Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

DOI: 10.1016/j.bmcl.2017.01.042

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Bioorganic and Medicinal Chemistry Letters p. 733 - 739 (2017)

Update date:2022-08-11

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Authors:

Wang, Yinhu

Mowla, Rumana

Guo, Liwei

Ogunniyi, Abiodun D.

Rahman, Taufiq

De Barros Lopes, Miguel A.

Ma, Shutao

Venter, Henrietta

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Article abstract of DOI:10.1016/j.bmcl.2017.01.042

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negativ

Full text of DOI:10.1016/j.bmcl.2017.01.042

Accepted Manuscript

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux

pump AcrB for the reversal of antimicrobial resistance

Yinhu Wang, Rumana Mowla, Liwei Guo, Abiodun D. Ogunniyi, Taufiq

Rahman, Miguel A. De Barros Lopes, Shutao Ma, Henrietta Venter

PII:

S0960-894X(17)30055-0

DOI:

http://dx.doi.org/10.1016/j.bmcl.2017.01.042

BMCL 24618

Reference:

Bioorganic & Medicinal Chemistry Letters

To appear in:

Received Date:

Accepted Date:

11 January 2017

13 January 2017

Please cite this article as: Wang, Y., Mowla, R., Guo, L., Ogunniyi, A.D., Rahman, T., De Barros Lopes, M.A., Ma,

S., Venter, H., Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for

the reversal of antimicrobial resistance, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/

10.1016/j.bmcl.2017.01.042

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Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug

efflux pump AcrB for the reversal of antimicrobial resistance

Yinhu Wang^1#, Rumana Mowla^2#, Liwei Guo¹, Abiodun D Ogunniyi³, Taufiq Rahman⁴,

Miguel A De Barros Lopes², Shutao Ma^1*& Henrietta Venter^2*

¹Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of

Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,

Jinan 250012, China

²School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University

of South Australia, SA 5000, Australia

³Australian Centre for Antimicrobial Resistance Ecology, School of Animal and Veterinary

Sciences, The University of Adelaide, SA 5371, Australia

⁴Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge

CB2 1PD, United Kingdom

^#These authors contributed equally

*Corresponding authors: Henrietta Venter, Email address: rietie.venter@unisa.edu.au and

Shutao Ma, Email address: mashutao@sdu.edu.cn

Running title: 2-Napthamide derivatives as efflux pump inhibitors

Keywords: 2-naphthamide, antimicrobial resistance, efflux pump inhibitor, drug efflux

pump, AcrB, multidrug resistance

ABSTRACT

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these

pumps important drug targets. We have synthesised a novel series of compounds based on a

2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative

bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux

pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds

were tested for their antibacterial action, ability to potentiate the action of antibiotics and for

their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial

against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux

indicating that they are substrates of the AcrB efflux pump. Three compounds were able to

synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-

(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to

the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the

MIC of the non-substrate rifampicin indicating that this compound acts specifically through

the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer

membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell

lines. Therefore, we have designed and synthesised a novel chemical compound with great potential

to further optimisation as inhibitor of drug efflux pumps.

Antibiotic-resistance is one of the most serious threats in healthcare today. Without new

strategies to address drug resistance, we are heading for a post-antibiotic era where small

injuries and minor infections will once again be fatal ¹. Recently, a few new antibiotics have

been approved for use against Gram-positive organisms such as Staphylococcus aureus.

However, infections caused by Gram-negative pathogens prove much harder to treat due to

their very high intrinsic drug resistance. In this group, intrinsic drug resistance is due to the

presence of an outer membrane - which acts as a permeability barrier - and the expression of

drug efflux pumps ^2-5. These efflux pumps remove antibiotics from the bacterial cell, thereby

lowering their concentrations to sub-inhibitory levels.

