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!!