From Noncovalent Fragment to (Non)covalent Leads Against PLPro

Key Takeaways

• NMR screen identified 77 fragment hits from 13,824 tested
• Non‑covalent series reached sub‑micromolar cellular potency
• Covalent analogs showed higher in‑vitro potency but weaker cell activity
• Hydrogen‑bond‑rich linker essential for covalent binding yet reduces permeability
• No ADME or PK data reported for any PLPro leads

Pulse Analysis

The papain‑like protease (PLPro) has emerged as a high‑value yet underexplored target in the SARS‑CoV‑2 drug landscape. Unlike the well‑characterized main protease, PLPro processes viral polyproteins and antagonizes host immune signaling, making it indispensable for viral replication. Its narrow substrate channel and solvent‑exposed subsites pose structural challenges, which is why early inhibitor programs stalled after the 2008 SARS effort. By employing protein‑observed NMR, the Fesik group rapidly mapped fragment binding hotspots, uncovering two distinct pockets that guided subsequent medicinal chemistry.

Building on the initial fragment hits, the team pursued a rule‑of‑three compliant scaffold that occupied the S4 pocket. Incremental modifications—adding methyl groups to improve hydrophobic contacts and introducing a basic nitrogen to engage a glutamic acid side chain—propelled potency from high‑micromolar to sub‑micromolar levels in cellular assays. Crystallographic data confirmed that these interactions lock the inhibitor in place, demonstrating how precise pocket exploitation can translate into meaningful antiviral activity without sacrificing drug‑like properties.

The covalent arm of the program sought to capitalize on irreversible binding, a strategy gaining traction for difficult enzymes. Warhead‑linked analogs displayed impressive enzymatic IC₅₀ values, yet the required diacetylhydrazine linker introduced multiple hydrogen‑bond donors that hampered cell permeability and overall efficacy. This paradox underscores a broader lesson: adding electrophilic warheads is not a guaranteed shortcut to potency, especially when the target’s architecture imposes strict steric and electronic constraints. Future efforts may benefit from screening dedicated covalent fragment libraries or exploring alternative, less polar linkers to balance reactivity with pharmacokinetic viability.