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Selective Small-Molecule Activator of Patient-Derived GPX4 Variant
Hengrui Liu1, Farhad Forouhar2, Russell Saneto3,4
1Department of Chemistry, Columbia University, New York, New York 10027, United States.
Abstract:
Glutathione peroxidase 4 (GPX4) is distinguished from other members of the GPX family as being the enzyme capable of reducing phospholipid hydroperoxides within cellular membranes and therefore protecting cells from ferroptosis, a form of iron-driven cell death involving lipid peroxidation. We previously identified a homozygous point mutation in the GPX4 gene, resulting in an R152H coding mutation and a substantial loss of GPX4 enzymatic activity, in patients with Sedaghatian-type spondylometaphyseal dysplasia (SSMD), an ultrarare progressive disorder. To explore whether selective binding and correction of the loss of enzyme activity observed with this variant is possible, we screened 2.8 billion compounds in a DNA-encoded chemical library and identified compounds with remarkably selective binding affinities with the R152H variant (GPX4R152H) over wild-type (GPX4WT). Our structural optimization of these compounds led to the identification of analogues with improved potency for R152H GPX4. The most promising compounds selectively restored the enzyme activity of GPX4R152H and specifically increased the viability of fibroblast and lymphoblast cells developed from an SSMD patient with the homozygous R152H variation but not control cells from a healthy parent or HEK293T cells undergoing ferroptosis induced by a wild-type GPX4 inhibitor. This approach represents a low-cost, high-throughput, and generalizable approach to identify targeted small-molecule therapeutics for missense variants, which features the potential to be broadly applied to diseases that bear point mutations on crucial proteins, including cancers.
Insights
Researchers identified small molecules that selectively bind and restore the activity of mutated Glutathione peroxidase 4 (GPX4) in Sedaghatian-type spondylometaphyseal dysplasia (SSMD). This approach offers a new therapeutic strategy for genetic disorders caused by missense variants.
Area of Science:
- Biochemistry
- Genetics
- Pharmacology
Background:
- Glutathione peroxidase 4 (GPX4) protects cells from ferroptosis by reducing lipid hydroperoxides.
- A homozygous R152H mutation in GPX4 causes Sedaghatian-type spondylometaphyseal dysplasia (SSMD) with loss of enzymatic activity.
- Targeting missense variants in crucial proteins is a significant challenge in developing therapeutics.
Purpose of the Study:
- To identify small molecules that selectively bind and restore the enzymatic activity of the GPX4 R152H variant.
- To evaluate the therapeutic potential of these molecules in patient-derived cells and disease models.
Main Methods:
- Screening of a 2.8 billion compound DNA-encoded chemical library for selective binding to GPX4 R152H.
- Structural optimization of identified lead compounds to improve potency.
- Assessing the restoration of GPX4 R152H enzymatic activity and cellular viability in patient-derived cells and ferroptosis models.
Main Results:
- Identified compounds with high selectivity for GPX4 R152H over wild-type GPX4.
- Optimized compounds demonstrated improved potency and restored GPX4 R152H enzymatic activity.
- Selected compounds specifically rescued the viability of SSMD patient-derived cells without affecting control cells or inducing ferroptosis.
Conclusions:
- A novel, high-throughput screening approach successfully identified targeted small-molecule therapeutics for a GPX4 missense variant.
- This strategy demonstrates potential for treating genetic disorders caused by point mutations in essential proteins.
- The findings offer a generalizable platform for developing precision medicines for a range of diseases, including cancers.
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