ABSTRACT
Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.
Subject(s)
Drug Discovery , Drug Evaluation, Preclinical , Proprotein Convertase 9/metabolism , Proteolysis/drug effects , Serine Proteinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Humans , Ligands , Models, Molecular , Molecular Structure , Serine Proteinase Inhibitors/chemistry , Small Molecule Libraries/chemistryABSTRACT
We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.
