Patent Application
20180023071
The subject matter disclosed generally relates to isolated or purified therapeutically effective hPCSK9 derived polypeptides. More specifically, the subject matter disclosed relates to isolated or purified therapeutically effective hPCSK9 polypeptides from its catalytic domain (aa153-421) of hPCSK9, and uses thereof (aa: Amino acid).
(b) Related Prior Art
Proprotein Convertase Subtilisin Kexin 9 (PCSK9) was first discovered in 2003 and was called Neural Apoptosis Regulated Convertase-1 (NARC-1). It is the 9th member of mammalian subtilase super family now collectively known as PCSK. The first 8 members of this family, PCSK1-8 have been implicated in the processing of a variety of inactive precursor proteins to generate functional and bioactive peptides, polypeptides and hormones that play important roles in maintaining growth, metabolism and overall general health, as well as a variety of illnesses. PCSK9 is unique in this respect since only very limited data is available about the proteolytic activity and its substrate. In fact its protease activity is not at all linked to its physiological action and biological activity. It acts primarily as a secreted soluble protein that mediates degradation of the membrane bound Low Density Lipoprotein receptor (LDL-R) by binding with its extracellular domain and targeting the receptor for degradation via rerouting to the lysosomal pathway.
LDL protein and its receptor LDL-R on plasma cell surface are the major carriers of cholesterol in the blood. LDL-receptors are particularly abundant in the liver, the primary organ responsible for removing excess cholesterol from the body. The number of LDL-receptors on the surface of liver cells determines how quickly cholesterol is removed from the bloodstream. Accumulation of LDL-Cholesterol (LDL-C) in the plasma is known to cause the formation of atherosclerotic plaque—a major risk factor of cardiovascular disease (CVD) which remains as the major killer worldwide particularly in western hemisphere. So hPCSK9 which is comprised of 12 exons that translate to 5 domains with 692 aa protein is considered as the third target gene besides Apo-B and LDL-R for cholesterol regulation. Following removal of signal peptide domain hPCSK9 is secreted as ˜74 kDa full length protein which is autocatalytically cleaved at VFAQ152↓SIP to generate ˜14 kDa prodomain and ˜60 kDa mature protein which remain strongly associated to one another. It is this complex that interacts and binds with the EGF-A domain of LDL-R leading to the ultimate degradation of latter and accumulation of LDL-C in the blood.
A large variety of PCSK9 mutations in human have been identified which cause increased (Gain of function mutation), decreased (Loss of function) or unaltered level of cholesterol in the serum. PCSK9 gain of function mutations lead to hypercholesterolemia and loss of function mutation leads to hypocholesterolemia. The two most profound gain of function mutations in hPCSK9 have been identified as Asp374/Tyr and Arg357/His. Interestingly these mutations are located within the catalytic domain (aa153-421) and the former variant is capable of binding to LDL-R with 25-fold stronger affinity leading to 23% decreased LDL-R and ˜38% decreased level of LDL-R internalization [Caneron J, Holla O L, Rauheim T, Kulseth M-A, Berge K E, Leren T P. Effect of mutations in the PCSK9 gene on the cell surface LDL receptors Hum Mol Genet. 15(9):1551-1558, 2006] responsible for ultimate increase in serum LDL-C. This mutation has been identified in 3% of the population with Autosomal Dominant Hypercholesterolemia (ADH). On the other hand most loss of function mutations has been characterized in African Americans with low levels of circulating LDL-C. Such mutations have resulted in a 30% increase in LDL-R, a truly protective effect against hypercholesterolemia and cardiovascular diseases. The most extreme loss of function mutation Arg46/Leu in the pro-domain, have resulted in ˜42% decrease in LDL-C.
