Testing novel Angiotensin II receptor compounds to evaluate their vascular effects in vitro

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Dnr 2020/06

Testing novel Angiotensin II receptor compounds to evaluate their vascular effects in vitro

Selin Hamid

Degree Project in Pharmaceutical pharmacology, 30 credits, Spring semester 2020

Supervisor: Prof. Robert Widdop Department of Pharmacology Monash University

Victoria, Australia

Examiner: Prof, Fredrik Jernerén, PhD

Department of Pharmaceutical Biosciences Division for Pharmacology

Faculty of Pharmacy Uppsala University

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Abstract

Introduction: Angiotensin 2 is a biologically active octapeptide known to bind with high affinity to two separate receptors, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). The two receptors can be seen having opposite effects when activated. The effects of AT2R activation oppose the pathophysiological effects of AT1R and causes

vasodilation, anti-inflammatory and anti-fibrotic effects. The first non-peptide selective AT2R agonist compound 21(C21) has as of recent entered phase two clinical trials for the indication idiopathic pulmonary fibrosis. Two novel newly synthesized non-peptide compounds MH280 and MH727 which exert AT2R selectivity have been evaluated. The data regarding these compounds is limited and the compounds are still being investigated. MH280 was tested as an antagonist to complement previous work, while MH727 was tested as an agonist and an antagonist on AT2R in mouse thoracic aortic vessels.

Method: Isolated mouse thoracic aortic vessels was used to assess the functional

cardiovascular effects of compounds MH280 and MH727 in vitro. The thoracic aortic vessel was used to determine whether the compound could contract the vessel indicating agonistic effect or blocking a vasorelaxant effect indicating antagonistic effect.

Result: MH280 could not be seen abolishing the effects of C21-mediated vasorelaxation in mouse thoracic aortic vessels. MH727 evoked a concentration-dependent relaxation in mouse thoracic aortic vessels. MH727 caused a relaxation of 89.9% which was very similar to the relaxations by C21 which was 86.2 ± 8.6%. The compound could not be seen abolishing the effects of C21 when tested for antagonistic effects. Additionally, the concentration-dependent vasorelaxant effect of MH727 was abolished in the presence of the AT2R antagonist

PD123319.

Conclusion: Both MH280 and MH727 compounds seem to exert agonistic behavior which is believed to be caused by the stimulation of AT2R in isolated thoracic aortic vessels.

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1.0 Introduction

1.1 The renin angiotensin aldosterone system

The renin angiotensin aldosterone system (RAAS) is intricately involved in regulating blood pressure and fluid-electrolyte balance (Isaksson et al., 2020). The RAAS pathway includes some important components such as renin, angiotensin converting enzyme (ACE),

angiotensinogen, angiotensin I (Ang-I) and angiotensin II (Ang-II). The well documented RAAS pathway involves two consecutive peptide conversions (Jones et al., 2008).

Angiotensinogen is the precursor to all angiotensin’s and is converted to Ang-I via renin released from the kidney. The second conversion from Ang-I to Ang-II is catalysed via ACE (Jones et al., 2008). The main effector peptide of RAAS is Ang-II (Bosnyak et al., 2010) and plays an pivotal role in cardiovascular control in regulating blood pressure and fluid and electrolyte homeostasis.

1.2 Angiotensin type 1 receptor and angiotensin type 2 receptor

AngII is a biologically active octapeptide known to bind with high affinity to two separate receptors, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R).

The two receptors can be seen having opposite effects when activated. When AT1R is activated it results in the classical actions of RAAS such as vasoconstriction, fibrosis, and inflammation (Bosnyak et al., 2011). The effects of AT2R activation oppose the pathophysiological effects of AT1R and cause vasodilation, anti-fibrotic and anti-inflammatory effects(Bosnyak et al., 2011;

Hallberg et al., 2018). There is a great level of AT2R expression in embryonic tissue and the level of expression decreases rapidly after birth. In adults, the AT2R has been seen expressed in heart, kidney, brain, endothelial and vascular smooth muscle cells of the vasculature. With occurrence of tissue damage, a upregulation of AT2R has be observed (Hallberg et al., 2018).

