Effect of VPA on Rat Long-Chain Acyl-CoA Synthetase

Jakob Avi ShimshoniNational Institute of Health National Institute on AgingBrain Physiology & Metabolism Section

Outline Part I: Introduction

Characterization of Acyl-CoA Synthetase FamilyCharacterization of Acyl-CoA Synthetase Family

Long Chain Acyl-CoA Synthetase SubfamilyLong Chain Acyl-CoA Synthetase Subfamily

Pharmacological Profile of VPAPharmacological Profile of VPA

Review of Fundamental Enzyme KineticReview of Fundamental Enzyme Kinetic

Study ObjectiveStudy Objective

Part II: Results

VPA’s Effect on ACSL Enzyme Kinetic VPA’s Effect on ACSL Enzyme Kinetic

Future ObjectivesFuture Objectives

Characterization of ACS Family ACS are membrane enzymes essential for de novo lipid synthesis, fatty acid metabolism

and remodeling of membranes.

The utilization of fatty acids requires an initial activation by a two-step reaction catalyzed by ACS:

fatty acid + ATP acyl-AMP + PPi acyl-AMP + CoA acyl-CoA +AMP

ACS Family is subdivided according to substrate specifity. Five subfamilies have been characterized:

1. Short-Chain ACS: C-2 to C-42. Medium-Chain ACS: C-4 to C-123. Long-Chain ACS: C-12 to C-204. Bubblegum ACS: C-14 to C-245. Very Long-Chain ACS: C-18 to C-26

A total of 26 ACS protein sequences have been identified in humans.

Soupene et al, Exp Biol Med, 2008;Watkins et al, J Lipid Res, 2007

Long Chain Acyl-CoA Synthetase (ACSL) Isoforms

5 ACSL genes were found in rodents and humans: ACSL-1, ACSL-3, ACSL-4, ACSL-5 and ACSL-6. ACSL-6 has 2 spliced variants 6v1 and 6v2. Each ACSL isoform direct the acyl-CoA product to a specific metabolic fate.

Isoform MW (kDa)

Tissue distribution Function Organelles

ACSL-1 78kDa Adipose, Liver, Muscle, heart Heart: TG-SynAdipose: TG-SynLiver: β-Ox

Plasma membraneMitochondriaER

ACSL-3 Brain, Testis

ACSL-4 80-74kDa Adrenal Gland, Liver, Brain Peroxisomes: β-Ox

ER: Phospholipid Synthesis

PeroxisomesERMitochondria

ACSL-5 73-76kDa Adipose, Intestinal epithelial cells, Liver

Liver: TG-Syn Mitochondria ,Plasma membraneER

ACSL-6 Brain, Testis Neurons: AA and DHA incorporation into phospholipids

Plasma membraneMitochondria

Mashek et al. J Lipid Res, 2006; Soupene et al, Exp Biol Med, 2008;Watkins et al, J Lipid Res, 2007

In Vitro Substrate Specifity of ACSL Isozymes

Soupene et al, Exp Biol Med, 2008

Structure and Catalytic Activity of human Medium-Chain ACS (ACSM)

ACSM consists of a large N-terminal (435 AA) and a small C-terminal (110 AA) domains, connected by a linker region with the active site in the domain interface.

Essential co-factor: Mg2+

Kochan et al., J Mol Biol, 2009

Valproic Acid (VPA)

Approved by the FDA for: epilepsy, bipolar disorder and migraine.

Mechanism of Action: -Inhibiting voltage gated NaInhibiting voltage gated Na++ channels channels

-Potentiation of GABAergic activity by GABA- -Potentiation of GABAergic activity by GABA-

transaminase inhibitiontransaminase inhibition

-Inhibition of T-type Ca-Inhibition of T-type Ca2+2+ channels channels

-Suppression of NMDA-mediated exitation-Suppression of NMDA-mediated exitation

-HDAC inhibition-HDAC inhibition

-Reduced Arachidonic acid turnover-Reduced Arachidonic acid turnover

Loescher, CNS Drugs, 2002; Rao et al, Mol Psychiatry, 2008; Yasuda et al, Mol Psychiatry, 2009

Pharmacokinetic Profile of VPAIn humans: Bioavailability: 100± 10%Urinary Excretion: 1.8 ± 2.4%Bound in Plasma: 95 ±1%Clearance: 7.7 ± 1.5 ml/minVolume of Distribution: 9.8 ± 1.4 L (restricted distribution to the circulation and rapidly exchangeable extracellular water)Half-life: 14 ± 3 h

Bialer et al, Epilepsy Behav, 2004; Loescher, CNS Drugs, 2002 ;Antiepileptic drugs, 4 th edition, Chapter 84, 2002

Review of Fundamental Enzyme Kinetic

Michaelis-Menten ModelA kinetic model describing the effect of substrate concentration on velocity .

