SuFEx-enabled high-throughput medicinal chemistry for developing potent tamoxifen analogs as Ebola virus entry inhibitors

Abstract

Ebola virus (EBOV) causes severe hemorrhagic fever with a high mortality rate in humans. In acute infection, an abnormal immune response results in excessive inflammatory cytokines and uncontrolled systemic inflammation that can result in organ damage and multi-organ failure. While vaccines and monoclonal antibody therapies are available, there is an urgent need for effective small-molecule antivirals against EBOV. Here, we report on the optimization of tamoxifen, an EBOV-glycoprotein (GP) binder that inhibits viral entry, using our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. Using a "Direct-to-Biology" approach, we generated a focused library of 2,496 tamoxifen analogs overnight and screened them in a cell-based pseudo-EBOV infection assay. The HTMC workflow enabled the development of a potent EBOV entry inhibitor with submicromolar EC₅₀ cellular antiviral activity and more than 50-fold improvement in binding affinity against EBOV-GP compared to the parent compound. Our findings underscore the use of SuFEx-enabled HTMC for rapidly generating and assessing potential therapeutic candidates against viral and immune-mediated diseases in a cell-based assay.

Keywords: Ebola; SuFEx; direct-to-biology; drug discovery; small molecule antiviral drugs.

Figure 1

HTMC-based diversification of EBOV entry lead inhibitor tamoxifen. (A) Schematics of SuFEx-based HTMC workflow. (B) Chemical structure and EC₅₀ of tamoxifen and the difluoride analog 1 against VSV-EBOV-GP (Vero cells). (C) Dose-response curves of VSV-EBOV-GP infection (blue) and Vero cell viability (red) for tamoxifen (●, solid lines) and compound 1 (○, dotted lines). Mean ± SD values are shown (n = 3).

Figure 2

Reaction schematics and screening results of HTMC against EBOV entry. (A) HTMC workflow from a difluoride functionalized lead compound to the improved compounds. Chemical structures and EC₅₀ (VSV-EBOV-GP, Vero cells) of representative hit molecules (compounds 2, 3, and 4) are shown. (B) Scatter plot for the tamoxifen-based HTMC library screening. Screening was performed at a small-molecule concentration of 200 nM. (C) Dose-response curves for representative hits identified from the HTMC library screening. Hit molecules were resynthesized in mg scale, purified, and chemically characterized, and their antiviral potency was measured against VSV-EBOV-GP using Vero cells. Mean ± SD values are shown (n = 3).

Figure 3

Characterization of the top tamoxifen analog 4S identified from the HTMC campaign. (A) The biophysical affinity of tamoxifen, 4R and 4S towards EBOV-GP as measured by MST. Error bars indicate the standard deviation of three technical replicates (n = 3). (B) Dose-response curves of VSV-EBOV-GP infection (○) and Vero cell viability (○) for compound 4S (left panel). Tamoxifen (●) and compound 4S (○) were tested for their ability to inhibit VLPs displaying the VSV-G glycoprotein (middle panel) and LASV-GP (right panel). Mean ± SD values are shown (n = 3).

Figure 4

Structure of EBOV-GP in complex with compound 4R. (A) Crystal structure of EBOV-GP trimer bound to 4R. The compound is depicted as sticks, and the protein as a cartoon representation. (B) 2Fo-Fc electron density map contoured at 1 sigma for 4R. (C) 4R bound within the hydrophobic binding pocket of EBOV-GP. The surface is colored according to hydrophobicity, with pale green indicating the most hydrophobic regions and green the least hydrophobic. (D) Electrostatic surface potential of the binding pocket, colored from red (most negative) to blue (most positive), with 4R bound. The color scale for electrostatics is shown at the bottom of the figure.