Proprotein convertase subtilisin/kexin-type 9 (PCSK9) is a member of the proprotein convertase endoproteinase family. Following its ribosomal synthesis, it undergoes autoproteolytic maturation and produces an enzymatically inactive complex between the mature protein with the cleaved propeptide which traffics to the extracellular space, where it binds to the EGF-A domain of the LDL receptor (LDLR), undergoes endocytosis with it and prevents the LDLR from being recycled by directing it towards lysosomes where it is degraded. By preventing LDLR recycling, PCSK9 decreases LDLR cell surface density which leads to an increase in LDL blood levels. Thus, PCSK9 became a prominent and novel target for the development of new treatments of hypercholesterolemia. Following the FDA approval (2015) of monoclonal antibodies that block PCSK9 from interacting with LDLR, it was suggested to selectively inhibit PCSK9 translation by directly binding small molecules to the ribosome RNA in the ribosome exit channel, inducing conformational change which inhibits the elongation of PCSK9 and stalling it in the exit channel. That mechanism was recently demonstrated by several structural studies and deposited in the Protein Data Bank (PDB).We picked from literature 59 molecules that act by the ribosomal stalling mechanism and diluted them by 59,000 molecules picked randomly on the basis of major similarities to these actives ("applicability domain"). We docked that set to six PDB structures in order to identify the one that distinguishes best between the actives and the expected inactives. That structure, 6OLE, was used to dock (on two docking levels) 3 million molecules from which only 17 could best fit strict geometry requirement together with good binding energy. We could get 9 out of these 17 candidates, and those were sent for in vitro testing. Five compounds were rejected due to their negative effects by 100mM in a 24-hour incubation with Huh7 cells. Of the remaining four, one compound (Z-023) reduced PCSK9 level in 24-h spent medium by nearly 60% (p < 0.01) and significantly reduced secreted PCSK9 by 42% and 64% at 100 μM and 128 μM, respectively. However, over a 2-h period of time. Z-023 affected 50% inhibition (IC 50 = 33.7 μM) and 90% inhibition (IC 90 = 116.4 μM). A half maximum viability (CC 50 ) of 160.2 μM was derived from an MTS assay, with a calculated Selectivity Index CC 50 /IC 50 = 4.8. Two other non-toxic compounds, Z-665 and Z-855, could be efficacious anti-PCSK9 anti-secretagogues as measured by cell confluence and mortality with low concentrations of 25μM, and the efficacy of these two compounds at 10 μM was evaluated using the “24 h incubation-2 h secretion” protocol. The PCSK9 level in spent media was reduced by 66.1% with -665 (p = 0.0022) and 50.6% with Z-855 (p = 0.011).
- PCSK9 blockers
- Ribosomal Synthesis inhibition
- Iterative Stochastic Elimination Algorithm
- Ligand-based modeling