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.
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.
Abstract only
Haloferax volcanii
, an extremophile species of Archaea, is a microorganism that incorporates functional amyloids into its biofilms; these proteins are proposed to contribute to the ...structural stability of this matrix. We developed a protocol based on the procedure described by Jordal et al. (2009) to isolate these proteins via differential centrifugation. The presence of amyloid proteins was monitored and quantified throughout this process using Thioflavin T, a molecule that can selectively quantify amyloids by fluorescing when bound to their cross‐beta motif. These results showed that a four‐week growth period was ideal and resulted in the highest amyloid fiber yield. The final supernatant contained the majority of the amyloid protein and had percent recoveries ranging from 41.2% to 63.4%, while the final pellet had recoveries from 6.3%‐18.1%. To further improve the efficiency of this procedure, we are utilizing the detergent Triton X‐100 during the isolation process. Preliminary findings show that this detergent slightly increases the amyloid percent recovery. Isolated proteins will ultimately be purified using preparatory SDS‐PAGE for atomic force microscopy imaging, deaggregated into their monomeric forms using formic acid, and sequenced to gain further information about their structure.
Support or Funding Information
SLU Fellowship Program & Strading Research Fund
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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