The biological activity of GYY4137

Among the number of newly introduced and developed donors of H2S, GYY4137 is

the best characterised and studied compound hitherto in term of the mode of H2S

generation and biological activity (summarised in Table 5.1). The release of H2S from

GYY4137 occurs through a hydrolysis pathway of two-steps; first with a direct sulfur- oxygen exchange to produce a molecule of H2S and arylphosphonamidothioate. The

second step, which is relatively slower, involves another exchange to produce H2S

P S- S N O +_H 2N O H3C

vasodilatory effect of GYY4137 in rat aortic ring. They demonstrated a long-lasting vasodilatory response to GYY4137 which was mediated through opening of KATP

channels. Using the same model, subsequent work by Bucci et al. (2012) showed that GYY4137-induced vasodilatation of aortic ring in a PKG-independent manner. Similarly, Chitnis et al. (2013) found that relaxant effect of GYY4137 mediated independently on eNOS signalling in bovine posterior ciliary artery. However, it has been suggested that GYY4137-released H2S possibly interacts with NO to generate

thiol-sensitive compound which exerts positive inotropic effect in rat cardiomyocytes (Yong et al., 2011). Interestingly, chronic treatment with GYY4137 reduced the blood pressure in normotensive rats and to a greater extent in SHRs (Li et al., 2008). A part from its actions on vascular smooth muscle, it has also been shown that GYY4137 mediates muscle relaxation on other nonvascular smooth muscles including human myometrium (Robinson and Wray, 2012) and primary human airway (Fitzgerald et al., 2014) through the opening of ATP-sensitive K+ _{channels. Other beneficial effects of}

GYY4137 on the cardiovascular system have been demonstrated including antithrombotic activity by inhibiting platelet activation in mouse (Grambow et al., 2014); anti-atherosclerotic action in apolipoprotein knockout mice fed with high fat diet (Li et al., 2013); and mitigated fibrosis progression in myocardial pathologies (Meng et al., 2015b, Lin et al., 2016).

The activity of GYY4137 therapy in cardiac pathologies has also been tested. It has recently been reported that GYY4137 therapy for 7 days following permanent ligation of the left descending coronary artery preserved cardiac contractility and attenuated remodelling via increased natriuretic peptide (ANP and BNP) release in rat myocardium (Lilyanna et al., 2015). Moreover, pre-ischaemia GYY4137 treatment for seven days elicited infarct limitation against myocardial ischaemia/reperfusion in vivo

by enhancing the expression of Bcl-2 and inhibiting the activity of MAPK, caspase-3 and Bax (Meng et al., 2015a). However, the therapeutically relevant time window for acute myocardial infarction implies administration as a postconditioning mimetic i.e. immediately prior to reperfusion since this is the time at which clinical therapeutic intervention can feasibly be made.

Table 5.1 Summary of the experimental studies using the slow-releasing hydrogen sulfide donor, GYY4137, listed in chronological order

Reference Experimental model(s) Main observed effect(s) Mechanism(s)

Li et al. (2008) Rat aortic ring, perfused rat kidney

and anaesthetised rat - Vasodilator and antihypertensive - Opened the vascular smooth muscle K

ATP

channels Li et al. (2009) Lipopolysaccharide (LPS)-induced

endotoxic shock in rat - Anti-inflammatory - Inhibited LPS-induced TNF-α production _{and increased IL-10 in rat blood} - Decreased the LPS-evoked rise in lung

myeloperoxidase activity and upregulation liver transcription factors (NF-КB and

STAT-3) Lee et al. (2011) Human cancer cell lines and

Xenograft study in mouse - Pro-apoptotic and cell _{cycle arrest} - Enhanced the generation of cleaved PARP _{and cleaved caspase 9.} - Partial G2/M arrest

Yong et al.

(2011) Rat cardiomyocytes - Positive _effect inotropic - Interaction with nitric oxide to generate _{thiol-sensitive compound} Bucci et al.