Very recently a novel loss of function mutation at the prodomain cleavage site of hPCSK9 (Gln152/His) has been discovered in a French Canadian family. This mutation resulted in impaired processing (no autocatalytic processing) and secretion in cell culture resulting in circulating PCSK9 and LDL-C concentrations of 79% and 48%, respectively, compared with unrelated non-carriers. The clinical and biochemical studies involving hPCSK9 variants as well as PCSK9 knock out, knock down and over-expressed mice studies all confirmed the key role of PCSK9 in cholesterol regulation As a result it has been identified as an important target for development of non-statin alternative cholesterol lowering agents. Statins have long been recognized as effective cholesterol lowering agents which inhibit HMG-CoA reductase-a crucial enzyme in the liver responsible for cholesterol synthesis. Statins work quite well in lowering LDL-C in most patients but a few do not have a strong response to statin treatment. In addition they may cause (i) Cognitive problems (˜2% patients), (ii) Increased risk of delirium in patients after surgery (up to 10% patients); (iii) Muscle pain/weakness & neuropathy; (1-5% patients), (iv) Elevation of liver enzymes Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) causing liver failure; (v) Increased risk of type 2 diabetes (˜9% population); (vi) Increase in colorectal and prostate cancers particularly in obese men.
Due to these findings, interest has grown to develop inhibitors of PCSK9 as potential cholesterol reducing agents alternative to statins. Various approaches have been pursued that include delivery of single stranded anti-sense DNA, RNAi and specific antibody preferably monoclonal (mAb) of PCSK9. Significant progress has been accomplished with the development of several mAbs by major pharmaceutical companies that include Sanofi-Regeneron Pharmaceuticals (REGN727 or Alirocumab), Amgen company (AMG145 or Evolocumab), Pfizer (RN 316 or Bococizumab) and Lilly (LY3015014). The first two of these antibodies have been proved to be most successful in human clinical trials and may soon be approved by Food and Drug Administration (FDA) as first PCSK9 targeted cholesterol lowering agents. Despite this success with mAbs, there is still a growing interest to develop small compound inhibitors of hPCSK9 which unlike the Abs are expected to be less expensive, more aqueous soluble, proteolytically & thermally stable as well as more bio-available. Currently only a limited number of data are available on small compound PCSK9 inhibitors. These include synthetic EGF-A peptide, Anexin-A2 and its derived peptides, PCSK9 prodomain derived peptides, the plant alkaloid barberine and the crude extract of Moringa Oleifera (59). More recently using phage-display library, a small linear peptide called Pep2-8 (13 mer long with sequence: Ac-TVFTSWEEYLDWV) (Ac=Acetyl) which structurally mimics EGF-A domain of LDL-R has been identified as the smallest PCSK9 inhibitor with IC50˜1.4-0.2 μM. Another study revealed that a peptide derived from EGF-A domain of LDL-R also exhibited potent PCSK9 inhibition.
The present invention concerns small molecule PCSK9 inhibitors based on peptides derived from its own catalytic domain (aa153-421) (
Research in this field is gaining tremendous importance for designing new cholesterol lowering agents. In fact two monoclonal antibodies, AMG145 by Amgen Company and Fab (Fragment antibody called REGN727/SAR236553 by Regeneron and Sanofi Companies directed against PCSK9 functional activity have successfully passed all short term phase and clinical trial studies including humans and will soon become first PCSK9 based therapeutics for cholesterol control. Two additional antibodies called RN316 by Pfizer Company and LGT209 by Novartis Company have either completed or in the process of Phase-II trial. Despite these success stories, research is still ongoing for identification of small compound inhibitors of PCSK9, both peptide and nonpeptide origins because of the many reasons as outlined above, but very little data are currently available in this field.
According to an 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.
The isolated or purified therapeutically effective hPCSK9 polypeptide may consist 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 consist 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 consist 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.
According to another embodiment, there is provided an isolated or purified therapeutically effective hPCSK9 polypeptide having the amino acid sequence:
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.
According to another embodiment, there is provided 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 the present invention, or combinations thereof, or a pharmaceutical composition of the present invention.
The method may comprise further 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.
According to 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 consist 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 consist 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 consist 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.
