Mechanistic Studies of Inhibitors of Dna Replication

MECHANISTIC STUDIES OF INHIBITORS OF DNA REPLICATION

RESTART PATHWAYS IN NEISSERIA GONORRHOEAE

Thesis

Submitted to

The College of Arts and Sciences of the

UNIVERSITY OF DAYTON

In Partial Fulfillment of the Requirements for

The Degree of

Master of Science in Chemistry

Dasharatha Radha Krishna C. Aduri

UNIVERSITY OF DAYTON

Dayton, Ohio

December, 2013

MECHANISTIC STUDIES OF INHIBITORS OF DNA REPLICATION

RESTART PATHWAYS IN NEISSERIA GONORRHOEAE

Name: Aduri, Dasharatha Radha Krishna C.

APPROVED BY:

______

Matthew E. Lopper, Ph.D. Faculty Advisor Assistant Professor Department of Chemistry

______Garry Crosson, Ph.D. Committee Member Assistant Professor Department of Chemistry

______Mark B. Masthay, Ph.D. Committee Member Associate Professor, Chair Department of Chemistry

______Shawn M. Swavey, Ph.D. Committee Member Associate Professor Department of Chemistry

ABSTRACT

MECHANISTIC STUDIES OF INHIBITORS OF DNA REPLICATION

RESTART PATHWAYS IN NEISSERIA GONORRHOEAE

Name: Aduri, Dasharatha Radha Krishna C. University of Dayton

Advisor: Dr. Matthew E. Lopper, Ph.D.

Complete and faithful replication of a cell’s genetic information is an essential process. Many enzymes are involved in the process of successfully duplicating a cell’s genetic information. Helicases, DNA polymerases, ligases, nucleases, and DNA binding proteins all play a role in DNA replication. However, the integrity of these enzymes can be compromised when they encounter DNA damage, which in general could be caused by chemical mutagens, ionizing radiations, or reactive oxidative species.

Bacterial cells use a pathway called “DNA replication restart” to resume DNA replication following a disruptive encounter of the DNA replication enzymes with DNA damage. This pathway is catalyzed by primosome proteins, including PriA, PriB, PriC,

DnaT, DnaB, DnaC, and DnaG. The importance of DNA replication restart for bacterial cell survival is demonstrated by the inability of strains that carry mutations in key primosome genes to grow and resist DNA damaging agents.

iii

Furthermore, this pathway is specific for bacterial cells: human cells don’t use the same replication restart pathway and they don’t encode genes for the primosome proteins that function in bacteria. Since DNA replication restart pathways are essential for bacterial cell growth and survival and are notably absent in human cells, we seek to answer the following question: can bacterial DNA replication restart pathways be targeted with novel antibacterial compounds?

In order to answer this question, we have developed an enzyme based assay for high-throughput inhibitor screening to identify compounds that block the function of the primosome proteins PriA and PriB. Several interesting lead compounds have already been identified from the preliminary screening. In this study, the lead compounds have been validated as legitimate inhibitors and characterized with respect to their potency and mechanism of action.

Dedicated to my family and friends.

ACKNOWLEDGEMENTS

First and foremost I would like to express my heartfelt gratitude to my advisor, Dr

Matthew E. Lopper. I attribute the successful completion of my thesis project and writing of my thesis to his invaluable guidance, effort, and encouragement. Starting life as a research student one simply could not wish for a better or friendlier supervisor. His enthusiasm for biochemistry, his communication and leadership skills will always be an inspiration to me.

I would like to thank my committee numbers Dr. Mark B. Masthay, Dr. Shawn M.

Swavey and Dr. Garry Crosson for their support. I am grateful to my instructors Dr.

David Johnson, Dr. Shawn Swavey, Dr. Gary Crosson, Dr. Mark Masthay and Dr.

Matthew Lopper for their invaluable teaching and guidance. I specially thank Dr. Kevin

Church for his help in selecting courses.

In addition, I would also like to offer my sincerest gratitude to Paula Keil, Dr.

Rochael Swavey for helping me with my teaching duties. I sincerely appreciate Connie

Schell and Margaret Goodrich for being patient with me and sending reminders whenever

I forgot to complete any office formality. On the whole I would like to thank Chemistry

Department at University of Dayton for supporting me throughout my masters.

TABLE OF CONTENTS

ABSTRACT ……………………………………………………………………iii

DEDICATION …………………………………………………………………v

ACKNOWLEDGEMENTS ……………………………………………………vi

LIST OF FIGURES …………………………………………………………….ix

LIST OF TABLES ……………………………………………………………...xi

CHAPTER I INTRODUCTION ………………………………………………..1

CHAPTER II METHODS ……………………………………………………....6

II-1 Purification of N. gonorrhoeae PriA ………………………………….....6

II-2 Purification of N. gonorrhoeae PriB ………………………………….....7

II-3 Purification of E. coli PriA ……………………………………………....8

II-4 Construction of Fork 2 DNA…………….……………………………....8

II-5 Helicase assays…………………………………………………………...9

II-6 ATP hydrolysis assays………………….………………………………..11

II-7 Crystallization ……………………………………………….…………..13

vii

II-8 Determination of IC50 value ……………………………………………14

CHAPTER III RESULTS ……………………………………………………...16

III-1 Effect of Inhibitor F0298-0039 ………………………………………..16

III-2 Effect of Inhibitor F0359-0046 ………………………………………..18

III-3 Effect of Inhibitor F0683-0441 ………………………………………..20

III-4 Effect of Inhibitor F2018-1489 ………………………………………..22

III-5 Effect of Inhibitor F0683-0207 ………………………………………..24

III-6 Mechanism of the Inhibition of DNA replication restart process of Neisseria gonorrhoeae by F0683-0207…………………………………………….26

III-6-1 ATP hydrolysis experiments- DNA titrations ………….…………….27

III-6-2 ATP hydrolysis experiments- ATP titrations…………………………30

III-7 Effect of F0683-0207 on E. coli PriA…………………………………..32

III-8 Determining the activity of F0683-0207 on E. coli (MG 1655)………..33

III-9 Crystallization ………………………………………………………….35

CHAPTER IV CONCLUSIONS ………………………………………………40

REFERENCES …………………………………………………………………42

viii

LIST OF FIGURES

Figure 1: DNA replication process………………………………….……………2

Figure 2a: Unwinding of duplex DNA by PriA helicase ………………………..9

Figure 2b: Decrease in the fluorescence polarization value …………………….10

Figure 3: PriA catalyzed ATP regeneration cycle ……………………………….13

Figure 4: Structure of the Inhibitor F0298-0039 ………………………………...16

Figure 5a: Average data of the effect of F0298-0039 on N. gon PriA……...... 17

Figure 5b: Average data of the effect of F0298-0039 on N. gon PriA and PriB...17

Figure 6: Structure of the Inhibitor F0359-0046………………………………….18

Figure 7a: Average data of the effect of F0359-0046 on N. gon PriA …………..19

Figure 7b: Average data of the effect of F0359-0046 on N. gon PriA and PriB…19

Figure 8: Structure of the Inhibitor F0683-0441………………………………….20

Figure 9: Average data of the effect of F0683-0441 on N. gonorrhoeae PriA ….21

Figure 10: Data showing the effect of F0683-0441 on intact DNA ……………..22

Figure 11: Structure of the Inhibitor F2018-1489 ………………………………..22

Figure 12: Data showing the effect of F2018-1489 on N. gon PriA……………...23

Figure 13: Data showing the effect of F2018-1489 intact DNA ………………...24

Figure 14: Structure of the Inhibitor F0683-0207 ………………………………...24

Figure 15a: Average data of the effect of F0683-0207 on N. gon PriA….………25

Figure 15b: Average data of the effect of F0683-0207 on N. gon PriA and PriB..25

Figure 16: DNA titrations…………………………………………………………29

Figure 17: ATP titrations ………………….………………………………………31

Figure 18: Plot displaying the effect of inhibitor F0683-0207 on E. coli PriA …...33

