MODULATION OF HUMAN MODULATION OF HUMAN PYRIDOXAL KINASE PYRIDOXAL KINASE ACTIVITY BY DRUGSACTIVITY BY DRUGS

Samuel AboagyeSamuel AboagyeHoward Hughes Medical Howard Hughes Medical

Institute Scholar.Institute Scholar.

ContentsContents IntroductionIntroduction

Expression and purification of human Expression and purification of human Pyridoxal Kinase (hPLK)Pyridoxal Kinase (hPLK)

Effects of drugs on PL kinase activityEffects of drugs on PL kinase activity

Vitamin B6Vitamin B6 Vitamin B6 is the most essential vitamin serving Vitamin B6 is the most essential vitamin serving

a vital role in the function of more than 100 a vital role in the function of more than 100 enzymes, including:enzymes, including:

-Neurotransmitter metabolism – dopamine, -Neurotransmitter metabolism – dopamine, serotonin, histamine, taurine, epinephrine serotonin, histamine, taurine, epinephrine

-Red blood cell formation - synthesis of heme-Red blood cell formation - synthesis of heme

-Amino acid metabolism -Amino acid metabolism

Diseases associated with Diseases associated with vitamin B6 deficiencyvitamin B6 deficiency

Neurological disorders Neurological disorders Anemia Anemia Blood sugar levels Blood sugar levels Cardiovascular disease Cardiovascular disease Immune disorders Immune disorders

N

CH2OH

CHO

HO

H3CN

CH2OH

CH2NH2

HO

H3CN

CH2OH

CH2OH

HO

H3C

N

CH2OPO3H2

CHO

HO

H3CN

CH2OPO3H2

CH2NH2

HO

H3CN

CH2OPO3H2

CH2OH

HO

H3C

Pyridoxine (PN) Pyridoxamine (PM) Pyridoxal (PL)

Pyridoxine-5'-phosphate (PNP) Pyridoxamine-5'-phosphate (PMP) Pyridoxal-5'-phosphate (PLP)

Figure 1: Structures of the six B6 vitamers

Six forms of Vitamin B6 Six forms of Vitamin B6

ATP ATP

PN, PM PL

ADP + Pi ADP + Pi

PL kinase

PNP, PMP PLPde novo synthesis

PNPOx PNPOx-PLP

salvage pathway

apo B6 Enzyme

holo B6 Enzyme

aminoacids

12

4

3

Figure 3: Scheme for the salvage pathway of vitamin B6

PL kinase4

Salvage pathway of vitamin Salvage pathway of vitamin B6B6

Pyridoxal Kinase (PLK)Pyridoxal Kinase (PLK)

PL Kinase catalyses the transfer of a phosphate PL Kinase catalyses the transfer of a phosphate group from ATP to PL, PN or PM to form PLP, PNP group from ATP to PL, PN or PM to form PLP, PNP and PMP, respectively.and PMP, respectively.

KinaseKinasePL + ATP PLP + ADPPL + ATP PLP + ADP

Cofactor for B6-Enzymes Cofactor for B6-Enzymes

ObjectivesObjectives hPLK activity seems to be the key site for some hPLK activity seems to be the key site for some

disorders and side effects of many drugs.disorders and side effects of many drugs.

- Protein production- Protein production

- Kinetic studies- Kinetic studies

- X-ray crystallography- X-ray crystallography

3D Structure of hPLK3D Structure of hPLK

EXPRESSION AND EXPRESSION AND

PURIFICATION PURIFICATION

PROTEIN EXPRESSIONPROTEIN EXPRESSION Preparation of Liquid broth (LB) mediumPreparation of Liquid broth (LB) medium - Bacto - Bacto

Tryptone; Yeast Extract; NaCl;KTryptone; Yeast Extract; NaCl;K22HPOHPO44 ; NaH ; NaH22POPO4 ;4 ;7L 7L Deionized Water .Deionized Water .

Growing of the cells- The protein clone (Growing of the cells- The protein clone (E.coli PdxK gene) E.coli PdxK gene) is is added to the LB medium and incubated for a minimum of 24 added to the LB medium and incubated for a minimum of 24 hours.hours.

Grown cell culture incubated for at least an hour. Presence of Grown cell culture incubated for at least an hour. Presence of grown cell cultures checked at absorbance of 1 or greater grown cell cultures checked at absorbance of 1 or greater with a spectrophotometer at 600nm. with a spectrophotometer at 600nm.

IPTG added to cell culture and incubated for 5 hours - IPTG added to cell culture and incubated for 5 hours - expressing the PL kinase protein.expressing the PL kinase protein.

Cells broken using the cell breaker column to release protein, Cells broken using the cell breaker column to release protein, and the presence of protein checked by Gel-electrophoresis. and the presence of protein checked by Gel-electrophoresis.