In Gram-negative bacteria clinical levels of resistance are conferred by transporters that

consist of tripartite assemblies that span the double membrane and periplasm of these

organisms such as the AcrAB-TolC efflux system from Escherichia coli ^{4, 6}. The AcrAB-

TolC system is widely distributed in Gram-negative organisms and is also a structural and

functional homologue of the MexAB-OprM drug efflux system from the notorious hospital-

acquired pathogen Pseudomonas aeruginosa ^7-9. In the AcrAB-TolC complex AcrB is the

inner membrane protein (IMP), TolC is the outer-membrane protein (OMP) and AcrA is the

periplasmic membrane fusion protein (MFP), which connects the first two proteins ^{10, 11}. The

inner membrane proteins are members of the resistance nodulation cell division (RND)-type

of transporters. They catalyse drug/H⁺antiport and is the part of the complex responsible for

drug selectivity ^12-15. AcrB is a homotrimeric asymmetric protein where the monomers cycle

through three conformational stages designated the loose/access, tight/binding and

open/extrusion stages ^16-19. These proteins recognise an extremely broad range of substrates

that are not limited to antibiotics but also include intracellular metabolites antiseptics,

5, 20-22

disinfectants, detergents, fatty acids, heavy metals, solvents and virulence factors

Hence, drug efflux pumps are also implicated in bacterial virulence and biofilm formation ^23-

²⁶. Moreover, functional efflux pumps are also necessary for the selection of drug-resistant

bacteria. Efflux pump inhibitors (EPIs) are therefore attractive targets to reverse multidrug

resistance, prevent the development of further resistance, inhibit biofilm formation, and

decrease virulence of clinically relevant pathogens ^{5, 27, 28}. Despite their crucial role in

bacterial pathogenesis and multidrug resistance, there are currently no inhibitors of drug

efflux pumps in clinical use. The first described EPIs for RND-type efflux pumps, the

peptidomimetic phenylalanylarginine-β-naphthylamide (PAβN) and its derivatives were

abandoned due to toxicity issues ^{3, 29, 30}

18, 31, 32

The recently solved structures of substrate- or inhibitor bound AcrB

provide a solid

basis for the development of high efficacy, target-specific inhibitors ^{5, 33, 34}. AcrB structures

containing the drugs minocycline and doxorubicin revealed two discreet multisite binding

pockets separated by a switch loop; the distal pocket in the binding/tight state and a proximal

pocket in the access/loose state ^{32, 35}. A structure of inhibitor-bound AcrB revealed that the

inhibitor binding site partially overlaps with the minocycline binding site in the distal binding

pocket, while another part of the inhibitor binds to a narrow hydrophobic pit lined by several

phenylalanine residues (F136, F178, F610, F615, and F628). Tight binding of the inhibitor to

residues in the pit prevents the functional rotation through the three stages; hence prevent the

efflux of antibiotics³¹.

The structural knowledge could be transferred to in silico screening to identify efflux pump

inhibitors. The feasibility of this approach has been demonstrated by our group ³⁶and various

other research groups^{28, 37, 38}. Recently, the structure of the periplasmic domain of AcrB was

produced in combination with several substrates and pyranopyridine inhibitors³⁹. These

results confirmed the importance of an interaction between inhibitors and residues from the

hydrophobic trap. It was also shown that the increased efficacy of the inhibitor MBX3135

was a result of a tight interaction between this compound and residues from the hydrophobic

trap³⁹.

In the past decades, several potent AcrB efflux pump inhibitors have been discovered. PAβN

is the first EPI with a broad-spectrum activity against RND-type efflux pumps, but it cannot

be developed for clinical use due to its toxicity . Besides, naphthylpiperazines (NMP) are

potent EPIs that can increase the activity of several antibiotics. However, they seem to be too

toxic for clinical use because of their “serotonin agonist’’ properties ⁴⁰. Another group of

quinoline EPIs show only full activity at high concentrations but their toxicity is unknown ⁴¹.

MBX3135 is the most active EPI in the sub-micromolar range as far ³⁹, but its target

identification and toxicity are unknown.

