@article{Huang2024b,

title = {Designed endocytosis-inducing proteins degrade targets and amplify signals},

author = {Buwei Huang and Mohamad Abedi and Green Ahn and Brian Coventry and Isaac Sappington and Cong Tang and Rong Wang and Thomas Schlichthaerle and Jason Z. Zhang and Yujia Wang and Inna Goreshnik and Ching Wen Chiu and Adam Chazin-Gray and Sidney Chan and Stacey Gerben and Analisa Murray and Shunzhi Wang and Jason O’Neill and Li Yi and Ronald Yeh and Ayesha Misquith and Anitra Wolf and Luke M. Tomasovic and Dan I. Piraner and Maria J. Duran Gonzalez and Nathaniel R. Bennett and Preetham Venkatesh and Maggie Ahlrichs and Craig Dobbins and Wei Yang and Xinru Wang and Danny D. Sahtoe and Dionne Vafeados and Rubul Mout and Shirin Shivaei and Longxing Cao and Lauren Carter and Lance Stewart and Jamie B. Spangler and Kole T. Roybal and Per Jr Greisen and Xiaochun Li and Gonçalo J. L. Bernardes and Carolyn R. Bertozzi and David Baker},

url = {https://www.nature.com/articles/s41586-024-07948-2, Nature [Open Access] },

doi = {10.1038/s41586-024-07948-2},

year  = {2024},

date = {2024-09-25},

urldate = {2024-09-25},

journal = {Nature},

publisher = {Springer Science and Business Media LLC},

abstract = {Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificity targeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand–receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody–drug and antibody–RNA conjugates.



},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Huang2024,

title = {De novo design of miniprotein antagonists of cytokine storm inducers},

author = {Buwei Huang and Brian Coventry and Marta T. Borowska and Dimitrios C. Arhontoulis and Marc Exposit and Mohamad Abedi and Kevin M. Jude and Samer F. Halabiya and Aza Allen and Cami Cordray and Inna Goreshnik and Maggie Ahlrichs and Sidney Chan and Hillary Tunggal and Michelle DeWitt and Nathaniel Hyams and Lauren Carter and Lance Stewart and Deborah H. Fuller and Ying Mei and K. Christopher Garcia and David Baker},

url = {https://www.nature.com/articles/s41467-024-50919-4, Nature Communications [Open Access]},

doi = {10.1038/s41467-024-50919-4},

year  = {2024},

date = {2024-08-16},

urldate = {2024-08-16},

journal = {Nature Communications},

publisher = {Springer Science and Business Media LLC},

abstract = {Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1β. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sun2024,

title = {Accurate de novo design of heterochiral protein–protein interactions},

author = {Ke Sun and Sicong Li and Bowen Zheng and Yanlei Zhu and Tongyue Wang and Mingfu Liang and Yue Yao and Kairan Zhang and Jizhong Zhang and Hongyong Li and Dongyang Han and Jishen Zheng and Brian Coventry and Longxing Cao and David Baker and Lei Liu and Peilong Lu},

url = {https://www.nature.com/articles/s41422-024-01014-2, Cell Research [Open Access]},

doi = {10.1038/s41422-024-01014-2},

year  = {2024},

date = {2024-08-14},

urldate = {2024-08-14},

journal = {Cell Research},

publisher = {Springer Science and Business Media LLC},

abstract = {Abiotic d-proteins that selectively bind to natural l-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein–protein interactions remain largely unknown. In this study, we present the de novo design of d-proteins consisting of 50–65 residues, aiming to target specific surface regions of l-proteins or l-peptides. Our designer d-protein binders exhibit nanomolar affinity toward an artificial l-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these d-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer d-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral d-protein–l-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical l-peptides and d-proteins, particularly elucidating a novel mode of heterochiral helix–helix interactions. Leveraging the design of d-proteins specifically targeting l-peptides or l-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{An2024,

title = {Binding and sensing diverse small molecules using shape-complementary pseudocycles},

author = {Linna An and Meerit Said and Long Tran and Sagardip Majumder and Inna Goreshnik and Gyu Rie Lee and David Juergens and Justas Dauparas and Ivan Anishchenko and Brian Coventry and Asim K. Bera and Alex Kang and Paul M. Levine and Valentina Alvarez and Arvind Pillai and Christoffer Norn and David Feldman and Dmitri Zorine and Derrick R. Hicks and Xinting Li and Mariana Garcia Sanchez and Dionne K. Vafeados and Patrick J. Salveson and Anastassia A. Vorobieva and David Baker},

