MULTI-ANTIBIOTIC RESISTANT BACTERIA ARE RAPIDLY INCREASING WORLDWIDE.Could a new antibiotic target alleviate this problem?

Nhi Hin

FtsZ monomer

Polymerisation with another FtsZ monomer

And again, to make an FtsZ protofilament

GTP

FtsZ plays a vital role in bacterial cell division. 1

Bacterial cell undergoing cytokinesis

Z-ring

Daughter cell Daughter cell

FtsZ protofilaments associate laterally

Cytokinesis (z-ring constricts)

(Erickson et al. 2010)

2

FtsZ – A promising, new antibiotic target FtsZ is essential for bacterial cell division

FtsZ is highly conserved among bacteria

Mammalian cells don’t use FtsZ when undergoing cytokinesis

No current antibiotic targets FtsZ

(Lin & Ma 2015; Erickson et al. 2010; Sass & Brötz-Oesterhelt 2013)

3

A functional z-ring requires…

⇌FtsZ monomers (cytoplasm) FtsZ protofilaments (z-ring)

(Erickson et al. 2010)

Bacterial cell

4

T7 loop helps to hydrolyse GTP on the next subunit

Hydrolysis of GTP GDPPolymerisation

(Ramírez-Aportela et al. 2014)

GTP

Conversion involves hydrolyzing GTP.

FtsZ protofilament FtsZ monomerFtsZ monomer

GTP binding site

5

PC190723

Two promising molecules that inhibit FtsZ activity

Berberine “2”

(Haydon et al. 2008)

A berberine derivative(Sun et al. 2014)

6

Both inhibit the growth of bacterial cells.

Organism MIC (μg/mL)PC190723 Berberine “2”

B. subtilis 0.5 4S. aureus 0.5 2S. aureus (MRSA) 0.5 2

S. epidermidis 0.5 2E. coli >64 32E. faecalis >64 4S. pneumoniae >64 64

(Sun et al. 2014)(Haydon et al. 2008)

Potent but narrow-spectrum Less potent but broad-spectrum

7

(Domadia et al. 2008)(Andreu et al. 2010)

No PC190723 With PC190723 No Berberine With Berberine

Both have different effects on FtsZ conversion

1000nm

Significantly more protofilaments compared to control

Significantly less protofilaments compared to control

8

FtsZ monomers FtsZ monomersFtsZ protofilaments FtsZ protofilaments

Both have different effects on FtsZ conversion

PC190723Equilibrium shifted to favour assembly of FtsZ = no functional z-ring!

BerberineEquilibrium shifted to favour disassembly of FtsZ = no functional z-ring!

9

Both molecules prevent FtsZ from hydrolyzing GTP

(berberine derivative)

(Sun et al. 2014)(Haydon et al. 2008)

10

X-ray crystallography crystal structures and STD NMR of each molecule complexed with FtsZ confirm these binding sites.

Since these binding sites are so similar, why are their effects on FtsZ conversion so different? PC190723 bound to FtsZ

(Matsui et al. 2012)

Both molecules bind to similar positions on FtsZ.

Berberine bound to FtsZ(Domadia et al. 2012)

(Haydon et al. 2008; Tan et al. 2012; Domadia et al. 2008; Ramírez-Aportela et al. 2014; Matsui et al. 2012)

11

Binding GTP induces a conformational change of the H7 helix

(Matsui et al. 2012; Elsen et al. 2012)

Non-GTP-bound form of FtsZ

New “cleft” has opened up next to the H7 helix

GTP

A recent breakthrough… 12

Bind GTP

H7 helix moves down

Polymerisation GTPGDP

H7 helix moves back up

T7 loop makes contact with GTP

Both molecules prevent vital steps in FtsZ function.

Berberine

PC190723

13

GTP

Differences in potency and spectrum activity of PC190723 and berberine are still not accounted for!

Most structures of inhibitor-FtsZ complexes are based on S. aureus FtsZ. How well is FtsZ structure conserved in other bacteria?

Haven’t yet identified functions of amino acid motifs in FtsZ

Other differences in bacteria that could account for differences (e.g. cell wall).

Current limitations & opportunities for future work

14

ReferencesAndreu, J. M., Schaffner-Barbero, C., Huecas, S., Alonso, D., Lopez-Rodriguez, M. L., Ruiz-Avila, L. B., & Martín-Galiano, A. J. (2010). The

antibacterial cell division inhibitor PC190723 is an FtsZ polymer-stabilizing agent that induces filament assembly and condensation. Journal of Biological Chemistry, 285(19), 14239-14246.

Domadia, P. N., Bhunia, A., Sivaraman, J., Swarup, S., & Dasgupta, D. (2008). Berberine Targets Assembly of Escherichia coli Cell Division Protein FtsZ. Biochemistry, 47(10), 3225-3234.

Elsen, N. L., Lu, J., Parthasarathy, G., Reid, J. C., Sharma, S., Soisson, S. M., & Lumb, K. J. (2012). Mechanism of action of the cell-division inhibitor PC190723: modulation of FtsZ assembly cooperativity. Journal of the American Chemical Society, 134(30), 12342-12345.

Erickson, H. P., Anderson, D. E., & Osawa, M. (2010). FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one. Microbiology and Molecular Biology Reviews, 74(4), 504-528.

Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., & Czaplewski, L. G. (2008). An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science, 321(5896), 1673-1675.

Li, X., & Ma, S. (2015). Advances in the discovery of novel antimicrobials targeting the assembly of bacterial cell division protein FtsZ. European Journal of Medicinal Chemistry, 95, 1-15.

Ramírez-Aportela, E., López-Blanco, J. R., Andreu, J. M., & Chacón, P. (2014). Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations. Biophysical Journal, 107(9), 2164-2176.

Sass, P., & Brötz-Oesterhelt, H. (2013). Bacterial cell division as a target for new antibiotics. Current Opinion in Microbiology, 16(5), 522-530.

Sun, N., Chan, F. Y., Lu, Y. J., Neves, M. A., Lui, H. K., Wang, Y., & Wong, K. Y. (2014). Rational design of berberine-based FtsZ inhibitors with broad-spectrum antibacterial activity. PloS ONE, 9(5), e97514.

Tan, C. M., Therien, A. G., Lu, J., Lee, S. H., Caron, A., Gill, C. J., & Roemer, T. (2012). Restoring methicillin-resistant Staphylococcus aureus susceptibility to β-lactam antibiotics. Science Translational Medicine, 4(126), 126ra35-126ra35.