In this study we aim to provide novel lead compounds that could be developed into EPIs for

antibiotic resistant Gram-negative bacteria. A series of compounds were synthesised based on

a 2-naphthamide core. These compounds were examined for their ability to inhibit the

AcrAB-TolC efflux pump from E. coli. The ability of the compound series to reverse the

resistance in a multidrug resistant strain of E. coli was investigated. Target specific activity

was also confirmed by measuring the inhibition of Nile Red efflux by AcrB and by ruling out

non-specific effects such as membrane permeabilisation. Finally, the cytotoxicity against Hep

G2 cells was determined for the most promising compound.

The 2-naphthamide derivatives (A1-A14) were synthesized from the commercially available

benzaldehyde (1) as outlined in Scheme 1. These compounds shared common features with

previously published EPIs against AcrB from E. coli ^42-46: All compounds have a binary

aromatic ring or aromatic heterocyclic ring structure and the connecting bridge between the

nucleus and the side chain is usually composed of 3-4 atoms. In addition, they have a similar

lipid-water partition coefficient logP.

The reaction of 1 with diethyl succinate gave (E)-2-benzylidenesuccinic acid (2) in the

presence of sodium methylate through the Stobbe condensation. 4-Hydroxy-2-naphthoic acid

(3) was obtained by dehydration of 2 with conc. H₂SO₄at room temperature. And then, the

hydroxyl group of 3 was acetylated with acetic anhydride in pyridine to give the acetate

product 4 in a good yield. The chlorination of 4 with thionyl chloride was followed by

reaction with aqueous ammonia to afford 4-hydroxy-2-naphthamide (6) in 75.6% yield. The

alkylation of 6 with corresponding alkyl or aryl halides in the presence of potassium

carbonate gave the 2-naphthamide derivatives (A1-A14) in yields ranging from 68.7% to

77.3%. The newly synthesized compounds were characterized by MS and H NMR, and all

the spectral data were in agreement with the proposed structures.

Scheme 1: Reagents and conditions:(a) Dimethyl succinate, CH₃ONa/CH₃OH, reflux, 3 h;

(b) 15% NaOH, reflux, 4 h, then HCl; (c) conc. H₂SO₄, 8 h, rt; (d) Ac₂O, pyridine, 1 h, rt; (e)

SOCl₂, THF, 70°C, 5 h; (f) aqueous ammonia, 18 h, rt; (g) R₁Br, K₂CO₃, DMF, 70 °C.

These ability of these compounds to bind to AcrB and reverse antimicrobial resistance was

investigated by assessing reversal of resistance, inhibition of efflux and target specific

activity ⁵.

Antibacterial activity

The antibacterial potential of the compounds were assessed by determining their MIC against

the wild type drug resistant strain of E. coli (BW 25113) and the antibiotic sensitive strain

with a deletion of the AcrB component of the AcrAB-TolC efflux pump (BW 25113 ∆AcrB).

None of the compounds showed any antibacterial activity against the resistant strain, while

only A3 were able to inhibit growth of the antimicrobial sensitive BW 25113 ∆AcrB strain

and only at the highest concentration tested (1024 μg/ mL) (results not shown).

A3 synergises with antibiotics and reverse drug resistance

The ability of the compounds to reverse antibiotic resistance conferred by the expression of

36, 47

the AcrAB-TolC efflux pump was determined with standard checkerboard assays

Rifampicin, which is not a substrate of AcrB, was also included as a control as an EPI should

not have an effect on the MIC of a compound that is not an efflux pump substrate ^{5, 47}

A3 was the most effective compound and reduced the MIC of erythromycin, chloramphenicol

and TPP to that of the level observed in the cells lacking an efflux pump (Table 1). A2 and

A9 synergised with erythromycin and chloramphenicol respectively (results not shown). All

the other compounds failed to synergise with any of the antibiotics tested (results not shown).

As compound A3 showed the most promise as an efflux pump inhibitor, it was investigated

further. To ascertain that A3 act on the efflux pump specifically, its effect on cells without an

efflux pump was tested. Syngergism was also tested in the presence of rifampicin which is

not a substrate of the efflux pump. Compound A3 had no effect on cells that lacked an efflux

pump or on the MIC of rifampicin (Table 1).

