Peptides derived from human PCSK9 catalytic domain and uses thereof for promoting LDL-R activity

Abstract

The present invention provides compositions comprising an isolated or purified therapeutically effective hPCSK9 polypeptide derived from the hPCSK9 catalytic domain, and their use in methods of treating hypercholesterolemia.

Claims

1. The isolated or purified therapeutically effective hPCSK9 polypeptide, consisting of the amino acid sequence of any one of SEQ ID NOS: 42 to 47, and SEQ ID NO: 56.

2. The isolated or purified therapeutically effective hPCSK9 polypeptide consisting of the amino acid sequence of any one of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 56 or combinations thereof.

3. The isolated or purified therapeutically effective hPCSK9 polypeptides of claim 2, consisting of the combination of the amino acid sequence SEQ ID NO: 36 and SEQ ID NO: 42 or SEQ ID NO: 36 and SEQ ID NO: 47.

4. An isolated or purified therapeutically effective hPCSK9 polypeptide consisting of the amino acid sequence of SEQ ID NO: 56: ##STR00010##

5. A pharmaceutical composition comprising a therapeutically effective amount of an isolated or purified therapeutically effective hPCSK9 polypeptide of claim 2, and a pharmaceutically acceptable carrier.

6. A method of preventing or treating hypercholesterolemia comprising administering to a subject in need thereof at least one of an isolated or purified therapeutically effective hPCSK9 polypeptide of claim 2, or combinations thereof.

7. The method of claim 6, further comprising administering a HMG-CoA reductase inhibitors (statin).

8. A kit for use for the prevention or the treatment of hypercholesterolemia in a subject in need thereof, the kit comprising: an isolated or purified therapeutically effective hPCSK9 polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs 42 to 47, and SEQ ID NO:56 and instructions on how to use the kit.

9. The kit of claim 8, wherein the isolated or purified therapeutically effective hPCSK9 polypeptide is consisting of the amino acid sequence of any one of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 56 or combinations thereof.

10. The kit of claim 9, wherein the isolated or purified therapeutically effective hPCSK9 polypeptides is consisting of the combination of the amino acid sequence SEQ ID NO: 36 and SEQ ID NO: 42 or SEQ ID NO: 36 and SEQ ID NO: 47.

11. The kit of claim 8, comprising an isolated or purified therapeutically effective hPCSK9 polypeptide consisting the amino acid sequence: ##STR00011##

12. The kit of claim 8, further comprising a HMG-CoA reductase inhibitors (statin).

13. A pharmaceutical composition comprising a therapeutically effective amount of an isolated or purified therapeutically effective hPCSK9 polypeptide of claim 4, and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

(2) FIG. 1 illustrates a schematic diagram showing the various characteristic domains of human (h) PCSK9 enzyme-cum-protein including its crucial catalytic domain. Upper panel: It shows the complete domain of h-PCSK9; Lower domain: Expanded catalytic domain of hPCSK9. The key catalytic triad residues D.sup.186, H.sup.226 and S.sup.386 along with the oxy-anion N.sup.317 residues are shown in bold characters and their locations indicated by thick vertical lines. The three known disulphide bonds within the catalytic domain as revealed by its crystal structure are indicated by dotted lines. The lone unpaired Cys.sup.301 residue is also indicated in the figure. Various gain of function (top side) and the loss of function (bottom side) mutations and their positions are depicted in the figure by regular vertical lines. The abbreviations are as follows: SP=Signal Peptide Domain; PD=Propeptide Domain, CD=Catalytic domain, CHRD=Cysteine-Histidine Rich Domain. The signal peptide and pro-peptide cleavage sites are indicated by scissors.

(3) FIG. 2 illustrates a schematic diagram showing the various proposed steps involved in the biosynthesis of functionally active mature hPCSK9 and its subsequent binding with hLDL-R leading to latter's degradation. The figure provides a rational mechanism and pathway for interaction of PCSK9 with LDL-R leading to re-routing of the latter from endosomal to lysosomal pathway for ultimate degradation. The figure demonstrates the initial formation of a strong complex via binding of cleaved prodomain segment (aa31-152) of hPCSK9 with the catalytic segment (shown in thick dark color line; aa153-421) of mature PCSK9 (aa153-692). Subsequently additional binding of this prodomain bound PCSK9 complex with the Epidermal Growth Factor-A (EGF-A) domain (aa314-355) of hLDL-R leading to latter degradation in the lysosome is also depicted in the figure. This binding interaction is most likely mediated via yet unknown region of catalytic domain of PCSK9 as revealed by various studies. (aa=Amino acid).

(4) FIG. 3 illustrates a chemical scheme for the synthesis of Fluorescein (FI) (Ia) and Biotin (Bio) (Ib) labeled EGF-A peptide (residue aa314-355 of hLDL-R). The dotted lines indicate the SS bond connectivity of 6 Cys residues present in the sequence. In all there are three disulphide bonds as indicated. The various reagents and coupling agents used are shown in each step. Three pairs of Cys side chain protecting groups namely Mmt, Trt and ACM were used in the synthesis in order to ensure the correct SS bond formations as present endogenously in the EGF-A domain of hLDL-R protein. The first SS bond formation was achieved while the protected peptide is still bound on the resin by deprotecting the Mmt group under mild condition. All side chain protecting groups except the two Cys-ACM groups were then fully deprotected by two 3 hour treatments with Reagent K (82.5% TFA+5% Phenol+5% Water+5% Thioanisole+2.5% EDT). This is then further cyclized by treatment with DMSO (NN Dimethyl sulphoxide) for 3 hours. The double SS bridges containing peptide thus obtained was then lyophilized and two protecting ACM groups were then removed by treatment with iodine in methyl alcohol (MeOH) which also led to the formation of third SS bond as indicated in the figure. These led to the formation of FI-EGF-A which was labeled either with 5-Carboxy Fluorescein or Biotin.

(5) FIG. 4 illustrates a scheme showing various steps involved in the chemical synthesis of FI-Bio-Ahx-Lys-Methyl ester (III)model compound used for fluorescence study. The synthesis was accomplished by first coupling Fmoc-Lys-methyl ester (IIIa) with 5-Carboxy Fluorescein N-Hydroxy succinimide followed by deprotection of Fmoc group with 20% Piperidene/DMF. The product thus obtained was then coupled with Biotin-Ahx NHSu. Ahx=Amino hexanoic acid (serves as a linker) for the biotin derivative.

(6) FIG. 5 illustrates SELDI-Tof mass spectrum of purified FI-EGF-A peptide. (Ia): Mass spectrum using Low (A, B) and High current (C) laser method conditions. The mass spectra were calibrated against standard h-Insulin added as internal standard (MW 5,807 Da) and conducted using sinapic acid (SPA) as matrix. Calculated MW of FI-EGF-A=4,962.45 Da (Dalton).

(7) FIG. 6A illustrates the proposed mechanism for the cleavage of FI-EGF-A peptide during laser-based mass spectrometry. It shows the possible mechanism for the breakdown of FI-EGF-A peptide under high laser power condition during SELDI-tof mass spectrometry. All atoms are shown in bold characters whereas the amino acids (single letter code) are indicated in regular character with Cys residue underlined.

(8) FIG. 6B illustrates a theoretical 3D model structure of FI-EGF-A peptide. It shows theoretical 3D energy minimised model structure of EGF-A peptide in vacuo based on Hyperchem software v11 program. The three SS bridges present are indicated by arrows. The sequence is derived from hLDL-R (aa314-355).

(9) FIG. 7A illustrates fluorescence scan spectroscopy of FI-Bio-Ahx-Lys-Methyl ester (IIId) in presence of avidin. The figure shows overlaid emission fluorescence spectra of FI-Bio-Ahx-Lys-Methyl ester (IIId) in absence and presence of varying concentrations of avidin protein at .sub.ex=520 nm.

(10) FIG. 7B illustrates fluorescence scan spectroscopy of FI-Bio-Ahx-Lys-Methyl ester (IIId) in presence of insulin as control peptide. The figure shows overlaid emission fluorescence spectra of FI-Bio-Ahx-Lys-Methyl ester (IIId) in absence and presence of varying concentrations of standard h-insulin externally added as control, .sub.ex=520 nm. For each experiment 5 l of 0.5 mM (IIId) peptide in water was used.