url = {https://www.science.org/doi/10.1126/science.adn3780, Science},

doi = {10.1126/science.adn3780},

year  = {2024},

date = {2024-07-19},

urldate = {2024-07-19},

journal = {Science},

publisher = {American Association for the Advancement of Science (AAAS)},

abstract = {We describe an approach for designing high-affinity small molecule–binding proteins poised for downstream sensing. We use deep learning–generated pseudocycles with repeating structural units surrounding central binding pockets with widely varying shapes that depend on the geometry and number of the repeat units. We dock small molecules of interest into the most shape complementary of these pseudocycles, design the interaction surfaces for high binding affinity, and experimentally screen to identify designs with the highest affinity. We obtain binders to four diverse molecules, including the polar and flexible methotrexate and thyroxine. Taking advantage of the modular repeat structure and central binding pockets, we construct chemically induced dimerization systems and low-noise nanopore sensors by splitting designs into domains that reassemble upon ligand addition.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Berger2024,

title = {Preclinical proof of principle for orally delivered Th17 antagonist miniproteins},

author = {Stephanie Berger and Franziska Seeger and Ta-Yi Yu and Merve Aydin and Huilin Yang and Daniel Rosenblum and Laure Guenin-Macé and Caleb Glassman and Lauren Arguinchona and Catherine Sniezek and Alyssa Blackstone and Lauren Carter and Rashmi Ravichandran and Maggie Ahlrichs and Michael Murphy and Ingrid Swanson Pultz and Alex Kang and Asim K. Bera and Lance Stewart and K. Christopher Garcia and Shruti Naik and Jamie B. Spangler and Florian Beigel and Matthias Siebeck and Roswitha Gropp and David Baker},

url = {https://www.cell.com/cell/fulltext/S0092-8674(24)00631-7, Cell [Open Access]},

doi = {10.1016/j.cell.2024.05.052},

year  = {2024},

date = {2024-06-26},

urldate = {2024-06-00},

journal = {Cell},

publisher = {Elsevier BV},

abstract = {Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Jiang2024,

title = {De novo design of buttressed loops for sculpting protein functions},

author = {Jiang, Hanlun

and Jude, Kevin M.

and Wu, Kejia

and Fallas, Jorge

and Ueda, George

and Brunette, T. J.

and Hicks, Derrick R.

and Pyles, Harley

and Yang, Aerin

and Carter, Lauren

and Lamb, Mila

and Li, Xinting

and Levine, Paul M.

and Stewart, Lance

and Garcia, K. Christopher

and Baker, David},

url = {https://www.nature.com/articles/s41589-024-01632-2, Nature Chemical Biology [Open Access]

https://www.bakerlab.org/wp-content/uploads/2024/05/s41589-024-01632-2.pdf, PDF},

doi = {10.1038/s41589-024-01632-2},

year  = {2024},

date = {2024-05-30},

urldate = {2024-05-30},

journal = {Nature Chemical Biology},

abstract = {In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9–14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sahtoe2024,

title = {Design of amyloidogenic peptide traps},

author = {Danny D. Sahtoe and Ewa A. Andrzejewska and Hannah L. Han and Enrico Rennella and Matthias M. Schneider and Georg Meisl and Maggie Ahlrichs and Justin Decarreau and Hannah Nguyen and Alex Kang and Paul Levine and Mila Lamb and Xinting Li and Asim K. Bera and Lewis E. Kay and Tuomas P. J. Knowles and David Baker},

url = {https://www.nature.com/articles/s41589-024-01578-5, Nature Chemical Biology [Open Access]},

doi = {10.1038/s41589-024-01578-5},

year  = {2024},

date = {2024-03-19},

urldate = {2024-03-19},

journal = {Nature Chemical Biology},

publisher = {Springer Science and Business Media LLC},

abstract = {Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein−peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1−42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mateos2024,

title = {Computational design of binders targeting the VSDIV from NaV1.7 sodium channel},

author = {Diego Lopez Mateos and Adam M. Murray and Hai M. Nguyen and Preetham Venkatesh and Brian Koepnick and David Baker and Heike Wulff and Vladimir Yarov-Yarovoy},

url = {https://www.cell.com/biophysj/abstract/S0006-3495(23)01470-4, Biophysical Journal},

