Celastrol derivatives ameliorate arthritis in AIA rats via modulating calcium signaling

Calcium (Ca²⁺) signaling is implicated in the pathogenesis of rheumatoid arthritis (RA). Our previous study found that celastrol (CEL), a natural product derived from herbal plant Tripterygium wilfordii Hook F, suppressed arthritic symptoms in AIA rats via modulating Ca²⁺ signaling pathway by inhibiting sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA). However, its high toxicity and narrow therapeutic window severely limit its clinical application. Therefore, developing safer and effective CEL derivatives are of utmost importance.

This study aims to develop CEL derivatives by structural modification and evaluate their anti-arthritic properties in vitro and in vivo, thus identifying derivatives with improved pharmacological properties for potential RA treatment.

CEL derivatives were prepared by modifying C29-carboxylic acid moiety. In vitro screening assessed cytotoxicity, intracellular Ca²⁺ dynamics, autophagy induction and anti-inflammatory effects. Selected compounds were evaluated in vivo in AIA rats. Mechanisms were explored by 3' RNA-sequencing on RAFLS. Pharmacokinetic (PK) study in SD rats and acute toxicity (up-and-down procedure) in ICR mice were studied.

Among six semi-synthetic CEL derivatives, COM5 and COM6 showed the lowest cytotoxicity, while exhibiting anti-inflammatory effects, induction of autophagic death and modulation of Ca²⁺ dynamics in vitro comparable to CEL. Notably, Ca²⁺ dynamic study also revealed that only COM5, similar to CEL, targeted SERCA. These two derivatives were as effective as CEL in repressing arthritic symptoms, bone erosion and pro-inflammatory cytokine expressions, and restoring Treg/Th17 balance in AIA rats. 3'RNA-seq found COM5 downregulated inflammatory cytokines more effectively and reduced toxicity by avoiding immediate early gene induction compared to CEL or COM6, while retaining unfolded protein response (UPR) activation in TNFα-stimulated RAFLS. Six UPR genes were validated by qPCR in RAFLS treated with compounds. Acute toxicity tests showed that both derivatives were ≥10 times less toxic than CEL. In PK study, COM5 and COM6 were surprisingly not found in rat serum, and several key metabolites were identified and analyzed semi-quantitatively.

Structurally modified C29-derivatives COM5 and COM6 exhibited potent anti-arthritic effects, comparable to CEL in vitro and in vivo, via modulating Ca²⁺-associated UPR pathway. They also demonstrate significantly reduced toxicity, highlighting their potential therapeutic use for RA.