Optimization of a direct Serum Response Factor inhibitor peptide and application in Heart Disease

Cardiovascular disease remains a leading cause of mortality and morbidity worldwide, and cardiac fibrosis is a central contributor to the loss of cardiac function. In 2019 approximately 18.6 million deaths globally were attributed to cardiovascular disease, and in the United States the estimated direct and indirect cost of cardiovascular disease for 2016-2017 was $363 billion. Cardiac fibrosis is characterized by activation of cardiac fibroblasts into myofibroblasts and remodeling of the extracellular matrix, resulting in increased tissue stiffness and diminished cardiac plasticity. Among molecular drivers of myofibroblast activation, the Serum Response Factor (SRF) transcription factor and its co-activator myocardin-related transcription factor A (MRTF-A) are key regulators; increased MRTF-A/SRF signaling has been shown to promote fibroblast-to-myofibroblast transition. Despite evidence implicating SRF over-activation in multiple disease pathways, no direct SRF inhibitor had been identified prior to the work described here.

This project focuses on optimization of a direct SRF inhibitor peptide derived from the B1 box motif of MRTF-A. A 21-amino-acid peptide corresponding to the MRTF-A B1 box motif was previously shown to bind SRF and inhibit MRTF-A/SRF complex formation in vitro. The research objective was to improve binding affinity and biochemical potency of this peptide and to evaluate whether a shorter, more drug-like peptide could effectively disrupt the MRTF-A/SRF protein:protein interaction.

To enable screening and characterization of peptide variants, the team developed a high-throughput biochemical assay using Amplified Luminescence Homogenous Proximity Assay (ALPHA). Screening with ALPHA allowed direct comparison of the inhibitory potency of different peptide lengths and sequence variants. Using this approach, the investigators found that a 14-mer peptide exhibited an IC50 nearly identical to the original 21-mer (1.6 µM versus 1.3 µM in the ALPHA assay), suggesting that a reduced-length peptide retains functional activity and may represent a smaller, potentially more drug-like lead.

Further optimization explored sequence substitutions informed by prior studies and computational analysis. Notably, a single amino acid change-substituting tyrosine with phenylalanine-in the 21-mer scaffold enhanced potency nearly two-fold in the biochemical assay, yielding an IC50 of 0.5 µM for the phenylalanine-containing 21-mer. These findings demonstrate both that shorter peptides can retain inhibitory activity and that targeted substitutions can materially improve potency against the MRTF-A/SRF interaction.

This work establishes a biochemical foundation for continued optimization of direct SRF inhibitor peptides, with implications for therapeutic strategies targeting cardiac fibrosis and other pathologies linked to SRF over-activation. The study was supported by the AHA predoctoral fellowship 23PRE1019204 and authored by Melissa Meschkewitz, Erika M. Lisabeth, and Richard R. Neubig at Michigan State University. By combining high-throughput biochemical screening with rational sequence modification, the project advances a promising peptide-based approach to modulating an important transcriptional regulator implicated in cardiac disease.