Inhibiting the interaction between the mitochondrial receptor Tom70 and SARS CoV 2 Orf9b with small molecules

Figures

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  Figure 1:. Overview of Orf9b homodimer crystallization system and fragment screening. A. Model for the Tom70-Orf9b equilibrium. Orf9b homodimer dissociates into Orf9b monomers which undergo a conformational change from β-sheet to α-helix. α-helical Orf9b binds to Tom70. B. Overview of Orf9b homodimer with modeled fragment hits. Orf9b homodimer surface is modeled with fragments as sticks including the number of modeled hits per binding site. Hydrogen bonds are shown as black dashes with a 4Å cutoff. C. Overview of binding site 1 showing residue side chains. Representative binding poses of modeled fragments bound at site 1. PanDDA event maps are shown contoured to 2σ. Hydrogen bonds are shown as black dashes with a 3.5Å cutoff. D. Representative binding poses of fragments identified at sites 3 and 4 corresponding to the homodimer central channel, the lipid molecule colored in green is modeled to provide orientation. PanDDA event maps are shown contoured to 3σ. Hydrogen bonds are shown as black dashes with a 3.5Å cutoff. E. Overview of binding site 2 showing representative fragments with PanDDA event maps contoured at 2σ. Fragment in structure PDB 13TT (ii) is shown with two conformations.

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  Figure 2:. Overview of Orf9b homodimer analog binding and binding behaviors. A. Structurally resolved fragment hit and analog compounds bound at site 1 with FTMap chemical probes superimposed. PDB codes for deposited structures are displayed in the top left corner of each panel. B. (Top) Kinetic traces of Orf9b homodimer binding to Tom70 in the presence of increasing x436 analog concentration. (Bottom left) Diagram of SPR experiment with immobilized Orf9b homodimer and x436 analog compound to measure binding at external dimer interface. (Bottom middle) Surface plasmon resonance sensorgram showing x436 analog compound binding to the Orf9b homodimer immobilized on the surface. (Bottom Right) Modeling SPR sensorgram equilibrium response with a non-linear regression to determine the affinity for the Orf9b homodimer of 17uM. C. (Top) Chai1 predictions of Orf9b homodimer co-folded with one copy of lipidated analog and ipTM scores in the Orf9b homodimer central channel. (Bottom) Chai1 predictions of Orf9b homodimer co-folded with two copies of the lipidated analog and ipTM scores. The original fragment and modeled lipid are highlighted in green with the Chai1 predicted analogs in cyan. D. Kinetic…

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  Figure 3:. High-throughput screen identifies Tom70-binding small molecules that block Orf9b binding. A. Diagram for generating fluorescent peptides derived from Orf9b for high throughput screening against Tom70. B. Overview of high throughput screen and triage process. C. Dose response curves for compounds identified by high throughput screen. D. Chai-1 prediction of Tom70 co-folded with HTS hits (green and blue). Orf9b (magenta) is superimposed with compounds illustrating potential mechanisms for blocking Orf9b binding to Tom70. E. Diagram of surface plasmon resonance experiments to measure binding of Tom70 to immobilized Orf9b peptide in the presence or absence of competitive binders derived from the Orf9b peptide. F. SPR sensorgrams for Tom70 binding to Orf9b peptide immobilized surface in the presence or absence of competitive binders.

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  Figure 4:. Summary of Tom70-binding analog compounds. A. Dose response of select HTS analog compounds in FP format. B. Table of purchased analogs showing the relationship between changes to the benzene group and affinity for Tom70. Compounds that had no measurable effect on FP were reported to have Ki >200uM. C. Chai-1 predictions of Tom70 co-folded with top binding analogs with ipTM scores. Original HTS hits co-folded with Tom70 are shown in blue and green and analog compounds are shown in cyan.