(11) FIG. 8 illustrates the relative binding affinities of 51 PCSK9 peptides towards FI-EGF-A peptide using fluorescence quench method. The figure shows binding results of all 51 synthetic PCSK9 catalytic peptides towards FI-EGF-A peptide based on fluorescence quenching. The fluorescence intensity of FI-EGF-A peptide was measured following incubation for one hour with each peptide at .sub.ex=492 nm, .sub.em=520 nm. The values were compared with that of control (C) in the absence of any peptide. The down vertical arrows indicated strong fluorescence quenching effects by peptides no P2-P5, P8, P16, P17, P22, P25, P26, P35, P37, P45, P48 and P49. The dotted horizontal line in bold character indicates the control fluorescence intensity when there is no peptide added. As before for each experiment 5 l of 0.5 mM FI-EGF-A peptide in water was incubated with 10 l, 0.5 mM hPCSK9 peptide solution for 1 h. For control the peptide was substituted by 10 l water.

(12) FIG. 9A illustrates the SELDI-TOF Mass Spectra of FI-EGF-A (Ib) following incubation with various PCSK9 catalytic peptides. The figure shows the mass spectrum of FI-EGF-A peptide (Ib) following incubation with selected PCSK9 catalytic peptides. Note the formation of a 1:1 complex between FI-EGF-A (Ib) (MW-4,962) and P18-P21 peptides leading to peaks in the mass spectrum at m/z 6,400-6,600 Da as indicated by thick vertical arrows. The thin arrows corresponded to the formation of 1:2 adduct formation (peaks ranging from 7,800-8,200 Da; shown underlined for calculated value). The calculated MW values of individual PCSK9 catalytic peptides are shown within a box while their expanded MS profiles in selected cases were shown within the inserts in the MS figure.

(13) FIG. 9B illustrates the SELDI-TOF Mass Spectra of FI-EGF-A (Ib) upon incubation with selected hPCSK9 catalytic peptides. Again 1:1 complex formation whenever observed was indicated by thick arrows while 1:2 complex formation was shown with thin arrows. The molecular weight of each peptide was shown within a box in the MS figure.

(14) FIG. 9C illustrates the SELDI-TOF Mass Spectra of control FI-EGF-A (Ib) (in triplicates) with no added peptide as well as in presence of selected PCSK9 catalytic peptides. As expected the mass spectrum of control FI-EGF-A peptide showed a broad single peak at 4,962 Da and no other peaks in the area 6,000-8,000 Da where the peaks for the adducts should normally appear.

(15) FIG. 10 illustrates native Gel Electrophoresis of FI-EGF-A (Ib) peptide showing adduct formation with selected PCSK9 catalytic peptides. The figure shows the native gradient gel electrophoresis of FI-EGF-A (Ib) peptide using 4-10-16% Tris/Tricine demonstrating the formation of 1:1 complex or adduct between FI-EGF-A (Ib) and selected PCSK9 catalytic peptides as highlighted by circles in the figure.

(16) FIG. 11 illustrates SDS-Gel electrophoresis of FI-EGF-A peptide (Ib). Various amounts of FL-EGF-A (Ib) were resolved in a 15% SDS-PAGE and monitored by fluorescence intensity measurement (Left) and Coomassie staining (Right). Appropriate marker proteins (fluorescent and non-fluorescent) were also loaded on the gels for molecular weight determination purpose.

(17) FIG. 12 illustrates SDS-Gel electrophoresis of FI-EGF-A peptide in presence and absence of recombinant hPCSK9 protein. 5 g of FI-EGF-A (Ib) was incubated with 1 g rec-hPCSK9 wild type (WT) for 2 h at room temperature. The samples were resolved in 10% SDS PAGE. Left: Fluorescence detection; Right: Western blot using anti-FLAG as primary antibody. There is a fluorescence positive band around 65 kDa (a mobility shift by 5 kDa) shown by thick arrow indicating complex formation between 60 kDa rec-hPCSK9 mature form with 5 kDa FI-EGF-A (Ib) (Left panel). The right panel shows the western blot profile of rec-PCSK9 in presence and absence of FI-EGF-A peptide, Note the presence of two bands at 74 (minor) and 60 kDa (major) for proPCSK9 and mature PCSK9 proteins respectively. It is also noticed that after incubation with FI-EGF-A peptide, the 74 kDa PCSK9 protein is not recognized by anti-FLAG antibody while the 60 kDa hPCSK9 protein is still recognized but with reduced potency.

(18) FIG. 13 illustrates (A) Western blot analyses showing the quantitation of LDL-R and PCSK9 levels under various conditions in HepG2 cells. These are western blot figures showing the effect of externally added FI-EGF-A peptide on exogenous levels of LDL-R and PCSK9 in growing HepG2 cells. The cells were harvested after 24 h and whole cell lysates were used for analysis of LDL-R, PCSK9 and actin levels both for control sample (DMSO) and FI-EGF-A done in duplicates. Actin was used as a house keeping control protein for quantitation purpose. (B): Rescue effect of FI-EGF-A peptide added in increasing concentration levels on LDL-R in growing HuH7 cells in the presence of added rec-PCSK9 protein. Rec-PCSK9 was incubated with various amounts of FI-EGF-A for 3 h at room temp and then applied to cells growing in 2 mL media to the concentrations as indicated in the figure. The cells were harvested 7 h later.

(19) FIG. 14 illustrates Western blots showing PCSK9 and LDL-R levels in the presence of various PCSK9 catalytic peptides in HepG2 cell. The figure shows the effect of various PCSK9 catalytic peptides on LDL-R level when added individually at 5.5 M concentration to the culture media of growing HepG2 cells. (A): P33-P43; (B): P44-P51 & (C): P36, P37 alone or in mixture with P42, P47 peptide respectively

(20) FIG. 15 illustrates (A): List of PCSK9 peptides with FI-EGF-A binding and/or LDL-R promoting activities based on various methods. FL=Fluorescence quench, MS=Mass spectrometry; CC=Cell culture; NG=Native gel. Two most potent gain of function mutations D.sup.374/Y and R.sup.357/H are shown with circles. (B): Location of above peptides within dotted boxes. SS bridges with solid lines. Catalytic residues D, H & S are in bold underlined character.

(21) FIG. 16 illustrates the effects of PCSK9 catalytic cyclic Loop-3 Peptide (CP3) and its acyclic Ala-mutant on LDL-R in HepG2 cells. The figure shows western blot analyses for endogenous LDL-R and the control the house keeping protein TR (Transferrin receptor) in cell lysates following treatment with peptides at two different concentrations as indicated. Bar graphs show the relative amounts of LDL-R with respect to TR.

(22) FIG. 17 illustrates the Energy minimized 3D model structure of CP3 peptide derived from hPCSK9 catalytic domain. This theoretically derived structure for disulphide bridged cyclic peptide CP3 [hPCSK9.sup.365-384: .sup.365GEDIIGASSDCSTCFVSQSG.sup.384) was generated in vacuo by using algorithms based on Hyperchem v11 software program. An expanded figure covering the central region (aa372-380) is also shown in the figure at the bottom section. The two key Cys residues implicated in SS bridges forming a cyclic structure are shown in the figure.

(23) FIG. 18 illustrates Raw fluorescence unit (RFU) values of FI-Bio-Ahx-Lys-Me-ester (IIId) measured at fixed .sub.ex=520 nm and .sub.em=492 nm (Duplicates) in the absence and presence of increasing concentrations of avidin protein as follows: Avidin:(IIId)=0.7:1, 0.9:1, 1.3:1, 3:1 and 5:1.

DETAILED DESCRIPTION

(24) In embodiments there is disclosed isolated or purified therapeutically effective hPCSK9 polypeptides derived from the amino acid sequence

(25) TABLE-US-00001 SEQIDNO:54) (CLYSPASAPEVITVGATNAQDQPVTGTLG1TNFGR) or SEQIDNO:55 (IIGASSDCSTCFVSQSGTSQAAAHV), or SEQIDNO:58 (CVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAA).

(26) In embodiments, the isolated or purified therapeutically effective hPCSK9 polypeptide of the present invention may consist of the amino acid sequence of any one of SEQ ID NOS: 35 (CLYSPASAPEVITVG), 36 (ASAPEVITVGATNAQ), 37 (VITVGATNAQDQPVT), 38 (ATNAQDQPVTLGTLG) (DQPVTLGTLGTNFGR), 42 (CVDLFAPGEDIIGAS). 43 (APGEDIIGASSDCST), 44 (IIGASSDCSTCFVSQ), 45 (SDCSTCFVSQSGTSQ), 46 (CFVSQSGTSQAAAHV), 47 (SGTSQAAAHVAGIAA) and 56 (GEDIIGASSDCSTCFVSQSG).