According to another embodiment, there is provided an isolated or purified therapeutically effective hPCSK9 polypeptide having the amino acid sequence:
for preventing or treating hypercholesterolemia.
The isolated or purified therapeutically effective hPCSK9 polypeptide of the present invention may be for use with 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.
According to 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 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.
According to 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 in a subject in need thereof.
The isolated or purified therapeutically effective hPCSK9 polypeptide may consist 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 consist 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 consist 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.
According to another embodiment, there is provided a use of an isolated or purified therapeutically effective hPCSK9 polypeptide having the amino acid sequence:
for preventing or treating hypercholesterolemia in a subject in need thereof.
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.
According to 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:
The isolated or purified therapeutically effective hPCSK9 polypeptide may consist 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 consist 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 consist 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.
According to 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:
The kit of the present invention 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 following terms are defined below.
The term <<composition>> as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to pharmaceutical composition or other compositions in general, is intended to encompass a product comprising the active ingredient(s) (i.e. the polypeptides of the present invention) and the inert ingredient(s) (i.e. the pharmaceutically acceptable carriers) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions or other compositions in general of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” or “acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
In some embodiments, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
The terms “inhibit”, “inhibition” or “inhibiting” as used herein in the context of the invention means to slow, hinder, restrain reduce or prevent. For example, “inhibiting growth” of a tumor cell as that term is used herein means to slow, hinder, restrain, reduce or prevent the tumor cell from growing.
The term “administering” as used herein refers to any action that results in exposing or contacting a composition containing a therapeutic IgE antibody specific for a cancer antigen, an antigen present in or around a stroma of a tumor, or a combination thereof, alone or in combination with at least one anti-cancer agent. As used herein, administering may be conducted in vivo, in vitro, or ex vivo. For example, a composition may be administered by injection or through an endoscope. Administering also includes the direct application to cells of a composition according to the present invention. For example, during the course of surgery, tumor cells may be exposed. In accordance with an embodiment of the invention, these exposed cells (or tumors) may be exposed directly to a composition of the present invention, e.g., by washing or irrigating the surgical site and/or the cells, or by direct intra-tumoral injection of the therapeutic IgE antibody specific for a cancer antigen, an antigen present in or around a stroma of a tumor, or a combination thereof with at least one anti-cancer agent individually or in a mixture.
A “subject” is preferably a human subject but can also be any mammal, including an animal model, in which modulation of an autoimmune reaction is desired. Mammals of interest include, but are not limited to: rodents, e.g. mice, rats; livestock, e.g. pigs, horses, cows, etc., pets, e.g. dogs, cats; and primates. A subject may also be referred to herein as a “patient”.
The terms “treatment”, “treat” and “treating” encompasses alleviation, cure or prevention of at least one symptom or other aspect of a disorder, disease, illness or other condition (collectively referred to herein as a “condition”), or reduction of severity of the condition, and the like. A composition of the invention need not affect a complete cure, or eradicate every symptom or manifestation of a disease, to constitute a viable therapeutic agent.
As is recognized in the pertinent field, drugs employed as therapeutic agents may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total, whether detectable or undetectable) and prevention of relapse or recurrence of disease. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a condition in order to constitute a viable prophylactic agent. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient.
“Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In one embodiment, an indication that a therapeutically effective amount of a composition has been administered to the patient is a sustained improvement over baseline of an indicator that reflects the severity of the particular disorder.
By a “therapeutically effective amount” of a composition of the invention is meant an amount of the composition which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect is sufficient to “treat” the patient as that term is used herein.
As used herein, the terms “co-administration” or administration “in combination” refers to administering to a subject, at least one 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 other therapeutic agent(s). The other therapeutic agent(s) and or purified therapeutically effective hPCSK9 polypeptide can be administered at the same time, separately, or sequentially, according to the methods disclosed herein.
Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
It is noted that terms like “preferably”, “commonly”, and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.
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:
In embodiments there is disclosed isolated or purified therapeutically effective hPCSK9 polypeptides derived from the amino acid sequence
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).
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:
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.
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.
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.
