HighPlay2: Structure-guided design of cyclic peptide candidates containing non-canonical amino acids.

HighMorph: De Novo Cyclic Peptide Sequence Design via Protein–Protein Interaction Recapitulation

Cyclic peptides have emerged as a compelling class of bioactive scaffolds, but de novo design of target-binding cyclic peptides from protein structures remains challenging. Here, we present HighMorph, an interaction-guided framework that combines protein–protein interaction information with artificial intelligence for rational cyclic peptide design. HighMorph integrates Monte Carlo tree search with a Transformer-based policy-value network to efficiently explore cyclic peptide sequence space, while incorporating explicit atomic-level hydrogen bond constraints extracted from reference protein–protein complexes to guide sequence optimization. The framework is systematically validated on two clinically relevant targets, programmed death-ligand 1 (PD-L1) and kallikrein-related peptidase 4 (KLK4). Notably, 33.3% and 40% of the generated candidates are active against PD-L1 and KLK4, respectively, with active cyclic peptides exhibiting micromolar binding affinities (approximately 10–6 M). These results validate our approach for cyclic peptide design. Additionally, interaction analysis provides insights for developing therapeutics targeting challenging protein interfaces.

NACraft: Programmatic nucleic-acid aptamer design via all-atom structure-model feedback

NACraft, a training-free and programmatic framework for all-atom nucleic-acid aptamer design based on backpropagation through structure-model feedback, is presented, demonstrating the effectiveness and versatility of NACraft and extending structure-model hallucination toward programmatic nucleic-acid aptamer design.

A ligand-property-guided computational framework for prioritizing de novo protein binders for small molecules

Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract

Seed-Guided De Novo Design Expands the Structural Diversity of Antitoxin Protein Binders

De novo design of protein binders targeting extended, multi-site interaction surfaces remains difficult for current generative methods, which often produce limited structural diversity and predominantly helical topologies. Here, we advance diffusion-based binder design by guiding protein backbone generation through “seeds,” which are PDB-derived fragments selected for geometric complementarity to the target surface. To test this approach, we computationally generated seed-guided binders of the bacterial toxin RelE. RelB, the native antitoxin of RelE, engages two distinct interfaces with high surface complementarity, making it an appropriate test case. Seed-guided RFdiffusion produced backbones with substantially higher structural diversity and more target contacts than RFdiffusion alone. Experimental screening of 1,402 designs in a high-throughput bacterial survival assay identified multiple functional binders, including variants with nanomolar to low-micromolar affinity and one design with RelE neutralization comparable to RelBpep. Computational structure prediction and mutational analyses support that the designed interfaces rely on seed-derived contacts and adopt binding modes distinct from RelB. Molecular dynamics simulations and hydrogen-deuterium exchange experiments further suggest that one high-affinity design undergoes a conformational change upon binding. Notably, successful designs exhibited reduced cross-reactivity to RelE orthologs compared with RelBpep, suggesting that the extensive interfaces generated through seed-guided design can enable enhanced selectivity. These results establish motif scaffolding of surface-complementing seeds as an effective strategy for overcoming current limitations of de novo generative models, enabling the design of proteins that can engage challenging interface sites.

Optimized Multiple Circular Sequence Alignment for Cyclic Peptide Motif Discovery

Head-to-tail (H2T) cyclized peptides are an increasingly important modality in drug discovery, combining high target affinity and selectivity with metabolic stability. Because their underlying chemistry is still that of a linear amino-acid chain, their linear sequence representation is the native input format of main-stream sequence generative models now driving de novo peptide design (Slough et al., 2018; Rettie et al., 2025a;b). Discovering the conserved motifs responsible for a family’s function across a library of such candidates requires a multiple sequence alignment (MSA). Because a cyclic peptide can be linearised at any residue, the alignment must additionally solve for the unknown rotation of each sequence, which is the multiple circular sequence alignment (MCSA) problem. However, leading MCSA heuristics (e.g. Ayad & Pissis, 2017) were tuned for the genomic regime (a few tens of long sequences) and become prohibitively slow on the cyclic peptide library regime (hundreds to thousands of shorter sequences). We close this gap by identifying quality-preserving optimisation opportunities, notably the library-scale preset tailored to short-sequence inputs (algorithmic details in Appendix A), and by adding an orthogonal multi-core and SIMD backend for further performance tuning, which gives near-linear thread scaling on the pairwise-comparison stage. We validate the pipeline on a library of 1,000 H2T cyclized peptides of length 18 targeting the oncoprotein Mouse double minute 2 human homolog (MDM2) produced by an internal peptide-design engine. In this practical setup, the optimised MCSA recovers the underlying positional motif of MDM2 binders at the same fidelity as the original MCSA implementation while running over 650× faster. Our optimised MCSA tool thus enables library-scale cyclic peptide sequence alignment and is publicly available at https://github.com/IVB-Generative-Biology/mars-turbo.

HighDB: a structure-annotated cyclic peptide database for comparative analysis, template retrieval, and design-oriented applications