(27) According to an embodiment, the isolated or purified therapeutically effective hPCSK9 polypeptide may be a disulphide bridged cyclic peptide. According to another embodiment, the isolated or purified therapeutically effective hPCSK9 polypeptide consists of the amino acid sequence SEQ ID NO:56 and may be a disulphide bridged cyclic peptide. For example, the isolated or purified therapeutically effective hPCSK9 polypeptide may have the amino acid sequence:

(28) ##STR00005##

(29) According to another embodiment, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an isolated or purified therapeutically effective hPCSK9 polypeptide of the present invention, or combinations thereof, and a pharmaceutically acceptable carrier.

(30) According to yet another embodiment, there is provided a method of preventing or treating hypercholesterolemia, and/or presumably preventing or treating associated cardiovascular diseases risks, by administering to a subject in need thereof at least one of an isolated or purified therapeutically effective hPCSK9 polypeptide of the present invention, or a pharmaceutical composition of the present invention. In embodiments, the method may further comprise administering a HMG-CoA reductase inhibitors (statin). The HMG-CoA reductase inhibitors (statin) may be for administration before, at the same time or after said hPCSK9 polypeptide. HMG-CoA reductase inhibitors (statin) include but are not limited to Pravastatin, Fluvastatin, Atorvastatin, Pravastatin, Lovastatin, Cerivastatin, Mevastatin, Pitavastatin, Rosuvastatin, Simvastatin.

(31) In another embodiment, there is provided an isolated or purified therapeutically effective hPCSK9 polypeptide derived from one of the amino acid sequence SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 58 for preventing or treating hypercholesterolemia. The isolated or purified therapeutically effective hPCSK9 polypeptide may be consisting of the amino acid sequence of any one of SEQ ID NOS: 35 to 39, SEQ ID NOS: 42 to 47, and SEQ ID NO: 56. The isolated or purified therapeutically effective hPCSK9 polypeptide may be consisting of the amino acid sequence of any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 56 or combinations thereof. The isolated or purified therapeutically effective hPCSK9 polypeptide may be consisting of the combination of the amino acid sequence SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 36 and SEQ ID NO: 42 or SEQ ID NO: 36 and SEQ ID NO: 47.

(32) According to another embodiment, the isolated or purified therapeutically effective hPCSK9 polypeptide having the amino acid sequence:

(33) ##STR00006##
for preventing or treating hypercholesterolemia in a subject in need thereof.

(34) The isolated or purified therapeutically effective hPCSK9 polypeptide of of the present invention may be for use with a HMG-CoA reductase inhibitors (statin). The HMG-CoA reductase inhibitors (statin) is for use before, at the same time or after said hPCSK9 polypeptide. HMG-CoA reductase inhibitors (statin) include but are not limited to Pravastatin, Fluvastatin, Atorvastatin, Pravastatin, Lovastatin, Cerivastatin, Mevastatin, Pitavastatin, Rosuvastatin, Simvastatin.

(35) According to yet another embodiment, there is provided a pharmaceutical composition for use in preventing or treating hypercholesterolemia, comprising a therapeutically effective amount of an isolated or purified therapeutically effective hPCSK9 polypeptide of the present invention, or combinations thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 17, further comprising a HMG-CoA reductase inhibitors (statin). The HMG-CoA reductase inhibitors (statin) may be for use before, at the same time or after said hPCSK9 polypeptide. HMG-CoA reductase inhibitors (statin) include but are not limited to Pravastatin, Fluvastatin, Atorvastatin, Pravastatin, Lovastatin, Cerivastatin, Mevastatin, Pitavastatin, Rosuvastatin, Simvastatin.

(36) According to yet another embodiment, there is provided a use of an isolated or purified therapeutically effective hPCSK9 polypeptide derived from one of the amino acid sequence SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 58 for preventing or treating hypercholesterolemia. The isolated or purified therapeutically effective hPCSK9 polypeptide is consisting of the amino acid sequence of any one of SEQ ID NOS: 35 to 39, SEQ ID NOS: 42 to 47, and SEQ ID NO: 56. The isolated or purified therapeutically effective hPCSK9 polypeptide is consisting of the amino acid sequence of any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 56 or combinations thereof. The isolated or purified therapeutically effective hPCSK9 polypeptide is consisting of the combination of the amino acid sequence SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 36 and SEQ ID NO: 42 or SEQ ID NO: 36 and SEQ ID NO: 47. The isolated or purified therapeutically effective hPCSK9 polypeptide having the amino acid sequence:

(37) ##STR00007##
for preventing or treating hypercholesterolemia.

(38) The use may further comprise a HMG-CoA reductase inhibitors (statin). The HMG-CoA reductase inhibitors (statin) may be for use before, at the same time or after said hPCSK9 polypeptide. The HMG-CoA reductase inhibitors (statin) may be for use before, at the same time or after said hPCSK9 polypeptide. HMG-CoA reductase inhibitors (statin) include but are not limited to Pravastatin, Fluvastatin, Atorvastatin, Pravastatin, Lovastatin, Cerivastatin, Mevastatin, Pitavastatin, Rosuvastatin, Simvastatin.

(39) According to yet another embodiment, there is provided a kit for use for the prevention or the treatment of hypercholesterolemia in a subject in need thereof, the kit comprising: an isolated or purified therapeutically effective hPCSK9 polypeptide derived from one of the amino acid sequence SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 58; and instructions on how to use the kit.

(40) The the isolated or purified therapeutically effective hPCSK9 polypeptide is consisting of the amino acid sequence of any one of SEQ ID NOS: 35 to 39, SEQ ID NOS: 42 to 47, and SEQ ID NO: 56. The isolated or purified therapeutically effective hPCSK9 polypeptide is consisting of the amino acid sequence of any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 56 or combinations thereof. The isolated or purified therapeutically effective hPCSK9 polypeptide is consisting of the combination of the amino acid sequence SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 36 and SEQ ID NO: 42 or SEQ ID NO: 36 and SEQ ID NO: 47. The isolated or purified therapeutically effective hPCSK9 polypeptide may have the amino acid sequence:

(41) ##STR00008##

(42) The kit may further comprise a HMG-CoA reductase inhibitors (statin). The HMG-CoA reductase inhibitors (statin) is for use before, at the same time or after said hPCSK9 polypeptide. The HMG-CoA reductase inhibitors (statin) may be for use before, at the same time or after said hPCSK9 polypeptide. HMG-CoA reductase inhibitors (statin) include but are not limited to Pravastatin, Fluvastatin, Atorvastatin, Pravastatin, Lovastatin, Cerivastatin, Mevastatin, Pitavastatin, Rosuvastatin, Simvastatin.

(43) Overall data presented herein indicates that the disulphide bridge containing Loop3 peptide from the catalytic domain of hPCSK9 (connecting the two Cys residues indicated in bold underlined character) defined by the sequence hPCSK9.sup.365-384 (.sup.365GEDIIGASSD[CSTC]FVSQSG.sup.384) (SEQ ID NO: 56) is an effective peptide with LDL-R promoting activity when added exogenously to the culture medium of growing HepG2 and HuH7 cells. Other effective peptides include peptides derived from SEQ ID NO: 54 (CLYSPASAPEVITVGATNAQDQPVTGTLG1TNFGR) or SEQ ID NO: 55 (IIGASSDCSTCFVSQSGTSQAAAHV), such as SEQ ID NOS: 35 (CLYSPASAPEVITVG), SEQ ID NO: 36 (ASAPEVITVGATNAQ), SEQ ID NO: 37 (VITVGATNAQDQPVT), SEQ ID NO: 38 (ATNAQDQPVTLGTLG), SEQ ID NO: 39 (DQPVTLGTLGTNFGR), SEQ ID NO: 42 (CVDLFAPGEDIIGAS), SEQ ID NO: 43 (APGEDIIGASSDCST), SEQ ID NO: 44 (IIGASSDCSTCFVSQ), SEQ ID NO: 45 (SDCSTCFVSQSGTSQ), SEQ ID NO: 46 (CFVSQSGTSQAAAHV) and SEQ ID NO: 47 (SGTSQAAAHVAGIAA). The SS bond between Cys.sup.375 and Cys.sup.378 (shown as bold underlined) in the SEQ ID NO: 56 peptide is believed to be critical for this activity, suggesting its role in providing a better binding opportunity with the EGF-A domain of LDL-R. Interestingly this peptide also contains the site for the most potent gain of function mutation, namely D.sup.374 (shown above in bold italic) to Y. Mimicking this mutation in the peptide (substitution of D.sup.374 by Y, i.e. .sup.365GEDIIGASSY[CSTC]FVSQSG.sup.384, SEQ ID NO: 57) may enhance its bioactivity and promote LDL-R level even more. The peptides of the present invention may represent the first generation of small compound agent which can be further exploited to enhance its LDL-R promoting activity and prevent or treat hypercholesterolemia, and/or presumably preventing or treating associated cardiovascular diseases risks.