1.3 AT

2

R signaling pathway

The AT2R signals via a G-protein coupled receptor and in recent years more knowledge has been gained about this receptor. This is mainly due to available selective non-peptide AT2R agonists which have made many studies in disease models possible (Colin Sumners et al.,

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2019). There are a number of signalling pathways for the stimulation of the AT2R, but the most frequently reported signalling pathways in vasculature is increased production of nitric oxide (NO), cyclic guanine 3´,5´-monophospate (cGMP) and bradykinin (BK) (Jones et al., 2008;

Wang et al., 2017). The increased NO and cGMP causes relaxation of the vascular smooth muscle cell such as blood vessels to relax. However, increased AT2R mediated cGMP is linked to natriuresis, which means that it is causing the opposite effect of the AT1R antinatriuretic effects (Widdop et al., 2003).

1.4 Drugs interfering with the RAAS

RAAS inhibiting drugs can effectively block different components of the system and therefore provide therapeutic advantages in heart failure, hypertension and other pathophysiological states. For the last 30 years RAAS inhibitors have become a fundamental treatment for cardiovascular diseases (Mirabito Colafella et al., 2019). In the 1990s, the development and synthesis of AT1 receptor blockers(ARB) namely Losartan occurred and thereafter numerous of ARBs have been synthesized and are available for the treatment of hypertension (Atlas A.

Steven, 2007). The first ever ACE inhibitor(ACEi) Captopril was introduced in 1978, ACEi can block the enzyme ACE and therefore inhibit the conversion from Ang-I to Ang-II (Atlas A.

Steven, 2007).

1.5 Present AT

2

R ligands

As mentioned earlier, the effects of AT2R activation oppose the pathophysiological effects of AT1R and cause vasodilation, anti-fibrotic and anti-inflammatory effects. However, the information about AT2 receptor mediated effects has been hindered by the lack of selective non-peptide AT2 receptor agonist and antagonist, particularly in vivo (Bosnyak et al., 2010).

Current studies lead us to the first selective non-peptide AT2R agonist named compound 21(C21). It has an agonistic effect that has been confirmed in numerous of in vivo studies (Isaksson et al., 2020). The compound has the highest affinity to the AT2R reported do date and it lacks affinity to the AT1R (Bosnyak et al., 2011; Hallberg et al., 2018). In human embryonic kidney cells, C21 showed a 4000-fold selectivity to AT2R (Matavelli and Siragy, 2015). As of recent, the compound has entered phase two clinical trials for the indication idiopathic

pulmonary fibrosis (Isaksson et al., 2020). Other AT2R ligands which are being studied is the antagonist EMA401 which has clinically been evaluated as a potential treatment for

neuropathic pain. C21 and EMA401 are in clinical trials for different indications which proves

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AT2R to be enigmatic (Isaksson et al., 2019). New selective AT2R compounds such as M132 from Uppsala University has been considered an AT2R selective antagonist (Wallinder et al., 2015).

1.6 Newly synthesized compounds

Current developments of AT2R ligands are the newly synthesized MH280 and MH727 by the medical chemistry department in Uppsala University. The data regarding these compounds is limited. In an earlier project at Monash University with Professor R. Widdop, MH280 was tested for potential AT2R agonistic effects. The results for MH280 showed a concentration- dependent relaxation of the pre-contracted mouse thoracic aortic vessel (Ablahad and Widdop, 2019). However, it is not known if MH280 could be used as an antagonist similar to the known antagonist PD123319 to test the antagonistic effects of the compound (Isaksson et al., 2020).

Such results would give more information to whether MH280 is an agonist, partial agonist or antagonist. MH727 has never been tested and therefore it is unclear if the compound is an agonist or antagonist, therefore, both modes of function have been investigated in preliminary studies. As mentioned earlier, the highly novel selective non-peptide AT2R compounds, MH280 and MH727, are compounds that are still being investigated and there are no other functional data at this stage. The pharmacologists involved with their testing have only been told they exhibit selective AT2R binding. Therefore, my study represents an unbiased

assessment of functional vascular effects of MH compounds from Uppsala University. Indeed, the characterization of these compounds is important since it can serve as a pharmacological research tool in the future and to further understand AT2R biology.

