Structure-activity relationship of S-adenosylmethionine analogs as pharmacological chaperones for cystathionine beta-synthase-deficient homocystinuria

Classical homocystinuria (HCU), primarily caused by pathogenic missense mutations in cystathionine beta-synthase (CBS) gene, is an inherited conformational disorder with limited treatment options. While some patients benefit from pyridoxine supplementation, many require strict dietary methionine restriction. S-adenosylmethionine (SAM) regulates CBS stability and activity, but unlike pyridoxine is unsuitable for treatment of HCU due to instability and metabolic side effects. Despite these drawbacks, we found that dietary methionine dose-dependently stabilized hepatic CBS I278T in transgenic mouse model of HCU confirming SAM ability to stabilize misfolded CBS variant in vivo. This study explores SAM analogs as potential pharmacological chaperones (PCs) to rescue misfolding of CBS variants. Using biophysical and cellular models, we evaluated 34 SAM analogs for their impact on CBS activity and stability. We found that sulfonium center is necessary for CBS activation similar to SAM. Non-activating SAM analogs, such as sinefungin (SAO) and AzaSAM, enhanced CBS stability without markedly increasing its activity. SAO and AzaSAM increased cellular CBS I278T levels by 3.5-4-fold, which translated into a rescued cellular CBS activity. Therefore, our data indicate direct action of the SAM analogs on CBS likely by inducing distinct conformational changes and oligomerization pattern of CBS consistent with recently described filamentation assemblies of human CBS. In conclusion, SAM analogs represent promising PCs for HCU by rescuing folding and conformational stability of CBS mutants, thus preventing their otherwise accelerated degradation and consequently rescuing CBS activity. This proof-of-concept highlights the potential of SAM analogs as a novel therapeutic strategy for pyridoxine-nonresponsive HCU.