(44) The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

Example 1

Experimental Description

(45) Materials

(46) All Fmoc amino terminal protected amino acids (L-configuration) with additional side chain protection as needed, peptide coupling reagents namely HBTU, PAL-PEG-PS resin for peptide synthesis, organic solvents such as Acetontrile (CAN, HPLC grade) and Dimethyl formamide (DMF, analytical grade) were obtained from Bachem Inc (Torrance, Ca, USA), Calbiochem Novabiochem Inc, (San Diego, Ca, USA), Neosystems Inc, (Strasbourg, France) and PE-Biosystems (Foster City, Ca, USA). TFA and all reagents such as phenol, TIS and EDT constituting Reagent B [Palmer Smith H, Basak A. Regulatory Effects of Peptides From the Pro and Catalytic domains of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) on LDL-R. Curr Med Chem, 17(20):2168-2182, 2010] for peptide deprotection and its cleavage from resin were purchased from Sigma-Aldrich Chemical (Milwaukee, USA). TCEP [Tris (2-carboxy ethyl) phosphine], Iodoacetamide as well as all other coupling agents and organic solvents were bought from Sigma-Aldrich, VWR or Fisher companies.

(47) Matrix Assisted Laser Desorption (MALDI) and Surface Enhanced Laser Desorption Ionization (SELDI) time of flight (tof) mass spectra (MS) were recorded using Voyageur (PE-Biosystems, Framingham, Ma, USA) and Ciphergen Protein Chips (Fremont, Ca, USA) respectively. The corresponding mass spectra plates, re-usable gold plates for SELDI and stainless plates for MALDI were purchased from the respective companies. -Cyano 4-hydroxy cinnamic acid (CHCA), 2,5-Di-hydroxy benzoic acid (DHB) and Sinapic (Sigma-Aldrich Chemical) were used as energy absorbing matrices for low and high molecular weight compounds respectively. Reagents for western blot and SDS-PAGE analyses were purchased from Bio-rad Labs (Hercules, Ca, USA). All chemi-luminescence reagents (Perkin Elmer LAS Inc, Shelton, Conn., USA) were used for detection of immuno-reactive bands. Images were captured using Kodak X-OMAT Blue autoradiography film (PerkinElmer LAS Inc., Waltham, Ma, USA).

(48) Antibodies

(49) Polyclonal antibodies against hLDL-R (#CY-M1033), hPCSK9 (#AF2148) and Transferrin receptor (#13-6800) were purchased from R&D Systems, Circulex and Invitrogen companies respectively. A-actin-HRP (Horse Radish Peroxidase) (#ab49900) and FLAG-HRP (#1238) primary antibodies were both bought from ABCAM Company. Goat-HRP (#ab6741) and mouse-HRP (#172-1011) secondary antibodies were obtained from ABCAM and Bio-rad company respectively.

(50) Peptide Synthesis

(51) hPCSK9 Catalytic Domain Derived Peptides.

(52) All 51 peptides were derived from hPCSK9 catalytic domain which is implicated in binding with EGF-A-domain of LDL-R. Each of these peptides is comprised of 15 aa except for the last one P51 which is 18 aa long (listed in Table 1). They were all synthesized on an automated solid-phase peptide synthesizer instrument (Intavis, Multipep model, Germany) using Fmoc (Fluorenyl methoxy carbonyl) mediated chemistry and HBTU [N,N,N,N-Tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate], HOBT (1-Hydroxy benzotriazole) in presence of DIEA (N, N-diisopropyl ethyl amine) as coupling reagent [Palmer Smith H, Basak A. Regulatory Effects of Peptides From the Pro and Catalytic domains of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) on LDL-R. Curr Med Chem, 17(20):2168-2182, 2010]. The following amino acid side chain protecting groups were used: Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) for Arg; tBu (tertiary butyl) for Ser, Thr, Tyr, Asp and Glu residues; Trityl (triphenyl methyl) for Cys, His, Gln and Asn and Boc (t-butyloxy carbonyl) for Lys. The synthesis started from carboxy (C) to amino terminus (N) end on an unloaded Fmoc-protected tentagel [a PAL-PEG {poly amino linker poly ethylene glycol} cross linked to PS (polystyrene)] resin. Following the end of synthesis, peptides were cleaved off from the resin and fully deprotected at the same time by 3 h treatment with Reagent B consisting of 90% TFA (Trifluoroacetic acid), 2.5% phenol, 5% water and 2.5% TIS (Tri-isopropyl silane) [Palmer Smith H, Basak A. Regulatory Effects of Peptides From the Pro and Catalytic domains of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) on LDL-R. Curr Med Chem, 17(20):2168-2182, 2010, Mishra, P et. al. In vitro regulatory effects of epidydimal serpin CRES on protease activity of Proprotein Convertase 4 (PC4). Current Molecular Medicine. 12, 1050-1067, 2012] for 3 h at ambient temperature. The crude peptides thus obtained were purified by RP-HPLC as described later and fully characterized by MALDI(Matrix Assisted Laser Desorption Ionization) (Voyageur, Applied Biosystems) or SELDI (Surface Enhanced Laser Desorption Ionization)-tof (time of flight) Mass Spectrometer (MS) (Protein chips, Ciphergen, Fremont, Calif., USA) using CHCA, DHB or SPA as an energy absorbing matrix.

(53) TABLE-US-00002 TABLE1 Listof51(P1-P51)peptides(15aalongwith 10aaoverlappingsequence)derivedfrom hPCSK9catalyticdomainandtheir molecularweights(MWs). CalcMW Peptide: FI-EGF-A Name Aminoacidsequence MW 1:1adduct P1 SIPWNLERITPPRYR 1898.0 6860.5 P2 LERITPPRYRADEYQ 1907.0 6895.5 P3 PPRYRADEYQPPDGG 1717.8 6680.3 P4 ADEYQPPDGGSLVEV 1575.7 6538.2 P5 PPDGGSLVEVYLLDT 1574.8 6537.3 P6 SLVEVYLLDTSIQSD 1681.9 6644.4 P7 YLLDTSIQSDHREIE 1818.9 6781.4 P8 SIQSDHREIEGRVMV 1755.9 6718.4 P9 HREIEGRVMVTDFEN 1831.9 6794.4 P10 GRVMVTDFENVPEED 1736.8 6699.3 P11 TDFENVPEEDGTRFH 1792.8 6755.3 P12 VPEEDGTRFHRQASK 1756.9 6719.4 P13 GTRFHRQASKCDSHG 1686.8 6649.3 P14 RQASKCDSHGTHLAG 1567.8 6530.3 P15 CDSHGTHLAGVVSGR 1495.7 6458.2 P16 THLAGVVSGRDAGVA 1409.8 6372.3 P17 VVSGRDAGVAKGASM 1404.7 6367.2 P18 DAGVAKGASMRSLRV 1630.9 6593.4 P19 KGASMRSLRVLNCQG 1619.9 6582.6 P20 RSLRVLNCQGKGTVS 1617.9 6580.4 P21 LNCQGKGTVSGTLIG 1447.8 6410.3 P22 KGTVSGTLIGLEFIR 1590.9 6553.4 P23 GTLIGLEFIRKSQLV 1674.0 6636.5 P24 LEFIRKSQLVQPVGP 1711.0 6673.5 P25 KSQLVQPVGPLVVLL 1590.0 6552.5 P26 QPVGPLVVLLPLAGG 1429.9 6392.4 P27 LVVLLPLAGGYSRVL 1570.0 6532.5 P28 PLAGGYSRVLNAA Q 1519.8 6482.3 P29 YSRVLNAA QRLARA 1691.9 6654.4 P30 NAA QRLARAGVVLV 1540.9 6503.4 P31 RLARAGVVLVTAAGN 1467.9 6430.4 P32 GVVLVTAAGNFRDDA 1504.8 6467.3 P33 TAAGNFRDDACLYSP 1600.7 6563.2 P34 FRDDACLYSPASAPE 1641.7 6604.2 P35 CLYSPASAPEVITVG 1506.8 6469.3 P36 ASAPEVITVGATNAQ 1428.7 6391.2 P37 VITVGATNAQDQPVT 1513.8 6476.3 P38 ATNAQDQPVTLGTLG 1485.8 6448.1 P39 DQPVTLGTLGTNFGR 1575.8 6538.3 P40 LGTLGTNFGRCVDLF 1612.8 6575.3 P41 TNFGRCVDLFAPGED 1640.7 6603.2 P42 CVDLFAPGEDIIGAS 1506.7 6469.2 P43 APGEDIIGASSDCST 1422.6 6385.1 P44 IIGASSDCSTCFVSQ 1517.7 6480.2 P45 SDCSTCFVSQSGTSQ 1536.6 6499.1 P46 CFVSQSGTSQAAAHV 1492.7 6455.2 P47 SGTSQAAAHVAGIAA 1311.7 6274.2 P48 AAAHVAGIAAMMLSA 1384.7 6347.2 P49 AGIAAMMLSAEPELT 1504.7 6467.2 P50 MMLSAEPELTLAELR 1703.9 6666.4 P51 EPELTLAELRQRLIHFSA 2123.2 7085.7

