ABSTRACT
Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.
Subject(s)
Bacterial Proteins , Bacterial Toxins , Hemolysin Proteins , Hemolysis , Streptococcus pneumoniae , Streptolysins , Streptolysins/metabolism , Streptolysins/chemistry , Humans , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/antagonists & inhibitors , Streptococcus pneumoniae/drug effects , Bacterial Toxins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/antagonists & inhibitors , Hemolysis/drug effects , Hemolysin Proteins/metabolism , Hemolysin Proteins/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , A549 Cells , Cholesterol/metabolism , Cryoelectron Microscopy , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Virulence Factors/metabolismABSTRACT
SARS coronavirus main proteases (3CL proteases) have been validated as pharmacological targets for the treatment of coronavirus infections. Current inhibitors of SARS main protease, including the clinically admitted drug nirmatrelvir are peptidomimetics with the downsides of this class of drugs including limited oral bioavailability, cellular permeability, and rapid metabolic degradation. Here, we investigate covalent fragment inhibitors of SARS Mpro as potential alternatives to peptidomimetic inhibitors in use today. Starting from inhibitors acylating the enzyme's active site, a set of reactive fragments was synthesized, and the inhibitory potency was correlated with the chemical stability of the inhibitors and the kinetic stability of the covalent enzyme-inhibitor complex. We found that all tested acylating carboxylates, several of them published prominently, were hydrolyzed in assay buffer and the inhibitory acyl-enzyme complexes were rapidly degraded leading to the irreversible inactivation of these drugs. Acylating carbonates were found to be more stable than acylating carboxylates, however, were inactive in infected cells. Finally, reversibly covalent fragments were investigated as chemically stable SARS CoV-2 inhibitors. Best was a pyridine-aldehyde fragment with an IC50 of 1.8â µM at a molecular weight of 211â g/mol, showing that pyridine fragments indeed are able to block the active site of SARS-CoV-2 main protease.