Figure 19: Plot displaying the growth rate of E. coli ……………………………...34

Figure 20: Tray setup for Hampton Crystal Screen ………………………………..36

Figure 21: Tray setup for Hampton Crystal Screen 2. …………………………….37

Figure 22: Tray setup for Hampton Index screen .……………………………..…..39

LIST OF TABLES

Table 1: Effect of mixed inhibitor on apparent Vmax and Km values……………..29

Table 2: DNA titrations: Kinetic parameters in the presence and absence of Inhibitor F0683-0207……………………………………………...……….………30

Table 3: ATP titrations: Kinetic parameters in the presence and absence of Inhibitor F0683-0207……………………………………….……………………...31

Table 4: Hampton Crystal Screen scoring sheet …………………………………..36

Table 5: Hampton Crystal Screen 2 scoring sheet……………………..…………..37

Table 6: Hampton Index Screen scoring sheet…………………………………….38

CHAPTER I

INTRODUCTION

The most essential aspect for the existence, survival and propagation of life is

DNA replication and this involves the duplication of cellular genome. In bacteria, this process is continually disrupted by obstructions on the DNA template such as single stranded nicks, double stranded breaks, and oxidized bases which arise due to environmental or cellular factors1. The progress of the DNA replication machinery

(replisome) is hindered by these barriers, causing it to stall or derail from the DNA template2. To ensure continuation of the DNA replication process, bacteria have evolved

“DNA replication restart pathways” which reload and reactivate the DNA replisome onto a repaired DNA replication fork3.

In E. coli, one of the several major pathways to activate stalled replication forks is

DnaA-catalyzed origin-dependent initiation of DNA replication4 (Figure 1). This is a very carefully regulated, sequence-specific event which is catalyzed by the initiator protein,

DnaA. DnaA recognizes and binds to a replication origin site and recruits the replicative helicase and the helicase loader protein, DnaB and DnaC, respectively. The replicative helicase unwinds the parental duplex DNA to facilitate replication of the template DNA strands4 (Figure 1).

Figure 1: (A). This is an origin-dependent replisome loading mechanism which starts at the origin of replication oriC. First DnaA (initiator protein) binds to the DNA and forms a loop. DnaB (the replicative helicase protein) then binds to the DNA. DnaC is the loader protein which helps in loading the DnaB onto the DNA and detaches itself from the complex after which the DnaB starts moving along the DNA strands, separating them. (B) An origin-independent mechanism of replisome loading in which PriA protein binds to the DNA strands when the above mechanism fails. PriB protein then binds to the DNA and also to the helicase domain of the PriA and stabilizes PriA on the DNA. DnaT binds to the PriA:PriB complex and releases the ssDNA on which the DnaB acts. (C) Another mechanism of origin-independent replisome loading. PriC binds to the DNA strand to which DnaB and DnaC bind and starts the replication process. In all the cases DnaC helps load DnaB onto the DNA strand. Origin-independent initiation of DNA replication (known as DNA replication restart) requires a distinct cellular machinery to reload the replisome at a repaired DNA replication fork5. This process of replication is initiated by the assembly of the primosome proteins, which include PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, and

Rep proteins5,6. All these proteins operate through two distinct pathways, one which requires DnaT, DnaB, DnaC, DnaG, PriA, and PriB, and one which requires DnaB,

DnaC, DnaG, Rep, and PriC6.

Primosome protein A (PriA), a 3’ to 5’ DNA helicase, serves to initiate the assembly of the primosome proteins4-6. It binds to two types of DNA structures with high

affinity, either a duplex DNA with a 3’-single stranded extension, or D-loop DNA4,8.

PriB then joins the PriA-DNA complex and is suggested to stabilize this complex6 and to stimulate PriA’s helicase activity via an interaction with single-stranded DNA7.

Association of DnaT on the PriA-PriB-DNA complex leads to the formation of the PriA-

PriB-DnaT-DNA complex8. DnaT appears to be interacting with PriB and allows it release its ssDNA. Reloading of the DnaB/DnaC complex onto the lagging strand template is associated with the dissociation of ssDNA from PriB and this is the point where DnaG and DNA polymerase III holoenzyme are recruited to resume DNA replication 5, 8. ATP hydrolysis is required for the process of loading DnaB9.

Earlier studies of DNA replication restart pathways were very much focused on E. coli and relatively very little is known about the actual mechanism of DNA replication restart and its biological importance in other species of bacteria. N. gonorrhoeae, the causative organism of gonorrhea, provides a nice illustration of the naturally-occurring variation that is found in DNA replication restart pathways across diverse bacterial species. N. gonorrhoeae is a gram-negative bacterium which shows a highly remarkable adaptation of survival towards oxidative damage to its genome caused by neutrophilic attack in infected individuals. This suggests that DNA replication restart might play an important role in its pathogenicity10.

There are quite distinct deviations in the mechanism of replication restart between

N. gonorrhoeae and E. coli. The affinity of interaction with ssDNA is high for E. coli

PriB and low for N. gonorrhoeae PriB. Apart from the ssDNA, the affinity of interaction between PriA and PriB is high in N. gonorrhoeae and low in E. coli. The lack of DnaT and PriC genes in Neisseria species clearly suggests that considerable mechanistic

differences might exist between N. gonorrhoeae and E. coli replication restart pathways11.

To facilitate the release of ssDNA from PriB within the primosome complex and also to assemble the primosome, DnaT might play a crucial role in E. coli. This release of ssDNA makes it possible for the replicative helicase to bind with ssDNA6, 12. There must be another source of weak interactions to acquire an equivalent level of stability, which is otherwise provided by the stabilizing weak interaction of DnaT. This requisite of primosome stabilizing binding energy must have been supplied by the notable strong interaction between PriA: PriB instead of DnaT in N. gonorrhoeae.

The high affinity interaction between the PriA and PriB of N. gonorrhoeae might indicate that these primosome proteins are constitutively complexed with one another in cells thereby allowing a more rapid response to DNA damage than could be evoked by the same primosome proteins that must foregather at a site DNA replication fork reactivation. As N. gonorrhoeae has evolved under selective pressure and high levels of oxidative damage to its genome, this kind of high affinity interaction could be particularly beneficial13. This explains the possibility for the differences between the two species. In addition to helping in understanding the features of affinity, we realized that studying DNA replication restart pathway in N. gonorrhoeae could also be used to develop novel antibiotics against this gonorrheal infection causing bacteria.

Bacterial resistance has made many existing anti-bacterial drugs ineffective towards treating bacterial infection. Thus by understanding the exact mechanism through which the bacterium is actually restarting the DNA replication process, we might be able to formulate a drug which can effectively combat bacterial growth by targeting the DNA

replication restart mechanism. This formulation would require a compound that inhibits the activity of primosome proteins.

We have already developed an enzyme-based assay to use in high-throughput screening (HTS) to identify inhibitors of DNA replication restart primosome protein function. HTS is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Thousands of pharmacological and chemical tests can be quickly conducted through HTS through data processing and control software. One can rapidly identify the active compound which can regulate a particular bimolecular pathway and these experiments provide starting points for drug design. Several interesting lead compounds have already been identified from the preliminary screening. In this study, the lead compounds have been validated as legitimate inhibitors and characterized with respect to their potency and mechanism of action.

