vpa inhibits acyl-coa-synthetase
DESCRIPTION
Acyl-CoA-synthatse 4:a novel antimanic targetTRANSCRIPT
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