doi = {10.1016/j.bpj.2023.11.770},

year  = {2024},

date = {2024-02-08},

urldate = {2024-02-00},

journal = {Biophysical Journal},

publisher = {Elsevier BV},

abstract = {Chronic pain affects about 20% of the US population, but safe treatments are limited. There is an urgent need for effective and non-addictive therapies for chronic pan conditions. Voltage-gated sodium (NaV) channel, NaV1.7, is a key player in pain signaling pathway, making it a promising target for novel pain therapeutics. Achieving high subtype selectivity when targeting NaV channels is of primary importance to avoid impairing vital physiological functions mediated by off-target channels. Efforts to selectively target NaV1.7 have been hindered by the difficulties in targeting NaV1.7 over other NaV channel subtypes. Peptidic gating modifier toxins (GMTs), such as Protoxin-II (ProTx2), are promising scaffolds for novel peptide design targeting ion channels with high potency and subtype selectivity. ProTx2 binds to the second and fourth voltage-sensing domains (VSDII and VSDIV) from NaV1.7 with moderate subtype selectivity and can modulate channel activation and inactivation. In this project, we modeled ProTx2 bound to human NaV1.7 VSDIV in an activated state. We used RoseTTAFold Diffusion and Protein MPNN protein design methods to generate protein binders inspired by ProTx2 binding motif with increased predicted binding affinity for human NaV1.7 VSDIV in an activated state. Additionally, we applied these protein design methods to create de novo binders targeting human NaV1.7 VSDIV in an activated state. We anticipate that trapping the VSDIV in an activated conformation will stabilize an inactivated state of the channel, as activation of VSDIV is coupled with channel fast inactivation. Initial electrophysiological screening of our top in silico binders identified promising candidates that inhibited NaV1.7 in the micromolar range. These binders will undergo further testing and optimization against NaV1.7 to create novel molecular tools to study NaV channel activity and effective and safe therapies for chronic pain.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38109936,

title = {De novo design of high-affinity binders of bioactive helical peptides},

author = {Susana Vázquez Torres and Philip J Y Leung and Preetham Venkatesh and Isaac D Lutz and Fabian Hink and Huu-Hien Huynh and Jessica Becker and Andy Hsien-Wei Yeh and David Juergens and Nathaniel R Bennett and Andrew N Hoofnagle and Eric Huang and Michael J MacCoss and Marc Expòsit and Gyu Rie Lee and Asim K Bera and Alex Kang and Joshmyn De La Cruz and Paul M Levine and Xinting Li and Mila Lamb and Stacey R Gerben and Analisa Murray and Piper Heine and Elif Nihal Korkmaz and Jeff Nivala and Lance Stewart and Joseph L Watson and Joseph M Rogers and David Baker},

url = {https://www.nature.com/articles/s41586-023-06953-1, Nature [Open Access]},

doi = {10.1038/s41586-023-06953-1},

issn = {1476-4687},

year  = {2023},

date = {2023-12-01},

urldate = {2023-12-01},

journal = {Nature},

abstract = {Many peptide hormones form an alpha-helix upon binding their receptors, and sensitive detection methods for them could contribute to better clinical management of disease. 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 peptides with helical propensity is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar affinity binders can be generated to helical peptide targets both by refining 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 optimisation. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimise by partial diffusion both natural and designed proteins, should be broadly useful.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{An2023,

title = {Hallucination of closed repeat proteins containing central pockets},

author = {An L, Hicks DR, Zorine D, Dauparas J, Wicky BIM, Milles LF, Courbet A, Bera AK, Nguyen H, Kang A, Carter L, Baker D},

url = {https://www.nature.com/articles/s41594-023-01112-6, Nature Structural & Molecular Biology [Open Access] },

doi = {10.1038/s41594-023-01112-6},

year  = {2023},

date = {2023-09-28},

urldate = {2023-09-28},

journal = {Nature Structural & Molecular Biology},

abstract = {In pseudocyclic proteins, such as TIM barrels, β barrels, and some helical transmembrane channels, a single subunit is repeated in a cyclic pattern, giving rise to a central cavity that can serve as a pocket for ligand binding or enzymatic activity. Inspired by these proteins, we devised a deep-learning-based approach to broadly exploring the space of closed repeat proteins starting from only a specification of the repeat number and length. Biophysical data for 38 structurally diverse pseudocyclic designs produced in Escherichia coli are consistent with the design models, and the three crystal structures we were able to obtain are very close to the designed structures. Docking studies suggest the diversity of folds and central pockets provide effective starting points for designing small-molecule binders and enzymes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Watson2023,

title = {De novo design of protein structure and function with RFdiffusion},

author = {Watson, Joseph L.

