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: valerie.defraine@kuleuven.be 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 1 0 + 5 0 µ M O f 1 0 + 1 0 0 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 5 0 + 5 0 µ M 2 0 SPI009 effectively clears intracellular P. aeruginosa infections R if 5 0 + 1 0 0 µ M 5 0 µ g /m L R if R if 5 0 + 5 0 µ M R if 5 0 + 1 0 0 µ M 2 0 µ g /m L O f O f 2 0 + 5 0 µ M O f 2 0 + 1 0 0 µ M 5 0 µ g /m L O f O f 5 0 + 5 0 µ M O f 5 0 + 1 0 0 µ M 1 0 µ g /m L O f O f 1 0 + 5 0 µ M O f 1 0 + 1 0 0 µ M 1 0 µ g /m L O f O f 1 0 + 5 0 µ M O f 1 0 + 1 0 0 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 .) * 6 0 0 0 0 6 0 0 0 0 0 2 * * * 4 0 0 0 0 4 0 0 0 0 0 0 1 5 1 0 2 0 2 0 0 0 0 C o n c e n tra tio n c ip ro flo x a c in (µ g m L ) - 1 2 0 0 0 0 0 Intracellular infection model³ c ip r o c ip r o + 2 0 µ M S P I0 0 9 c ip r o + 3 0 µ M S P I0 0 9 2 5 µ M 5 0 µ M 1 0 0 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 + 5 0 µ M S P I0 0 9 O f + 1 0 0 µ M S P I0 0 9 P o lB P o lB + 5 0 µ M P o lB + 1 0 0 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≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 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.0413.10), 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):699-709. 3 Buyck et al.(2013). Antimicrobial Agents and Chemotherapy, 57(5): 2310-8