In my research I have tested the efficiency of several inhibitors on inhibiting the

DNA replication restart pathways in N. gonorrhoeae. I used helicase assays and steady state enzyme kinetic experiments to understand the mechanistic features of inhibition of

DNA replication restart pathway in N. gonorrhoeae using various inhibitors.

CHAPTER II

METHODS

II–1Purification of N. gonorrhoeae PriA

N.gonorrhoeae PriA was purified from BL21 (DE3) E.coli harboring the pET28b:N.gonorrhoeae-PriA plasmid. Cells were grown in Luria-Bertani (LB) medium containing 50 mg/L Kanamycin at 37 °C until an OD600 of 0.6 was reached. PriA was expressed by inducing it with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for

4 hours and cells were centrifuged at 5,500 × g at 4 °C for 25 min and were stored at -80

°C. This was followed by lysing of the cells in 10% (v/v) glycerol, 10 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7, 0.5 M NaCl, 10 mM imidazole, 1 mM phenyl methane sulphonyl fluoride (PMSF) , 1 mM β-mercaptoethanol by sonication on ice, using 5 × 30 sec pulsed bursts (pulse=1 sec on, 1 sec off) at 70% power.

The lysate was clarified by centrifugation at 40,000 × g for 20 min at 4 °C. His- tagged PriA was bound to nickel-nitrilo-triacetic acid (NTA) agarose beads (Qiagen) and in 10 mM HEPES pH 7, 10% glycerol, 100 mM NaCl, 250 mM imidazole.

The nickel-NTA agarose eluate was dialyzed against 10 mM HEPES pH 7, 10% glycerol,

100 mM NaCl and 1mM β-mercaptoethanol and incubated with thrombin to remove the

His-tag, leaving a Gly-Ser-His sequence at the amino-terminus directly preceding the first methionine residue. Residual His-tagged PriA that was not cleaved by thrombin, as well as contaminating E.coli proteins, were depleted by incubating the thrombin-cleaved PriA solution with nickel-NTA agarose. Thrombin-cleaved PriA was concentrated and incubated in 0.1 M 2-(N-morpholino) ethane sulfonic acid (MES) pH 6, and the protein solution was loaded onto a HiPrep SPFF 16/10 ion-exchange column pre-equilibrated with 10 mM MES pH 6, 10% (v/v) glycerol, 100 mM NaCl, 1 mM β-mercaptoethanol.

The SPFF column was resolved at 0.5 mL/min using a ten column volume linear gradient of 0%-100% Buffer B, which contains 10 mM MES pH 6, 10% (v/v) glycerol, 1 M NaCl,

1 mM β-mercaptoethanol. Appropriate fractions containing PriA were collected and concentrated overnight by centrifugation in a CentriPrep YM-10 concentrator at 2,643 × g at 4°C. The concentrated protein solution was aliquoted and stored at -80 °C.

II–2 Purification of N. gonorrhoeae PriB

N. gonorrhoeae PriB was purified as described previously14, the detailed protocol is as follows. N. gonorrhoeae PriB protein was purified from BL21 (DE3) E. coli harboring the pET28b:N.gon-priB plasmid. Cells were grown in LB medium containing

50 mg/L kanamycin at 37 °C until an OD600 of 0.6 was reached. PriB was expressed by inducing it with 0.5 mM IPTG for 4 h and cells were centrifuged at 5000 × g. This was followed by lysing of the cells in 0.1 M NaCl, 10 mM Tris–HCl pH 8.5, 10% (v/v) glycerol, 10 mM imidazole, 1 mM PMSF, 1 mM β-mercaptoethanol, by sonication on ice.

The lysate was clarified by centrifugation at 40 000 × g. His-tagged PriB was bound to nickel-NTA agarose (Qiagen) and eluted in 1mM β-mercaptoethanol, 10 mM

Tris–HCl pH 8.5, 10 % (v/v) glycerol, 250 mM imidazole and 0.1 M NaCl. The nickel-

NTA agarose eluate was dialyzed against 0.1 M NaCl, 10 % (v/v) glycerol, 0. 10 mM

Tris–HCl pH 8.5, 1 mM β-mercaptoethanol and was incubated with thrombin in order to remove the His-tag. This leaves a Gly-Ser-His sequence at the amino-terminus directly preceding the first methionine residue. Contaminating E. coli proteins as well as residual

His-tagged PriB that was not cleaved by thrombin were depleted by incubating with nickel-NTA agarose. PriB, which is thrombin-cleaved was concentrated and purified using a HiPrep HR 16/10 Sephacryl S-100 size exclusion column (GE Healthcare) in 10 mM Tris–HCl pH 8.5, 10% (v/v) glycerol, 0.5 M NaCl, 1 mM β-mercaptoethanol. PriB fractions were pooled, concentrated and stored at -80 ºC.

II-3 Purification of E. coli PriA

The PriA gene of E. coli strain MG1655 was cloned and the recombinant PriA protein was purified as previously described23.

II-4 Construction of Fork 2 DNA

The DNA substrate used in the duplex DNA unwinding assays was constructed by annealing complementary DNA oligonucleotides, one of which is labeled with fluorescein at the 3’ end. The oligonucleotides were suspended in 10 mM Tris. HCl pH 8,

50 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) at a 2:1 molar ratio of non- labeled DNA to fluorescein–labeled DNA. The DNAs were incubated at 95 ºC for 5 min, slow-cooled to 70 ºC and incubated at that temperature for 60 min, and slow-cooled to 25

ºC. The duplex DNA was gel purified through a 6 % polyacrylamide gel using 100 mM

Tris-borate pH 8.3, 2 mM EDTA as the electrophoresis buffer. The DNA was excised from the polyacrylamide gel, electroeluted using the same electrophoresis buffer, dialyzed against 10 mM Tris-HCl pH 8, 5 mM MgCl2, aliquoted and stored at -20 ºC.

II-5 Helicase assays

DNA helicases are proteins that use the energy of ATP hydrolysis to unwind duplex DNA. A helicase assay is used for the detection of PriA’s helicase activity. This assay involves the unwinding of a forked DNA substrate by PriA on the lagging strand arm, which was labeled with fluorescein. The unwound ssDNA and the intact DNA fork differ greatly in size, resulting in a sharp decrease in fluorescence polarization value, which in turn, reveals PriA’s helicase activity (Figure 2).

Figure 2: a) Duplex DNA is unwound by PriA helicase using energy released by ATP hydrolysis. b) The intact DNA substrates have been unwound to smaller molecules as the unwinding reaction proceeds, resulting in a sharp decrease in fluorescence polarization value.

In these experiments 0 nM to 50 nM of PriB or its variants were used with 1 nM

DNA and 2 nM PriA in the helicase assay buffer which consists of 20 mM Tris HCl pH

8, 50 mM NaCl, 3 mM magnesium chloride, and 1 mM β mercaptoethanol. 1 mM ATP was added to initiate the reaction and the reaction mixture was incubated for 37ºC for 10 min. The reaction was stopped by adding sodium dodecyl sulfate (SDS) and the fluorescence polarization values (mx) of the reaction samples were measured at 25ºC in a

Beacon TM 2000 variable temperature fluorescence polarization system. After the measurements, the solution was incubated at 95ºC for 25 sec and cooled on ice for 25 sec so that the entire DNA will be unwound. The fluorescence anisotropy values (mss) of the completely unwound DNA samples were then measured.

where mx represents the experimental fluorescence anisotropy for each measurement, mss represents the fluorescence anisotropy of DNA when heated to 95°C and then cooled back to 25ºC (completely unwound) and mo represents the fluorescence anisotropy of the

DNA in the absence of PriA (fully intact DNA duplex substrates). For some experiments, PriB was included in the reaction mixture at concentrations ranging from 0 to 50 nM.

