The PipX Protein, When Not Bound to Its Targets, Has Its Signaling C‑Terminal Helix in a Flexed Conformation

PipX, an 89-residue protein, acts as a coactivator of the global nitrogen regulator NtcA in cyanobacteria. NtcA–PipX interactions are regulated by 2-oxoglutarate (2-OG), an inverse indicator of the ammonia abundance, and by P_II, a protein that binds to PipX at low 2-OG concentrations. The structure of PipX, when bound to NtcA or P_II, consists of an N-terminal, five-stranded β-sheet (conforming a Tudor-like domain), and two long α-helices. These helices adopt either a <i>flexed conformation</i>, where they are in close contact and in an antiparallel mutual orientation, also packing against the β-sheet, or an <i>open conformation</i> (observed only in the P_II–PipX complex) where the last α-helix moves apart from the rest of the protein. The aim of this work was to study the structure and dynamics of isolated PipX in solution by NMR. The backbone chemical shifts, the hydrogen-exchange, and the NOE patterns indicated that the isolated, monomeric PipX structure was formed by an N-terminal five-stranded β-sheet and two C-terminal α-helices. Furthermore, the observed NOEs between the two helices, and of α-helix2 with β-strand2 suggested that PipX adopted a <i>flexed conformation</i>. The β-strands 1 and 5 were highly flexible, as shown by the lack of interstrand backbone–backbone NOEs; in addition, the ¹⁵N-dynamics indicated that the C terminus of β-strand4 and the following β-turn (Phe42-Thr47), and the C-cap of α-helix1 (Arg70-Asn71) were particularly mobile. These two regions could act as hinges, allowing PipX to interact with its partners, including PlmA in the newly recognized P_II–PipX–PlmA ternary complex

C‑2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV‑2 through Interaction with the Viral Spike (S) Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19

C‑2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV‑2 through Interaction with the Viral Spike (S) Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19