2.0 Aim

The aim of this project was to test the vascular effects of the two novel newly synthetized compounds, MH280 and MH727, that are reported to exhibit AT2R binding. MH280 was tested as an antagonist to complement previous work, while MH727 was tested as an agonist and an antagonist on AT2R in mouse thoracic aortic vessels.

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3.0 Material and Methods

3.1 Krebs solution

The Krebs solution is a physiological salt solution used to preserve the thoracic aortic vessel during experiments. Krebs Solution is a solution made from powdered sodium chloride (Merck Millipore, Germany), potassium chloride (Merck Millipore, Germany), magnesium sulphate (Sigma), monopotassium phosphate (Merck Millipore, Germany), sodium hydrogen carbonate (Sigma, USA), glucose (Amresco, USA) and calcium chloride dehydrate(Sigma, Japan).

The amount of each component was 68.9 g Sodium chloride (NaCl), Potassium chloride (KCl) 3.5g, Magnesium sulphate (MgSO4) 2.91g, Monopotassium phosphate (KH2PO4) 1.63 g, sodium hydrogen carbonate (CHNaO3) 21.0 g and glucose 9.01 g. The final concentration of each component of the Krebs solution was: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4 , 1.2 mM KH2PO4, 24.9 mM CHNaO3, 2.5 mM CaCl2, 11.1 mM glucose.

All of the components except CaCl2 were dissolved with 1 L distilled water (dH2O) while being stirred in a 1000 ml bottle to make a [10x] stock solution. The stilled solution was then stored in refrigerator at 4 °C (Spiers and Padmanabhan, 2005).

For each day an experiment was performed the stocked solution was distilled to obtain the low concentration [x1M], this was done by taking 100 mL from the stock solution and dissolving with 900 mL dH2O. For each 1 L of stock solution, 2.5 mL of CaCl2 was added. The solution was placed in a 14 L digital water bath (Ratek, Australia) heated to 37°C with bubbled carbogen (95% O2, 5% CO2, BOC, Australia).

3.2 Animals

Male FVB/N (Monash Animal Research Platform) mice approximately 11-12 week old from Monash Animal Research Laboratories were kept in the Monash University Pharmacolog Animal House. Animals were maintained at 21 °C ± 1°C- 5 °C with 12 h- light/ 12-h dark cycle and all the mice had access to food and water ad libitum. All experiment were approved by the School of Biomedical Science Animal Ethics Committee.

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3.3 Aorta dissection

The mice was sacrificed with isoflurane inhalation, the thoracic aortae and heart was removed and placed in a petri dish with ice cold Krebs solution buffer. The petri dish was placed under a dissecting microscope and with the use of fine scissors and forceps the thoracic aortae was cut transversely into eight ring segment with a size of approximately 1,5-2 mm (Spiers and Padmanabham, 2005).

3.4 Wire Myograph

The lumen of each aortic ring were threaded through two wires of 100 μm of stainless steel connected to an isometric force inducer (FT- 03; Grass Instruments, Astro-Med Inc, R.I, USA).

The Multi Myograph, model 610M (DMT, Australia) with four organ baths was connected to a Powerlab 8/35 channel recorder (ADInstruments, Australia). In every organ bath, 7 ml of Krebs solution was added with bubbled carbogen and the temperature in the bath was set to 37°C by a built-in heater.

3.5 In vitro reactivity

When the aortic rings were placed in each organ bath, the tension of the vessels was set to 0.5 g resting tension by stretching for approximately 30 minutes and allowed to equilibrate for 15 minutes. Following equilibration, 21 μl 10^-4M of the thromboxane A2 receptor agonist U46619 was added to attain maximum contractile response (70-80%). U44419 is a potent

vasoconstrictor and causes smooth muscle cells to contract (Nakahata, 2008). In result of the vessel contracting the graph line increases until plateau is reached, the tissue was washed several times with Krebs solution to reach baseline and then equilibrated for approximately 30 minutes. After reaching baseline and maximum response was determined, a sub-maximum contractile response (30%) was achieved using different concentrations of U44619 until plateau was reached again.