II-6 ATP hydrolysis assays

Steady state ATP hydrolysis catalyzed by PriA was measured using a spectrophotometric assay. This includes an ATP regeneration system that converts ADP to ATP in a reaction that is coupled to the conversion of NADH to NAD+ 15. This coupled reaction can be detected spectrophotometrically by measuring the decrease of absorbance at 340 nm due to NADH oxidation according to Beer-Lambert law:

A=Ɛbc

Because the molar extinction coefficient of NADH is 6,220 M-1 cm-1, the initial rate of ATP hydrolysis was calculated by converting steady-state ΔA340 nm/Δt rates to Δ

[ATP]/ Δt from the linear region in the time courses. To compare DNA-dependent ATP hydrolysis profiles, purified PriA at 10 nM was mixed with certain concentrations of dT36 ranging from 0-200 nM in 20 mM HEPES (pH 8.0), 50 mM NaCl, 7 mM β-

mercaptoethanol, 0.1 mg/ml bovine serum albumin (BSA), and 1 mM ATP in the presence and absence of 100 nM PriB.

To compare ATP-dependent ATP hydrolysis profiles, purified PriA at 10 nM was mixed with 0-1000 μM ATP in the absence and presence of 100 nM PriB and 100 μM dT36, the mixture solution also contained 7 mM β- mercaptoethanol, 0.1 mg/ml BSA,

50mM NaCl, 20 mM HEPES (pH 8.0). For the ATP regeneration system, 7 units/mL of pyruvate kinase, 2 mM phosphoenol pyruvate (PEP), 0.1 mM NADH and 10 units/mL of lactate dehydrogenase (LDH) were also included in each reaction mixture. Reactions were incubated at 37°C in the presence of 15 μM F0863-0207 or an equivalent volume of

DMSO and the absorbance at 340 nm was measured over a period of 800±200 s. Steady- state Δ [NADH]/Δt rates were calculated using the molar extinction coefficient of 6220

M-1 cm-1 for NADH, and these rates are equivalent to Δ[ATP]/Δt. The kinetic parameters

Km, Kact, and Vmax were determined by fitting the rates of ATP hydrolysis from individual experiments to the Michaelis−Menten equation

V = (Vmax [ATP])/ (Km + [ATP])

For experiments in which ATP was varied or to the equation

V = (Vmax [dT36])/ (Kact + [dT36])

For experiments in which dT36 was varied (Curve Expert version 1.3). The relationship

between apparent Vmax and KI was determined using the equation

Vmax (app) = Vmax/ (1 + [I]/KI′).

The relationship among apparent Km, KI, and KI′ was determined using the equation

Km (app) = Km (1 + [I]/KI)/ (1 + [I]/KI′).

The relationship among apparent Kact, KI, and KI′ was determined using the equation

Kact (app) = Kact (1 + [I]/KI)/ (1 + [I]/KI′)

Figure 3: When PriA unwinds dsDNA, it requires energy released by ATP hydrolysis. During this process, ATP is converted to ADP, which in turn reacts with PEP in the presence of pyruvate kinase to form ATP. PEP present in the solution is converted to pyruvate, which is reduced by NADH to lactate, catalyzed by LDH. during this process, NADH is oxidized to NAD+. Conversion of ATP to ADP by PriA is the rate- limiting step. The rate of ATP hydrolysis is measured by the disappearance of NADH which can be monitored spectrophotometrically at 340 nm. II-7 Crystallization These experiments were performed to study the physical interaction between the

PriA and the inhibitor F0683-0207. The technique used was the hanging drop vapor diffusion method, which was achieved by using commercially-available sparse matrix kits from Hampton Research.

Before the crystallization experiments were setup, microdialysis of N. gonorrhoeae

PriA was performed in a minimal buffer (10 mM MES pH 6 and 0.5 M Ammonium acetate). For efficient crystallization the ratio of F0863-0207 and N. gonorrhoeae PriA should be 1:1.2. Accordingly, the final concentration PriA used was 25.8 µM and that of the inhibitor F0683-0207 was 31 µM. A 24-well plate was set up and each well was filled

with 500 µL of each reservoir solution. The well was covered with a plastic coverslip on which 1 µL of F0683-0207, 1 µL of reservoir solution and 1 µL of minimal buffer were pipetted and mixed. The drop was observed under the microscope after adding all the reservoir solutions and the data was recorded in a screening sheet.

II-8 Determination of IC50 value

The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This measure indicates the quantity of a particular drug or other substance (inhibitor) needed to inhibit a given biological process by fifty percent.

Dose−response curves were generated by titrating F0683-0207 into mixtures of

PriA and PriB proteins from N. gonorrhoeae. These DNA unwinding assays are similar to the one used for the HTS, except that the concentrations of PriA and PriB were varied as described in the main text to account for slight differences in enzyme activity between the two bacterial PriA homologues. In each case, the concentrations of primosome proteins were chosen to maximize the full dynamic range of the assay while avoiding saturating the assay with excess enzyme. Serial dilutions of F0683-0207 were made into

DMSO from a 10 mM stock solution, and the concentration of DMSO was fixed at 5%

(v/v) for all experimental points in the F0683-0207 titrations. Fluorescence anisotropies were measured at 25 °C with a Beacon 2000 fluorescence polarization system. The concentration of F0683-0207 required for 50% inhibition (IC50) was determined using a four-parameter sigmoidal model

y = (a − d)/[1 + (x/c)−b] + d

where a is the minimal enzyme activity, b is the slope, c is the IC50, d is the maximal enzyme activity, y is the fraction of DNA unwound, and x is the molar concentration of inhibitor (CurveExpert version 1.3).

CHAPTER III

RESULTS

In order to investigate the effect of inhibition of DNA replication restart pathways in N. gonorrhoeae, compounds were screened from the Life Chemicals 2 library through high throughput screening (HTS) to identify potential lead compounds which could work as inhibitors of DNA replication restart pathway of N. gonorrhoeae. Five lead compounds were identified and chosen for further investigation to determine their potency, specificity, and mechanism of action. Below, I report the results of my experiments with these five different inhibitors.

III-1 Effect of Inhibitor F0298-0039:

Figure 4: Structure of the inhibitor F0298-0039. Molecular weight 470.96 g/mol

The primary goal was to identify the prime target of F0298-0039 among PriA and

PriB. In order to investigate this, we have developed an enzyme based assay based on

DNA unwinding property as mentioned in the methods section. The DNA unwinding activity of 2 nM PriA was determined in the presence and absence of PriB and F0298-

0039. One set of experiments were done with PriA alone and receiving either F0298-

0039 or DMSO as solvent control. The other set of experiments had both PriA (2 nM concentration as monomer) and PriB (10 nM concentration as a monomer), with F0298-

0039 and DMSO as a solvent control. In this way the primary goal, the percentage inhibition of F0298-0039 in the presence and absence of PriB was studied.

