Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (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 were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concentration (IC
) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC
~ 0.16 nanograms per milliliter). 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.
Proteins fold into unique native structures stabilized by thousands of weak interactions that collectively overcome the entropic cost of folding. Although these forces are “encoded” in the thousands ...of known protein structures, “decoding” them is challenging because of the complexity of natural proteins that have evolved for function, not stability. We combined computational protein design, next-generation gene synthesis, and a high-throughput protease susceptibility assay to measure folding and stability for more than 15,000 de novo designed miniproteins, 1000 natural proteins, 10,000 point mutants, and 30,000 negative control sequences. This analysis identified more than 2500 stable designed proteins in four basic folds—a number sufficient to enable us to systematically examine how sequence determines folding and stability in uncharted protein space. Iteration between design and experiment increased the design success rate from 6% to 47%, produced stable proteins unlike those found in nature for topologies where design was initially unsuccessful, and revealed subtle contributions to stability as designs became increasingly optimized. Our approach achieves the long-standing goal of a tight feedback cycle between computation and experiment and has the potential to transform computational protein design into a data-driven science.
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 a challenge
. Here we ...describe a general solution to this problem that starts with a broad exploration of the vast 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 the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with 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 closely match 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 improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.
Recently it has become possible to de novo design high affinity protein binding proteins from target structural information alone. There is, however, considerable room for improvement as the overall ...design success rate is low. Here, we explore the augmentation of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency.
To create new enzymes and biosensors from scratch, precise control over the structure of small-molecule binding sites is of paramount importance, but systematically designing arbitrary protein pocket ...shapes and sizes remains an outstanding challenge. Using the NTF2-like structural superfamily as a model system, we developed an enumerative algorithm for creating a virtually unlimited number of de novo proteins supporting diverse pocket structures. The enumerative algorithm was tested and refined through feedback from two rounds of large-scale experimental testing, involving in total the assembly of synthetic genes encoding 7,896 designs and assessment of their stability on yeast cell surface, detailed biophysical characterization of 64 designs, and crystal structures of 5 designs. The refined algorithm generates proteins that remain folded at high temperatures and exhibit more pocket diversity than naturally occurring NTF2-like proteins. We expect this approach to transform the design of smallmolecule sensors and enzymes by enabling the creation of binding and active site geometries much more optimal for specific design challenges than is accessible by repurposing the limited number of naturally occurring NTF2-like proteins.
De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and ...screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.
Computational design of a synthetic PD-1 agonist Bryan, Cassie M; Rocklin, Gabriel J; Bick, Matthew J ...
Proceedings of the National Academy of Sciences - PNAS,
07/2021, Letnik:
118, Številka:
29
Journal Article
Recenzirano
Odprti dostop
Programmed cell death protein-1 (PD-1) expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate ...balance is shifted in either direction. Tumor cells often take advantage of this pathway by overexpressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result. Using a combination of computation and experiment, we designed a hyperstable 40-residue miniprotein, PD-MP1, that specifically binds murine and human PD-1 at the PD-L1 interface with a K
of ∼100 nM. The apo crystal structure shows that the binder folds as designed with a backbone RMSD of 1.3 Å to the design model. Trimerization of PD-MP1 resulted in a PD-1 agonist that strongly inhibits murine T cell activation. This small, hyperstable PD-1 binding protein was computationally designed with an all-beta interface, and the trimeric agonist could contribute to treatments for autoimmune and inflammatory diseases.
Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to have a global impact. Previously, ...we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 spike receptor-binding domain. Here, we investigated the capacity of modified versions of one lead miniprotein, LCB1, to protect against SARS-CoV-2-mediated lung disease in mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung when given as many as 5 days before or 2 days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.
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•SARS-CoV-2 RBD-specific miniproteins confer 100% survival against lethal challenge•Miniproteins prevent SARS-CoV-2-induced lung inflammation and pathology•One miniprotein dose protects when given between D-5 and D+3 relative to infection•Miniproteins inhibit historical, circulating, and emerging SARS-CoV-2 strains
Case et al. show that de novo-designed SARS-CoV-2 RBD-specific miniproteins administered as prophylaxis or therapy protect mice against SARS-CoV-2-mediated disease, inflammation, and pathology. Injected or intranasal administration of miniproteins showed efficacy when given any time over an 8-day treatment window.
General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as ...most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone
. Here, inspired by natural and re-engineered protein-peptide systems
, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone
. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.
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 was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the 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 in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.
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