Each day one experiment was conducted. A total of eight baths were prepared with Krebs solution each day. In the presence of a potential antagonist in an experiment, the compound hypothesized to exert antagonistic behavior was added before pre-contraction to a sub- maximum contractile response with U46619 to ensure that it had equilibrated for at least 30 minutes (figure 1). The experiment could be conducted in two different ways depending on if

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the compound was tested as an agonist or an antagonist. The compounds that were tested as potential antagonists in this study was PD123319, M132, MH280 and MH727 and the compounds were added before adding U46619 to allow incubation for at least 30 minutes.

After equilibration, C21 was added in increasing concentrations (see table 1) to evoke relaxation of the thoracic aortic vessel. To test a compound for agonistic effects, only the vasorelaxant compound would be added to evoke relaxation, both C21 and MH727 were used to obtain a vasorelaxation of the thoracic aortic vessel. The concentration of the vasorelaxant added is presented in table 1. The concentration of antagonist tested was 10 μM and 100 μM of PD123319, 1 μM and 100 μM of M132, 1 μM and 10 μM of MH280 and MH727 as 1 μM.

Sodium nitroprusside was added in the final step to further relax the vessel and check the smooth muscle cells integrity.

Table I. Amount of C21 and MH727 added Concentration added (M)

7 μl of 10^-6 M 63 μl 10^-6 M 63 μl 10^-5 M 63 μl 10^-4 M 63 μl 10^-3 M 63 μl 10^-2 M

Amount of C21 and MH727 added as a vasorelaxant in each bath, starting from a bath concentration of 10^-9 until a final bath concentration of 10^-2is reached.

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Figure 1. Myograph protocol. For each bath, the thoracic vessel ring segment were stretched to 0.5 g and equilibrated for 30 minutes in 37°C. A maximum contractile function was achieved by adding U444169. The tissue was washed several times with Krebs solution to reach baseline. A sub-maximal contractile response was achieved by adding U44169 again in different concentrations. If a potential antagonist was added and incubated, the compound would be added just before adding U44169. After reaching a sub-maximal contractile response, the potential vasorelaxant compound was added in the concentrations 10^-9-10^-4 M, each time a concentrations was added a plateau had to be reached. At the end SNP was added.

3.6 Statistical analysis

All of the data are presented as mean responses ± standard deviation (SD) of mean. With the use of GraphPad Prism the differences in vasorelaxation between different treatments were analyzed. This was done using a mixed two-way ANOVA as this is the default method in which Prism handles missing and unequal n-values between groups. A P-value of P < 0.05. was considered to be significant.

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4.0 Results

4.1 C21-mediated vasorelaxation in the absence and presence of PD123319

Dose-response curves to compound 21 performed in mice thoracic aortic vessels in the absence (C21) and presence of PD123319. The relaxation is presented as a reversal of precontraction by U46619. The relaxation was dose-dependent, by using higher concentrations of C21, a greater relaxation was achieved. As seen in Figure 1, the maximal percentage of relaxation obtained from C21 alone at the concentration 1x10^-4 M was 86.2 ± 5.6%. The relaxation by C21 when incubated with 10 μM or 100 μM was 60.7 ± 12% or 10.8 ± 21% respectively. A significant difference could be observed between C21 alone and C21+ PD 10 μM (P<0.001), and between C21 alone and C21+ 100 μM (P<0.01), such that the effect of C21 was attenuated in the presence of PD123319.

Figure 1. Dose-response curve to compound 21 (C21) performed on mice thoracic aortic vessels with functional endothelium. The compound was tested in absence and presence of PD (10 µM and 100 µM). C21 was added in increasing concentration (10^-9-10^-4 M). Values are presented as mean responses ± SE. **P<0.01 for C21 alone versus C21+PD 100 μM, ***P<0.001 for C21 versus C21+PD 10 μM, mixed ANOVA (two-way) analysis.

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4.2 C21-mediated vasorelaxation in the absence and presence of M132

Dose-response curves to compound 21 performed in mice thoracic aortic vessels in the

absence(C21) and presence of M132. The relaxation is presented as a reversal of precontraction by U46619. The relaxation was dose-dependent, by using higher concentrations of C21, a greater relaxation was achieved. As seen in Figure 2, the maximal percentage of relaxation obtained from C21 alone at the concentration 1x10^-4 M was 80.4 ± 12.5%. The relaxation evoked by C21 when incubated with M132 1 μM at the concentration 1x10^-4 was 51.7 ± 19.7%.