5(a) 5(b)

Figure 5: (a) Average data of three trials showing the effect of inhibitor F0298-0039 on N. gonorrhoeae PriA alone. (b) Average data of three trials showing the effect of inhibitor F0298-0039 on PriA and PriB combined and error bars represent one standard deviation of the mean. When F0298-0039 was added to PriA in the absence of PriB, I observed a dose dependent decrease in the amount of DNA unwound. F0298-0039 exhibited similar levels of inhibiton even in the presence of PriB (Figure 5). Thus it can be stated that F0298-

0039 activity is independent of PriB and it seems that PriA is the target of the inhibitor.

The inhibitor can be regarded as a weak inhibitor. Though the fraction unwound is low at higher concentrations of the inhibitor (at 300 µM, fraction unwound was found to be 0.20 relative to the fraction unwound value of 0.74 in the absence of inhibitor), the level of inhibition was insignificant at lower concentrations (at 1 µM inhibitor, the fraction unwound was found to be 0.74). As very high concentration of the inhibitor is not recommended (higher concentration of the inhibitor may lead to either no inhibition or reverse effect), F0298-0039 is not a potent inhibitor. We were looking for a potent inhibitor because a highly specific and potent inhibitor ensures that a drug will have few side effects and thus low toxicity.

III-2 Effect of Inhibitor F0359-0046:

Figure 6: Structure of the inhibitor F0359-0046. Molecular weight 411.29 g/mol

This is the second inhibitor which was selected to study its effect on N. gonorrhoeae. A 10 mM stock solution was prepared from the compound and several concentrations of the inhibitor have been prepared from the stock solution to test the efficiency of inhibitor on the PriA and PriB. But, precipitation of the sample at concentrations greater than 300 µM was observed. Thus, the maximum concentration of

200 µM was used for this particular inhibitor. In order to identify the primary target of the inhibitor a series of DNA unwinding assays has been performed. The DNA unwinding activity of 2 nM PriA was determined in the presence and absence of PriB and

F0359-0046. One set of experiments was done with PriA alone and receiving either

F0359-0046 or DMSO as solvent control. The other set of experiments had both PriA (2 nM concentration as monomer) and PriB (10 nM concentration as monomer), with

F0359-0046 and DMSO as a solvent control.

7(a) 7(b)

Figure 7: (a) Average data of three trials showing the effect of inhibitor F0359-0046 on N. gonorrhoeae PriA alone. (b) Average data of three trials showing the effect of inhibitor F0359-0046 on PriA and PriB combined and error bars represent one standard deviation of the mean. The inhibitor F0359-0046 exhibited similar levels of inhibition in the presence and absence of PriB. Thus it can be inferred that F0359-0046 is independent of PriB and it seems that PriA is the target of the inhibitor.

This inhibitor can be regarded as a weak inhibitor. The fraction unwound value at concentration of 1 µM was found to be 0.73 whereas the value was 0.72 in the absence of

F0359-0046. Hence, the compound F0359-0046 is not a potent inhibitor in general and also when compared to the inhibitor F0359-0039.

III-3 Effect of Inhibitor F0683-0441:

Figure 8: Structure of the inhibitor F0683-0441. Molecular weight 434.66 g/mol

F0683-0441 was the next compound under consideration. The main idea was to find its potency based on the enzyme based DNA unwinding assay as mentioned in the methods section. It had some solubility issue initially as it was not soluble in DMSO at a stock concentration of 10 mM, which was the common solvent for all the other inhibitors.

DMSO was later replaced with methanol to investigate its solubility. Even with the methanol, the compound was not soluble, thus the solution was heated up to 45 ºC and the same process was followed with DMSO and the results appeared same. I started diluting down the sample to various concentrations and at about 0.5 mM concentration the sample appeared to be particle free. The maximum concentration of the inhibitor in the assay will be maintained at 50 µM in order to avoid the precipitation.

As the maximum concentration will be maintained at 50 µM, I decided to perform the assay only with PriA and no PriB. I have assumed that, in this particular case, using a

10 nM concentration of PriB and 2 nM PriA along with such a low concentration of inhibitor might result in masking the actual effect of the inhibitor. In order to identify the primary target of the inhibitor, DNA unwinding assay has been performed. The DNA unwinding activity of 5 nM PriA was determined in the absence of PriB. One set of experiments was done with PriA alone and receiving either F0683-0441 or DMSO as solvent control. The results below indicate the unwinding activity of DNA in presence and absence of F0683-0441.

Figure 9: Average data of three trials showing the effect of inhibitor F0683-0441 on N. gonorrhoeae PriA alone and error bars represent one standard deviation of the mean. The final outcome of this experiment appeared to be deviating from the general trend seen in all the other inhibitors. The expected result was decrease in the fraction unwound values as the concentration of the inhibitor increased, but the actual result was completely different. When there was no inhibitor the fraction unwound value appeared to be around 0.3 and as the concentration increased the values tend to be negative. This made us think about the primary target of the inhibitor and we have doubted whether the inhibitor was targeting the intact DNA instead of PriA.

Based on this observation, I performed an assay with DNA and the inhibitor but without PriA or PriB. I have used five different concentration of the inhibitor and maintained a constant concentration of the DNA. The results for this assay are as below,

Figure 10: Data showing the effect of inhibitor F0683-0441 on DNA alone. Note that the scale of x-axis here is different from the scale of x-axis in figure 9. The fraction unwound value of the DNA showed a significant decrease in the absence of PriA and at various low concentrations of the inhibitor F0683-0441. The anisotropy values of the intact DNA and the sample with 1 µM inhibitor were almost the same. Based on this data, we have come to a conclusion that inhibitor F0863-0441 might be affecting the DNA itself irrespective PriA and PriB. Thus, I eliminated this lead compound from further study.

III-4 Effect of Inhibitor F2018-1489:

Figure 11: Structure of the inhibitor F2018-1489. Molecular weight 565.53 g/mol

F2018-1489 was expected to show a stimulating effect on DNA unwinding as per the results obtained from high-throughput screening. In order to identify the primary target of F2018-1489, DNA unwinding assay has been performed. The DNA unwinding activity of 5 nM PriA was determined with either F2018-1489 or DMSO as a solvent control. The results indicate the unwinding activity of DNA in the presence and absence of F2018-1489 (Figure 12).

Figure 12: Data showing the effect of F2018-1489 on N. gonorrhoeae PriA alone.

As seen from the results above, the compound F2018-1489 did not show any signs of stimulation but proved to be a weak inhibitor of unwinding, requiring 300 µM of the compound to give significant levels of inhibition. The fraction unwound values have decreased as the concentration of the compound was increased. We wanted to see the effect of F2018-1489 on the intact DNA without any PriA or PriB in it, so that it might show any stimulating effect in the absence of the primosome proteins. I titrated F2018-

1489 with intact DNA without any primosome proteins and even in this case the compound didn’t show any stimulating effect as suggested by the high-throughput screening. The compound didn’t even show the inhibiting effect on the unwinding of

DNA as the fraction unwound values were negative (Figure 13). We have come to a conclusion that compound F2018-1489 might be acting directly on the DNA.

Figure 13: Data showing the effect of F2018-1489 on intact DNA without primosome proteins.

III-5 Effect of Inhibitor F0683-0207:

Figure 14: Structure of the inhibitor F0683-0207. Molecular weight 410.77 g/mol

This was our final compound to be tested on N. gonorrhoeae and also the most interesting compound because F0683-0207 has shown quite promising results in the HTS among all the other compounds which we have selected. I have started out by investigating the prime target of the compound through DNA unwinding assay.