According to another embodiment, the isolated or purified therapeutically effective hPCSK9 polypeptide having the amino acid sequence:
for preventing or treating hypercholesterolemia in a subject in need thereof.
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.
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.
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:
for preventing or treating hypercholesterolemia.
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.
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:
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:
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.
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 hPCSK9365-384 (365GEDIIGASSD[CSTC]FVSQSG384) (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 S—S bond between Cys375 and Cys378 (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 D374 (shown above in bold italic) to Y. Mimicking this mutation in the peptide (substitution of D374 by Y, i.e. 365GEDIIGASSY[CSTC]FVSQSG384, 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.
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.
Materials
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.
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. α-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).
Antibodies
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. Aβ-actin-HRP (Horse Radish Peroxidase) (#ab49900) and αFLAG-HRP (#1238) primary antibodies were both bought from ABCAM Company. Goat-HRP (#ab6741) and α mouse-HRP (#172-1011) secondary antibodies were obtained from ABCAM and Bio-rad company respectively.
Peptide Synthesis
hPCSK9 Catalytic Domain Derived Peptides.
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 15aa except for the last one P51 which is 18aa 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.
Q
QRLARA
QRLARAGVVLV
Table 1 lists of 51 (P1-P51) peptides (15 aa long with 10aa 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 Fl-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 D186, H226 and S386 and the oxyanion N317 residue were shown with underline whereas the crucial D374 whose natural mutation to Y leads to most potent gain of function is depicted in bold. The second most potent gain of function mutation R357 to H is also shown in bold. The single presumed unpaired Cys residue at position 301 is indicated in bold italics character.
Fl/Bio-EGF-A peptide (Ia and Ib).
The synthesis of fluorescein labeled EGF-A peptide (Fl-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
Fl-Bio-Ahx-Lys-Methyl Ester (IIId).
Fluorescent biotinylated lysine methyl ester [Fl-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
Peptide Purification by RP-HPLC.
All crude peptides except Fl-Bio-Ahx-Lysine methyl ester (IIIb) were purified by Reverse Phase High Performance Layer Chromatography (RP-HPLC) using C18 Silica gel analytical column (Varian, 1×25 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.
Cell Culture
The human hepatic HepG2 cells were maintained at 37° C. with 5% CO2 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 μM 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.
Protein Assay
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.
Western Blot Analysis and SDS-PAGE
In general 20 μg 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), αβ-actin-HRP (Horse Radish Peroxidase) (abcam #ab49900) and αFLAG-HRP (abcam #1238) primary antibodies and detected with α 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.on
Recombinant hPCSK9 WT and D/Y
Recombinant PCSK9 wild type (WT) as well as D374/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.
In Vitro Binding Assays
Binding assays of various PCSK9 peptides were carried out by incubating 15 μg of each peptide (˜0.008 mM) with 20 μg of FL-EGF-A in a final volume of 20 μl of 25 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl2 for 2 h at 37° C. Similar study was also performed using recPCSK9 wild type as well as D/Y variants against Fl-EGF-A. Each sample along with the control without any added peptide was assessed for any binding between Fl-EGF-A and peptide using fluorescence study, Mass spectrometry and Native gel studies
Fluorescence study: The fluorescence intensity of Fl-EGF-A peptide solution in water (typically 5 μl of 0.5 mM concentration) was measured in the absence and presence of aqueous solution (10 μl, 0.5 mM concentration) of each hPCSK9 catalytic peptide (P1-P51) solution at λex and λem 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 μl capacity) using fluorescence spectrophotometer (Molecular Devices Co, USA).
SELDI-Tof-Mass Spectrometry:
SELDI-Tof mass spectrum was performed on gold plate chips with 2 μl of sample and 2 μl 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).
Native Gel Electrophoresis: Native gel electrophoresis was performed on each incubated sample under SDS free condition in Tris-Glycine gel. Typically 5 μl of each peptide sample was pre-incubated with 20 μl 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.