The effect of C21 was significantly less at 1x10^-4 M in the presence of M132 (1 μM ). No relaxation could be observed by C21 when incubated with 100 μM of M132, although this was a single experiment observation.

Figure 2. Dose-response curve to compound 21 (C21) tested on mice thoracic aortic vessels with functional endothelium. The compound was tested in absence and presence of M132 (1 µM and 100 µM). C21 was added in increasing concentration (10^-9-10^-4 M). Values are presented as mean responses ± SE ****P<0.0001 only for C21 versus C21+M132 1 μM reveals a difference only at 10^-4M C21 concentration. Mixed ANOVA (two-way) analysis.

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4.3 C21-mediated vasorelaxation in the presence and absence of MH280 and Time-control

Dose-response curves to compound 21 performed in mice thoracic aortic vessels in the absence(C21) and presence of MH280. The relaxation is presented as a reversal of precontraction by U46619. The relaxation was dose-dependent, by using a higher

concentrations of C21, a greater relaxation was achieved. As seen in Figure 3, the maximal percentage of relaxation obtained from C21 alone at the concentration 1x10^-4 M was 79.3 ± 12.9%. The relaxation by C21 when incubated with MH280 1 μM at the concentration 1x10^-4 was 58.3 ± 14.1%. The maximal relaxation evoked by C21 when incubated with MH280 10 μM at the concentration 1x10^-4 was 74.9 ± 18.9%. No statistical significant difference were observed between C21 alone and C21+MH280 1 μM or 10 μM. No relaxation could be

observed by time-control. A significant difference could be observed between C21 alone versus time-control (P<0.0001) and C21+MH280 1 μM versus time-control (P<0.0001). A significant difference could also be observed between C21+MH280 10 μM versus time-control (P<0.001).

Figure 3. Dose-response curve to compound 21 (C21) tested on mice thoracic aortic vessel with functional endothelium. The compound was tested in absence and presence of MH280(1 µM AND 10 µM). C21 was added in increasing concentration (10^-9-10^-4 M). Time control was used as a control and therefore no addition of C21 was made. Values are presented as mean responses ± SE. ****P<0.0001 for C21 alone versus time-control and also for C21 versus C21+MH280 1 μM, ***P<0.001 for C21 versus C21+MH280 10 μM. Mixed ANOVA (two-way) analysis.

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4.4 C21 and MH727-mediated vasorelaxation and Time-control

The dose-response curves to compound 21 performed in mice thoracic aortic vessels in the absence(C21) and presence of MH280. The relaxation is presented as a reversal of

precontraction by U46619. The relaxation was dose-dependent, by using a higher concentrations of C21, a greater relaxation was achieved. C21 and MH727 cause a dose-

dependent relaxation and the final concentration 1x10-4 M gave an average relaxation of 86.2%

± 8.6 for C21 and 89.9% for MH727. No relaxation could be observed by time-control.

Figure 4. Dose-response curve to compound 21 (C21) and MH727 tested on mice thoracic aortic vessel with functional endothelium. C21 and was added in increasing concentration (10^-9-10^-4 M). Values are presented as mean ± SE. Time control was used as a control and therefore no addition of C21 was made.

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4.5 C21-mediated vasorelaxation in absence and presence of MH727

Dose-response curves to compound 21 performed in mice thoracic aortic vessels in the absence(C21) and presence of MH280. The relaxation is presented as a reversal of precontraction by U46619. The relaxation was dose-dependent, by using a higher

concentrations of C21, a greater relaxation was achieved. From the Figure 4, the maximal percentage of relaxation obtained from C21 alone at the concentration 1x10-6 M was 81.6%

and the relaxation evoked by C21 when incubated with 1 μM MH727 was 75.6%.

Figure 5. Dose-response curve to compound 21 (C21) tested on mice thoracic aortic vessel with functional endothelium. The compound was tested in absence and presence of MH727(1 µM). C21 was added in increasing concentration (10^-9-10^-4 M). Values are presented as mean responses ± SE.