Enzymes, 2th edition, Chapters 5 and 8, 2000

Competitive Inhibition:

Inhibitor and substrate compete for the same active site

Enzymes, 2th edition, Chapters 5 and 8, 2000

Noncompetitive Inhibition:

Inhibitor binds to free enzyme and enzyme-substrate complex with similar affinity.

Enzymes, 2th edition, Chapters 5 and 8, 2000

Uncompetitive Inhibition:

Inhibitor binds only to the ES complex.

I

Enzymes, 2th edition, Chapters 5 and 8, 2000

Substrate Inhibition:

A second substrate binds to the ES complex to form an inactive ternary complex

Enzymes, 2th edition, Chapters 5 and 8, 2000

Study Objectives

Richard and colleges showed that VPA non-competitively inhibited microsomal ACSL isolated from rat brain (Richard et al., Psychopharmacol, 2006). The calculated ki value was: 14mM and substrate specific to AA. However an in-vivo study in rats clearly showed that a chronic VPA treatment did not change ACSL activity as compared to

control (Richard et al., Psychopharmacol, 2006) .

Objective: To determine which of the ACSL isozymes was specifically inhibited by VPA and to characterize its substrate specifity and the inhibiton kinetics of VPA.

Methods

Expression of the brain abundant ACSL-isozymes (3,4,6vi and 6vii) in E. Coli according to published procedures (Caviglia et al, JBC, 2004).

The enzyme activity was determined using radioisotop measurement of labeled 14C-AA-CoA as described in Caviglia et al, 2004.

Results

ACSL-3 ACSL-6vi

ACSL-6vii

VPA effect on ACSL-3, 6vi and 6vii

VPA effect on ACSL-4

The model best describes ACSL-4 kinetic is substrate inhibition. VPA’s effect on ACSL-4 kinetics, cab be elucidated at lower AA concentrations from the ascending part of the curve.

VPA effect on ACSL-4: Uncompetitive Inhibitor

Double-Reciprocal Plot

-1.0 -0.5-0.01

0.01

0.02

0.03

0.04

0.050mM VPA30mM VPA60mM VPA100mM VPA

1/S

1/V

Ki=25mMKm=5.2µMVmax=146nmol/min/mg

Li effect on ACSL-4 at 30mM

Li +at 30mM has no effect on ACSL-4

ACSL-4 is specifically inhibited by thiazolidinediones, a medication class used since the late 90s as antidiabetic drugs .FDA approved thiazolidinediones:

1 .Troglitazone (Rezulin®): possess antidiabetic and anti-inflammatory properties. Removed from market due to liver toxicity.

2 .Rosiglitazone (Avandia®): blockbuster antidiabetic drug; possess anti-inflammatory properties. Recent research suggests that Avandia® may be beneficial to a subset of patients with Alzheimer disease not expressing ApoE4 allele.

3 .Pioglitazone (Actos®): antidiabetic drug

Mode of Action :-Potent activators of PPARα and γ .

-anti-inflammatory effect due to decresed NFκB

ACSL-4 known Specific Inhibitors

Landreth et al, Neurotherapeutics, 2008; Risner et al, Pharmacogenomics J, 2006; Sarafidis, Fundam Clin Pharmacol, 2008

ACSL-4 Polymorphism

Mutations and deletions in ACSL-4 have been established in X-linked mental retardation and hematologic malignancies. Female ACS-4+/- mice showed increased uterine PGE2 and PGF2α, consistent with a defiency in arachidonoyl-CoA metabolism.

Cho et al., Biochem Biophys Res Commun, 2001

Determine substrate specifity of VPA inhibition (DHA, Palmitate)

Determine ACSL-4 distribution in Brain using immunocytochemistry

Screen potent CNS-active VPA-derivatives as potential ACSL-4 inhibitors

Measure AA and DHA turnover of CNS-active VPA-derivatives in rats

Establish a correlation between ACSL-4 inhibitory potency and turnover activity

Future Objectives

Acknoledgments

Dr. Mireille Basselin

Dr. Jagadeesh Rao

Dr. Hyung-Wook Kim

Dr. Stanely Rapoport

Thank you very much

Prof. Rosalin Coleman

Prof. Margaret Weis