These results indicate that A3 exerts its effect by inhibition of the efflux pump and not by a

non-specific mechanism. For the three substrates used, the ΣFIC of A3 was 0.25 which

indicated synergistic activity.

Table 1: MIC in the absence and presence of 4-isopentyloxy-2-naphthamide (A3) showing

synergetic activity with different antibiotics and TPP against the wild-type drug-resistant

strain of E. coli.

Minimum Inhibitory Concentration (MIC)* (µg/ mL)

A3 concentration (µg/ mL)

128 256

PAβN

(μg/ mL)

200

512

Antibiotic

(+)

(-)

(+)

(-)

(+)

(-)

(+)

(-)

(+)

AcrB

AcrB AcrB AcrB AcrB AcrB AcrB AcrB AcrB

ERY

CAM

TPP

128

1024

512

128

RIF

ERY = Erythromycin; CAM = Chloramphenicol; TPP = Tetraphenyl phosphonium chloride;

RIF = Rifampicin

*Data is representative of assays done in duplicate on four different days using different

batches of cells each time.

A3 is able to inhibit drug transport

An EPI should be able to inhibit the transport of an efflux pump substrate ^{5, 47}. The ability of

the compounds to inhibit pump-mediated efflux was assessed by measuring the reduction in

the efflux of the fluorescent substrate Nile Red ^{36, 48}. Nile Red is only weakly fluorescent in

aqueous solutions, but undergoes a significant increase in fluorescent quantum yield when in

non-polar environments such as the cell membrane. Cells were de-energised by incubation

with the ionophore CCCP and pre-loaded with Nile Red in the absence or presence of the

compounds. Upon energisation of the cells by the addition of glucose, the efflux of Nile Red

could be observed as a drop in fluorescence. A3 inhibited Nile Red efflux at 50 µM and at

100 µM was able to abolish efflux to the level of the pump-deleted strain (Figure 1). A12,

which failed to show synergism with any of the antibiotics tested, inhibited Nile Red efflux at

200 µM while A2 and A9 were also able to inhibit Nile Red efflux but only at much higher

concentrations of 500 µM. Most of the compounds were able to reduce Nile Red efflux to an

extent (Figure 1), even the compounds that were not able to synergise with any of the

antibiotics tested. However, their inhibitory activity was only achieved at the fairly high

concentration of 500 μM indicating that they act as competing substrates instead of inhibitors.

The wide ranging substrate recognition profile of drug efflux proteins means that many

inhibitors act through competitive inhibition ^{29, 49}. For instance the inhibitor PAβN is also a

transported substrate of the AcrB and MexB efflux pumps . It is clear from the inhibitor

bound structures of AcrB that there is a discrete difference in binding between a substrate and

an inhibitor. Substrates such as minocycline would occupy the deep binding pocket of the

binding protomer of AcrB, while inhibitors should also interact with a phenylalanine rich

groove, named the Phe trap ^{5, 6, 18, 39}

A10

A11

A12

Figure 1: Inhibition of Nile Red efflux. Wild-type-resistant cells with active pump (thick

black line), ΔAcrB drug-sensitive cells (grey line) or wild-type cells in the presence of the

compounds (dotted line) were preloaded with Nile Red before the start of fluorescence

measurements. Efflux was triggered at 100 sec by the addition of 0.2% glucose (indicated by

arrow). Representative fluorescent traces are shown for experiments with different batches of

cells done on three different days.