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4.6 MH727-mediated vasorelaxation in the absence and presence of PD123319

Dose-response curves to compound MH727 performed in mice thoracic aortic in the absence(MH727) and presence of PD123319. The relaxation is presented as a reversal of precontraction by U46619. The relaxation was dose-dependent, by using a higher

concentrations of MH727, a greater relaxation was obtained. The final concentration of MH727 (1x10^-4 M) alone resulted in a 43.05 ± 12.6%. The maximal relaxation evoked by MH727 when incubated with 10 μM or 100 μM was s 53 ± 24.5% or 0% respectively. While n is low, there was no difference between these effects.

Figure 6. Dose-response curve to compound MH727 in the presence and absence of PD123319 ( 10 μM or 100 μM) tested on mouse thoracic aortic tissue with functional endothelium. MH727 was added in increasing concentration (10^-9-10^-4 M). Time control was used as a control and therefore no addition of C21 was made.

Values are presented as mean responses ± SE.

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5. Discussion

In the present study, the novel compounds MH280 and MH727 were synthesized by the Medicinal Chemistry Department in Uppsala University and given to Prof. Widdop at the Pharmacology Department in Monash University. MH727 is a novel newly synthesized compound which has never been tested before, it is hypothesized that MH727 is an AT2R selective ligand although it is not known whether it acts as an AT2R agonist or antagonist. As a starting point, MH727 was tested as a AT2R antagonist (R.Widdop, personal communication).

The compound was then tested as an agonist and finally it was tested in the presence of PD123319 which would give more information about the binding specificity towards AT2R.

MH280 has previously been tested by an Uppsala University student at Monash University, and has been tested for the compounds potential agonistic behaviour although further studies

needed to be made for the characterization of MH280. As the project started, I was informed that MH280 potential antagonistic effects needed to be tested in a vascular assay. As mentioned earlier, MH727 has never been tested and therefore it was necessary to test both modes of function. In a previous study by Bosnyak et al, the vascular effects of C21 was tested using mouse isolated aortic vessel and similar tissue was used for my experiments (Bosnyak et al., 2010). Although for this study, a higher concentration of vasorelaxant was used which gave a bigger relaxation response. This was done to achieve a proper relaxation of the thoracic vessels for the experiments.

Time control was used during experiment in a parallel tissue/ bath in which only U46619 was added. This ensured that time did not affect how the blood vessel was contracting. During experiments, time-control did not have tissue relaxation which means that time did not affect the vessels relaxation. This enables comparison when examining the experiments results between time-control and the compound tested. Krebs solutions was added in all baths, including time-control and therefore it could clearly be observed that Krebs solution did not affect relaxation.

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5.1 C21 in the presence and absence of PD123319

The project started with using PD123319 as an antagonist with C21, PD123319 is a known AT2R antagonist (Isaksson et al., 2020). In a study by Bosnyak et al, we could see that C21 causes a concentration-dependent vasorelaxant effect. C21 in the presence of PD123319 has been seen abolishing the concentration-dependent vasorelaxation effects caused by C21.

(Bosnyak et al., 2010) This allowed us to start of the vascular assay with C21 and PD1233619 to ensure that PD123319 could essentially block the effects of C21. PD123319 was used in this project as an AT2R antagonist in the presence of a potential agonistic compound to gain

additional knowledge in the compounds AT2R binding specificity. If the AT2 receptors were blocked by PD123319 it would block the potential agonistic compounds vasorelaxant effect.

MH727 was the only compound tested as an agonist in this project and therefore it was tested in the presence of PD123319.

When a lower concentration of 10 μM PD1233 was incubated it gave a small inhibitory effect, from Figure 1 a slight shift in the curve can be observed and potentially a C21 blockade. The C21 dose dependent vasorelaxation was abolished by incubating with a much higher

concentration of 100 μM PD123319. Statistically, a significant difference could be observed between C21 alone versus C21+ 100 μM PD123319 (P<0.01). Indeed, the results indicates that the effect of C21 was attenuated in the presence of PD123319

5.2 C21 in the presence and absence of M132

In a similar way to C21 in the presence of PD, M132 is an antagonist from the Medicinal chemistry department of Uppsala University. The compound was used and tested since it could serve as a further AT2R antagonist in this study other than PD123319. In the presence of the vasorelaxant C21 it could be seen blocking the effects of C21. When 1 μM of M132 was incubated, there could be seen a small shift of the curve indicating a small blockade of C21.