The DNA unwinding activity of 5 nM PriA was determined in the presence and absence of PriB and F0683-0207. One set of experiments was done with PriA alone and receiving either F0683-0207 or DMSO as solvent control. The other set of experiments had both PriA (2 nM as a monomer) and PriB (10 nM concentration as a dimer), with

F0683-0207 and DMSO as a solvent control. The stock concentration used for the sample was 1 mM in DMSO since the stock concentration of 10 mM in DMSO has particles of the compound floating in the solvent and at 1 mM concentration the compound was completely soluble in DMSO. The maximum concentration in the assay was maintained to be 50 µM because the concentration above it has shown precipitation. In this way the primary goal, the percentage inhibition of F0683-0207 in the presence and absence of

PriB was studied. The results indicate the unwinding activity of DNA in presence and absence of F0683-0207 (Figure 15).

15 (a) 15(b)

Figure 15: (a) Average data of three trials showing the effect of inhibitor F0683-0207 on N. gonorrhoeae PriA alone. (b) Average data of three trials showing the effect of inhibitor F0683-0207 on PriA and PriB combined and error bars represent one standard deviation.

From the graph it can be seen that, both in the presence and absence of PriB the pattern of unwinding the DNA looks similar. From the results above, we have come to a conclusion that the inhibitor is not affecting PriB and likely targets PriA. The potency of

this particular inhibitor is the highest among the five lead compounds. It has a measured

IC50 value of 1.64 µM. The average fraction unwound value was 0.23 at 50 µM concentration of the inhibitor which was as high as 0.74 in the absence of the inhibitor.

The IC50 value indicates that this is a potent inhibitor and can be used for future studies in understanding the mechanism through which it inhibits the DNA unwinding.

F0683-0207 has structural similarity with the compound F0683-0441, thus we have thought that it might be showing some kind of effect directly on the DNA. An assay has been performed with F0683-0207 and DNA alone without any PriA and PriB. But it didn’t show any kind of action on the DNA (data not shown).

III-6 Mechanism of the inhibition of DNA replication restart process of Neisseria gonorrhoeae by F0683-0207:

Based on the DNA unwinding assays we have decided that PriA was the main target of the inhibitor F0683-0207 and have decided to study the mechanism through which it inhibits the DNA unwinding activity of Neisseria gonorrhoeae PriA. Since

PriA’s ATPase activity applies energy gained from ATP hydrolysis for unwinding the duplex DNA, I wanted to determine if the effects of F0683-0207 are due to perturbations that affect how PriA binds and hydrolyzes ATP. ATPase assays were used as a tool to determine the mode of inhibition through which the inhibitor shows its effect and also to measure the rates of PriA-catalyzed ATP hydrolysis. The ATPase assay includes an ATP regeneration system that converts ADP-ATP in a reaction that is coupled to the conversion of NADH to NAD+. This coupled reaction can be detected

spectrophotometrically by measuring the decrease of absorbance at 340 nm due to NADH oxidation.

III-6-1 ATP hydrolysis experiments- DNA titrations:

The rate of ATP hydrolysis was determined in these experiments. Rates of PriA- catalyzed ATP hydrolysis were measured as a function of ATP concentration and as a function of dT36 concentration using a spectrophotometric assay that couples ATP hydrolysis to oxidation of NADH. In order to determine the ideal concentration of the inhibitor which allows us to calculate the kinetic parameters (Km and Vmax) for the PriA catalyzed reaction, several concentrations of the inhibitor were tested. Initially, the titration was performed with DMSO alone as a solvent control and the ATPase activity was found to be 136.43 nM/sec (+/- 8.3 St.Dev.) and at the inhibitor IC50 concentration the ATPase activity is 105.30 nM/sec (+/- 6.2 St.Dev). Although the difference between solvent control and inhibitor is small, it does indicate that the inhibitor is blocking ATP hydrolysis. In order to obtain values for Km and Vmax in the presence of the inhibitor that are both reliable and significantly different from the solvent control, I chose to test several different inhibitor concentrations. At 50 µM inhibitor the ATPase activity was too low (4.10 nM/sec) to make realible measurements, so that I couldn’t calculate the Km and

Vmax values with confidence. At 15 M inhibitor, the ATPase activity was high enough to be measureable and significantly lower than the solvent control, thus I chose this concentration of inhibitor for the kinetics analysis.

A concentration of 15 µM inhibitor was used in the titrations and steady state kinetics of ATP hydrolysis of 10 nM PriA was studied in the presence of 100 nM PriB as monomers both in the presence and absence of the inhibitor F0683-0207. Different concentrations of dT36 ranging from 0 nM to 100 nM were used. The average Vmax value decreased from 278.6 + 12.2 nM/sec to about 205.6 + 36.4 nM/sec in the presence of

F0683-0207. The Km value increased from 18.5 + 1.6 nM to 89.0 + 19 nM in the presence of F0683-0207. This effect of F0683-0207 in its presence and absence allows us to state that it is exhibiting a mixed mode of inhibition.

A mixed inhibitor binds at a site distinct from the substrate active site and it could bind either to the PriA-PriB enzyme complex or to the PriA-PriB-DNA-ATP enzyme- substrate complex. The rate equation describing mixed inhbition is

where,

Table 1 shows the effect of a mixed inhibitor on apparent Vmax and apparent Km values.

Table 1: Effect of mixed inhibitor on apparent Vmax and Km values.

Apparent Vmax Apparent Km

’ ’ Mixed inhibitor Vmax / α αKm / α

The plot shows the effect of the inhibitor F0683-0207 on ATP hydrolysis (Figure

16). The Vmax value decreases and Km value increases with the addition of F0683-0207, indicating that it is a mixed type of inhibitor with respect to DNA.

Figure 16: PriA’s ATPase activity is affected by F0683-0207 – DNA titrations: F0683-0207 exhibits a mixed mode of inhbition with respect to DNA: DNA dependent ATP hydrolysis rates catalyzed by 10 nM PriA in presence of 100 nM PriB and in absence and presence of 15 μM of F0683-0207. The DNA used was dT36 DNA . The measurements are reported in triplicate and the error bars represent one standard deviation of the mean.

The table below shows the calculated Kact and Vmax values in the presence and absence of ihibitor F0683-0207.

Table 2: DNA titrations: Kinetic parameters in the presence and absence of inhibitor F0683-0207.

-F0683 0207 +F0683 0207

Vmax, DNA, nM/sec 278.63 + 12.2 205.5 + 36.4

Kact, DNA, nM 18.54 + 1.6 89 + 19

As mentioned above in the methods above, the relationship among apparent Km, Ki and

’ Ki was determined using the equations

Vmax (app) = Vmax/ (1 + [I]/KI′)

Km (app) = Km (1 + [I]/KI)/ (1 + [I]/KI′).

The calculated average Ki value was found to be 2.73 + 1.2 µM and the calculated

’ average Ki was found to be 42.8 + 23.4 µM. Since the average Ki value is lower than the

’ calculated average Ki value, it indicates that inhibitor has stronger affinity towards the free enzyme than the enzyme:ATP complex.

III-6-2 ATP hydrolysis experiments- ATP titrations:

I have also examined the steady state kinetics of ATP hydrolysis by PriA while using different concentrations of ATP in the presence and absence of 15 μM F0683-0207.

The concentrations of PriA and PriB were similar to DNA titration experiments and different concentrations of ATP ranging from 0 μM to 1000 μM were used. The Vmax value has decreased from 241.3 + 13.1 nM/sec to 118.25 + 10 nM/sec in the presence of

F0683-0207. The Km value has increased from 26.35 + 8.2 µM in the presence of

F0683-0207 to 35.33 + 3.5 µM in the absence. This effect of F0683-0207 on the kinetic parameters suggests a mixed mode of inhibition with respect to ATP.