The predicted binding site of A3 overlaps with that of the inhibitor MBX2319

Docking analysis was performed to explore how A3 could bind to AcrB. The structure of the

periplasmic domain of AcrB bound to the inhibitor MBX2319 (PDB code 5ENO) was used

as a template ³⁹. The binding of compound A3 overlaped with that of MBX2319 and showed

multiple hydrophobic interactions with various side chains located in the distal binding

pocket (Figure 2). The isoamyl group at the 4-position of the naphthalene ring was anchored

to the hydrophobic trap via hydrophobic interaction with Phe178 and Val 612. The flexible

isoamyl side chain induced a conformational flexibility in the inhibitor that allowed π-π

stacking interaction between the naphthalene core and AcrB residue Phe628, which might be

important for tight binding. The naphthalene core interacted with hydrophobic residues

including Phe610 and Val139. The amide group at 2-position could form H-bonding

interactions with Pro326 which would further stabilize inhibitor positioning in the active site

(Figure 2). Overall, the predicted docking site of A3 correlated well with the binding of

MBX2319 to AcrB as obtained by crystal structure where Phe628, Phe178, and Phe610 were

also some key residues for interaction with MBX2319 ³⁹.

Moreover, A3 containing a 2-naphthamide core with a flexible substituent appears to be a

novel structural EPI because it is not similar to the EPIs reported previously in the structure.

The activity for the 2-naphthamide derivatives is related to the extension of the C-4 side

chains in the hydrophobic cavity. For example, A3 shows higher activity than A2 because it

longer side chain may be closer to the critical amino acid Phe178 and form a stronger

hydrophobic interaction than that of A2. The efflux inhibition and docking results were very

encouraging. However, target specific interaction with AcrB still had to be verified. In order

to rule out non-specific effects due to permeabilisation of the bacterial membranes, the

integrity of the bacterial outer- and inner- membranes were determined with nitrocefin and

the fluorescent membrane probe DIOC₂(3) respectively.

Figure 2: Molecular docking of A3. (A) Chemical structure of 4-(isopentyloxy)-2-

naphthamide (A3). (B) Overlay of A3 (yellow) on the crystal structure of AcrB (PDB code

5ENO) with MBX2319 (magenta). (C) The interaction of A3 with the amino acids of AcrB.

A3 does not permeabilise the outer membrane

The outer membrane of Gram-negative organisms act as a permeability barrier. Hence

compounds that permeabilise the bacterial membrane would also synergise with antibiotics ²⁷.

The effect of the three compounds on the bacterial outer membrane was assessed by

examining the rate of hydrolysis of a chromogenic β-lactam, nitrocefin, in E. coli. Hydrolysis

of nitrocefin by the β-lactamase releases a coloured compound that can be measured at 486

nm. The rate of nitrocefin hydrolysis is faster if the rate of diffusion across the outer

membrane is immediate due to outer membrane permeabilisation ⁴⁷. The effect of A2, A3 and

A9 on the rate of nitrocefin hydrolysis was determined and the rate was comparable to the

untreated cells when A3 was present (Figure 3). On the other hand, the presence of A2 and

A9 caused faster rates of nitrocefin hydrolysis compared to the control. It is not clear why

compound A2 should permeabilise the outer membrane given the structural similarity

between A2 and A3. However, we could conclude that the synergism of A3 with known

antibiotics observed was not due to secondary mechanisms such as outer membrane

permeabilisation.

Figure 3: Compound A3 does not permeabilise the outer membrane of E. coli but

compounds A2 and A9 act non-specifically on the outer membrane. E. coli wild-type cells

were treated with 10 µM CCCP to inhibit the efflux of nitrocefin. Nitrocefin was added to the

cells that received no compound (blue circles) or were treated with the outer membrane

permeabiliser polymyxin B nonapeptide (PMBN, green squares) as positive control or the

test compounds at 512 µg/ml (red triangles). Nitrocefin hydrolysis by the periplasmic β-

lactamase was observed as an increase in A₄₉₀. Compounds that permeabilise the outer

membrane, such as PMBN, shows a fast initial rate of nitrocefin hydrolysis in comparison

with the untreated cells. Representative traces are shown from experiments done in different

days.

