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.
Abstract
Motivation
As more data of experimentally determined protein structures are becoming available, data-driven models to describe protein sequence–structure relationships become more feasible. ...Within this space, the amino acid sequence design of protein–protein interactions is still a rather challenging subproblem with very low success rates—yet, it is central to most biological processes.
Results
We developed an attention-based deep learning model inspired by algorithms used for image-caption assignments to design peptides or protein fragment sequences. Our trained model can be applied for the redesign of natural protein interfaces or the designed protein interaction fragments. Here, we validate the potential by recapitulating naturally occurring protein–protein interactions including antibody–antigen complexes. The designed interfaces accurately capture essential native interactions and have comparable native-like binding affinities in silico. Furthermore, our model does not need a precise backbone location, making it an attractive tool for working with de novo design of protein–protein interactions.
Availability and implementation
The source code of the method is available at https://github.com/strauchlab/iNNterfaceDesign
Supplementary information
Supplementary data are available at Bioinformatics online.
Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria‐causing parasite putting 2.5 billion people ...at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood‐stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. A unique characteristic of P. vivax is that it depends on the Duffy antigen/receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease‐resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.
We describe a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. Favorable interactions between disembodied amino acid residues and ...the target surface are identified and used to anchor de novo designed interfaces. The method was used to design proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus. After affinity maturation, two of the designed proteins, HB36 and HB80, bind H1 and H5 HAs with low nanomolar affinity. Further, HB80 inhibits the HA fusogenic conformational changes induced at low pH. The crystal structure of HB36 in complex with 1918/H1 HA revealed that the actual binding interface is nearly identical to that in the computational design model. Such designed binding proteins may be useful for both diagnostics and therapeutics.
Computational design provides the opportunity to program protein–protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding ...protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a K d of ∼4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.
Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide ...range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework. RosettaScripts provides access to protocol-level functionalities, such as rigid-body docking and sequence redesign, and allows fast testing and deployment of complex protocols without need for modifying or recompiling the underlying C++ code. We illustrate these capabilities with RosettaScripts protocols for the stabilization of proteins, the generation of computationally constrained libraries for experimental selection of higher-affinity binding proteins, loop remodeling, small-molecule ligand docking, design of ligand-binding proteins, and specificity redesign in DNA-binding proteins.
Despite undergoing allogeneic hematopoietic stem cell transplantation (HCT), patients with acute myeloid leukemia (AML) with internal tandem duplication mutation in the
like tyrosine kinase 3 gene (
...ITD) have a poor prognosis, frequently relapse, and die as a result of AML. It is currently unknown whether a maintenance therapy using FLT3 inhibitors, such as the multitargeted tyrosine kinase inhibitor sorafenib, improves outcome after HCT.
In a randomized, placebo-controlled, double-blind phase II trial (SORMAIN; German Clinical Trials Register: DRKS00000591), 83 adult patients with
ITD-positive AML in complete hematologic remission after HCT were randomly assigned to receive for 24 months either the multitargeted and FLT3-kinase inhibitor sorafenib (n = 43) or placebo (n = 40 placebo). Relapse-free survival (RFS) was the primary endpoint of this trial. Relapse was defined as relapse or death, whatever occurred first.
With a median follow-up of 41.8 months, the hazard ratio (HR) for relapse or death in the sorafenib group versus placebo group was 0.39 (95% CI, 0.18 to 0.85; log-rank
= .013). The 24-month RFS probability was 53.3% (95% CI, 0.36 to 0.68) with placebo versus 85.0% (95% CI, 0.70 to 0.93) with sorafenib (HR, 0.256; 95% CI, 0.10 to 0.65; log-rank
= .002). Exploratory data show that patients with undetectable minimal residual disease (MRD) before HCT and those with detectable MRD after HCT derive the strongest benefit from sorafenib.
Sorafenib maintenance therapy reduces the risk of relapse and death after HCT for
ITD-positive AML.
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.
Infection with the protozoan parasite Trypanosoma cruzi elicits substantial CD8+ T cell responses that disproportionately target epitopes encoded in the large trans-sialidase (TS) gene family. Within ...the C57BL/6 infection model, a significant proportion (30-40%) of the T. cruzi-specific CD8+ T cell response targets two immunodominant TS epitopes, TSKb18 and TSKb20. However, both TS-specific CD8+ T cell responses are dispensable for immune control, and TS-based vaccines have no demonstrable impact on parasite persistence, a determinant of disease. Besides TS, the specificity and protective capacity of CD8+ T cells that mediate immune control of T. cruzi infection are unknown. With the goal of identifying alternative CD8+ T cell targets, we designed and screened a representative set of genome-wide, in silico-predicted epitopes. Our screen identified a previously uncharacterized, to our knowledge, T cell epitope MUCKb25, found within mucin family proteins, the third most expanded large gene family in T. cruzi. The MUCKb25-specific response was characterized by delayed kinetics, relative to TS-specific responses, and extensive cross-reactivity with a large number of endogenous epitope variants. Similar to TS-specific responses, the MUCKb25 response was dispensable for control of the infection, and vaccination to generate MUCK-specific CD8+ T cells failed to confer protection. The lack of protection by MUCK vaccination was partly attributed to the fact that MUCKb25-specific T cells exhibit limited recognition of T. cruzi-infected host cells. Overall, these results indicate that the CD8+ T cell compartment in many T. cruzi-infected mice is occupied by cells with minimal apparent effector potential.
Many pathogenic viruses rely on class I fusion proteins to fuse their viral membrane with the host cell membrane. To drive the fusion process, class I fusion proteins undergo an irreversible ...conformational change from a metastable prefusion state to an energetically more stable postfusion state. Mounting evidence underscores that antibodies targeting the prefusion conformation are the most potent, making it a compelling vaccine candidate. Here, we establish a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. With this protocol, we stabilize the fusion proteins of the RSV, hMPV, and SARS-CoV-2 viruses, testing fewer than a handful of designs. The solved structures of these designed proteins from all three viruses evidence the atomic accuracy of our approach. Furthermore, the humoral response of the redesigned RSV F protein compares to that of the recently approved vaccine in a mouse model. While the parallel design of two conformations allows the identification of energetically sub-optimal positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens.
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