(2012) Phenylephrine-contracted mouse aortic ring - Vascular _{muscle relaxation} smooth - PKG-independent mechanism Robinson and

Wray (2012) Human and rat myometrium - Smooth _relalxation muscle - opening of KATP-channels Fox et al. (2012) Primary human articular chondrocyte

and mesenchymal progenitor cells - Cytoprotection - Unknown Chitnis et al.

(2013) Phenylephrine-contracted bovine posterior ciliary artery - Vascular _{muscle relaxation} smooth - The activity was dependent on endogenous _{production of both prostanoids and H2S} and mediated by KATP channels with no role

Li et al. (2013) Human synoviocytes and articular chondrocytes and Complete Freund’s adjuvant (CFA) model of acute joint inflammation in mouse

- Pro-inflammatory when given before CFA

- Anti-inflammatory when administrated after CFA

- Inhibited TNF-α, IL-1β, IL-6 and IL-8.

Lencesova et al.

(2013) HeLa cell - Pro-apoptotic - Upregulated the expression of IP3R1 and _{IP3R2 on both mRNA and protein levels} - Increased cytosolic Ca2+ _{and depleted}

reticular Ca2+ _{in concentration dependent}

manner

- Increased endoplasmic reticulum stress markers, such as X-box, CHOP and ATF4 Liu et al. (2013b) High fat fed apolipoprotein (ApoE-/-₎

mice - Anti-atherosclerotic - Abrogated TNF-α, ICAM-1and IL-6 _{expression and activated aortic Akt and} eNOS

Grambow et al.

(2014) Platelets activation and venular thrombus formation - Anti-thrombotic - Increased S-sulfhydration of platelet _proteins

- Reducted TRAP-induced adhesion molecule expression

Fitzgerald et al.

(2014) Primary human airway smooth muscle - Smooth muscle _relaxant - Opened sarcolemmal KATP channels Wei et al. (2014) Diabetic cardiomyopathy in H9c2

cells - Cytoprotection - Activated AMPK/mTOR signalling pathway

Burguera et al.

Lu et al. (2014) Human hepatocellular carcinoma cell line and subcutaneous HepG2

xenograft model

- Anti-cancer - Inhibited STAT3 phosphorylation and altered its downstream proteins including Bcl-2, Mcl-2, VEGF, surviving, HIF1-α and cyclin D1

Lee et al. (2014) Human cancer cell and normal cell _{- Selective anti-cancer - Increased lactate accumulation and impair} cellular pH regulation mechanisms, leading to metabolic acidosis

Wang et al.

(2015) Neck model of itch in mice - No scratching _{behaviour induced} compare to NaHS

- Unknown

Meng et al.

(2015b) Spontaneous hypertensive rat - Anti-fibrotic - Inhibited oxidative stress, TGF-β1/Smad2 _{pathway and α–SMA expression} Lilyanna et al.

(2015) Acute myocardial infarction model in rat - Cardioprotection _{when GYY4137 was} given pre-ischaemia

- Activated endogenous natriuretic peptide (ANP and BNP) at early post-ischaemic phase

Meng et al.

(2015a) Acute myocardial infarction model in rat - antioxidant and anti-_{apoptosis when} GYY4137 was given pre-ischaemia

- Suppressed MAPK phosphorylation,

activity of caspase-3 and expression of Bax, while increased the expression of Bcl-2 in the myocardium

Wu et al. (2015) Coxsackie virus B3-induced myocarditis in rat-derived cardiomyocytes

- Anti-inflammatory - Suppressed NF-КB and MAPK signalling

pathway Kubickova et al.

(2016) Cholinergic neuronal cell line - Differentiating factor - Unknown Lin et al. (2016) Unilateral ureteral obstruction model

Chen et al.

(2016b) Patients with spinal cord ischaemia/reperfusion injury and TNF-α-stimulated neural human cell lines (AGE1.HN and SY-SH5Y)

- Anti-apoptotic - Reduced serum TNF-α and, consequently, suppressed the activity of miR-485- 5p/TRADD axis

Chen et al.