(54) Table 1 lists of 51 (P1-P51) peptides (15 aa long with 10 aa overlapping sequence) (SEQ ID NO: 1 to 51) derived from hPCSK9 catalytic domain and their molecular weights (MWs). The calculated (Calc) MWs of 1:1 complex between peptide and FI-EGF-A (MW=4962.5 Da) which range from 6274.2 (for P47) and 7085.7 Da (for P51) were also shown. The catalytic triads D.sup.186, H.sup.226 and S.sup.386 and the oxyanion N.sup.317 residue were shown with underline whereas the crucial D.sup.374 whose natural mutation to Y leads to most potent gain of function is depicted in bold. The second most potent gain of function mutation R.sup.357 to H is also shown in bold. The single presumed unpaired Cys residue at position 301 is indicated in bold italics character.

(55) FI/Bio-EGF-A peptide (Ia and Ib).

(56) The synthesis of fluorescein labeled EGF-A peptide (FI-EGF-A) was carried out by using unloaded Fmoc-protected tentagel PS resin and Fmoc-mediated solid phase peptide chemistry with minor modification of triple couplings in each cycle instead of usual double coupling as described previously [Mishra, P et al. In vitro regulatory effects of epidydimal serpin CRES on protease activity of Proprotein Convertase 4 (PC4). Current Molecular Medicine. 12, 1050-1067, 2012]. Three pairs of Cys protecting groups namely the highly acid labile Mmt group, ACM and Trt were used as indicated in FIG. 3 during the synthesis. This will allow SS bridge connections between right Cys residues (namely Cys.sup.1-Cys.sup.3, Cys.sup.2-Cys.sup.4 and Cys.sup.5-Cys.sup.6, numbered from N-terminal end) as present physiologically in the EGF-A domain of hLDL-R protein. Following the completion of the peptide assembly, the final attachment (via the peptide's free amino terminal) of fluorescein group using activated 5-Carboxy fluorescein or Biotin moiety was accomplished by HBTU/HOBT activating agent. The fully protected labeled peptide still bound on the resin was then treated with 0.5% TFA/DMF for 5 min (2 times) to remove specifically the acid labile Mmt protecting group on Cys.sup.6 and Cys.sup.5. On resin cyclization via SS bridge formation between these two free Cys residues were performed by treatment with 1.5M excess of 0.4 M thallium trifluoracetate/DMF for 1 h following the protocol described before [Angeletti R H, et al. Formation of a Disulfide Bond in an Octreotide-Like Peptide: A Multicenter Study, Techniques in Protein Chemistry VII, Academic Press, Inc. 1996.]. The resin is next treated with Reagent B for 3 h to remove all the protecting groups except the two ACM groups on Cys.sup.2 and Cys.sup.4 and cleave the peptide from the resin. The recovered material following lyophilisation was treated with DMSO to induce SS bridge cyclization between the two free Cys.sup.1 and Cys.sup.3 residues. Next the two ACM protecting groups were removed from Cys.sup.2 and Cys.sup.4 by treatment with 12 in MeOH and cyclised at the same time to furnish FI/Bio-EGF-A peptide (Ia and Ib). The peptides were purified by RP-HPLC and fully characterized by mass spectrometry

(57) FI-Bio-Ahx-Lys-Methyl Ester (IIId).

(58) Fluorescent biotinylated lysine methyl ester [FI-Bio-Ahx-Lys-Methyl ester] (IIId), used as a model compound in the current study was synthesized by using the steps and reagents shown in FIG. 4. Here Ahx (epsilon amino hexanoic acid) serves as a linker. Typically Fmoc-Lysine Methyl ester (Bechem Inc) (1 mmol) was first labeled with 5-Carboxy Fluorescein via its side chain free amino group by reacting with 5-carboxy fluorescein-NHSu (N-hydroxy succinimide). The labeled product (IIIb) was then treated with 30% piperidine in DMF to remove the protecting Fmoc group. The resulting free amino derivative (IIIc) thus obtained was finally reacted with Biotin-Ahx-NHSu leading to the formation of FI-Bio-Ahx-Lys-Methyl ester (IIId), which was purified by silica gel column chromatography and fully characterized by mass spectrometry (Calculated MW=877, observed MW=878 (M++H).

(59) Peptide Purification by RP-HPLC.

(60) All crude peptides except FI-Bio-Ahx-Lysine methyl ester (IIIb) were purified by Reverse Phase High Performance Layer Chromatography (RP-HPLC) using C.sub.18 Silica gel analytical column (Varian, 125 cm size). During RP-HPLC purification, proteins were separated using a linear gradient of Solvent B from 10% to 90% in Solvent A [Solvent B=0.1% TFA in ACN and Solvent A=0.1% TFA in water]. Fractions were collected and analyzed as the elution was monitored on-line by UV detector with wavelength fixed at 230 nm. Peaks were collected, lyophilized and subjected to mass spectrometry for their identifications.

(61) Cell Culture

(62) The human hepatic HepG2 cells were maintained at 37 C. with 5% CO.sub.2 in Dulbecco's modified Eagle's medium (DMEM) (Wisent #319-005-CL) supplemented with 10% fetal bovine serum (Wisent #080-350) as well as penicillin and streptomycin (Wisent #450-201-EL). The number passages of cell lines used were 6-8 times until they are at least 80% confluent as determined by microscope. For each culture experiment, nearly 1 million cells were seeded in a petri dish. Each synthetic peptide was dissolved in DMSO solvent at 1 mM concentration and stored at 20 C. before use. The peptide treatments (final concentration 10 M unless otherwise specified) were carried out by adding the solution in the fresh culture medium. The cells were grown for additional for 16 h. The medium was removed and the cells were washed twice with PBS buffer. The residual cells were finally collected in PBS buffer using cell scrapers. It was the centrifuged and the cell pellet thus obtained was lysed in modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40) containing protease and phosphatase inhibitors (Sigma Aldrich #P8340 and #P5726). All culture samples including whole cell lysate (WCL) were analysed for their protein contents using Pierce BCA (Bicinchoninic acid) reagent method or Bradford assay (Bio-Rad #500-0205) as described later.

(63) Protein Assay

(64) Total protein content in a sample was measured by using Bradford's optical density or BCA methods. Each sample was mixed with Coomassie reagent (Bio-rad) and optical density (OD) value was measured using Multiskan Spectrum (Thermo) plate following the protocol of the manufacturer.

