Title : Pathoblockers: A novel anti-virulence approach to combat bacterial infections by targeting cholesterol-dependent cytolysins
Abstract:
Cholesterol-dependent cytolysins (CDC), a protein family of analogous pore-forming toxins that bind to cholesterol-rich membranes and exacerbate infections through diverse mechanisms, including direct toxicity and immune evasion. CDC are major source of virulence in various gram- positive bacteria (Streptococcus, Clostridium, Listeria, etc.), and independently cause the deleterious responses when released by bacteria during an infection. Thus, CDC are a legitimate health threat during invasive bacterial infection, responsible for nearly 2.5 million deaths globally. Conventional antibiotics cannot curtail CDC, therefore, new therapeutic strategies are required. Our objective is to develop targeted small molecule inhibitors of homologous CDC that could be used as an anti-virulence therapy, alone or adjunct to antibiotics against various CDC-containing bacterial infections.
For the development of inhibitors, we utilized the archetypical CDC toxin pneumolysin (PLY) from
S. pneumoniae, which is an acknowledged drug target and a key contributor in the pathogenesis of bacterial pneumonia. S. pneumoniae is responsible for nearly 1 million fatalities among children under 5 years old worldwide. We employed a combination of virtual screening and in- vitro testing to obtain our first inhibitor named, Pathoblocker-1 (PB-1). Through structural optimization, PB-2 emerged, exhibiting a tenfold increase in potency. Subsequent optimization rounds culminated in the synthesis of PB-3, a more potent molecule exhibiting substantial efficacy in blocking CDC-mediated cytotoxicity during bacterial infection. Moreover, we identified the mechanism of PB molecules that enabled the application of PB compounds toward homologous CDC. Consequently, we validated the efficacy of PB inhibitors against perfringolysin, a CDC, homologous to PLY and belonging to C. perfringens. Our proof of concept has been recently published (U.B.A. Aziz et al. Nat. Commun. 15 (1), 3537 (2024), https://rdcu.be/dJX20).
We are continuing to enhance the potency and drug-like attributes of our lead and consequently, we synthesized a chemically distinct and more drug-like PB inhibitor than PB-3, which is under investigation in bacterial infection assays and subsequent animal studies. Our project has significantly advanced from the early preclinical studies to verge of animal studies and the promising developments indicate a trajectory towards future applicability of PB molecules.