(2016a) Mouse model of endotoxemia - Cytoprotective - Inhibited the elevated levels of TNF-α/IFN-_{γ in the plasma} Salvi et al. (2016) Isolated superfused bovine iris-ciliary

bodies - Inhibited sympathetic _{neurotransmission} - Opening of KATP channels Ivanciuc et al.

(2016) Respiratory syncytial virus-induced lung disease in wild and CSE knockout mice

- Antiviral and anti-

inflammatory effects - Attenuated inflammatory mediators and cells infiltration Meng et al.

(2016) SHR, human hypertrophic myocardium and neonatal rat cardiomyocytes

- Mitigated cardiac

hypertrophy - Abrogated KLF5-induced cardiac hypertrophy by S-sulfhydrating its transcriptional factor SP1 at Cys664

Xie et al. (2016) STD-induced LDLr-_/- _mice,

LDLr-_/-_Nrf2-_/- _{mice and HG/ox-LDL-}

treated endothelial cells

- Attenuated diabetes- accelerated

atherosclerosis

- Enhanced Nrf2 activity via S-sulfhydration of Keap1 at Cys151 _{and attenuated oxidative}

stress van den Born et

al. (2016) HCA plaque and HMEC-1 CSE

-_/- _{cell - Destabilised}

atherosclerotic plaque - Stimulated micro-angiogenesis to create intraplaque microvessels Coavoy-Sanchez

et al. (2016) Murine model of pruritus - Alleviated itching - NO/cGMP-independent inhibition of PAR-2 through opening KATP channel

Grambow et al.

(2016) Human whole blood - Antithrombotic - Enhanced endogenous thrombolysis by disturbing platelet-leukocyte aggregation Rodrigues et al.

Lobb et al.

(2016) Renal I/R injury in mice and NRK-52E - Anti-apoptotic - Decreased mito-ROS generation and improved mitochondrial membrane potential

VEGF vascular endothelial growth factor; HIF1-α hypoxia-inducible factor-1α, MAPK mitogen activated protein kinases; TNF-α tumor necrosis factor-alpha; SHR spontaneous hypertensive rat; KLF5 Krüppel‐Like Factor 5; SP1 specificity factor 1; STD Streptozotocin; LDLr low-density lipoprotein receptor; Nrf2 nuclear factor erythroid 2-related factor; HG high glucose; ox-LDL oxidised LDL; Keap1 Kelch-like ECH-associated protein 1; HCA Human carotid atherosclerotic; HMEC-1 human dermal microvascular endothelial cell, NO nitric oxide; cGMP cyclic guanosine monophosphate; PAR-2 proteinase-activated receptor-2.

5.1.3 Aim

The aims of this study were to:

1- Investigate for the first time the injury-limiting effects of GYY4137 at early reperfusion when given specifically as an adjunct to reperfusion in a rat model of acute myocardial infarction.

2- Characterise whether GYY4137 triggers the RISK pathway at early reperfusion to limit infarct size.

5.1.4 Hypotheses

We hypothesised the following:

1- GYY4137 will limit reperfusion injury when given just prior to reperfusion, thereby limiting ultimate infarct size.

2- The protective action of GYY4137 is due to H2S-releasing capacity.

3- GYY4137 replies on activation of key components of the RISK signalling cascade, namely PI3K/Akt and eNOS, at the first minutes of reperfusion to elicit its postconditioning effect.

5.1.5 Objectives

The aforementioned hypotheses were tested through the following specific experimental objectives:

1- Perform a dose-response study to determine the optimum cardioprotective dose of GYY4137 using an in vivo rat model of myocardial regional ischaemia/reperfusion injury.

2- Apply depleted (D-GYY4137), as a negative control, at reperfusion to investigate whether the parent structure of GYY4137 retains any cardioprotection.

3- Investigate the activity of the key kinases and effector proteins of the RISK pathway at the first minutes of reperfusion using Western blot analysis.

5.2 Materials and Methods

5.2.1 Animals

Male Sprague Dawley rats, 300-350 g (9-11 weeks old), were purchased from Harlan, UK and used to characterise the cardioprotection of GYY4137. Rats handling, housing and reporting are all explained in Chapter 2, section 2.2.