(65) Western Blot Analysis and SDS-PAGE

(66) In general 20 g of WCLs derived from various peptide treated HepG2 cell experiments was resolved by conventional 12% SDS-PAGE (Laemmli, 1970 #2). Resolved samples were then transferred to polyvinylidene fluoride (Bio-Rad #162-0177) and probed for LDL-R (R&D Systems #AF2148), hPCSK9 (Circulex #CY-M1033), Transferrin Recepter (TR) (Invitrogen #13-6800), -actin-HRP (Horse Radish Peroxidase) (abcam #ab49900) and FLAG-HRP (abcam #1238) primary antibodies and detected with goat-HRP (abcam #ab6741) or a mouse-HRP (Bio-Rad #172-1011) secondary antibodies and visualized on a Bio-Rad versa dock imaging system using Clarity ECL Western Substrate (Bio-Rad #170-5060). All cell culture and western blot experiments have been repeated three times and the average results and data were shown.

(67) Recombinant hPCSK9 WT and D/Y

(68) Recombinant PCSK9 wild type (WT) as well as D.sup.374/Y mutant both containing a C-terminal FLAG tag (Sequence: DYKDDDDK) was expressed and purified. The protein was characterized by SDS-gel electrophoresis, western blot and mass spectral analyses.

(69) In Vitro Binding Assays

(70) Binding assays of various PCSK9 peptides were carried out by incubating 15 g of each peptide (0.008 mM) with 20 g of FL-EGF-A in a final volume of 20 l of 25 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl.sub.2 for 2 h at 37 C. Similar study was also performed using recPCSK9 wild type as well as D/Y variants against FI-EGF-A. Each sample along with the control without any added peptide was assessed for any binding between FI-EGF-A and peptide using fluorescence study, Mass spectrometry and Native gel studies

(71) Fluorescence Study:

(72) The fluorescence intensity of FI-EGF-A peptide solution in water (typically 5 l of 0.5 mM concentration) was measured in the absence and presence of aqueous solution (10 l, 0.5 mM concentration) of each hPCSK9 catalytic peptide (P1-P51) solution at .sub.ex and .sub.em fixed at 490 and 514 nm respectively following incubation for 1 h at 37 C. with shaking (70 rpm). The experiment was conducted in a 96-microtitre well plate (black color, 50 l capacity) using fluorescence spectrophotometer (Molecular Devices Co, USA).

(73) SELDI-tof-Mass Spectrometry:

(74) SELDI-Tof mass spectrum was performed on gold plate chips with 2 l of sample and 2 l of SPA matrix as described in Palmer et al. and Mishra et al. Each spectrum of peptide sample was calibrated against hlnsulin (MW 5,807 Da) both as internal and external standards. For mass spectrum of samples containing rec-hPCSK9, the calibration was performed against BSA (MW 66,120 Da) and Carbonic Anhydrase (MW 16,998 Da).

(75) Native Gel Electrophoresis:

(76) Native gel electrophoresis was performed on each incubated sample under SDS free condition in Tris-Glycine gel. Typically 5 l of each peptide sample was pre-incubated with 20 l of buffer at room temperature under mild condition and loaded onto Tris-Tricine gel (1 mm thick) along with appropriate standards. The bands were revealed upon staining with Coomassie blue dye.

(77) Fluorescence Gel Electrophoresis:

(78) This is performed on the samples (typically 5 l) containing FI-EGFA+rec-PCSK9 WT or its D/Y variants as well as the corresponding control consisting of FI-EGF-A alone in buffer. The bands were resolved on Native-PAGE as indicated before and revealed under UV light in the dark. A mixture of fluorescent standard markers was also run in parallel.

(79) MTT Assay

(80) MTT assays were carried out in 96 well plates in triplicate according to the manufacturer's protocol (Biotium #30006). These provide a measurement of cell viability. This is a highly sensitive method which measures cell proliferation based on the reduction of 3 [4,5-Dimethylazol-2-yl] 2,5 diphenylTetrazolium Bromide Tetrazolium salt (MTT).

(81) Statistical Analysis

(82) Unless otherwise indicated, results were compared using Student's t test. A p value of less than 0.05 was considered significant. Each experiment was performed in triplicates and the data were used for statistical purposes.

Example 2

Peptide Design, Purification and Characterization

(83) hPCSK9 Catalytic Peptides (P1-P51)

(84) The binding of hPCSK9, a secreted soluble protein with the membrane bound receptor hLDL-R has been the subject of intense investigation in recent years. Studies now revealed that hPCSK9 binds to hLDL-R via the extracellular 42 aa long EGF-A domain of the latter that also possesses a strong Ca.sup.+2 binding site. Interestingly the precise binding segment of the other partner molecule namely hPCSK9 has not been fully ascertained although all indications point to its catalytic domain (aa153-421). This was finally confirmed by the crystal structures of recombinant hPCSK9 complex with LDL-R or synthetic EGF-A peptide. However which specific peptide sequence/s of PCSK9 catalytic segment is associated with this binding remained unclear.

(85) In an effort to define this a series of peptides (P1-P51; SEQ ID NOS:1 to 51) of 15 mer length (except the 18-mer last peptide P51) has been designed that encompass the entire catalytic domain of hPCSK9. Moreover each of these peptides shares 10 aa overlapping sequences with the immediate preceeding one. The list of these peptides with their amino acid sequences and locations are shown in Table-1. The four important catalytic residues D.sup.186, H.sup.226, N.sup.317 and S.sup.386 as well as the two most potent gain of function mutations (D.sup.374/Y (and R.sup.357/H) in these peptides were highlighted. In addition, the seven Cys residues, 6 of which are inter-linked via SS bridges were also highlighted in underlined regular character. Following purification by RP-HPLC, these peptides exhibited in their mass spectra peaks at m/z values consistent with the calculated value (Table-1). Table-1 also shows the calculated molecular weights of 1:1 complex of these peptides with synthetic FI-EGF-A (see later).

(86) FI-EGF-A (Ia) and Bio-EGF-A (Ib)

(87) This peptide is designed from the EGF-A domain of hLDL-R which comprises the segment (aa314-355). A 5-Carboxy Fluorescein moiety is attached to the free amino terminus of this peptide while it is still resin bound with all amino acid side chain functions protected. The fluorescein labeled free fully cyclized peptide (FIG. 3) (see above for details) is then purified by RP-HPLC and characterized by SELDI-tof mass spectrum (FIG. 5) which showed a peak at m/z4,958. This is consistent with the calculated value of m/z 4,962. The presence of three SS bridges in the peptide was confirmed by alkylation and reductive alkylation experiments. As revealed by mass spectra (not shown), FI-EGF-A remains unreacted upon treatment with iodoacetamide.

(88) However following reduction with TCEP [Tris (2-carboxy ethyl) phosphine], it reacted with 6 molecules of iodoacetamide leading to hexa-acetamidyl derivative (increase of 657=342 Da in MW) as revealed by mass spectrum (not shown). This result confirmed the presence of 3 SS bonds in the starting peptide. Furthermore it is noted that FI-EGF-A upon high-power laser treatment during mass spectrum exhibited two additional broad peaks at m/z2,980 and 1,987. It is proposed that these two peaks are likely generated through breakdown of FI-EGF-A molecule via a 6-member transition state mechanism as shown in FIG. 6A. This proposed fragmentation provided additional support to the structure and the presence of 3 SS bridges as shown in (Ia). In a similar manner Bio-EGF-A (Ib) was also prepared by attaching biotin at the N-terminus and fully characterized. A 3D energy minimized model structure in vacuo based on Hyperchem v11 software program (FIG. 6B) revealed the rigid geometry of the molecule stabilized by 5-H-bonds, 2 within the SS bond involving Cys.sup.12 and Cys.sup.25

(89) FI-Bio-Ahx-Lys-Methyl Ester (IIId)

(90) This model bis-functional Lysine derivative was synthesized in 4 steps as described in FIG. 4, then purified by silica-gel chromatography and fully characterized by mass spectrometry (calculated MW=877 Da; Observed MW=878 (M.sup.++H).

Example 3

In Vitro Binding Study

(91) hPCSK9 Catalytic Peptides Vs FI-EGF-A

(92) Fluorescence Quenching.