and Juergens, David

and Bennett, Nathaniel R.

and Trippe, Brian L.

and Yim, Jason

and Eisenach, Helen E.

and Ahern, Woody

and Borst, Andrew J.

and Ragotte, Robert J.

and Milles, Lukas F.

and Wicky, Basile I. M.

and Hanikel, Nikita

and Pellock, Samuel J.

and Courbet, Alexis

and Sheffler, William

and Wang, Jue

and Venkatesh, Preetham

and Sappington, Isaac

and Torres, Susana Vázquez

and Lauko, Anna

and De Bortoli, Valentin

and Mathieu, Emile

and Ovchinnikov, Sergey

and Barzilay, Regina

and Jaakkola, Tommi S.

and DiMaio, Frank

and Baek, Minkyung

and Baker, David},

url = {https://www.nature.com/articles/s41586-023-06415-8, Nature

https://www.bakerlab.org/wp-content/uploads/2023/07/s41586-023-06415-8_reference.pdf, PDF (29MB)},

doi = {10.1038/s41586-023-06415-8},

year  = {2023},

date = {2023-07-11},

journal = {Nature},

abstract = {There has been considerable recent progress in designing new proteins using deep learning methods1–9. Despite this progress, a general deep learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher order symmetric architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modeling but limited success when applied to protein modeling, likely due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding, and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold Diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryo-EM structure of a designed binder in complex with Influenza hemagglutinin which is nearly identical to the design model. In a manner analogous to networks which produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kim2023,

title = {De novo design of small beta barrel proteins},

author = {Kim, David E.

and Jensen, Davin R.

and Feldman, David

and Tischer, Doug

and Saleem, Ayesha

and Chow, Cameron M.

and Li, Xinting

and Carter, Lauren

and Milles, Lukas

and Nguyen, Hannah

and Kang, Alex

and Bera, Asim K.

and Peterson, Francis C.

and Volkman, Brian F.

and Ovchinnikov, Sergey

and Baker, David},

url = {https://www.pnas.org/doi/10.1073/pnas.2207974120, PNAS (Open Access)},

doi = {10.1073/pnas.2207974120},

year  = {2023},

date = {2023-03-10},

urldate = {2023-03-10},

journal = {Proceedings of the National Academy of Sciences},

abstract = {Small beta barrel proteins are attractive targets for computational design because of their considerable functional diversity despite their very small size (<70 amino acids). However, there are considerable challenges to designing such structures, and there has been little success thus far. Because of the small size, the hydrophobic core stabilizing the fold is necessarily very small, and the conformational strain of barrel closure can oppose folding; also intermolecular aggregation through free beta strand edges can compete with proper monomer folding. Here, we explore the de novo design of small beta barrel topologies using both Rosetta energy–based methods and deep learning approaches to design four small beta barrel folds: Src homology 3 (SH3) and oligonucleotide/oligosaccharide-binding (OB) topologies found in nature and five and six up-and-down-stranded barrels rarely if ever seen in nature. Both approaches yielded successful designs with high thermal stability and experimentally determined structures with less than 2.4 Å rmsd from the designed models. Using deep learning for backbone generation and Rosetta for sequence design yielded higher design success rates and increased structural diversity than Rosetta alone. The ability to design a large and structurally diverse set of small beta barrel proteins greatly increases the protein shape space available for designing binders to protein targets of interest.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Agarwal2022,

title = {Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor},

author = {Agarwal, Dilip Kumar

and Hunt, Andrew C.

and Shekhawat, Gajendra S.

and Carter, Lauren

and Chan, Sidney

and Wu, Kejia

and Cao, Longxing

and Baker, David

and Lorenzo-Redondo, Ramon

and Ozer, Egon A.

and Simons, Lacy M.

and Hultquist, Judd F.

and Jewett, Michael C.

and Dravid, Vinayak P.},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9211039/, Analytical Chemistry},

year  = {2022},

date = {2022-06-06},

urldate = {2022-06-06},

journal = {Analytical Chemistry},

abstract = {New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (<5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hunt2022,

