Small Molecule Inhibitors of the PCSK9·LDLR Interaction

The protein–protein interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) is a relatively new, and extremely important, validated therapeutic target for treatment and prevention of heart disease. Experts in the area agree that the first small molecules to disrupt PCSK9·LDLR would represent a milestone in this field, yet few credible leads have been reported. This paper describes how side-chain orientations in preferred conformations of carefully designed chemotypes were compared with LDLR side chains at the PCSK9·LDLR interface to find molecules that would mimic interface regions of LDLR. This approach is an example of the procedure called EKO (Exploring Key Orientations). The guiding hypothesis on which EKO is based is that good matches indicate the chemotypes bearing the same side chains as the protein at the sites of overlay have the potential to disrupt the parent protein–protein interaction. In the event, the EKO procedure and one round of combinatorial fragment-based virtual docking led to the discovery of seven compounds that bound PCSK9 (SPR and ELISA) and had a favorable outcome in a cellular assay (hepatocyte uptake of fluorescently labeled low-density lipoprotein particles) and increased the expression LDLR on hepatocytes in culture. Three promising hit compounds in this series had dissociation constants for PCSK9 binding in the 20–40 μM range, and one of these was modified with a photoaffinity label and shown to form a covalent conjugate with PCSK9 on photolysis

BBK32 _(21–205) induces a conformational change in plasma Fn.

<p>(a) Purified plasma Fn and Fn aggregates induced by anastellin or BBK32 _(21–205) were digested with thermolysin. The digested products were fractionated by SDS-PAGE and peptides that were only found in BBK32 _(21–205) /Fn (lane 3) or anastellin/Fn (lane 2) digests and not present among the peptides obtained from purified plasma Fn (lane 1) were excised and identified by N-terminal sequencing. The sequences obtained, SSPVVID, AVEENQE, and ITETIP, were located to the end of ¹³F3, middle of ³F3, and the start of ¹F3, respectively for both BBK32/Fn and anastellin/Fn. Pre-stained protein standards with indicated M_w are shown. (b) Schematic of Fn. Stars denote cryptic cleavage sites in Fn after aggregation due to both BBK32 and anastellin.</p

Forest plot of the expression of HIF-1α in borderline versus that in nomal tissue. (<i>I</i>² = 57%).

<p>Forest plot of the expression of HIF-1α in borderline versus that in nomal tissue. (<i>I</i>² = 57%).</p

BBK32 _(153–175) aggregates plasma Fn.

<p>(a) BBK32 _(21–205), BBK32 _(146–166), BBK32 _(153–175), and BBK32 _(175–202) were titrated into 1 µM purified plasma Fn and the optical density at 600 nm was measured. (b) BBK32 _(153–175) is the minimal amino acid sequence required to affect Fn matrices assembled by NHDFs. Cells were allowed to grow for 48 hours and then were incubated with 5 µM BBK32 _(153–166), BBK32 _(146–166), BBK32 _(153–166), and BBK32 _(175–202). Treated cells were incubated for 20 hours and probed with Alexa Fluor 488-anti-Fn and monoclonal antibody IST 9. Images were taken using LSM 510 Confocal Microscope, objective 63X/1.4 oil. Bar = 10 µm. Staining with monoclonal antibody IST 9 indicates that only the BBK32 peptide containing residues 153–175 changes the conformation of Fn matrices. (c) BBK32 _(153–175) is the minimal amino acid sequence required to inhibit HUVEC proliferation. Cells were seeded onto tissue culture plates in full media and allowed to adhere for two hours. After two hours, BBK32 _(21–205), BBK32 _(153–175), BBK32 _(175–202), and anastellin were added in increasing concentrations (in triplicate) to the wells. Cells were then incubated for 72 hours at 37°C, 5% CO₂. After 72 hours, cells were washed, trypsinized, and counted (three counts/well, three wells/dose) using a hemacytometer.</p

BBK32 _(21–205) inhibits the proliferation of HUVECs.

<p>Cells were seeded onto tissue culture plates in full media and allowed to adhere for two hours. After two hours, BBK32, anastellin, and FnbpA peptide D3 were added in increasing concentrations (in triplicate) to the wells. Cells were then incubated for 72 hours at 37°C, 5% CO₂. After 72 hours, cells were washed, trypsinized, and counted (three counts/well, three wells/dose) using a hemacytometer.</p

BBK32 _(21–205) effects on Fn matrix assembled by NHDFs.

<p>Cells were incubated for 48 hours and then incubated with 5 µM BBK32 _(21–205), 5 µM anastellin, or 5 µM FnbpA peptide D3. Treated cells were incubated for 20 hours and probed with Alexa Fluor 488-anti-Fn and monoclonal antibody IST 9. Images were taken using LSM 510 Confocal Microscope, objective 63X/1.4 oil. Bar = 10 µm.</p

Den svenska regleringen av hatmotiverade brott : i linje med internationella normer?

<p>Forest plot of the expression of HIF-1α in Grade 2 tissue versus that in Grade 1 tissue. (<i>I</i>² = 6%).</p