Peptide Design for Targeting BTB Domain Homodimerization of BACH2: Complementary In Silico and In Vitro Approaches

Potential activation of NRF2 via targeting its nuclear suppressor BACH2 represents an unexplored field, forming a gap yet to be addressed. Besides, considering the pathological role of BACH2 in tumor immunosuppression, an attempt to target this repressor was deemed necessary. Despite this background, a peptide-based modulator that could interfere with the BACH2 BTB domain has not been reported. Accordingly, using in silico and in vitro approaches, a BACH2 inhibitory peptide was identified via determining its affinity for BACH2 and demonstrating its negative impact on homodimerization. Specifically, a preliminary study was conducted to generate a peptide library that could target BACH2 BTB homodimerization. Initially, members of this peptide library were tested in terms of their capacity to bind the dimerization surface of the BACH2 BTB domain using docking programs. Second, selected BACH2 BTB domain-peptide complexes were analyzed for persistence of binding through 500 ns molecular dynamics simulations, and trajectories were also subjected to estimation of relative binding free energy levels. Following the computational approach, selected peptides, peptide 13 and control peptide 1, were ranked according to their dissociation constants to full-length BACH2 in vitro using supernatant depletion assay, with peptide 13 showing a trend toward higher affinity. Additionally, the inhibitory effect of peptides on homodimerization of the BACH2 BTB domain was investigated through coimmunoprecipitation studies, revealing peptide 13 as a potential disruptor of homodimerization. Based on these observations, peptide 13 has been identified as a putative BACH2 negative regulator. Furthermore, limitations and future directions are also discussed.