6) d 8.53 (s, 1H), 8.74 (bs, 1H), 8.19 (bs, 1H).

117

4-amino-2-methoxypyrimidine-5-carbonitrile (14) To 116 mg of sodium was added 10 mL of methanol followed by addition of (1 g, 5.mmol) of (21). The reaction mixture was refluxed for 15 min at 60 °C and then evaporated under reduced pressure and filtered. The residue was dried and repeatedly triturated with water to remove the acid. The methoxy substituted nitrile , (443 mg, 59%) was obtained as a faint

1 yellow solid. H NMR 500 MHz (DMSO-d6) d 8.48 (s, 1H), 8.6 (bs, 1H), 8.18 (bs, 1H),

3.84 (s, 3H)

(4-amino-2-methoxypyrimidin-5-yl)methanol (15) A mixture of

(78 mg,0.58 mmol) (14), sulfuric acid (2 mL) and 10% Pd/C (50 mg) in water 10 mL was stirred and hydrogenated at room temperature at an initial pressure of 50 psi for 24 h.

The reaction mixture was filtered through celite and the pH adjusted to~8 with NH3 TLC

(CHCl3/EtOH: 10:1) showed two spots (alcohol and aldehyde). The crude product was subjected to silica gel chromatography. Elution of column with the same solvent system as TLC afforded the desired alcohol as an off white solid 11 mg in 14% yield. m.p.170-

1 172 °C. H NMR 500 MHz (DMSO-d6) d 7.81 (s, 1H), 6.64 (bs, 2H), 4.97 (s, 1H), 4.29-

13 4.28 (s, 2H), 3.74 (s, 3H) C NMR 125 MHz (DMSO-d6) d 165.04, 164.03, 155.05,

111.65, 58, 54.04.

118

Propionimidamide (16) (1 g, 13 mmol) of NH4Cl was suspended in toluene under argon. The mixture was cooled and to 0 °C followed by addition of addition of 10 mL 25% w/w AlMe3 and (1 g, 18.1 mmol) of propionitrile. After reflux overnight the clear solution turned milky white. Filtering the solid and triturating crude product with

Hexane/Ethylacetate (10:90) got rid of any impurity to afford the desired amidine 1.22 g

1 of product in 93% yield. H NMR 500 MHz (DMSO-d6) d 2.41-2.37 (q, J = 6.87 Hz, 2H),

13 1.15-1.19 (t, J = 6.87 Hz, 3H). C NMR 125 MHz (DMSO-d6) d 172.8, 25.71, 11.68.

4-amino-2-ethylpyrimidine-5-carbonitrile (17) (224 mg, 9.72 mmol) of sodium was allowed to dissolve in 12 mL methanol. This was followed by addition of the amidine, (16) (0.7 g, 9.72 mmols) and malononitrile (1.19 g, 9.72 mmol). The mixture was stirred for 1 h at room temperature. The yellow precipitate was filtered and washed with 15 mL methanol. TLC Hexane/EtOAc/MeOH (20:70:10) indicated that the crude product (1.44 g, 42%) was pure enough to go to the next step. 1H NMR 500 MHz

(DMSO-d6) d 8.55-8.52 (s, 1H), 7.77 (bs, 2H), 2.64-2.61 (q, J = 7.45 Hz, 2H), 1.18-1.15

13 (t, J = 7.45 Hz, 3H) C NMR 125 MHz (DMSO-d6) d 174.60, 163.19, 161.88, 116.44,

87.53, 32.84, 12.63.

Methyl 4-amino-2-ethylpyrimidine-5-carboxylate (18): (0.27 g,

1.78 mmol) of the nitrile (17) was dissolved in a mixture of 5 mL methanol and 1 mL

H2SO4 and heated at 140 °C for 9 h. The reaction mixture was neutralized with saturated

119

Na2CO3, extracted with ethylacetate (50 mL). The organic later was dried and concentrated under reduced pressure to obtain the crude material. Further purification was done with silica gel chromatography Hexane/Ethylacetate/Methanol (40:55:5) to

1 obtain the ester as an off white solid, (0.32 g, 44%) yield. H NMR 500 MHz (DMSO-d6) d 8.82 (s, 1H), 7.76 (bs, 1H), 6.13 (bs, 1H), 3.89 (s, 3H), 2.78-2.73 (q, J = 7.45 Hz, 2H),

1 1.30-1.27 (t, J = 7.45 Hz, 3H) H NMR 500 MHz (DMSO-d6) d 175.08, 166.45, 162.36,

159.16, 102.09, 51.69, 32.38, 11.93.

(4-amino-2-ethylpyrimidin-5-yl)methanol (19) (0.09 g, 0.5 mmol) of the ester (18) was dissolved in diethyl ether (15 mL) and the mixture was cooled to 0

°C under argon atmosphere. LiAlH4 (0.04 g, 1 mmol) was slowly added. After addition the reaction was stirred at room temperature overnight. TLC (hexane/ethyl acetate/methanol 20:70:10) indicated disappearance of starting material. The reaction mixture was then quenched with 0.5 mL saturated NH4Cl. Following filtration, the material was washed with methanol while further purification was achieved through silica gel chromatography to obtain the desired alcohol (0.014 g,18%) as an white solid. 1H

NMR 500 MHz (DMSO-d6) d 7.92 (s, 1H), 6.58 (bs, 2H), 5.27 (t, J = 5.12 Hz, 1H), 4.29-

4.28 (d, J = 5.5 Hz, 2H), 2.56-2.50 (q, J = 7.45 Hz, 2H), 1.18-1.14 (t, J = 7.45 Hz, 3H).

120

Appendix A

List of Abbreviations

121

ADP Adenosine diphosphate

AMP Adenosine monophosphate

AIR 5-aminoimidazole ribonucleotide

ATP Adenosine triphosphate

Da Dalton

DCM Dichloromethane

DIBAL-H Diisobutylaluminum hydride

DIEA Diisopropylethylamine

DMF N,N-Dimethylformamide

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

DXP 1-Deoxy-D-xylulose 5-phosphate

FAD Flavin adenine dinucleotide

FDA Food and Drug Administration

HOMO Highest occupied molecular orbital

122

HMP 4-Amino- 2-methyl-5-hydroxymethylpyrimidine

HMP-P 4-Amino- 2-methyl-5-hydroxymethylpyrimidine phosphate

HMP-PP 4-Amino- 2-methyl-5-hydroxymethylpyrimidine phosphate pyrophosphate

HMP(P)K 4-Amino- 2-methyl-5-hydroxymethylpyrimidine phosphate kinase

HPLC High-performance liquid chromatography

HTS High-throughput screening

IC50 Half maximal inhibitory concentration

LC-MS Liquid chromatography-mass spectrometry

NRPS Nonribosomal peptide synthetase

PDB Protein data bank

PIESI Paired-Ion electrospray ionization

PLP Pyridoxal-5’-phosphate

PDxY Pyridoxal kinase

PPi Pyrophosphosphate

RNA Ribonucleic acid

SAR Structure–activity relationship

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Appendix B

NMR spectra of compounds

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

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Biographical Information

Diego A. Lopez received his B.S in Biological Chemistry in 2010 from The

University of Texas at Arlington. He continued his graduate education at the same institution, earning a Ph.D. in Biorganic Chemistry under the mentorship of professor

Frank W. Foss Jr. His research areas focus in antibiotic drug discovery and high throughput assay development for the detection and monitoring of kinase systems. Diego accepted a post-doctoral position with AZYP LLC under the supervision of professor

Daniel W. Armstrong where he will be applying his bio-organic expertise towards the development and manufacturing of chiral stationary phases.

168