Discovery of a Potent and Selective Inhibitor of Human NLRP3 with a Novel Binding Modality and Mechanism of Action
Posted on: 13 February 2025
Preprint posted on 22 December 2024
Categories: biochemistry, immunology, pharmacology and toxicology
Background
Altered cellular homeostasis due to infections or disease states, can activate intracellular sensors known as inflammasomes, including the NLRP3 inflammasome. NLRP3 activation has been observed in Alzheimer’s disease, Parkinson and Gout. Inflammasomes are central molecular complexes that govern inflammation by inducing secretion of key cytokines, like Il-1β and Il-18, as well as induction of a form of cell death called pyroptosis (Broz and Dixit, 2016). Upon detection of pathogens of cellular damage, NLRP3 inflammasome oligomerizes, forming the so-called ASC speckles. In turn, NLRP3 oligomerization activates caspase 1, which is responsible for starting the cascade leading to Il-1β and Il-18 release (Blevins et al. 2022). At present, targeted therapies to blunt pathogenic inflammation due to NLRP3 activation are limited to antibodies against downstream effectors such as Anakinra (Il-1 receptor antagonist) and Canakinumab (anti-Il-1b) (Coll et al, 2022). Small molecules directly targeting NLRP3 have been developed. However, early stages clinical trials with such molecules (i.e. MCC950-derived compounds), showed off-target effects and inefficiency against some NLRP3 mutations. The authors of the present study developed BAL-0028, a novel human-specific NLRP3 inhibitor.
Key findings
- BAL-0028 efficiently blocks NLRP3-mediated inflammation
The authors previously performed a screen for potential NLRP3 inhibitors and identified BAL-0028 as a top hit. of. In this preprint, they evaluate the effect of BAL-0028 using several human primary cell types and cell lines and compared BAL-0028 efficacy to the known NLRP3 inhibitor MCC950 (Fig.1). BAL-0028 was observed to efficiently block NLRP3-mediated inflammation at a nanomolar range of concentration, providing a good basis for further analysing the activity of this novel inhibitor.
Figure 1 – Comparison of MCC950 and BAL-0028 inhibition of Il-1β release in human monocytes. Figure adapted from the preprint where it is available under a CC-BY-NC-ND 4.0 International license
- BAL-0028 prevents NLRP3 oligomerization
To directly visualize upstream events leading to NLRP3 inflammasome activation, the authors used fluorescent microscopy to monitor ASC-speck formation following NLRP3 induction. BAL-0028 was observed to actively inhibit NLRP3 oligomerization, thus preventing its activation. Surprisingly, the authors found that, while MCC950 was active against NLRP3 of different species, BAL-0028 was highly specific for primate NLRP3.
- BAL-0028 binds to the NACHT domain of NLRP3 inflammasome
To further dissect the mechanism of action of BAL-0028, the authors performed different experiments to monitor its binding to various NLRP3 regions. Like MCC950, BAL-0028 was found to bind to the NACHT domain of NLRP3. In contrast to MCC950 however, BAL-0028 did not inhibit the ATPase activity of NLRP3, indicating that the binding occurs at a different location within the NACHT domain.
- Generation of BAL-0598 for in-vivo studies
Despite these promising properties of BAL-0028, its in-vivo applications are limited by bad pharmacokinetics and high binding to plasma proteins. To overcome this issue, the authors developed a derivative compound, named BAL-0598, with improved pharmacokinetics features. BAL-0598 was proven to inhibit NLRP3 in in-vivo setting, revealing a novel promising inhibitor of the NLRP3 inflammasome.
- Generation of BAL-0028 is relevant for disease-specific NLRP3 mutations
To conclude their investigation, the authors proved that BAL-0028, but not MCC950, was specifically able to inhibit disease specific mutant NLRP3 (Fig.2); suggesting BAL-0028 may have superior abilities and future applications on disease-relevant mutations affecting the NLPR3 inflammasome.
Figure 2 – BAL-0028 inhibit disease specific mutations of NLRP3 inflammasome, whereas MCC950 does not. Figure adapted from the preprint where it is available under a CC-BY-NC-ND 4.0 International license.
Why I chose this preprint
The discovery of novel small molecules with improved specificity and activity to target inflammatory pathways is of great interest for immunology and clinical settings. This paper provides a nice example of small molecule discovery and validation. Moreover, it highlights the complexity of immune signaling and the importance of considering many variables, including species-specific differences, when developing good and efficient therapeutic compounds.
Questions to the authors
· Is BAL-0598 also able to inhibit disease specific mutations of the NLRP3?
· What are common parameters and strategies commonly used to improve pharmacokinetics and decrease plasma sequestration of small molecules?
References
· Broz, P., and V.M. Dixit. 2016. Inflammasomes: mechanism of assembly, regulation and signaling. Nat Rev Immunol 16:407-420.
· Blevins HM, Xu Y, Biby S, Zhang S. The NLRP3 Inflammasome Pathway: A Review of Mechanisms and Inhibitors for the Treatment of Inflammatory Diseases. Front Aging Neurosci. 2022 Jun 10;14:879021. doi: 10.3389/fnagi.2022.879021.
· Coll, R.C., K. Schroder, and P. Pelegrin. 2022. NLRP3 and pyroptosis blockers for treating inflammatory diseases. Trends Pharmacol Sci 43:653-668.
doi: https://doi.org/10.1242/prelights.39630
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