Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor.
PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 μm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 μm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the β-strand and a discontinuous short α-helix, and it engaged in the same β-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.
From the Departments of Early Discovery Biochemistry.
Yingnan
Y
Zhang
Charles
C
Eigenbrot
Lijuan
L
Zhou
Steven
S
Shia
Wei
W
Li
Clifford
C
Quan
Jeffrey
J
Tom
Paul
P
Moran
Paola
P
Di Lello
Nicholas J
NJ
Skelton
Monica
M
Kong-Beltran
Andrew
A
Peterson
Daniel
D
Kirchhofer
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D019151:Peptide Library; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009842:Oligopeptides; D019151:Peptide Library; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
Crystal Structure; LDL Receptor; PCSK9; Peptides; Phage Display; Protease; Protein-Protein Interactions