the fibrate gemfibrozil is a no- and haem independent...
TRANSCRIPT
The fibrate gemfibrozil is a NO- and haem independent activator of soluble guanylyl cyclase: in vitro studies
Introduction
Soluble guanylyl cyclase (sGC)
Soluble guanylyl cyclase
(Pan et al., 2013)haem-NO/oxygen binding domain
coil-coil elements
Per/Arnt/Sim –like (PAS) domain GC catalytic domains
Cellular signaling with NO and cGMP
(Murad, 2006)
cGKs: cGMP-dependent protein kinase (PKG)IRAG: inositol receptor cyclic GMP kinase substrateVASP: Vasodilator-stimulated phosphoprotein
Phototransduction
Smooth-muscle relaxation
Platelet inhibition
Cell growth and differentiation
eNOS: Endothelial nitric oxide synthaseMLCK: Myosin light chain kinaseMLCP: Myosin Light Chain PhosphataseGTP: Guanosine triphosphatecGMP: Cyclic guanosine monophosphatePDE: PhosphodiesterasePKG: Protein Kinase G
NO-sGC pathway regulates vascular smooth muscle cell relaxation
PKG
MLCP
sGC dysfunction in hypertension
○:Young WKY●:Young SHR△:Old WKY▲:Old SHR
(Kloss et al., 2000)
Diminished sGC activity and expression exacerbate endothelial dysfunction, decrease vascular plasticity.
(Ruetten et al., 1999)
sGC α-subunit
sGC α-subunit sGC β-subunit
sGC β-subunit
NO-sGC pathway regulates platelet aggregation
SNP: sodium nitroprussidePG: prostaglandin
(Barsom et al., 2003)
platelet aggregation
glycoprotein IIb/IIIa
fibrinogen
sGC beta-subunit knock out in platelet aggregation
(Guoying et al., 2011)
sGC plays important roles NO-dependent inhibition of platelet activation.
Platelet-specific sGC-β1 deficient C57BL/6J mice.
SNP: NO donorSNAP1: NO donorForskolin: adenylate cyclase activator
sGC dysfunction in platelet accelerate thrombus formation
Time to visible thrombus formation Time to visible thrombus complete occlusion
Thrombus formation was accelerated in α1-/- mice after indometacin. Inhibition of NO enhanced thrombus formation.
Indo: indomethacin (prostaglandin inhibitor)L-NA: NOS inhibitor
(Erdmann et al., 2013)
BL6 mice
Agents that increase cGMP levels
NO donors
Angina
Heart failure
Phosphodiesterase inhibitors
Erectile dysfunction
Pulmonary hypertension
sGC stimulators
Pulmonary hypertension
sGC activators
(Murad, 2006)
sGC stimulators and activators
sGC stimulators Riociguat (BAY 63-2521, trade
name Adempas)
sGC activators Ataciguat (HMR 1766)
Cinaciguat (BAY 58-2667)
(Johannes et al., 2011)
Gemfibrozil
Agent used in dyslipidemiaSubclass, Drug Mechanism of Action Effects Clinical Applications
STATINS• Atorvastatin, simvastatin,
rosuvastatin, pitavastatinInhibit HMG-CoA reductase Reduce cholesterol synthesis and up-
regulate low-density lipoprotein (LDL) receptors on hepatocytes • modest reduction in triglycerides
Atherosclerotic vascular disease (primary and secondary prevention) • acute coronary syndromes
• Fluvastatin, pravastatin, lovastatin: Similar but somewhat less efficacious
FIBRATES• Fenofibrate, gemfibrozil Peroxisome proliferator-activated
receptor-alpha (PPAR-α) agonistsDecrease secretion of very-low-density lipoproteins (VLDL) • increase lipoprotein lipase activity • increase high-density lipoproteins (HDL)
Hypertriglyceridemia, low HDL
BILE ACID SEQUESTRANTS• Colestipol Binds bile acids in gut • prevents
