Fig. 6: Development of lead stapled lipopeptide inhibitors of RSV, Ebola, and Nipah viruses.

a–c Structures of the conserved 6-HB assemblies of RSV (a), Ebola (b), and Nipah (c) viruses (PDB IDs 1G2C, 1EBO, and 1WP7, respectively), with the HR2 domain that formed the basis for stapled lipopeptide inhibitor (SLI) designs colored in red. d–f Staple scanning panels of RSV (d), Ebola (e), and Nipah (f) viruses were tested in the corresponding in vitro infectivity assays against RSV (A549 cells) and Ebola (HeLa cells) live viruses and Nipah (293T cells) pseudovirus at screening doses of 2, 10, or 5 μM, respectively. Data are mean ± SEM for assays performed in at least technical triplicate (RSV, n = 8; Ebola, n = 3; Nipah, n = 4), and then repeated with similar results. The gray shading highlights those lipopeptides that inhibited infectivity to <25% of infected cells (data points colored red). g–i Further optimization, including iteration of PEG-lipid linker length, yielded lead SLIs with low nanomolar activity against RSV (A549 cells) and Ebola (HeLa cells) live viruses and Nipah (293T cells) pseudovirus, as assessed by in vitro infectivity assays. Data are mean ± SEM for assays performed in at least technical triplicate (RSV, n = 4; Ebola, n = 3; Nipah, n = 10) and then repeated with similar results. IC₅₀ values were calculated by nonlinear regression analysis of the dose-response curves. j A workflow for the development of SLIs for highly pathogenic viruses, incorporating staple scanning of HR2 domain sequences, iterative optimization of PEG-lipid composition, biophysical analyses, functional testing in in vitro pseudovirus and live-virus assays, PK analyses in respiratory tissues and blood, and in vivo efficacy testing in animal models of infection and transmission.