The effect of C21 was significantly less at 1x10^-4 M in the presence of M132 (1 μM) (P<0.0001). At a higher concentration of 100 μM of M132 the shift of the curve was much

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bigger indicating that the effects of C21 are being blocked, although this was a single experiment observation.

Both PD123319 and M132 in the presence of C21 could indicate a blockade of the vasorelaxant effect of C21. The effect can be explained as PD123319 and M132 blocking the AT2R receptor and therefore, not allowing C21 to bind to AT2R and causing vasorelaxation. These results together with the experiment were C21 was incubated with PD123319 (figure 1) confirm the use of this assay to demonstrate AT2R function However, additional experiments are required to increase the number of replicates.

5.3 MH280

As previously mentioned, the novel compound MH280 potential agonistic effects has previously been tested. The results suggested that MH280 may be an AT2R agonist. In this study, the compound has further been tested for the potential antagonistic effect (figure 3). By incubating with 1 μM and 10 μM it could be observed that the curve in Figure 3, had not really been altered suggesting that C21 had not been blocked by MH280. No significant difference between C21 alone and C21+MH280 1 μM and 10 μM could be observed. However, a

significant difference could be observed between C21 alone versus time-control (P<0.0001). A significant difference could also be observed for both C21+MH280 1 μM versus time-

control(P<0.0001) and between C21+MH280 10 μM versus time-control (P<0.001) could be seen. This indicates that the amount of time-controls are enough.

The observed behavior by MH280 suggests that it not an AT2R agonist, although more

experiments are required using a higher MH280 concentration (as used with PD123319). In an earlier project, MH280 caused a relaxation similar to C21 effects (Ablahad and Widdop, 2019).

Therefore, collectively, this data suggest that MH280 is not an antagonist and therefore more likely to be an agonist.

5.4 MH727

MH727 was tested as an agonist in Figure 4, the compound could be seen relaxing the thoracic aortic vessels similar to C21. MH727 evoked a relaxation of 89.9% similar to that of C21 which was 86.2 ± 8.6% assessed in parallel tissue. Although the results are not statistically

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also able to block the MH727-induced vasorelaxation response (figure 6), although not many experiments were conducted. To examine if the vasorelaxant effects observed by MH727 were AT2R -mediated, the antagonist PD123319 was incubated in presence of MH727. The effects of MH727 was abolished when incubated with the higher concentration of 100 μM. It is suggestive that the effects observed by MH727 is a result by the stimulation of AT2R.

MH727 was tested as an antagonist with a concentration of 1 μM in presence of C21 (figure 5) however it did not block the effects of C21 and the number of replicates are too low. The results suggests that MH727 is in fact an AT2R agonist and not an antagonist. Therefore, using an unbiased assessment, it seems that the results obtained suggests both compounds may be acting as agonists. However, more experiments are needed to confirm these preliminary studies.

5.5 Ethical aspect of performing animal experiments

All mice were sacrificed humanely with isoflurane overdose. An approval from the School of Biomedical Science Animal Ethics Committee for all experiment was given. The experiments were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. By minimizing the amount of animals to one mouse per experiment we could make sure that we used the absolute minimum animals necessary for the experiment. It is always important to consider the necessity of the use of Animals. For an experiment like this, it is not possible to measure the vasorelaxant effect by another method such as an in silico method.

5.6 Limitations

It was planned that four compounds from the medicinal chemistry department would be tested, only two of the compounds that were of most interest was tested due to shortage of time. The project at Monash University was stopped early due to COVID-19 pandemic. This has also made the amount of experiments limited, it was planned that each trial would consist of n=8 replicates. The lack of replicates and that the data is preliminary limits the opportunity to evaluate data statistically.

