Work

Mechanism of action and impact of thiol homeostasis on efficacy of an enzyme replacement therapy for classical homocystinuria

Year: 2024

Type: article

Abstract: Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is characterized by elevated plasma and tissue homocysteine levels. There is no cure, but HCU is typically managed by methionine/protein restriction and vitamin B 6 supplementation. Enzyme replacement therapy (ERT) based on human CBS has been developed and has shown significant efficacy correcting HCU phenotype in several mouse models by bringing plasma total homocysteine below the clinically relevant 100 μM threshold. As the reactive nature of homocysteine promotes disulfide formation and protein binding, and ERT is unable to normalize plasma total homocysteine levels, the mechanism of action of ERT in HCU remains to be further characterized. Here we showed that only a reduced homocysteine serves as a substrate for CBS and its availability restricts the homocysteine-degrading capacity of CBS. We also demonstrated that cells export homocysteine in its reduced form, which is efficiently metabolized by CBS in the culture medium. Availability of serine, a CBS co-substrate, was not a limiting factor in our cell-based model. Biological reductants, such as N-acetylcysteine, MESNA or cysteamine, increased the availability of the reduced homocysteine and thus promoted its subsequent CBS-based elimination. In a transgenic I278T mouse model of HCU, administration of biological reductants significantly increased the proportion of protein-unbound homocysteine in plasma, which improved the efficacy of the co-administered CBS-based ERT, as evidenced by significantly lower plasma total homocysteine levels. These results clarify the mechanism of action of CBS-based ERT and unveil novel pharmacological approaches to further increase its efficacy. Graphical abstract. Schematic representation of the proposed mechanism of action of the simultaneous application of CBS ERT and NAC . ( Left ) Application of CBS45 and NAC in cell culture. CBS45 catalyzes the condensation of free, reduced Hcy with Ser or Cys to form cystathionine. This eliminates Hcy released from the cells during their growth and Hcy released from homo-/hetero-disulfides or protein conjugates by NAC. ( Right ) Application of CBS ERT and NAC in the plasma of HCU mice. Exogenous NAC, endogenous or dietary reducing agents can release Hcy from proteins and disulfides. This makes them available for renal clearance or catalysis by CBS ERT. In addition, CBS ERT eliminates the constant flow of endogenously produced and exported reduced Hcy from tissues. • Mechanism of Hcy degradation by CBS-based ERT in HCU is not fully understood. • First, ERT degrades any free reduced Hcy available in plasma. • Second, tissues export reduced Hcy, which is instantly degraded by ERT present in circulation. • Third, ERT eliminates reduced Hcy released from dimers and plasma proteins. • Alteration of plasma redox dynamics by the biological reductants could increase efficacy of the ERT.

Source: Redox Biology

DOI: 10.1016/j.redox.2024.103383