De novo design of high-affinity protein binders to bioactive helical peptides

Abstract

Many peptide hormones form an alpha-helix upon binding their receptors, and sensitive detection methods for them could contribute to better clinical management. De novo protein design can now generate binders with high affinity and specificity to structured proteins. However, the design of interactions between proteins and short helical peptides is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that with the RFdiffusion generative model, picomolar affinity binders can be generated to helical peptide targets either by noising and then denoising lower affinity designs generated with other methods, or completely de novo starting from random noise distributions; to our knowledge these are the highest affinity designed binding proteins against any protein or small molecule target generated directly by computation without any experimental optimization. The RFdiffusion designs enable the enrichment of parathyroid hormone or other bioactive peptides in human plasma and subsequent detection by mass spectrometry, and bioluminescence-based protein biosensors. Capture reagents for bioactive helical peptides generated using the methods described here could aid in the improved diagnosis and therapeutic management of human diseases.

De novo designed proteins neutralize lethal snake venom toxins

“…Synthetic genes encoding 44 designs targeting ScNTx were screened via yeast surface display (YSD), and one candidate was identified to bind to ScNTx with a dissociation constant (K d ) of 842 nM as confirmed by bio-layer interferometry (BLI) (Supplementary Figure S1). Partial diffusion optimization 42 improved the binding affinity of the ScNTx binder (SHRT) to 0.9 nM, as determined by surface plasmon resonance (SPR) following the screening of 78 designs (Figure 2c, top row; a very similar value of 0.7 nM was obtained by BLI (Supplementary Figure S2)). The optimized binder displayed a single monomeric peak on size exclusion chromatography (SEC), characteristic αβ-protein circular dichroism (CD) spectra, and thermal stability with a melting temperature (T m ) of 78 °C (Figure 2d, top row).…”

Design of stimulus-responsive two-state hinge proteins

“…Like transistors in electronic circuits, we can couple the switches to external outputs and inputs to create sensing devices and incorporate them into larger protein systems to address a wide range of outstanding design challenges. Hinges containing a disulfide that locks them in state X couple the input “red/ox state” to the output “target binding,” where the target can be a peptide or a protein, and our FRET-labeled hinges couple the input “target binding” to the output “FRET signal.” Our approach can be readily extended such that state switching is driven by naturally occurring rather than designed peptides: recently designed extended peptide binding proteins ( 39 ) resemble the state X of our hinges, and recent designs that bind glucagon, secretin, or neuropeptide Y ( 40 ) resemble the state Y of our hinges. Hinges based on such designs could thus provide new routes to applications in sensing and detection.…”

Engineering of Therapeutic and Detoxifying Enzymes

“…High-throughput experimental pipelines, increasingly utilising microfluidics, ensure efficient functional screening and biochemical characterisation of target enzymes, [30c,53b] ultimately facilitating the development of effective therapeutic enzymes. Furthermore, the advent of next-generation protein design tools like RF Diffusion, [57] DiffDock, [58] and ProteinMPNN [59] showcases immense potential, particularly in the realm of protein and enzyme therapeutics. These cutting-edge tools empower researchers to design customised proteins with enhanced functionality, specificity, and stability, paving the way for groundbreaking advancements in therapeutic enzyme research.…”