Design and Engineering of Miniproteins

Ożga, Katarzyna; Berlicki, Łukasz

doi:10.1021/acsbiomedchemau.2c00008

Cited by 11 publications

(7 citation statements)

References 90 publications

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“…The rapid development of the de novo design of proteins through deep learning models has also stoked the ambition to create small proteins or miniproteins with desired functions 15, 44, 45 . Small proteins have considerable advantages over larger ones: their genes are easier to synthesize, they take up fewer cell resources to express, they can be engineered to be highly stable, and they are potentially able to penetrate into tissues and cells.…”

Section: Discussionmentioning

confidence: 99%

Replicating enzymatic activity by positioning active sites with synthetic protein scaffolds

Ding,

Zhang,

Hess

et al. 2024

Preprint

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Evolutionary constraints significantly limit the diversity of naturally occurring enzymes, thereby reducing the sequence repertoire available for enzyme discovery and engineering. Recent breakthroughs in protein structure prediction and de novo design, powered by artificial intelligence, now enable us to create enzymes with desired functions without relying on traditional genome mining. Here, we demonstrate a computational strategy for creating new-to-nature PET hydrolases by leveraging the known catalytic mechanisms and implementing multiple deep learning algorithms and molecular computations. This strategy includes the extraction of functional motifs from a template enzyme (here we use leaf-branch compost cutinase, LCC), regeneration of new protein scaffolds, computational screening, experimental validation, and sequence refinement. We successfully replicate PET hydrolytic activity with designer enzymes that are at least 30% shorter in sequence length than LCC. Among them, RsPETase 1 stands out due to its robust expressibility. It exhibits comparable activity to IsPETase and considerable thermostability with a melting temperature of 56 °C, despite sharing only 34% sequence similarity with LCC. This work suggests that enzyme diversity can be expanded by recapitulating functional motifs with computationally built protein scaffolds, thus generating opportunities to acquire highly active and robust enzymes that do not exist in nature.

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Section: Discussionmentioning

confidence: 99%

Replicating enzymatic activity by positioning active sites with synthetic protein scaffolds

Ding,

Zhang,

Hess

et al. 2024

Preprint

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“…Second, the candidate miniproteins selected using the calculated binding affinity to V600E mutant BRAF 15mut were further screened according to the aqueous solubility to exclude the insoluble and poorly soluble miniproteins in the early stage of discovery. This step seemed to be necessary because miniproteins comprising several helices tend to be hydrophobic due to the abundance of nonpolar residues such as Val and Leu at the interface between helices [18].…”

Section: De Novo Design Of Candidate Miniprotein Inhibitors Of V600e ...mentioning

confidence: 99%

Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF

Ham,

Kim,

Kim

et al. 2024

IJMS

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Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

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“…Zinc fingers 16,216,217 are involved in specific double-stranded DNA recognition and are the most common class of DNA-binding proteins throughout biology, and their folding properties have been extensively studied. Folding and metal bonding are closely related.…”

Section: Mimics Of Zinc Fingermentioning

confidence: 99%