(93) Previously a number of studies reported that the interaction between two ligands one of which is fluorescence labeled can be followed by studying fluorescence intensity. In general the fluorescence intensity is suppressed with or without shift of emission peak position when there is a strong interaction between the two ligands. Greater the suppression or quenching of fluorescence intensity, greater is the strength of binding. In order to further confirm the above notion, a study was carried out by using a fixed concentration of fluorescent biotinylated peptide (IIId) and increasing doses of avidin protein. As more and more avidin binds with biotin (K.sub.d10.sup.15 m), a gradual suppression of fluorescence intensity was observed until it is 98% quenched at 8:1 molar ratio of avidin:(IIId) (FIG. 7A). As expected similar results were also obtained with streptavidin (K.sub.d10.sup.4 M), Captavidin (K.sub.d10.sup.15 M) and Nutravidin (K.sub.d10.sup.15 M) [Molecular Probes: The handbook, Chapter 7 available, Invitrogen Corporation] with maximal of 81.4%, 75.5% and 85% quenching respectively (Data not shown). These avidin analogs with varying binding affinity towards biotin have been developed and made available through commercial organizations [Polyscience Inc., Technical Data Sheet Report-779]. On the contrary, when the same experiment was conducted with h insulin which does not bind with biotin, a maximum of only 30% quenching at 8:1 molar ratio of h insulin:(IIId) was noticed (considered as non-specific binding) (FIG. 7B). This study confirms that fluorescence quenching is an indicator of binding between two ligands one of which is fluorescently labeled.

(94) Additional fluorescence quenching studies were conducted using FI-EGF-A peptide and each of the 51 synthetic hPCSK9 catalytic peptides, one at a time. The results are depicted in FIG. 8 which revealed strong fluorescence quenching effect in the presence of peptides P2-P5, P8, P16, P17, P22, P25, P26, P35, P37, P45, P48 and P49 (SEQ ID NOS: 2-5, 8, 16-17, 22, 25, 26, 35, 37, 45, 48 and 49) suggesting their interactions with FI-EGF-A but no conclusion could be made about their relative binding affinities.

(95) Mass Spectrum.

(96) SELDI-Tof mass spectra of FI-EGF-A peptide following 1 h incubation with each of the 51 peptides (P1-P51, SEQ ID NOS:1-51) revealed formation of 1:1 and in some cases weak 1:2 adducts with selected peptides as shown in FIGS. 9A-C. It is noted that formation of such complexes or adducts were not observed as expected in the mass spectrum of FI-EGF-A alone (see FIG. 9C done in triplicate) and for samples with other peptides. It is likely that above non-covalent adducts were strong enough to survive dissociation during mass spectrometry laser bombardment. Overall the data suggested qualitative binding of FI-EGF-A with the peptides P18-P21, P28-P30, P33-P36 and P43-P46 (SEQ ID NO: 18-21, 28-30, 33-36 and 43-46). However the data did not reveal the comparative strength of binding of these peptides with FI-EGF-A peptide.

(97) Native Gel Electrophoresis

(98) In order to gather further evidence for the above findings, native gel electrophoresis was performed on each incubated sample under SDS free non-denaturing condition in Tris-Glycine gel with appropriate standards as described [77]. The results were shown in FIG. 10 for some selected samples which showed that the peptides P32, P35-P38 and P44-P48 (SEQ ID NO: 32, 35-38 and 44-48) all form 1:1 stable adducts with FI-EGF-A peptide (MW5 kDa) leading to the formation of an additional band at 7 kDanot seen in the control sample consisting of FI-EGF-A alone with no added peptide.

Example 4

Recombinant FLAG-HPCSK9 vs FL-EGF-A

(99) In addition to investigating the binding potential of PCSK9 catalytic peptides towards FI-EGF-A, affinity study for rec FLAG-PCSK9 protein against FI-EGF-A were also conducted. Purified rec-FLAG-PCSK9 protein WT or D/Y mutant were used. SDS-PAGE performed on FI-EGF-A peptide incubated alone and in the presence of recombinant FLAG-PCSK9 protein at various concentrations using fluorescence and coomassie staining detection methods were shown in FIGS. 11 and 12. As expected, FI-EGF-A exhibited a band at 5 kDa consistent with its calculated MW which increases in intensity with dose in both fluorescence and staining intensity (FIG. 11). It may be pointed out that the exact position of the band for FI-EGF-A peptide as measured by fluorescence intensity (left) and coomassie stain (right) is in fairly good agreement and the minor difference noted were due to diffused nature of the band due to low molecular weight of FI-EGF-A (Mol Wt5 kDa) in SDS-scale. When incubated with recPCSK9, FI-EGF-A exhibited a fluorescent positive band at 65 kDa (indicated by a big arrow, FIG. 12, left panel) likely due to the formation of adduct between 60 kDa mature recPCSK9 and 5 kDa FI-EGF-A peptide. As expected this fluorescent band is absent in recPCSK9 alone lane which showed immunoreactive bands at 74 kDa and 60 kDa for pro-PCSK9 and mature PCSK9 forms respectively when probed against anti-FLAG antibody (FIG. 12, right panel). The data shows that the recPCSK9 forms a stable complex with FI-EGF-A peptide but it is likely that only the mature PCSK9 (MW 60 kDa) binds with FI-EGFA to produce an adduct detectable by both fluorescence (left panel) and western blot (right panel). However, it is also possible that the pro-PCSK9 form (74 kDa) which is the minor one, may also bind to FI-EGFA but relatively weakly thereby not detectable by fluorescence at all. However it is detectable by western blot though with reduced intensity. This may be explained by the fact that upon binding with FI-EGFA, PCSK9 is recognized by FLAG antibody only with reduced potency. Unfortunately similar experiments could not be performed to study the interactions of various synthetic hPCSK9 peptides with FI-EGF-A since the expected MWs of each adduct (varying from 6.3-7 kDa) are too close to the molecular weight of unbound FI-EGF-A peptide (5 kDa) and therefore will be difficult to be detected in the large background of mass intensity of unreacted FI-EGF-A

Example 5

Cell Culture Study

(100) The above binding affinities of selected PCSK9 catalytic peptides and rec-PCSK9 protein towards FI-EGF-A peptide indicated that they may regulate LDL-R level when applied to the culture medium of growing hepatic cells such as HepG2 and HuH7 which express both PCSK9 and LDL-R. This expectation is based on the fact that binding of PCSK9 with LDL-R via latter EGF-A domain is the key event for LDL-R degradation.

(101) Effect of FI-EGF-A on LDL-R in HepG2/HuH7 Cell Lines

(102) The first set of results are shown with FI-EGF-A in FIG. 13A which demonstrates that a 24 h treatment of HepG2 cells with this peptide solution in DMSO (12 g/ml) resulted in a significant up regulation of the LDL-R (lanes 3 and 4) as compared to control treated only with DMSO (lanes 1 and 2). The results were based on western blot analysis of whole cell lysates as standardized against the house-keeping protein actin. The above effects were accompanied by a marked up regulation of PCSK9 suggesting an auto regulatory feedback loop. It also suggested that the actual effect may be much more significant than what is revealed by western blot data (FIG. 13A). To further confirm the data and to find out whether our synthetic FI-EGF-A peptide is capable of restoring LDL-R level following it's degradation by PCSK9, we added rec-PCSK9 (20 g/ml) to the culture medium of growing HuH7 cells in the absence and presence of increasing amounts of FI-EGF-A peptide. The results are shown in FIG. 13B by western blot analysis for PCSK9, LDL-R and actin contents in cell lysates. The data suggested that following addition of recPCSK9 protein, LDL-R level as expected decreased significantly which was then partly restored gradually upon addition of FI-EGF-A peptide in a dose-dependent manner.

Example 6

Effect of hPCSK9 Catalytic Peptides on LDL-R in HepG2 Cells

(103) Next we examined the effects of all 51 hPCSK9 catalytic peptides on LDL-R and PCSK9 levels in HepG2 cells using a fixed 5.5 M concentration level which was found to be most optimum and non-toxic based on MTT test (data not shown) and other data which revealed that most peptides begin to exhibit toxic effect at >25-50 M, depending on the peptide's nature. The data based on western blot analysis of cell lysates for LDL-R and PCSK9 as compared to the house keeping protein Transferrin Receptor (TR), suggested that the peptides P35-P39, P42, P43, P46 and P47 (SEQ ID NOS: 35-39, 42, 43, 46 and 47) differentially enhance LDL-R level without significantly affecting PCSK9 level (FIGS. 14A-C). Interestingly addition of an equimolar mixture of two peptides often results in synergistic effect on LDL-R promoting activity. So far the highest synergistic LDL-R promoting activity (3-fold higher than control) was noted with P36 and P37 mixture (SEQ ID NOS: 36-37) (FIG. 14C). This is followed by P36+P47 (SEQ ID NOS: 36 and 47) and P36+P42 (SEQ ID NOS: 36 and 42) mixture respectively. This likely suggests that P36/P37 as well as P46/P47 segments were equally effective in promoting LDL-R level based on HepG2 cell study.