A3 does not disrupt the inner membrane

RND-type drug efflux pumps such as AcrB are powered by the proton motive force (pmf)

across the bacterial inner membrane . In order to distinguish between a direct inhibition of

AcrB and the disruption of the pmf by permeabilisation of the inner membrane the effect of

the active compounds on the integrity of the inner membrane was assessed. The ability of E.

coli cells to establish a membrane potential (Δψ) when challenged with 512 μg.mL^-1A3 was

determined using the fluorescent membrane potential probe 3,3-diethyloxacarbocyanine

iodide (DiOC₂(3)). We previously used this probe to measure the magnitude and stability of Δψ

in bacterial cells and proteoliposomes ⁵². Bacterial cells were energized by the addition of

glucose to establish a proton motive force (negative and basic inside the cell). This led to an

increase in fluorescence associated with aggregation of the DiOC₂(3). Upon addition of the

ionophore CCCP, the Δψwas dissipated and the fluorescence intensity dropped to the level

before addition of glucose. The magnitude and stability of Δψ in the presence of A3 was

comparable to that of untreated cells (Figure 4). Therefore, A3 mediated synergism does not

involve disruption of the inner membrane of bacteria.

Figure 4: Compound A3 does not permeabilise the inner membrane of E. coli. Bacterial

suspensions were either left untreated (solid blue line) or exposed to 512 μg/mL A3 (broken

red line) for 10 min after which the potentiometric probe, DiOC²(3) was added and the

fluorescence monitored until it plateaued. Cells were then re-energized with 0.5% glucose

and the establishment of a membrane potential (inside negative) was measured as an increase

in fluorescence until it plateaued. The membrane potential was then disrupted by the addition

of the proton ionophore CCCP (observed as a sharp drop in fluorescence intensity). After pre-

treatment of the cells with A3, ∆Ψ was maintained indicating that A3 does not act on the

inner membrane.

A3 is not toxic to mammalian cells

The final test to asecertain the feasibility of A3 to be developed into an efflux pump inhibitor

was to ascertain that this compound is not cytotoxic to human cells. Cytotoxicity was

assessed using the using the RealTime–Glo MT Cell Viability assay kit from Promega on

HepG2 (liver) and HEK293 (kidney) cell lines. The MT Cell Viability Substrate is reduced to

a NanoLuc substrate by metabolically active cells. The NanoLuc substrate diffuses from cells

into the surrounding culture medium and is rapidly used by NanoLuc enzyme to produce a

luminescent signal. The signal correlates with the number of viable cells. Dead cells do not

reduce the substrate and therefore produce no signal. The relative luminescence unit (RLU)

for untreated cells and cells treated with A3 at a concentration required for efflux pump

inhibition (as well as a twofold higher concentration) were indistinguishable from the

untreated cells over the 19 hours tested, indicating that A3 is not cytotoxic (Figure 5).

Figure 5: A3 is not cytotoxic to mammalian cell lines. Real-time cell viability measurements

for Hep G2 (A) and kidney cells (B) after treatment with 512 μg/mL (red line) or 1024 μg/mL

(orange line) A3. Cell viability was measured every 60 min for 20 h at 37°C and 5% CO²on a

Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) using the RealTime-Glo™ MT Cell

Viability Assay reagent (Promega).

In conclusion, A3 is the best candidate of the fourteen compounds synthesised, because it

potentiates the antibacterial activity of erythromycin, chloramphenicol and

tetraphenylphosphonium against drug resistant E. coli (Table 1) and inhibits Nile red efflux

(Figure 1). This compound also acts specifically on the efflux pump (Table 1), does not

permeabilise the bacterial outer membrane (Figure 3), does not harm the bacterial inner

membrane (Figure 4) and is not cytotoxic (Figure 5). Moreover, A3 containing a 2-

naphthamide core with a flexible substituent appears to be a novel structural EPI because it is

not similar to the EPIs reported previously in the literature. Therefore, A3 is a promising hit

compound for further optimization to find EPIs with higher efficacy.

ACKNOWLEDGEMENTS

This research was supported financially by the National Natural Science Foundation of China

(81673284), the Natural Science Foundation of Shandong (ZR2015BM019), Major Project of

Research and development of Shandong Province (2016GSF201202) and China-Australia

Centre for Health Sciences Research grant (2014GJ06 and 2016GJ, HV and SM) and

Australian Postgraduate Award (RM).

Supplementary material

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Figure Captions