Expression of ProteinExpression of Protein

Before IPTG After IPTG

hPLK Band Corresponding to marker position at Band 6 ( Mol Wt. 34.)

hPLK Purification hPLK Purification ProceduresProcedures

First column purificationFirst column purification

Sample loaded onto a 5x9cm TMAE column pre-Sample loaded onto a 5x9cm TMAE column pre-equilibrated with 20mM KPi pH 7.2. Protein eluted equilibrated with 20mM KPi pH 7.2. Protein eluted after Abs of 278 is below 0.4 with the equilibration after Abs of 278 is below 0.4 with the equilibration buffer and 40mM KPi 200mM Nacl pH 6.8. buffer and 40mM KPi 200mM Nacl pH 6.8.

Collected fractions checked at Abs of 278, followed by Collected fractions checked at Abs of 278, followed by gel electrophoresis to determine the fraction gel electrophoresis to determine the fraction containing the PL kinase.containing the PL kinase.

hPLK Band Corresponding to marker position at Band 6 ( Mol Wt. 34.)

Second Column PurificationSecond Column Purification Sample from 1Sample from 1stst column loaded onto a phenyl column loaded onto a phenyl

sepharose column (2.5x5cm) that is pre-equilibrated sepharose column (2.5x5cm) that is pre-equilibrated with 20% ammonium sulfate in 20mM KPi pH 7.2 with with 20% ammonium sulfate in 20mM KPi pH 7.2 with 5mM Mercaptoethanol and 0.2mM EDTA.5mM Mercaptoethanol and 0.2mM EDTA.

Equilibration buffer used to wash away excess DNA Equilibration buffer used to wash away excess DNA

until Abs 260nm is less than 0.1. until Abs 260nm is less than 0.1.

The enzyme eluted with the equilibration buffer and The enzyme eluted with the equilibration buffer and 20nM Na BES pH 7.5, 3% propylene glycol. 20nM Na BES pH 7.5, 3% propylene glycol.

hPLK Band Corresponding to marker position at Band 6 ( Mol Wt. 34.)

Purification columnsPurification columns

KINETIC STUDIESKINETIC STUDIES

How PL activity is measuredHow PL activity is measuredSpectrophotmetric AnalysisSpectrophotmetric Analysis

KinaseKinasePLPL PLPPLP MgATPMgATP

Change in absorbance at 388 nm

Initial Activity of ProteinInitial Activity of Protein

BUFFERBUFFER EnzymeEnzyme

(10mg/(10mg/ml)ml)

MGATPMGATP

(20MM)(20MM)PLPL

(4MM)(4MM)Ave. Ave. RATERATE

878 uL878 uL 10 ul10 ul 100 ul100 ul 12ul 12ul 0.1835 0.1835 minmin

Activity after compound is added Activity after compound is added THEOPYLLINE.THEOPYLLINE.

BufferBuffer EnzymeEnzyme

(10mg/(10mg/ml)ml)

InhibitoInhibitorr

(20MM)(20MM)

MGATPMGATP

(20MM)(20MM)PLPL

(4MM)(4MM)Ave.Ave.

RATERATE

863 ul863 ul 10 ul10 ul 15 ul15 ul 100 ul100 ul 12 ul12 ul 0.047 0.047 min.min.

848 ul848 ul 10 ul10 ul 30 ul30 ul 100 ul100 ul 12 ul12 ul 0.0285 0.0285 min.min.

833 ul833 ul 10 ul10 ul 45 ul45 ul 100 ul100 ul 12 ul12 ul 0.0233 0.0233 min.min.

Inhibition by TheophyllineInhibition by Theophylline

00.02

0.040.06

0.080.10.12

0.140.16

0.180.2

0 5 10 15 20 25 30 35 40 45 50

Volume of Theophylline

rate Series1

Activity after compound is Activity after compound is added added

CAFFIENECAFFIENEBufferBuffer EnzymeEnzyme

(10mg/(10mg/ml)ml)

InhibitoInhibitor(20Mr(20MM)M)

MGATPMGATP

(20MM(20MM))

PLPL

(4MM)(4MM)Ave.Ave.

RATERATE

863 ul863 ul 10 ul10 ul 15 ul15 ul 100 ul100 ul 12 ul12 ul 0.105 0.105 min.min.

848 ul848 ul 10 ul10 ul 30 ul30 ul 100 ul100 ul 12 ul12 ul 0.063 0.063 min.min.

833 ul833 ul 10 ul10 ul 45 ul45 ul 100 ul100 ul 12 ul12 ul 0.051 0.051 min.min.

Inhibition by CaffeineInhibition by Caffeine

00.02

0.040.06

0.080.10.12

0.140.16

0.180.2

0 5 10 15 20 25 30 35 40 45 50Volume of Caffiene

rate Series1

X-RAY CRYSTALLOGRAPHYX-RAY CRYSTALLOGRAPHY

Drugs that show inhibition such as Drugs that show inhibition such as theophylline are co-crystallized with theophylline are co-crystallized with PL Kinase to see where it binds. PL Kinase to see where it binds.

I hope to work on this in the future. I hope to work on this in the future.