Example 7

Design of SS Bridge Loop Peptides from PCSK9 Catalytic Domain

(104) So far all results taken together (summarized in FIG. 15, top panel) suggest that four specific peptide segments of hPCSK9 catalytic domain as indicated exhibit modest to strong binding affinity towards synthetic FI-EGF-A derived from LDL-R. Peptides derived from these segments possess LDL-R promoting activity in varying degrees based on studies in culturing HepG2 and HuH7 hepatoma cells. The precise location of these peptides within the catalytic domain of PCSK9 is shown in FIG. 15, bottom panel. There are three SS bridge loop domains that consist of (aa223-255) (Loop-1), (aa323-358) (Loop-2) and (aa363-392) (Loop-3). Interestingly the two most potent LDL-R promoting peptide groups (P34-P37) and (P44-P47) (SEQ ID NOS: 34-37 and 44-47, respectively) were found to be located within Loop-2 and Loop-3 regions which also bear the two most potent gain of function mutations namely R.sup.257/H and D.sup.374/Y respectively.

(105) The presence of SS bond may be crucial in terms of binding to LDL-R as it imparts a rigid structure and conformation to the molecule. In order to examine this notion and to develop even more potent LDL-R promoting agents, we synthesized SS bridged cyclic hPCSK9.sup.365-384 (CP3) and noncylic Cys/Ala mutant (CP3-C/A) (Table 2). These peptides were tested at 5.5 M concentration as before in HepG2 cells for their effects on LDL-R. FIG. 16 showed that SS bridged cyclic CP3 peptide at 50 M promoted LDL-R level by 3.5-fold (the highest so far) when added to growing HepG2 cells. This effect is completely lost for the corresponding acyclic peptide where Cys is substituted by Ala (CP3-C/A), suggesting the crucial role of SS bond derived cyclic structure in LDL-R promoting activity.

(106) TABLE-US-00003 TABLE 2 S-S bridged cyclic loop-3 peptide derived from the catalytic domain of hPCSK9 and its acyclic Ala mutant as indicated. Name Position Amino acid sequence CP3 (Loop-3 peptide) Cyclic- hPCSK9.sup.365-384 CP3-C/A Cyclic- .sup.365GEDIIGASSDASTCFVSQSG.sup.384 Loop-3 hPCSK9.sup.365-384 Ala-Mutant

(107) The crucial D.sup.374 whose natural mutation to Y leads to the most potent gain of function and severe hypercholesterolemia, is depicted in bold italics character. The Cys pair with SS connection as well as the mutation [Cys.sup.375 to Ala (bold underlined)] were indicated in the figure.

Example 8

3D Model Structure of CP3

(108) Owing to the critical role of SS bond on biological activity (LDL-R promoting) as observed with CP3, we conducted 3D molecular model analysis of this peptide using Hyperchem v 11.0 software program (FIG. 17). The figure revealed that CP3 within its central core structure contains a small loop consisting of 14-atoms. This theoretical geometry developed in vacuo is supported by 2-H-bonds as indicated by dotted lines. With Ala-mutant this structural feature is lost which may contribute to the loss of LDL-R promoting activity.

Example 9

Discussion

(109) LDL-R Binding Linear and Cyclic Peptides from hPCSK9

(110) Using 51 synthetic linear peptides covering the entire catalytic sequence of hPCSK9 (aa153-421) and various in vitro studies based on mass spectrometry, fluorescence quench method and Native-gel electrophoresis as well as western blot based cellular studies indicated that multiple specific peptide segments of hPCSK9 catalytic domain bind to synthetic 42-mer EGF-A peptide mimicking hLDL-R (aa314-355). This sequence has been implicated in the binding of LDL-R to PCSK9 catalytic domain as established by various studies including the crystal structure [Piper D E et al. The crystal structure of PCSK9: A regulator of plasma LDL-cholesterol. Structure 15:545-552, 2007]. Although these studies are not fully consistent with one another in terms of their ultimate binding conclusions (FIG. 15A), but the strong binding interaction of at least two domains characterized by the peptides P35-P39 and P44-P46 (SEQ ID NOS: 35-39 and 44-46 respectively) of hPCSK9 have been found to be supported by all the studies. In addition these peptides as expected also exhibited LDL-R promoting activity when added exogenously to growing HepG2 and/or HuH7 cells. In addition when some of these peptides are added in combination to the cells, a synergistic promoting effect on LDL-R has been noted (FIG. 14). This observation suggested that there exist multiple domains (at least 2) within PCSK9 catalytic region which bind to EGF-A leading to enhancement of LDL-R level in cells.

(111) Upon close examination of the location of these active peptides within hPCSK9 catalytic segment, it appears that they actually represent two SS bridge cyclic loop domains of the protein (FIG. 15B). Sequence revealed that hPCSK9 catalytic domain (aa153-421) contains 7 Cys residues, namely at 223, 255, 301, 323, 358, 375 and 378. Among these, crystal structure and modeling studies revealed the following SS bonds (aa223-255), (aa323-358) and (aa375-378) with the connectivity of Cys.sup.301 remained unknown. Thus hPCSK9 catalytic segment is characterized by the presence of three SS loop domains termed as Loop-1 (C.sup.223C.sup.255), Loop-2 (C.sup.323C.sup.358) and Loop-3 (C.sup.375C.sup.378). Among them, Loop-3 contains a short cyclic structure and creates a small bump in the backbone structure. This structural feature appears more crucial and interesting. Moreover it also encompasses the sequence represented by P44-P47 peptides (SEQ ID NOS: 44-47) which individually promotes LDL-R level when added to growing HepG2 cells at a fixed concentration.

(112) Our designed Loop-3 peptide, CP3 (having sequence SEQ ID NO: 56), exhibited a modest but significant LDL-R promoting activity when it was exogenously added to the culture medium of growing HepG2 cells at 5.5 M concentration (FIG. 16). It is likely that this physiological effect is mediated via its competing effect with PCSK9 catalytic domain for LDL-R. This leads to reduced ability of PCSK9 to interact with LDL-R and degrade the latter. It will thus result in less LDL-C accumulation in the blood serum and thereby provide intervention of hypercholesterolemiaa serious risk factor of cardiovascular disease. It may also be pointed out that crystal structures of hPCSK9 bound to LDL-R or its derived EGF-A peptide revealed physical contact and interaction of various amino acid residues of EGF-A with D.sup.374 and F.sup.379 residues of hPCSK9 catalytic domain. In addition to these two amino acids, R.sup.194 of hPCSK9 also plays a critical role in binding with EGF-A domain of LDL-R involving its D.sup.331 residue located within EGF-A domain of LDL-R (ND.sup.331 LK, see peptides Ia/b). Interestingly both D.sup.374 and F.sup.379 residues of hPCSK9 are present within CP3 sequence and this may explain its observed LDL-R promoting activity.

(113) So far the data and findings are based upon binding experiments using synthetic EGF-A peptide.

(114) Mutation and Consequence

(115) This study identified the SS bond containing cyclic loop peptide, hPCSK9.sup.365-384 (CP3) as a potent region that can enhance LDL-R level upon its exogenous administration to the culture medium of growing HepG2 or HuH7 cells. Using 50 M concentration of CP3, a 3.5-fold increase in LDL-R level was observed (FIG. 16). It is also noted that this peptide can rescue LDL-R level to a significant level following its degradation by external addition of PCSK9 protein. This is highly significant and promising since it has the potential in clearing more LDL-C from circulation and may represent a lead peptide sequence for further future study towards development of small molecule PCSK9 inhibitors as non-statin alternative cholesterol lowering agents. The rigid cyclic geometry of this peptide and its model structure so crucial for its bio-activity may be utilized for development of future nonpeptide PCSK9 based cholesterol suppressing agents. In this connection, it is interesting to highlight that the above peptide sequence contains two key PCSK9 gain of function mutations namely D.sup.374/Y and R.sup.357/H sites which one can take advantage of for future development of more potent PCSK9 inhibitors.

(116) While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.