A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

目前研究最为透彻的Toll样受体4（Toll-like receptor 4, TLR4）配体为脂多糖（lipopolysaccharide, LPS）及其经化学修饰脱毒的变体单磷酰脂质A（monophosphoryl lipid A, MPL）。尽管二者对人源TLR4均具有激活活性，但基于转染细胞系与两种物种原代细胞的实验数据，二者对小鼠TLR4均表现出更强的活性偏好。
通过对小分子化合物库开展高通量筛选（high throughput screening），我们发现了一类全新的TLR4激动剂，其物种偏好谱与MPL截然不同。经四组分Ugi合成反应（Ugi synthesis reaction）得到的产物可强效激活转染了人源TLR4的HEK细胞（HEK cells），但无法激活小鼠TLR4；不过当人源MD2与小鼠TLR4共表达时，可部分恢复该类化合物的激活活性。与脂多糖不同，Ugi类化合物发挥最优激活活性时无需共表达CD14。
该系列Ugi化合物的物种偏好谱实验结果显示：其对人源及食蟹猴（cynomolgus monkey）原代细胞具有极强的激活活性；对豚鼠原代细胞的活性虽有所降低，但仍保持较高水平；而对小鼠、大鼠、兔、雪貂及棉鼠的原代细胞几乎无激活活性。不过，若在缺失内源性TLR4/MD2的小鼠体内表达人源TLR4与MD2，则Ugi化合物可在体内外均表现出显著的激活活性。
本研究拓展了可用于开发TLR4激动剂的化合物谱系，同时发现了一类可逆转MPL对小鼠TLR4相较于人源TLR4的激活偏好的新型分子。这类化合物有望被开发为比传统MPL类佐剂（adjuvants）更强效的人源特异性TLR4配体（TLR4L, Toll-like receptor 4 ligand）类佐剂。