De novo design of dual-target mini-binders simultaneously neutralizing Streptococcus equi subspecies zooepidemicus M-like protein and host TNFR1 confers complete protection against lethal infection.

Computationally designed mini-binders have shown great promise, yet current strategies primarily yield molecules targeting a single target. For complex infections, simultaneously engaging multiple pathogenic pathways may offer superior efficacy. Streptococcus equi subspecies zooepidemicus (SEZ) infection, characterized by a highly reproducible and rapid lethal phenotype in mice, serves as an ideal model to test this hypothesis. Based on our previous success with SzM- and TNFR1-targeting mini-binders, which conferred partial protection alone and enhanced survival in combination, we developed a RFdiffusion-based platform to de novo design dual-target binders. Two distinct methods, Superimposition-Optimization (SIO) and Fusion-Diffusion (FD), were developed to explore various configurations of the designed binders. All binders exhibited micromolar affinities to both targets. Functional validation confirmed their abilities to inhibit SEZ adhesion to host cells and block TNFα-TNFR1 signaling. SIO_b5 and SIO_b40 conferred complete protection against lethally infected mice, with a significant reduction in bacterial loads in organs and a suppression of serum TNFα. This work established an effective RFdiffusion-based strategy for designing dual-target mini-binders, providing a novel therapeutic strategy for combating SEZ infection and a generalizable platform for developing multi-target therapeutics against diverse complex diseases.