title = {Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice},

author = {Andrew C. Hunt and James Brett Case and Young-Jun Park and Longxing Cao and Kejia Wu and Alexandra C. Walls and Zhuoming Liu and John E. Bowen and Hsien-Wei Yeh and Shally Saini and Louisa Helms and Yan Ting Zhao and Tien-Ying Hsiang and Tyler N. Starr and Inna Goreshnik and Lisa Kozodoy and Lauren Carter and Rashmi Ravichandran and Lydia B. Green and Wadim L. Matochko and Christy A. Thomson and Bastian Vögeli and Antje Krüger and Laura A. VanBlargan and Rita E. Chen and Baoling Ying and Adam L. Bailey and Natasha M. Kafai and Scott E. Boyken and Ajasja Ljubetič and Natasha Edman and George Ueda and Cameron M. Chow and Max Johnson and Amin Addetia and Mary Jane Navarro and Nuttada Panpradist and Michael Gale and Benjamin S. Freedman and Jesse D. Bloom and Hannele Ruohola-Baker and Sean P. J. Whelan and Lance Stewart and Michael S. Diamond and David Veesler and Michael C. Jewett and David Baker},

url = {https://www.science.org/doi/abs/10.1126/scitranslmed.abn1252, Science Translational Medicine

https://www.bakerlab.org/wp-content/uploads/2022/04/scitranslmed.abn1252.pdf, Download PDF},

doi = {10.1126/scitranslmed.abn1252},

year  = {2022},

date = {2022-04-12},

urldate = {2022-04-12},

journal = {Science Translational Medicine},

abstract = {New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy with a homo-trimeric version of the 75-residue angiotensin converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. In the cryo-electron microscopy structure, TRI2 formed a tripod on top of the spike protein which engaged all three receptor binding domains (RBDs) simultaneously as in the design model. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than that of monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies and native receptor traps. By comparison, the designed proteins have resistance to viral escape and antigenic drift by construction, precisely tuned avidity, and greatly reduced chance of autoimmune responses. Computationally designed trivalent minibinders provide therapeutic protection in mice against emerging SARS-CoV-2 variants of concern.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Cao2022,

title = {Design of protein binding proteins from target structure alone},

author = {Cao, Longxing and Coventry, Brian and Goreshnik, Inna and Huang, Buwei and Park, Joon Sung and Jude, Kevin M. and Marković, Iva and Kadam, Rameshwar U. and Verschueren, Koen H. G. and Verstraete, Kenneth and Walsh, Scott Thomas Russell and Bennett, Nathaniel and Phal, Ashish and Yang, Aerin and Kozodoy, Lisa and DeWitt, Michelle and Picton, Lora and Miller, Lauren and Strauch, Eva-Maria and DeBouver, Nicholas D. and Pires, Allison and Bera, Asim K. and Halabiya, Samer and Hammerson, Bradley and Yang, Wei and Bernard, Steffen and Stewart, Lance and Wilson, Ian A. and Ruohola-Baker, Hannele and Schlessinger, Joseph and Lee, Sangwon and Savvides, Savvas N. and Garcia, K. Christopher and Baker, David},

url = {https://www.nature.com/articles/s41586-022-04654-9, Nature

https://www.bakerlab.org/wp-content/uploads/2022/03/Cao_etal_Nature2022_Design_of_binders_from_target_structure_alone.pdf, Download PDF},

doi = {10.1038/s41586-022-04654-9},

year  = {2022},

date = {2022-03-24},

urldate = {2022-03-24},

journal = {Nature},

abstract = {The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains an outstanding challenge1–5. We describe a general solution to this problem which starts with a broad exploration of the very large space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate its very broad applicability by de novo design of binding proteins to 12 diverse protein targets with very different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and following experimental optimization bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five are very close to the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvement of both. Our approach now enables targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hosseinzadeh2021,

title = {Anchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites},

author = {Hosseinzadeh, Parisa

and Watson, Paris R.

and Craven, Timothy W.

and Li, Xinting

and Rettie, Stephen

and Pardo-Avila, Fátima

and Bera, Asim K.

and Mulligan, Vikram Khipple

and Lu, Peilong

and Ford, Alexander S.

and Weitzner, Brian D.

and Stewart, Lance J.

and Moyer, Adam P.

and Di Piazza, Maddalena

and Whalen, Joshua G.

and Greisen, Per Jr.

and Christianson, David W.