Figure 17: F0683-0207 exhibits a mixed mode of inhbition with respect to ATP: ATP dependent ATP hydrolysis rates catalyzed by 10 nM PriA in the presence of 100 nM PriB and in the absence and presence of 15 μM of F0683-0207. The DNA used was dT36. The measurements are reported in triplicate and the error bars represent one standard deviation of the mean.

Table 3 shows the calculated Km and Vmax values in the presence and absence of inhibitor F0683-0207.

Table 3: ATP titrations: Kinetic parameters in the presence and absence of inhibitor F0683-0207

-F0683 0207 +F0683 0207

Vmax, ATP, nM/sec 241.36 + 13.1 118.25 + 10

Km, ATP, mM 35.33 + 3.5 26.35 + 8.2

’ The relationship among apparent Km, Ki and Ki was determined using the equations

Vmax (app) = Vmax/ (1 + [I]/KI′)

and

Km (app) = Km (1 + [I]/KI)/ (1 + [I]/KI′).

The calculated average Ki value was found to be 28.8 + 14.2 µM and the calculated

’ average Ki was found to be 14.4 + 2.4 µM. Since the average Ki value is greater than the

’ average Ki value, it indicates that inhibitor has stronger affinity towards the enzyme-

DNA complex than the free enzyme.

III-7 Effect of F0683-0207 on E. coli PriA:

After investigating the mechanism by which F0683-0207 affects the N. gonorrhoeae PriA, in order to investigate the species specifity of the inhibitor a similar

DNA unwinding assay has been performed with E. coli PriA. Various concentrations of

F0683-0207 were titrated against 10 nM E. coli PriA. It can be seen from the plot that

F0683-0207 affects the DNA unwinding activity of E. coli PriA (Figure 18). The fraction unwound value of the intact DNA is about 0.75 in the absence of the inhibitor and 0.27 at

3 µM concentration of the inhibitor. Based on the results obtained the IC50 value was calculated and found to be 1.72 µM + 1, which is very close to the IC50 value of N. gonorrhoeae (1.64 µM + 1). It can be inferred that the inhibitor F0683-0207 is not a species specific inhibitor and it is also showing a relatively potent inhibiting effect on the

E. coli PriA.

Figure 18: Plot displaying the effect of inhibitor F0683-0207 on E. coli PriA. The fraction unwound value of DNA in the absence of the inhibitor is approximately 0.7 and as the concentration of the inhibitor increases the fraction unwound value decreases. Data are reported in triplicate and error bars reresent one standard deviation of the mean. III-8 Determining the activity of F0683-0207 on E. coli (MG1655):

As it was already found that the inhibitor F0683-0207 is not species specific, I wanted to determine if it would affect bacterial cell growth in culture. The concentration of bacteria in a liquid culture can be determined spectrophotometrically by measuring the optical density at 600 nm. In order to determine the activity of the inhibitor on E. coli, we have decided to treat the culture of E. coli cells with the inhibitor and record the absorbance value at 600 nm wavelength.

Inoculation of E. coli cells

Initially, two samples (one with LB medium and DMSO and the other with LB medium and F0683-0207) were inoculated with strain MG1655 E. coli. Various concentrations of F0683-0207 ranging from 0.01 µM to 3 µM were used. The cells were examined under the microscope after overnight inoculation. It was found that the culture of cells with DMSO have shown rod shaped cells as expected (data not shown). The culture with the inhibitor contained clumps of cells (data not shown). Despite the clumping behavior, in the presence of the inhibitor a notable growth of bacterial cells is

taking place. This indicates that the inhibitor is not capable of affecting the growth of E. coli cells in liquid culture to a great degree. I next conducted a timecourse experiment in which the optical density at 600 nm was measured at various time intervals following inoculation of liquid cultures with MG1655 E. coli either in the presence or absence of inhibitor. The absorbance of the DMSO solvent control culture and the culture with the inhibitor F0683-0207 as a function of time are shown in Figure 19.

Figure 19: Plot displaying the growth rate of E. coli in the presence and absence of the inhibitor.

It can be clearly seen from the plot that at every time interval the absorbance values of both the cultures were almost same and even the shape of the graph looks similar. The absorbance values of the culture with inhibitor in it would have been less when compared to the values of the culture without the inhibitor, if F0683-0207 had shown an effect on cell growth. Thus, it can be concluded that F0683-0207 has no effect on the growth of strain MG1655 E. coli. What remains uncertain is whether the inhibitor F0683-0207 is getting inside the cells (a cell permeability issue), or if PriA is capable of reactivating replisomes without its helicase activity to a degree that would allow normal levels of cell growth. These questions could be with the topic of further studies.

III-9 Crystallization:

Cocrystallization experiments were setup to study the physical interaction between

N. gonorrhoeae PriA and the inhibitor F0683-0207. These experiments should provide information about the location of the inhibitor binding site on PriA, and reveal how inhibitor binding affects PriA’s structure. Cocrystallization was attempted using the hanging drop vapor diffusion method and commercially-available sparse matrix screens from Hampton Research. The Hampton Research Crystal Screen, Crystal Screen 2, and

Index Screen reagent kits are designed to provide a highly effective and rapid screening method for the crystallization of macromolecules. This is a simple and practical screen for finding initial crystallization conditions. Using these screens, conditions of crystallization for proteins, oligonucleotides, and many small molecules have been determined.

Hampton Crystal Screen has 50 reagents and all the reagents were used. The total drop volume was 3 µL, which contains the sample, reservoir and additive each of volume

1µL. The sample used was a combination of inhibitor F0683-0207 and N. gonorrhoeae

PriA, the sample buffer used was minimal buffer (10 mM MES pH 6, 0.5 M Ammonium acetate) and the total reservoir volume was 500 µL. Immediately after setting up the crystal screen a carefull obseravtion of drops was made under the microscope. I have recorded all observations and I was particularly careful to scan the focal plane for small crystals. Observations were made once each day first week, then once a week there after.

Records also indicated whether the drop is clear, contains precipitate, or crystals. Clear drops indicate that either the relative supersaturation of the sample and reagent is too low or the drop has not yet completed equilibration. Drops containing precipitate indicate that

either the relative supersaturation of the sample and reagent is too high, the sample has denatured, or the sample is heterogeneous. If the drop contains a macromolecular crystal the relative supersaturation of the sample and reagent is good.

On the first day almost all of the reagents have exhibited a clear drop or a low magnitude brown precipitate. After a week, most of the drops have moved from being clear to a low magnitude brown precipitate, except a few which have shown signs of potentially being microcrystals and needles. After a week, the reagents which have shown potential microscrystals and needles were as listed below:

Table 4: Hampton Crystal Screen scoring sheet showing the reagents which lead to potential microcrystals and needles.

Reagent Formulation Observation made after two weeks 07) 1.4 M Sodium Acetate, 0.1 M Na Cacodylate pH 6.5 Microcrystal and needles 16) 1.5 M Lithium Sulfate, 0.1 M Na Hepes pH 7.5 Microcrystal and needles 17) 30% PEG4000, 0.1 M Tris HCl, 0.2 M Lithium Sulfate Microcrystal and needles 25) 1 M Sodium Acetate, 0.1 M Imidazole pH 6.5 Microcrystal and needles 28) 30% PEG8000, 0.1 M Na Cacodylate, 0.2 M Na Acetate Microcrystal and needles 31) 30 % PEG 4000, 0.2 M Ammonium Sulfate. Microcrystal and needles

Tray-1 (1-24) Tray-2 (24-48) Tray-3 (49-50)

1 2 3 4 5 6 25 26 27 28 29 30 49 50

7 8 9 10 11 12 31 32 33 34 35 36

13 14 15 16 17 18 37 38 39 40 41 42 19 20 21 22 23 24 43 44 45 46 47 48

Figure 20: Tray setup for Hampton Crystal Screen. The blue color represents the formation of microcrystals and needles in those particular wells and the reagent formulations responsible for the formation of needles and microcrystals are given in the table above. The colorless wells indicate either a clear drop or formation of precipitate.