CONCLUSIONCONCLUSION

These studies suggest that with theophylline, there is These studies suggest that with theophylline, there is a significant drop in activity as compared to caffeine. a significant drop in activity as compared to caffeine.

These finding suggests that certain drugs, such as These finding suggests that certain drugs, such as theophylline has antagonistic effect on hPLK.theophylline has antagonistic effect on hPLK.

Other drugs with similar chemical structure as Other drugs with similar chemical structure as

theophylline are known to cause neurological theophylline are known to cause neurological disorders. disorders.

We hypothesize that these drugs namely nicotine, We hypothesize that these drugs namely nicotine, theobromine and enprofylline may also inhibit PL theobromine and enprofylline may also inhibit PL Kinase activity.Kinase activity.

ReferencesReferences

1.Musayev et al,(1999) Crystallization and preliminary X-ray crystallographic 1.Musayev et al,(1999) Crystallization and preliminary X-ray crystallographic analyses of pyridoxine 5’-phosphate oxidase complexed with flavin analyses of pyridoxine 5’-phosphate oxidase complexed with flavin mononucleotide.J.Struct.Biol. 127,88-91. mononucleotide.J.Struct.Biol. 127,88-91.

2.Safo et al(2000) X-ray structure of Escherichia coli pyridoxine 5’-phosphate 2.Safo et al(2000) X-ray structure of Escherichia coli pyridoxine 5’-phosphate oxidase complexed with FMN at 1.8 A resolution. Structure 8, 751-762. oxidase complexed with FMN at 1.8 A resolution. Structure 8, 751-762.

3.Safo et al(2001) X-ray structure of Escherichia coli pyridoxine 5’-phosphate 3.Safo et al(2001) X-ray structure of Escherichia coli pyridoxine 5’-phosphate oxidase complexed with Pyridoxal 5’-phosphate oxidase complexed with oxidase complexed with Pyridoxal 5’-phosphate oxidase complexed with pyridoxal 5’-phosphate at 2.0 A resolution. J. Mol. Biol.310, 817-826 pyridoxal 5’-phosphate at 2.0 A resolution. J. Mol. Biol.310, 817-826

4.Di Salvo et al(2002) J.Mol.Bio. 315,385-397 4.Di Salvo et al(2002) J.Mol.Bio. 315,385-397 5.Safo et al(2003) Structure and properties of Recombinant Human Pyridoxine 5.Safo et al(2003) Structure and properties of Recombinant Human Pyridoxine

5’-phosphate Oxidase. Protein Sci.2003, In press. 5’-phosphate Oxidase. Protein Sci.2003, In press. 6.Martino et al(2003) Structure and mechanism of Escherichia Coli pyridoxine 6.Martino et al(2003) Structure and mechanism of Escherichia Coli pyridoxine

5’-phosphate Oxidase.Biochimica et Biopysica Acta Proteins& Proteomics, 5’-phosphate Oxidase.Biochimica et Biopysica Acta Proteins& Proteomics, 1647, 76-82 1647, 76-82

7.Safo et al(2003) Role of Proline Residues in the Folding of Serine 7.Safo et al(2003) Role of Proline Residues in the Folding of Serine Hydroxymethyltransferase. J. Biol.Chem., In Press.Hydroxymethyltransferase. J. Biol.Chem., In Press.

8.Cessac et al, Mechanisms of the Inhibition of Human Erythrocyte Pyridoxal 8.Cessac et al, Mechanisms of the Inhibition of Human Erythrocyte Pyridoxal Kinase by Drugs. Biochemical Pharmacology,Vol. 54 pp. 863-870,1997 Kinase by Drugs. Biochemical Pharmacology,Vol. 54 pp. 863-870,1997

9.The American Heritage Science Dictionary . 9.The American Heritage Science Dictionary . 10.Linus Pauling Institute at Oregon State University. 10.Linus Pauling Institute at Oregon State University.

http://LPi.oregonstate.edu/infocenter/vitamins/vitaminB6/http://LPi.oregonstate.edu/infocenter/vitamins/vitaminB6/

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS Howard Hughes Medical Institute and Howard Hughes Medical Institute and

organizers of the summer scholars program. organizers of the summer scholars program. Dr. Allison Johnson, Dr. Greg BuckDr. Allison Johnson, Dr. Greg Buck Dr. Martin Safo, Mentor, Assistant Professor Dr. Martin Safo, Mentor, Assistant Professor

Department of Medicinal Chemistry Department of Medicinal Chemistry Dr. Musayev Faik, Researcher, Institute of Dr. Musayev Faik, Researcher, Institute of

Structural Biology and Drug Discovery Structural Biology and Drug Discovery Amit Gandhi , PHD candidate, Department of Amit Gandhi , PHD candidate, Department of

Medicinal Chemistry Medicinal Chemistry Ashwani Goswami , PHD candidate, Ashwani Goswami , PHD candidate,

Department of Medicinal Chemistry.Department of Medicinal Chemistry.