Ultrapotent influenza hemagglutinin fusion inhibitors developed through SuFEx-enabled high-throughput medicinal chemistry

Abstract

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC₅₀ cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.

Keywords: Sulfur-Fluoride Exchange; click chemistry; high-throughput screening; influenza hemagglutinin inhibitor; x-ray crystallography.

Fig. 1.

HTMC-based diversification of HA lead inhibitor F0045(S). (A) Reaction schematics of HTMC used in this study, including SuFEx-based and amide-based processes. (B) Diversification of F0045(S) via SuFEx and amide-bond formation with fragment libraries. Molecules were synthesized with the difluoride concentration at 200 µM (compounds 1 and 2), 1,000 µM (compound 3), and amine and carboxylic acid concentration at 300 µM for amide-based diversification. The reaction mixtures were used directly for biochemical activity measurement after overnight incubation with 100-fold dilution into the FP-based biochemical assay. Scatterplots depict % inhibition of all analogs derived from primary (green) and secondary (purple) amine or amide-coupling (orange) fragments. Data points corresponding to compound 4 or an azetidine derivative of compound 1 are circled with blue or green, respectively. (C) Structures of improved molecules 4, 5, and 6(R), their biochemical IC₅₀ values of the resynthesized and purified molecules, and the representative dose–response curves. The IC₅₀ values are derived from manually resynthesized compounds and performed in two independent experiments in triplicate. Representative dose-dependent response curves are shown for improved compounds 4 (blue squares) and 6(R) (pink triangles) in the biochemical FP-based assay compared to F0045(S) (red circles).

Fig. 2.

Structural characterization of improved HA inhibitors from HTMC. (A) Structure and potency of the potent SuFEx-based F0045(S) analog 4 identified through the HTMC campaign. Mean (±SD) values of IC₅₀ as measured by biochemical FP assay are shown. (B) Overview of HA trimer structure (HA1 in cyan, HA2 in green. Compound 4 is colored with magenta carbons, blue nitrogens, yellow sulfur, green chlorine, and cyan fluorine. (C) The HA electrostatic surface potential is depicted with positive potential (≥5 mV) in blue; neutral potential (0 mV) in white; and negative potential (≤−5 mV) in red. (D) The CH⋯O/N/F interactions between 4 (colored as in panel C) and its measured distances to the side-chain hydroxyl of HA1 Ser291 (orange carbons), HA2 Asn53 (light purple carbons), HA2 Ile56 (green carbons) or HA2 Thr49 (cyan carbons) are depicted in yellow. (E-H) Corresponding panels for SuFEx-based F0045(S) analog 6(R) to those shown in A-D for analog 4. Mean (±SD) values of IC₅₀ as measured by biochemical FP assay, or an EC₅₀ value as measured by cellular antiviral assay against H1PR8 strain are shown (E). The carbon position number is labeled in blue. Due to the size similarity between oxygen and fluorine, the absolute configuration of the sulfur chiral center could not be determined based on the cocomplex x-ray structures. The measured distances inherently encompass some uncertainty due to the resolution (2.05 to 3.11 Å) of some of the structures and thus necessitate cautious interpretation.

Fig. 3.

Filling additional pockets and multiple weak hydrogen bonds appear to contribute to the improved affinity of HA inhibitors. X-ray structures of the inhibitor-H1/PR8 complex are shown (colored as in Fig. 2). Potential CH⋯O/N/F interactions are shown in yellow with 4Å cutoff in panel A, and the distance is shown in orange between HA protein and the inhibitors in panel B. The measured distances inherently encompass some uncertainty due to the resolution (2.05 to 3.11 Å) of some of the structures and thus necessitate cautious interpretation.

Fig. 4.

Cellular anti-influenza activity and biochemical affinity of ultrapotent HA inhibitor 7. (A) Summary of the design of compound 7. (B) Concentration–response curve of 7 in a cellular viral neutralization assay using MDCK-SIAT1 cells against a panel of group 1 influenza strains, including A/Puerto Rico/8/1934 (H1/PR8), A/Beijing/262/1995 (H1/Beijing95), A/Marton/1943 (H1/Marton43), A/California/04/2009 (H1/Cal04), and A/USSR/90/1977 (H1/USSR77). No MDCK-SIAT1 cellular toxicity was observed for compound 7 (red squares) in the dose range used (2 nM to 2 µM). EC₅₀ values are derived from manually synthesized and purified compounds and based on two independent assays in triplicate, and mean (±SD) values of EC₅₀ are shown. (C) Steady-state affinity of 7 against HAs as measured by SPR. Biotinylated HA was immobilized on streptavidin chip, and the affinity was calculated based on steady-state analysis. Left panel: H1/PR8. Middle panel: H1/Beijing95. Right panel: H1/Marton43.