Hampton Crystal Screen 2 has 48 reagents and all the reagents were used. The total drop volume was 3 µL, which contains the sample, reservoir and additive each of volume

1 µL. The sample used was a combination of inhibitor F0683-0207 and N. gonorrhoeae

PriA, sample buffer used was minimal buffer (10 mM MES pH 6, 0.5 M Ammonium acetate) and the total reservoir volume was 500 µL. On the first day almost all of the reagents have exhibited a clear drop or a low magnitude brown precipitate. After a week, most of the drops have moved from being clear to a low magnitude brown precipitate, except a few which have shown signs of being potential microcrystals and needles. Later a week after, the reagents which have shown potential microscrystals and needles were as listed below:

Table 5: Hampton Crystal Screen 2 scoring sheet showing the reagents which lead to potential microcrystals and needles.

Reagent Formulation Observation made after two weeks 14) 2.0 M Ammonium Sulfate, 0.1 M Na Citrate pH 5.6 Needles

15) 1.0 M Li Sulfate, 0.1 M Na Citrate, 0.5 M NH4 sulfate Needles 24) 30% JeffamineM-600, 0.1 M MES pH 6.5, 0.05 M CsCl Needles 28) 1.6 M Na Citrate pH 6.5 Needles 35) 70 % MPD, 0.1 M Hepes pH 7.5 Needles 36) 4.3 M NaCl, 0.1 M Hepes pH 7.5 Microcrystal and needles Tray-1 (1-24) Tray-2 (24-48)

1 2 3 4 5 6 25 26 27 28 29 30

7 8 9 10 11 12 31 32 33 34 35 36

13 14 15 16 17 18 37 38 39 40 41 42

19 29 21 22 23 24 43 44 45 46 47 48

Figure 21: Tray setup for Hampton Crystal Screen 2. The blue color represents the formation of potential microcrystals and needles in those particular wells and the reagent formulations responsible for the formation of needles and microcrystals are given in the table above. The green color represents the formation of only needles. The colorless wells indicate either a plain drop or formation of precipitate.

Hampton Index Screen is a 96 reagent crystallization screen that utilizes a broad,

yet refined portfolio of crystallization reagent systems, which include the following: (1)

traditional salts versus pH; (2) neutralized organic acids; (3) high salt concentration

mixed with low polymer concentration as well as high polymer concentration mixed with

low salt concentration and; (4) low ionic strength using polymers versus pH. These

reagent systems are formulated across a sparse matrix and incomplete factorial of

concentration ranges, sampling a pH range of 3 to 9.24 Hampton Index Screen has 96

reagents but only 48 reagents were used in the experiment. The total drop volume was 3

µL, which contains the sample, reservoir and additive each of volume 1µL. The sample

used was a combination of inhibitor F0683-0207 and N. gonorrhoeae PriA, the sample

buffer used was minimal buffer (10 mM MES pH 6, 0.5 M Ammonium acetate) and the

total reservoir volume was 500 µL. On the first day almost all of the reagents have

exhibited a clear drop. After a week, most of the drops have moved from being clear to a

low magnitude brown precipitate, except few which have shown signs of being potential

microcrystals and needles. The reagents which have shown potential microscrystals and

needles were as listed below:

Table 6: Hampton Index Screen scoring sheet showing the reagents which lead to potential microcrystals and needles.

Reagent Formulation Observation made after two weeks

07) 0.1 M Citric acid pH 3.5, 3 M NaCl Needles 09) 0.1 M BIS-TRIS pH 5.5, 3 M NaCl Needles 11) 0.1 M Hepes pH 7.5, 3 M NaCl Microcrystal and needles 22) 0.8 M Succinic acid pH 7.0 Microcrystal and needles 23) 2.1 M DL-Malic acid pH 7.0 Needles 24) 2.8 M Sodium acetate trihydrate pH 7.0 Needles

Tray-1 (1-24) Tray-2 (24-48)

1 2 3 4 5 6 25 26 27 28 29 30

7 8 9 10 11 12 31 32 33 34 35 36

13 14 15 16 17 18 37 38 39 40 41 42

19 29 21 22 23 24 43 44 45 46 47 48

Figure 22: Tray setup for Hampton Index Screen. The blue color represents the formation of potential microcrystals and needles in those particular wells and the reagent formulations responsible for the formation of needles and microcrystals are given in the table above. The green color represents the formation of only needles. The colorless wells indicate either a plain drop or formation of precipitate.

CHAPTER IV

CONCLUSIONS

In this study, we have adapted a fluorescence polarization-based DNA unwinding assay for use in HTS to identify small molecule inhibitors of PriA helicase activity.

Among all the five lead compounds, F0683-0207 was the most promising in terms of its potency according to the DNA unwinding assays. Thus, I selected it for further study in order to understand the mechanism by which it inhibits the DNA unwinding activity of N. gonorrhoea PriA. Though the HTS assay includes both PriA and PriB, I determined that

F0683-0207 targets only PriA and not PriB. Furthermore, the inhibitory effects of F0683-

0207 are not restricted to primosome proteins from the bacterial species whose PriA and

PriB proteins were used in HTS. While the HTS made use of N. gonorrhoeae PriA and

PriB proteins, I have found that E. coli PriA is also inhibited by F0683-0207. Thus, the binding site on PriA that is targeted by F0683-0207 is likely a fairly well-conserved structural feature of the enzyme.

The mechanism of inhibition studied through the steady-state enzyme kinetic analysis indicates that F0683-0207 exhibits a mixed mode of inhibition with respect to

ATP and with respect to DNA. This indicates that F0683-0207 binds to PriA at a site that

is distinct from the primary ATP and DNA binding sites and presumably blocks necessary conformational changes in the helicase or distorts the conformation of the helicase in a manner that makes it less conducive to binding DNA and ATP and coupling

ATP hydrolysis to duplex DNA unwinding. In general, helicases undergo conformational changes as the ATP is being hydrolyzed and duplex nucleic acid is being unwound.

Differences in the values of the inhibitor equilibrium binding constants were observed for the binding of F0683-0207 to the PriA·PriB·ATP complex (Ki, obtained from the DNA titrations) and the PriA·PriB·dT36 complex (Ki’, obtained from the ATP titrations). Since the average Ki value is lower than the calculated average Ki’ value for DNA titrations

(values shown in the results part), it indicates that inhibitor has stronger affinity towards the free enzyme than the enzyme:ATP complex. And since the average Ki value is greater than the average Ki’ value in ATP titrations, it indicates that inhibitor has stronger affinity towards the enzyme-DNA complex than the free enzyme. These values provide indirect evidence that conformational changes accompany binding of DNA and ATP to

PriA.

In order to identify the loaction where F0683-0207 binds to the N. gonorrhoeae

PriA, I have attempted to crystallize the lead compound and the primosome component mixture. Unfortunately, the outcome was not satisfactory since quality crystal growth was not obtained, and hence the binding location has not been established. Future work is required to study the other lead compounds and identify a potential candidate to use it to target the DNA replication restart mechanism in this gonorrheal infection causing pathogen.

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