and Baker, David},

url = {https://www.nature.com/articles/s41467-021-23609-8, Nature Communications

https://www.bakerlab.org/wp-content/uploads/2021/06/Hosseinzadeh_etal_NatureComms2021_AnchorExtention.pdf, Download PDF},

doi = {10.1038/s41467-021-23609-8},

year  = {2021},

date = {2021-06-07},

urldate = {2021-06-07},

journal = {Nature Communications},

abstract = {Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational “anchor extension” methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sahtoe2021,

title = {Transferrin receptor targeting by de novo sheet extension},

author = {Sahtoe, Danny D. and Coscia, Adrian and Mustafaoglu, Nur and Miller, Lauren M. and Olal, Daniel and Vulovic, Ivan and Yu, Ta-Yi and Goreshnik, Inna and Lin, Yu-Ru and Clark, Lars and Busch, Florian and Stewart, Lance and Wysocki, Vicki H. and Ingber, Donald E. and Abraham, Jonathan and Baker, David},

url = {https://www.pnas.org/content/118/17/e2021569118, PNAS

},

doi = {10.1073/pnas.2021569118},

year  = {2021},

date = {2021-04-27},

urldate = {2021-04-27},

journal = {Proceedings of the National Academy of Sciences},

abstract = {The de novo design of proteins that bind natural target proteins is useful for a variety of biomedical and biotechnological applications. We describe a design strategy to target proteins containing an exposed beta edge strand. We use the approach to design binders to the human transferrin receptor which shuttles back and forth across the blood{textendash}brain barrier. Such binders could be useful for the delivery of therapeutics into the brain.The de novo design of polar protein{textendash}protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood{textendash}brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.Crystal structures have been deposited in the RCSB PDB with the accession nos. 6WRX, 6WRW, and 6WRV. Additional supporting data has been deposited in the online Zenodo repository (https://zenodo.org/record/4594115) (47). All other study data are included in the article and/or supporting information.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Cao2020,

title = {De novo design of picomolar SARS-CoV-2 miniprotein inhibitors},

author = {Cao, Longxing and Goreshnik, Inna and Coventry, Brian and Case, James Brett and Miller, Lauren and Kozodoy, Lisa and Chen, Rita E. and Carter, Lauren and Walls, Alexandra C. and Park, Young-Jun and Strauch, Eva-Maria and Stewart, Lance and Diamond, Michael S. and Veesler, David and Baker, David},

url = {https://science.sciencemag.org/content/early/2020/09/08/science.abd9909

https://www.bakerlab.org/wp-content/uploads/2020/09/Cao_etal_Science_COVID_spike_binders.pdf},

doi = {10.1126/science.abd9909},

year  = {2020},

date = {2020-09-09},

journal = {Science},

abstract = {Targeting the interaction between the SARS-CoV-2 Spike protein and the human ACE2 receptor is a promising therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer generated scaffolds were either built around an ACE2 helix that interacts with the Spike receptor binding domain (RBD), or docked against the RBD to identify new binding modes, and their amino acid sequences designed to optimize target binding, folding and stability. Ten designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked ARS-CoV-2 infection of Vero E6 cells with IC 50 values between 24 pM and 35 nM; The most potent, with new binding modes, are 56 and 64 residue proteins (IC 50 ~ 0.16 ng/ml). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Brunette2020,

title = {Modular repeat protein sculpting using rigid helical junctions},

author = {Brunette, TJ and Bick, Matthew J. and Hansen, Jesse M. and Chow, Cameron M. and Kollman, Justin M. and Baker, David},

url = {https://www.bakerlab.org/wp-content/uploads/2020/04/Brunette2020_Junctions.pdf

https://www.pnas.org/content/early/2020/04/02/1908768117},

doi = {10.1073/pnas.1908768117},

year  = {2020},

date = {2020-04-02},

journal = {Proceedings of the National Academy of Sciences},

abstract = {The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ∼10-nm-diameter “L” and “V” shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Silva2018,

title = {Structures and disulfide cross‐linking of de novo designed therapeutic mini‐proteins},

author = {Silva, Daniel-Adriano and Stewart, Lance and Lam, Kwok-Ho and Jin, Rongsheng and Baker, David},

url = {https://febs.onlinelibrary.wiley.com/doi/abs/10.1111/febs.14394

},

doi = {10.1111/febs.14394},

year  = {2018},

date = {2018-02-01},

journal = {FEBS Journal},

volume = {285},

number = {10},

pages = {1783-1785},

abstract = {Recent advances in computational protein design now enable the massively parallel de novo design and experimental characterization of small hyperstable binding proteins with potential therapeutic activity. By providing experimental feedback on tens of thousands of designed proteins, the design-build-test-learn pipeline provides a unique opportunity to systematically improve our understanding of protein folding and binding. Here, we review the structures of mini-protein binders in complex with Influenza hemagglutinin and Bot toxin, and illustrate in the case of disulfide bond placement how analysis of the large datasets of computational models and experimental data can be used to identify determinants of folding and binding.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}