Fluorescence Gel Electrophoresis:
This is performed on the samples (typically 5 μl) containing Fl-EGFA+rec-PCSK9 WT or its D/Y variants as well as the corresponding control consisting of Fl-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.
MTT Assay
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 diphenyl—Tetrazolium Bromide Tetrazolium salt (MTT).
Statistical Analysis
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.
hPCSK9 Catalytic Peptides (P1-P51)
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 42aa long EGF-A domain of the latter that also possesses a strong Ca+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.
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 10aa 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 D186, H226, N317 and S386 as well as the two most potent gain of function mutations (D374/Y (and R357/H) in these peptides were highlighted. In addition, the seven Cys residues, 6 of which are inter-linked via S—S 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 Fl-EGF-A (see later).
Fl-EGF-A (Ia) and Bio-EGF-A (Ib)
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 (
However following reduction with TCEP [Tris (2-carboxy ethyl) phosphine], it reacted with 6 molecules of iodoacetamide leading to hexa-acetamidyl derivative (increase of 6×57=342 Da in MW) as revealed by mass spectrum (not shown). This result confirmed the presence of 3 S—S bonds in the starting peptide. Furthermore it is noted that Fl-EGF-A upon high-power laser treatment during mass spectrum exhibited two additional broad peaks at m/z ˜2,980 and ˜1,987. It is proposed that these two peaks are likely generated through breakdown of Fl-EGF-A molecule via a 6-member transition state mechanism as shown in
Fl-Bio-Ahx-Lys-Methyl Ester (IIId)
This model bis-functional Lysine derivative was synthesized in 4 steps as described in
hPCSK9 Catalytic Peptides Vs Fl-EGF-A
Fluorescence Quenching.
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 (Kd˜10−15 μm), a gradual suppression of fluorescence intensity was observed until it is ˜98% quenched at 8:1 molar ratio of avidin:(IIId) (
Additional fluorescence quenching studies were conducted using Fl-EGF-A peptide and each of the 51 synthetic hPCSK9 catalytic peptides, one at a time. The results are depicted in
Mass Spectrum.
SELDI-Tof mass spectra of Fl-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
Native Gel Electrophoresis
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
In addition to investigating the binding potential of PCSK9 catalytic peptides towards Fl-EGF-A, affinity study for rec FLAG-PCSK9 protein against Fl-EGF-A were also conducted. Purified rec-FLAG-PCSK9 protein WT or D/Y mutant were used. SDS-PAGE performed on Fl-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
The above binding affinities of selected PCSK9 catalytic peptides and rec-PCSK9 protein towards Fl-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.
Effect of Fl-EGF-A on LDL-R in HepG2/HuH7 Cell Lines
The first set of results are shown with Fl-EGF-A in
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 μM 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 μM, 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 (
So far all results taken together (summarized in
The presence of S—S 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 S—S bridged cyclic hPCSK9365-384 (CP3) and noncylic Cys/Ala mutant (CP3-C/A) (Table 2). These peptides were tested at 5.5 μM concentration as before in HepG2 cells for their effects on LDL-R.
365GEDIIGASSDASTCFVSQSG384
The crucial D374 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 S—S connection as well as the mutation [Cys375 to Ala (bold underlined)] were indicated in the figure.
Owing to the critical role of S—S 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 (
LDL-R Binding Linear and Cyclic Peptides from hPCSK9
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 (
Upon close examination of the location of these active peptides within hPCSK9 catalytic segment, it appears that they actually represent two S—S bridge cyclic loop domains of the protein (
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 μM concentration (
So far the data and findings are based upon binding experiments using synthetic EGF-A peptide.
Mutation and Consequence
This study identified the S—S bond containing cyclic loop peptide, hPCSK9365-384 (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 μM concentration of CP3, a ˜3.5-fold increase in LDL-R level was observed (
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.
This application claims priority of U.S. provisional patent application 62/109,790, filed on Jan. 30, 2015, the specification of which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2016/050080 | 1/29/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62109790 | Jan 2015 | US |
