Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target the E-box DNA Site

Inamoto, Ichiro; Sheoran, I. S.; Popa, Serban C.; Hussain, Montdher; Shin, Jumi A.

doi:10.1021/acschembio.0c00684

Cited by 12 publications

(7 citation statements)

References 78 publications

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“…This could be because Myc is only boosted by E-box sequences through preferential but not absolute specificity and may bind at all available promoters. , To overcome this, other Myc-inhibiting miniproteins may be explored for ApP tagging and intracellular delivery. Recently, two such proteins, dubbed MEF and Mad, have surfaced with the latter exhibiting greater inhibition of Myc-mediated transcription than Omomyc. In control studies, we observed that free Omomyc and Omomyc-E30 exhibited no inhibitory effect and negligible cytosolic delivery despite reports of Omomyc possessing intrinsic cell-penetrating behavior.…”

Section: Discussionmentioning

confidence: 99%

Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc

et al. 2022

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The ability to deliver small protein scaffolds intracellularly could enable the targeting and inhibition of many therapeutic targets that are not currently amenable to inhibition with small-molecule drugs. Here, we report the engineering of small protein scaffolds with anionic polypeptides (ApPs) to promote electrostatic interactions with positively charged nonviral lipid-based delivery systems. Proteins fused with ApPs are either complexed with off-the-shelf cationic lipids or encapsulated within ionizable lipid nanoparticles for highly efficient cytosolic delivery (up to 90%). The delivery of protein inhibitors is used to inhibit two common proto-oncogenes, Ras and Myc, in two cancer cell lines. This report demonstrates the feasibility of combining minimally engineered small protein scaffolds with tractable nanocarriers to inhibit intracellular proteins that are generally considered “undruggable” with current small molecule drugs and biologics.

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Section: Discussionmentioning

confidence: 99%

Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc

et al. 2022

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“…MEF displays slightly increased E-box binding and strong cooperative binding. [74] In recent work Moellering and co-workers used the basic helix along with the minimal zipper helix from Max to target E-Box DNA with high affinity. The individual helices harbor olefin containing amino acids allowing for side-chain stapling by ring-closure-metathesis and appropriate amino acids for ligation with a maleimide linker.…”

Section: Max-derived Mini Proteinsmentioning

confidence: 99%

“…This yields MEF which they term a ‘franken‐protein’ as it is composed of domains from different classes of TFs, preventing heterodimerization. MEF displays slightly increased E‐box binding and strong cooperative binding [74] …”

Section: Targeting Of Dna With Synthetic Peptidesmentioning

confidence: 99%

Synthetic Peptides: Promising Modalities for the Targeting of Disease‐Related Nucleic Acids

Ellenbroek,

Kahler,

Evers

et al. 2024

Angewandte Chemie

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DNA and RNA play pivotal roles in life processes by storing and transferring genetic information, modulating gene expression, and contributing to essential cellular machinery such as ribosomes. Dysregulation and mutations in nucleic acid‐related processes are implicated in numerous diseases. Despite the critical impact on health of nucleic acid mutations or dysregulation, therapeutic compounds addressing these biomolecules remain limited. Peptides have emerged as a promising class of molecules for biomedical research, offering potential solutions for challenging drug targets. This review focuses on the use of synthetic peptides to target disease‐related nucleic acids. We discuss examples of peptides targeting double‐stranded DNA, including the clinical candidate Omomyc, and compounds designed for regulatory G‐quadruplexes. Further, we provide insights into both library‐based screenings and the rational design of peptides to target regulatory human RNA scaffolds and viral RNAs, emphasizing the potential of peptides in addressing nucleic acid‐related diseases.

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“…We have used raRonal design and phage-assisted evoluRon to discover small proteins that target specific 6 base-pair (bp) DNA sequences. [1][2][3][4] Simply put, our proteins comprise modules from various transcripRon factors (TF) families that we cut-and-paste together. Our 60-90 amino-acid (aa) frankenproteins cooperaRvely bind as dimers to the 6 bp E-box sequence 5'-CACGTG with low nM binding affiniRes, without binding to nonspecific DNA controls even at micromolar concentraRons.…”

Section: Introductionmentioning

confidence: 99%

HinZip, a designed frankenprotein that combines Hin recombinase and FosW, mimics the structure and DNA-binding function of the HD-Zip plant transcription factor family

Akel,

Edaibis,

Shin

2024

Preprint

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Small bespoke proteins that bind a desired DNA sequence in a cell's genome could be a powerful tool in various applications. Examples include using genetically encoded tools to control gene circuits in synthetic biology, or serve as a protein drug that inhibits a disease network impacting human health. We designed HinZip to bind a specific target comprising at least 24 base pairs with high affinity and DNA sequence specificity, because a larger DNA sequence is likely to be unique in a genome, thereby minimizing off-target effects. We took inspiration from the HD-Zip, a transcription factor family only found in plants. No high-resolution structures exist for HD-Zip: genome-wide analyses indicate that they use a homeodomain to bind DNA and leucine zipper for dimerization. HinZip is a fusion of the Hin recombinase DNA-binding domain and FosW leucine zipper. HinZip binds cooperatively as a dimer to DNA targets comprising two 12 base-pair hixC half-sites. Using bacterial one-hybrid and quantitative electrophoretic mobility shift assays, we tested spacings of 0-9 base pairs between half-sites to optimize the orientation and space parameters that FosW needs for coiled-coil dimerization. HinZip binds cooperatively to a 29 base-pair inverted hixC palindrome with Kd 17 nM and no binding to nonspecific DNA up to 2 µM protein. Hin—which lacks ability to dimerize—was previously shown to bind hixC with Kd 34 nM, showing similar binding to half- or full-sites and no cooperativity. HinZip/LA, where the Leu residues responsible for dimerization were replaced with Ala, showed virtually no benefit from cooperative binding at any full-site, and bound first as a monomer, and then as two monomers. Circular dichroism and dynamic light scattering indicate that only HinZip is capable of forming a coiled-coil dimer. HinZip demonstrates that even in the absence of guidance from structural information, small frankenproteins designed from cut-and-paste of unrelated protein modules can specifically target long DNA sequences with broad applications toward orthogonally controlling gene networks in a wide variety of organisms.

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Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target the E-box DNA Site

Cited by 12 publications

References 78 publications

Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc

Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc

Synthetic Peptides: Promising Modalities for the Targeting of Disease‐Related Nucleic Acids

HinZip, a designed frankenprotein that combines Hin recombinase and FosW, mimics the structure and DNA-binding function of the HD-Zip plant transcription factor family

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