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6.0 Future studies

For future experiments, a good continuation to this project would be to test MH280 and MH727 as antagonists at higher concentrations of 100 μM since the higher concentration of PD123319 could be seen abolishing the vasorelaxant effect of C21. It would be interesting to test these same compounds in another assay such as measuring an increase in cGMP, since this is a well- known signaling pathway (se introduction). If successful, it would be exciting to proceed to in vivo experiments if these compounds turn out to be agonist positive compounds. It should be noted that these in vitro studies realistically are not comparable with in vivo studies and

therefore it would be exciting to continue to examine the effects of MH280 and MH727 in an in vivo setting. It would also be interesting to see if they modify inflammation or fibrosis, which are studies routinely done in Prof. Widdop´s laboratory

7.0 Conclusion

The experiments from this project provide new data suggesting that MH727 is an AT2R agonist and not an antagonist in these preliminary data. Previous data by a student at Monash

University showed concentration-dependent vasorelaxation for MH280. Therefore, collectively data from this project where MH280 did not block the effects of C21, suggests that the

compound is an AT2R agonist and not an antagonist. As such these findings provide

preliminary information to suggest that both compounds could be useful research tools to study AT2R biology.

8.0 Acknowledgements

I would like to express my sincere gratitude to my supervisor Prof. Widdop for his enthusiasm, motivation and immense knowledge. A special thank you to Iresha Spizzo for the guidance and help during lab.

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9.0 Svensk populärvetenskaplig sammanfattning

Renin angiotensin aldosteron systemet (RAAS) är ett viktigt system som kontrollerar blodtryck och vätska elektrolytbalansen. I RAAS finns olika viktiga komponenter däremot anses

angiotensin 2 vara den huvudsakliga effektiva peptiden hos RAAS som påverkar blodtryck och vätska elektrolytbalansen. Peptiden kan binda till två olika receptorer, angiotensin typ 1

receptor (AT1R) och angiotensin typ 2 receptor (AT2R). De två receptorerna kan ses ha

motsatta effekter vid aktivering, där aktivering av AT1R ger de klassiska effekterna hos RAAS som vasokontstriktion, inflammation och fibros. Vid aktivering av AT2R kan man istället se effekter som vasodilatation, anti-inflammation och anti-fibroseffekt.

Compund 21 (C21) som nyligen befinner sig i en fas II studie för indikation idiopatisk lungfibros är den första icke-peptidliknande selektiva AT2R agonist. Andra AT2R selektiva föreningar som nyligen syntetiserats av Uppsala Universitet är MH280 och MH727.

Informationen kring dessa föreningar är begränsad, man vet att de är selektiva AT2R ligander och man har i ett tidigare arbete studerat MH280 för dess potentiella effekt som agonist och sett att den likt C21 kunna orsaka vasorelaxation hos isolerade blodkärl från mus. Man har valt att studera MH280 för dess antagonistiska effekter i denna studie genom att se hur bra den kan blockera agonisten C21:s effekt. Föreningen MH727 är ny förening och har aldrig testats tidigare, man har därför valt att testa föreningen som både agonist och antagonist. I denna studie använde man sig av isolerade blodkärl från möss, blodkärlen har skurits till små ring segment som man trätt på två metalltrådar i ett Wire Myograph instrument som mäter

spänningen hos blodkärlen. Blodkärleken kontraherades med föreningen A2 tromboxane för att sen kunna mäta föreningarnas förmåga att relaxera blodkärlen (testat som agonist) alternativt kunna blockera en förenings vasorelaxativa effekt(testat som antagonist).

Resultat från studien visade att när MH280 användes som en AT2R antagonist, observerades ingen inhibering av C21:s effekt som orsakar vasorelaxation hos blodkärlen. När man testat MH727 som agonist visade MH727 likt C21 orsakade vasorelaxation, MH727 och C21 resulterade i en vasorelaxation på 89.9% respektive 86.2 ± 8.6%. Hos båda föreningar kunde en koncentrations-beroende relaxation av blodkärl observeras. Detta tyder på att båda

föreningarna med stor sannolikhet är AT2R agonister. Karakterisering av dessa föreningar är viktig, eftersom de kan tjäna som viktigt verktyg inom AT2R biologi.

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YYYY-MM-DD Dnr xxxx

Testing novel Angiotensin II receptor compounds to evaluate their vascular effects in vitro