Nature Microbiology                          volume  6, pages  1188–1198 (2021 )Cite this article

SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution

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2021-11-25 19:00:14

Nature Microbiology volume  6, pages 1188–1198 (2021 )Cite this article

SARS-CoV-2 variants of interest and concern will continue to emerge for the duration of the COVID-19 pandemic. To map mutations in the receptor-binding domain (RBD) of the spike protein that affect binding to angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, we applied in vitro evolution to affinity-mature the RBD. Multiple rounds of random mutagenic libraries of the RBD were sorted against decreasing concentrations of ACE2, resulting in the selection of higher affinity RBD binders. We found that mutations present in more transmissible viruses (S477N, E484K and N501Y) were preferentially selected in our high-throughput screen. Evolved RBD mutants include prominently the amino acid substitutions found in the RBDs of B.1.620, B.1.1.7 (Alpha), B1.351 (Beta) and P.1 (Gamma) variants. Moreover, the incidence of RBD mutations in the population as presented in the GISAID database (April 2021) is positively correlated with increased binding affinity to ACE2. Further in vitro evolution increased binding by 1,000-fold and identified mutations that may be more infectious if they evolve in the circulating viral population, for example, Q498R is epistatic to N501Y. We show that our high-affinity variant RBD-62 can be used as a drug to inhibit infection with SARS-CoV-2 and variants Alpha, Beta and Gamma in vitro. In a model of SARS-CoV-2 challenge in hamster, RBD-62 significantly reduced clinical disease when administered before or after infection. A 2.9 Å cryo-electron microscopy structure of the high-affinity complex of RBD-62 and ACE2, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

Humans can be infected by SARS-CoV-2 either through inhalation of airborne viral particles or by touching contaminated surfaces. Structural and functional studies have shown that a single RBD of the SARS-CoV-2 homotrimer spike glycoprotein interacts with ACE2, which serves as its receptor1,2. Binding of spike (S) protein to ACE2 and subsequent cleavage by the host protease transmembrane serine protease 2 (TMPRSS2) results in cell and virus membrane fusion and cell entry1. Blocking of the ACE2 receptor by specific antibodies prevents viral entry1,3,4,5. In vitro binding measurements have shown that SARS-CoV-2 S protein binds ACE2 with an affinity of around 10 nM, which is about tenfold tighter than the binding of the SARS-CoV S protein2,4,6. It has been suggested that this is, at least partially, responsible for the higher infectivity of SARS-CoV-27. Recently, three major SARS-CoV2 variants of concern have emerged and mutations in the RBD of the spike proteins of these variants have further strengthened this hypothesis. Deep-mutational scanning of the RBD domain showed that the N501Y mutation in the Alpha variant to enhances binding to ACE27. The Beta variant has three altered residues in the ACE2-binding site (K417N, E484K and N501Y), and has spread extremely rapidly, becoming the dominant lineage in the Eastern Cape and Western Cape Provinces within weeks8. The Gamma variant, with independent K417T, E484K and N501Y mutations, similar to the B.1.351 variant is spreading rapidly from the Amazon region9. Another S mutation associated with increased SARS-CoV-2 infectivity is S477N, which became dominant in many regions10.

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