Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.

Hydrogen sulfide (HS) is now recognized as an important gaseous transmitter that is involved in a variety of biological processes. Here, we report the design and synthesis of a luminescent lanthanide biosensor for HS, LP2-Cu(II)-Ln(III), a heterobinuclear metal complex that uses Cu(II) decomplexation to control millisecond-scale-lifetime-Tb(III)- or Eu(III)-emission intensity. LP2-Cu(II)-Ln(III) responded rapidly, selectively, and with high sensitivity to aqueous HS. The probe's potential for biological applications was verified by measuring the HS generated by the slow-releasing chemical-sulfide-donor GYY4147, by cystathionine γ-lyase (CSE), and by NaS-stimulated HeLa cells.

Herein, we report the design, synthesis and characterization of a lanthanideIII complex-based probe for the time-gated luminescence detection of hydrogen sulfide (H2S) in aqueous media. The probe’s unique sensing mechanism relies on the selective reduction of azide to amine by sulfide, followed by intramolecular cyclization to form a quinolinone. The quinolinone is a sensitizer that absorbs near-UV light and transfers excitation energy to coordinated TbIII or EuIII ions to trigger a strong “turn-on” luminescence response with ms-scale lifetimes characteristic of lanthanide complexes. Using this probe, we developed a robust, high throughput screening (HTS) assay for detecting H2S generated by cystathionine γ-lyase (CSE), one of the main producers of H2S in mammalian cells. In a 240-compound screen to identify potential CSE inhibitors, the EuIII analog of the sensor showed a low false positive rate and high Z′-factor (> 0.7).

Lanthanide Probes Hydrogen sulfide (H2S) functions as a signaling second messenger, and inhibition of H2S‐producing enzymes may be a promising therapeutic strategy. The emissive lanthanide probe LLPS‐LnIII generates strong turn‐on luminescence via a unique mechanism. H2S‐mediated reduction of an aryl azide moiety triggers an intramolecular cyclization to form a chromophore that sensitizes TbIII or EuIII luminescence. Time‐gated detection with LLPS‐LnIII enabled high‐throughput screening for inhibition of H2S‐producing cystathionine γ‐lyase. For more details, see the Communication by L. W. Miller et al. on page 752 ff.