reabsorption • increases cholesterol catabolism • up-regulates LDL receptors
Decreases LDL Elevated LDL, digitalis toxicity, pruritus
• Cholestyramine, colesevelam: Similar to colestipol
STEROL ABSORPTION INHIBITOR• Ezetimibe Blocks sterol transporter NPC1L1 in
intestine brush borderInhibits reabsorption of cholesterol excreted in bile • decreases LDL and phytosterols
Elevated LDL, phytosterolemia
NIACINDecreases catabolism of apo AI • reduces VLDL secretion from liver
Increases HDL • decreases lipoprotein(a) [Lp(a)], LDL
Low HDL • elevated VLDL, Lp(a); elevated LDL in statin-unresponsive or intolerant patients
• Extended-release niacin: Similar to regular niacin• Sustained-release niacin (not the same as extended-release product): Should be avoided
HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme ANPC1L1 : Niemann-Pick C1-Like 1
Mechanism of fibrates on lipid and lipoprotein metabolism
Fibrate
PPAR-α
LPL: lipoprotein lipase
Fibrates
Extent of cardiovascular and coronary benefit
(Rubins et al., 1999)
Extent of cardiovascular and coronary benefit
(Rubins et al., 1999)
(34 mg vs. 32 mg, P<0.001)
(170 vs. 177 mg, P<0.001)
(115 mg vs. 166 mg, P<0.001)
Extent of cardiovascular and coronary benefit
Gemfibrozil is effective for the prevention of myocardial infarction and death from coronary heart disease.
(Rubins et al., 1999)
Incidence of Death from Coronary Heart Disease and Nonfatal Myocardial Infarction in the Gemfibrozil and Placebo Groups
sGC is activated by gemfibrozil, but not by other fibrates.
Values are mean ± SEM (n = 9).
*P < 0.05 versus control (basal activity).(Michael et al.,2011)
Aim
GTPcGMP
cGKs
Smooth muscle relaxation Platelet inhibition
Gemfibrozil
sGC activatorsGC stimulator
sGC
𝐹𝑒2+ 𝐹𝑒3+
Oxidation or haem loss
GTP
N O
Materials and Methods
Materials
Bay 41-2272: sGC stimulator
Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate : DEA-NO (NO donor)
𝑴𝒏𝟐+ : sGC cofactor
Tween 20: sGC haem depletion
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one: ODQ (sGChaem oxidation)
protoporphyrin IX : PPIX (sGC activator)
Cinaciguat: sGC activator
Ataciguat: sGC activator
Recombinant human sGC enzyme
Vector: pBacPAK9pBacPak-αpBacPak-βpBacPak-αΔ269 pBacPak-βΔ200
Sf9 cells
competent cell: E. coli
CaCl2Electroporation
Assay of sGC activity
𝑃32
𝑃32
sGC
𝐹𝑒2+
(Schultz, 1974)
β-radiation
GTP cGMP
Aortic ring relaxation
AortaSacrificeSD
(Danish Myo Technology, Aarhus, Denmark)
Multiwire myograph system
14–16 weeks old,300–350 g
Platelets aggregation
Washed human plateletsPre-incubated with the different agent
Aggregation was initiated by 0.1 μM ADP
Western blot analysis
anti-Phospho-𝑉𝐴𝑆𝑃𝑆𝑒𝑟239
anti-β-actin
Results
Gemfibrozil-dependent activation of sGC and its effect of NO stimulation
Activity of purified sGC in the presence of indicated concentrations of gemfibrozil.
□𝐸𝐶50=94 [75-117] μM
▲𝐸𝐶50=48 [37-67] μM
Values are mean ± SEM (n = 5). *P < 0.05 versus control.
Gemfibrozil blunts maximal response to NO without affecting the 𝐸𝐶50 value.
Values are mean ± SEM (n = 6). *P < 0.05 versus control.
Gemfibrozil’s mechanism of action is different from that of described sGC stimulators.
