Toward Unveiling Putative Binding Sites of Interleukin-33: Insights from Mixed-Solvent Molecular Dynamics Simulations of the Interleukin‑1 Family

The interleukin (IL)-1 family is a major proinflammatory
cytokine
family, ranging from the well-studied IL-1s to the most recently discovered
IL-33. As a new focus, IL-33 has attracted extensive research for
its crucial immunoregulatory roles, leading to the development of
notable monoclonal antibodies as clinical candidates. Efforts to develop
small molecules disrupting IL-33/ST2 interaction remain highly desired
but encounter challenges due to the shallow and featureless interfaces.
The information from relative cytokines has shown that traditional
binding site identification methods still struggle in mapping cryptic
sites, necessitating dynamic approaches to uncover druggable pockets
on IL-33. Here, we employed mixed-solvent molecular dynamics (MixMD)
simulations with diverse-property probes to map the hotspots of IL-33
and identify potential binding sites. The protocol was first validated
using the known binding sites of two IL-1 family members and then
applied to the structure of IL-33. Our simulations revealed several
binding sites and proposed side-chain rearrangements essential for
the binding of a known inhibitor, aligning well with experimental
NMR findings. Further microsecond-time scale simulations of this IL-33-protein
complex unveiled distinct binding modes with varying occurrences.
These results could facilitate future efforts in developing ligands
to target challenging flexible pockets of IL-33 and IL-1 family cytokines
in general.

白细胞介素（IL）-1家族是一类主要的促炎细胞因子家族，其成员从研究较为深入的IL-1s延伸至最近发现的IL-33。作为研究的新焦点，IL-33因其关键的免疫调节作用而受到广泛的关注，并促使研究人员开发出具有临床潜力的单克隆抗体。针对破坏IL-33/ST2相互作用的低分子量化合物的研究仍然备受青睐，但由于其界面浅薄且缺乏特征，面临着诸多挑战。相关细胞因子的研究信息表明，传统的结合位点识别方法在定位隐秘位点时仍然存在困难，亟需动态方法来揭示IL-33上的可药物结合位点。在本研究中，我们采用了混合溶剂分子动力学（MixMD）模拟，并结合具有不同性质的探针来绘制IL-33的热点区域并识别潜在的结合位点。该协议首先利用已知的两种IL-1家族成员的结合位点进行验证，随后应用于IL-33的结构。我们的模拟揭示了多个结合位点，并提出了对已知抑制剂结合至关重要的侧链重排，这与实验NMR结果吻合良好。进一步进行的微秒时间尺度模拟揭示了IL-33-蛋白质复合物的不同结合模式，其发生频率各异。这些结果有望促进未来针对IL-33及其家族细胞因子的挑战性柔韧性位点开发配体的努力。