Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• NLRP3 inflammasome

• Inhibitor BAL-0028

• Species-specific mode of action

• CAPS syndrome

Main Findings

This study characterizes a previously disclosed compound (by the same authors), BAL-0028, which was identified as an inhibitor of the NLRP3 inflammasome via DNA-encoded library screens. NLRP3 inflammasome is a key driver of inflammation in various diseases, including cryopyrin-associated periodic syndromes (CAPS). While existing inhibitors like MCC950 effectively suppress NLRP3 activation they have not successfully passed clinical trials as of today. The authors convincingly show that BAL-0028 selectively and potently inhibits human and primate NLRP3, but exhibits poor activity against other mammalian species, suggesting a species-specific interaction. It is demonstrated that, unlike MCC950 that inhibits ATPase activity, BAL-0028 effectively blocks IL-1β via binding to the NACHT domain of NRLP3, highlighting a novel binding modality. Subsequently, they develop a derivative of BAL-0028, with improved pharmacokinetic properties, that showed in vivo efficacy in a humanized mouse model of peritonitis. Finally, BAL-0028 was tested in a series of disease-associated NRLP3 mutations, resistant to MCC950, suggesting a therapeutic strategy for previously undruggable NLRP3-driven autoinflammatory disorders.

The authors describe the following findings and always in comparison with the “standard” inhibitor MCC950:

1. Compared to MCC950, BAL-0028 has a reduced potency, but potentially broader clinical applicability, for instance via better inhibition of rare disease-causing NLRP3 variants (resistant to MCC950).

2. BAL-0028 selectively inhibits the NLRP3 inflammasome in different cell types without interfering with other inflammasomes (AIM2, NLRC4, NLRP1), or signaling pathways such as those triggered by LPS.

3. The compound shows primate-specific activity, highlighting the importance of sequence variation in certain NLRP3 domains between species.

4. Compared to previously described NLRP3 inflammasome inhibitors, BAL-0028 binds the NACHT domain and does not affect ATPase activity.

5. BAL-0028 suppresses activation caused by clinically relevant gain-of-function mutations in NLRP3, underscoring its therapeutic potential for CAPS and other NLRP3-related diseases.

Limitations & Suggestions

• The authors convincingly show the activity of their inhibitor in a range of several human cell types, yet do not go into detail when introducing BAL-0598 for in vivo studies. The use of a humanized mouse is an elegant approach to overcoming the limitation of primate-specific action of their compound, however does not present the most disease-relevant model. If primate models (i.e. studies directly in non-human primates, or primate organoid models) are unavailable, the authors may consider testing patient-derived cells, such as dermal fibroblasts, directly.

• Are there any roles of NLRP3 in chronic inflammation? Could this be recapitulated in vivo (ie repeated LPS stimulation)? Is it possible to test animal models with the specific NRLP3 mutations? Authors could discuss these limitations further.

• While it is clear that BAL-0028 was chemically modified to improve pharmacokinetics, the data is very limited, with just mentioning some (minimal) improvement in plasma protein binding. If the chemical structure cannot be disclosed (ie due to patenting) the authors could at least be more specific on additional improved properties (pharmacokinetics/pharmacodynamics, half live, tissue distribution etc). In addition, no comparison with the initial compound BAL-0028 is provided – is BAL-0598 more or less efficient in human cells? Why do authors go back to BAL-0028 in figure 5 where they test the NLRP3-AID mutants and do not use the improved compound?

• The identification of the NACHT domain as binding pocket is very interesting. However, the authors do not fully elucidate the mechanism of action. What are the direct consequences of binding? The authors may want to comment on this in more detail, about downstream effects (i.e. inhibition of caspase-activation platform oligomerization, cleavage inhibition, inhibition of interaction with other modulators).

• In the discussion, it’s mentioned that structural studies are ongoing. If not successful, potential in silico modelling using alphafold or docking models would be informative. (maybe use Alphafold 3 or Chai-1).

• It would be interesting to understand why certain disease-causing mutations can and cannot be targeted with different compounds. Are these located predominantly in certain NLRP3 domains (i.e. NACHT domain)?

• The authors show a set of NLRP3 mutations that are not targeted by MCC950 but BAL-0028. The authors, however, do not show data on the most common CAPS mutations. It would be of general interest to add this data, even if negative, to the study.

• Species specificity is intriguing. The authors already show that there are certain sets of amino acids that are unique in primates vs the other mammals. Site-directed mutagenesis of these mutations could reveal the exact binding site.

• While BAL-0028 is compared to MCC950 for potency in vitro and in certain mutant contexts, it would be informative to see how it compares against other advanced NLRP3 inhibitors in clinical pipelines (e.g., dapansutrile/OLT1177, DFV890, Inzomelid) if possible.

• The need for a small molecule to pass the blood-brain-barrier was identified, yet this was not actively studied with BAL-0028. Do the authors expect improved BBB properties for BAL-0028/0598?

The data presented in this preprint is novel and of broad interest. However, to be published in very high-impact journal, additional experiments, especially on exact elucidation of the mechanism (e.g. through more structural work and mutagenesis assays), and more physiological disease models are required.

Significance/Novelty

This work describes a novel binding-site and different mode of action compared to previous NLRP3 inflammasome inhibitors. This may have implications for the design of next-generation compounds for the general treatment of hyperinflammation. BAL-0028/0598 may be able to target currently undruggable NLRP3 variants, but this requires further mechanistic elucidation and validation in a disease-relevant in vivo setting. Given that the compound BAL-0028 was already disclosed, including more details regarding the improved compound BAL-0598 and adding data showing the better efficacy both in vitro and in vivo could contribute to the novelty.

Credit

Reviewed by Chrysanthi Kagiou and Lukas Englmaier as part of a cross-institutional journal club between the Vanderbilt University Medical Center (VUMC), the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.