1. A small molecule candidate for antibiotic co-therapy in the fight against persistence
Valerie Defraine1, V. Liebens1, T. Swings1, R. Corbau2, A. Marchant2, P. Chaltin2, 3, F. Van Bambeke4, A. Anantharajah4, M. Fauvart1,5 and J. Michiels1
1Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium
2CISTIM Leuven, vzw, Leuven, Belgium. 3Centre for Drug Design and Discovery, KU Leuven, Leuven, Belgium
4Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium
5Smart Systems and Emerging Technologies Unit, Department of Life Science Technologies, imec, Leuven, Belgium.
For correspondence:
CENTRE
of MICROBIAL
and PLANT GENETICS
Introduction
Identification of SPI009 and initial research
Background
Pseudomonas aeruginosa, one of the notorious ESKAPE pathogens, is best known for the life-threatening infections it causes in cystic fibrosis patients and its rapidly increasing antibiotic resistance. Infections caused by multidrug-resistant pathogens are increasingly difficult to treat and, predicted to cause 10 million deaths each year by 2050, pose a serious public health threat. Contributing to treatment failure is the presence of a small fraction of persister cells, which transiently tolerate treatment with high doses of antibiotics. The development of novel therapies, which also eliminate persister cells, could greatly improve patient outcome.²
The quest for novel anti-persister therapies
We recently identified a new antibacterial compound SPI009 that, in combination with different classes of conventional antibiotics, poses an excellent candidate for novel anti- persister co-therapies in the fight against P. aeruginosa and other relevant pathogens.
Screening of 23909
compounds
Selection of 3 compound families
Testing of 48 chemical analogues
1 0
SPI009
SPI009 significantly reduces the persister fraction of
P. aeruginosa in combination with ofloxacin
SPI009 reduces the persister fraction in combination with different classes of antibiotics
L o g 1 0 ( C F U /m L ) 1 8 6 4 2
SPI009 directly kills persister cells
The combination therapy with SPI009 can
completely eradicate bacterial populations
C o n tro l
1 0 µ g / m L O f
O f µ M
O f µ M
Further characterization
Determination of the mode of action
SPI009 shows a broad spectrum activity
SPI009 inhibits all major macromolecular synthesis processes
E n te ro c o c c u s
S ta p h y lo c o c c u s
K le b s ie lla
A c in e to b a c te r
E n te ro b a c te r
E s c h e r ic h ia
1 0
fa e c iu m
a u re u s
p n e u m o n ia e
b a u m a n n ii
a e ro g e n e s
c o li 8 6 *
c ip ro f lo x a c in
r ifa m p ic in
1 0 0
N D N D
L o g 1 0 ( C F U / m L )
% in c o rp o ra t io n
fo s fo m y c in
e ry th ro m y c in **
***
6 0 2 2
*** 2 2 2 2
m e lit t in
Indication of membrane damage
N D
N D
N D
4 0
5 0 µ g /m L R if
R if µ M
2 0
SPI009 effectively clears intracellular P. aeruginosa infections
R if µ M
5 0 µ g /m L R if
R if µ M
R if µ M
2 0 µ g /m L O f
O f µ M
O f µ M
5 0 µ g /m L O f
O f µ M
O f µ M
1 0 µ g /m L O f
O f µ M
O f µ M
1 0 µ g /m L O f
O f µ M
O f µ M
D N A
R N A
p ro te in
fa tty a c id s
p e p tid o g ly c a n e
L o g 1 0 ( C F U / m g p ro te in )
1 0
Eukaryotic THP-1 cell 8
SPI009 damages outer and inner membrane 6 5h
ciprofloxacin (+ SPI009) *
N P N
S Y T O X G re e n
PAO1 WT cells 4
* * *
F lu o re s c e n c e in te n s ity ( a .u .)
F lu o re s c e n c e in te n s ity (a .u .) * 2 *
* *
C o n c e n tra tio n c ip ro flo x a c in (µ g m L )
- 1
Intracellular infection model³
c ip r o
c ip r o µ M S P I0 0 9
c ip r o µ M S P I0 0 9
2 5 µ M
5 0 µ M
µ M
2 5 µ M
5 0 µ M
1 0 µ g /m L
1 0 0 µ M
1 µ g /m l p o lB
2 µ g /m L p o lB
m e llitin e
S P I0 0 9
S P I0 0 9
SPI009 enables treatment of resistant P. aeruginosa strains
N-phenyl-1-napthylamine (hydrophobic)
Nucleic acid stain; impermeant to intact membranes
SPI009 alters the bacterial membrane
Control
Membrane-binding FM4-64 dye after 10’ treatment
o f lo x a c in re s is ta n t P A 6 2
p o ly m y x in B re s is ta n t 9 B R *
1 0 **
****
1 0
****
****
****
L o g 1 0 ( C F U /m l) 8
L o g 1 0 ( C F U /m l) 8
SPI009 6 6
O f
O f µ M S P I0 0 9
O f µ M S P I0 0 9
P o lB
P o lB µ M P o lB µ M 4 4 2 2
N D N D
N D N D
n o 1 x M I C a n tib io tic
4 x M I C
8 x M I C
n o a n tib io tic
1 x M I C
4 x M I C
Mode Of Action?
SPI009 causes severe membrane damage leading to bacterial cell death and facilitating the effects of other antibiotics
* P≤ 0.05 ** P≤ *** P ≤ **** P ≤
Conclusion
Extensive research has shown that SPI009
possesses a broad spectrum activity against different clinically relevant pathogens and clinical isolates
allows the design of strain-specific combination therapies
enables treatment of multidrug-resistant strains
extensively damages the bacterial membrane, tackling both normal and persister cells
is capable of clearing intracellular infections without damaging eukaryotic cells and shows no hemolytic activity
Crucially, SPI009 shows great clinical potential and may serve as the starting point for the development of novel antibacterial and anti-persister therapies in the fight against chronic infections. Genetic analysis of resistant mutants and assessment of antibacterial activity in different model systems will provide additional information about the mode of action and clinical applicability of our compound.
Acknowledgments and references
This work was supported by grants from the KU Leuven Research Council (IDO/09/010), the Fund for Scientific Research – Flanders (FWO G ), KU Leuven Excellence Center Financing (PF/2010/07) and the Interuniversity Attraction Poles Program initiated by the Belgian Science Policy Office. VD, VL and TS are recipients of a fellowship from the Agency for Innovation by Science and Technology (IWT).
1 O’neill (2016). Tackling Drug-Resistant Infections Globally: Final Report and Recommendations – The review on Antimicrobial Resistance.² Fauvart et al.(2011). Journal of Medical Microbiology, 60(6): Buyck et al.(2013). Antimicrobial Agents and Chemotherapy, 57(5):