Bay 41-2272: sGC stimulator
DEA-NO:diethylammonium salt (NO donor)
Such additive effect strongly suggests non-overlapping binding sites for BAY41-2272 and gemfibrozil.
Gemfibrozil targets the haem-binding domain
Gemfibrozil activates only sGC variants with intact β HNOX domain.
Values are mean ± SEM (n = 6) *P < 0.05.
The haem-binding domain is necessary for gemfibrozil-dependent activation of sGC.
𝑴𝒏𝟐+ : sGC cofactor
Effect of sGC haem depletion or oxidation on sGCactivation
Values are mean ± SEM from two independent experiments performed in triplicate
*P < 0.05 vs. control; #P < 0.05 vs. basal control.
Tween 20: sGC haem depletionODQ: sGC haem oxidationPPIX: protoporphyrin IX (Haem precursor)Cinaciguat: sGC activatorAtaciguat: sGC activatorDEA-NO: diethylammonium salt (NO donor)
Gemfibrozil is characteristic for haem-mimickingsGC activators ataciguat and cinaciguat
□𝐸𝐶50=14 [12-16] nM
●𝐸𝐶50=26 [22-32] nM
Values are mean ± SEM (n = 6). *P < 0.05 versus control.
□𝐸𝐶50=1.1 [0.9-1.3] μM
●𝐸𝐶50=1.7 [1.1-3.1] μM
Values are mean ± SEM (n = 6). *P < 0.05 versus control.
Activity of human sGC treated with cinaciguat or ataciguat in the presence of gemfibrozil.
These effects are consistent with competition for overlapping sites.
Gemfibrozil induces phosphorylation of VASP protein in platelets
Gemfibrozil inhibits ADP-induced platelet aggregation
Platelet aggregation induced by 0.1 μM ADP and light scatter.
value represents the mean ± SEM (n = 5) *P < 0.05 versus untreated control; #P < 0.05
Gemfibrozil is a vasoactive agent
Relaxation of aortic rings in response to different
concentrations of fibrates.
Values are mean ± SEM (n = 5) *P < 0.05 versus gemfibrozil
Gemfibrozil is a vasoactive agent
Relaxation of pre-constricted rat aortic rings with denuded or intact endothelium in response to different concentrations of gemfibrozil alone or in the presence of 100 nM BAY41-2272 or 10
μM ODQ.
■𝐸𝐶50=61 [37–100] μM
▽𝐸𝐶50=56 [33–81] μM
▲𝐸𝐶50=25 [10–52] μM
◇𝐸𝐶50= 27 [9–61] μM
Data are mean ± SEM (n = 5) *P < 0.05 versus control
Gemfibrozil is a vasoactive agent.
Bay 41-2272: sGC stimulator
Structure-activity relationship studies
Structure-activity studies. Activity of purified sGC was determined in the presence of 100 μM gemfibrozil or tested compounds containing the phenoxy or alkyl carboxyl moieties. Inset: structures of tested compounds.
Values are mean ± SEM (n = 6)*P < 0.05 versus control
dimethylpentanoic acid
phenoxy group
Vasoactive effects of gemfibrozil-like compounds
Relaxation of precontracted rat aortic rings in response to different concentration of gemfibrozil-like compounds.
■𝐸𝐶50=61 [37-100] nM
▲𝐸𝐶50=20 [10-36] nM
○𝐸𝐶50=108 [36-362] nM
Values are mean ± SEM (n = 5) *P <0.05 versus gemfibrozil
Compound 3
Compound 2
(log)
Molecular modelling of gemfibrozil binding site to HNOX domain
Molecular modelling of gemfibrozil binding site to HNOX domain. Close-up view of the human HNOX haem-binding pocket with bound cinaciguat (A) or two distinct molecules of gemfibrozil (B).
Conclusion
GTPcGMP
cGKs
Smooth muscle relaxation Platelet inhibition
Gemfibrozil
sGC activator
𝐹𝑒2+ 𝐹𝑒3+
Oxidation or haem loss
GTP
Thanks for attention!!