Patent Application
20190002511
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 255352004700SeqList.txt, date recorded: Apr. 23, 2018, size: 11,207 bytes).
The present invention is related to isolated peptides, variants and fragments thereof which specifically bind thrombin that causes coagulopathies in humans. There is also provided uses of such peptides in methods of diagnosis and treatment of thrombosis-related diseases, and for coating of medical devices.
Haemostasis is the physiological process that minimizes extravasation of blood following a vascular injury. Blood coagulation, a part of this process involves activation of circulating zymogens by limited proteolysis in a sequential manner and culminates in the formation of a fibrin clot. Thrombin (FIIa) plays pivotal roles in haemostasis (Stubbs M. T. and Bode W. Throm Res 69, 1-58 (1993)). In its pro-haemostatic role: (a) it cleaves soluble fibrinogen to fibrin monomers, which polymerize to form a nascent clot (Versteeg H. H., et al., Physiol Rev 93, 327-358 (2013)); (b) it activates the transglutaminase (FXIII) that covalently crosslinks fibrin monomers to stabilize the clot; (c) it activates non-enzymatic cofactors (FV and FVIII) required for its own amplification; (d) it activates FXI which in turn activates the intrinsic pathway (Versteeg H. H., et al., Physiol Rev 93, 327-358 (2013)); and (e) it activates platelets by cleaving protease-activated receptors leading to their shape change, degranulation and aggregation (Monroe D. M., et al., Arterioscler Thromb Vasc Biol 22, 1381-1389 (2002)). Conversely, thrombin also plays a significant role as an anticoagulant by down regulating the progression and amplification of the blood coagulation process: upon binding to thrombomodulin, it activates protein C, which in turn inactivates both cofactors FVa and FVllla to mitigate further thrombin generation (Di Cera E. Mol Aspects Med 29(4), 203-254 (2008)). These paradoxical procoagulant and anticoagulant roles of thrombin maintain a balance between uncontrolled bleeding and formation of obstructive thrombi, with sufficient thrombus formation when required.
Cardiovascular disease is the single largest killer worldwide and is a hefty contributor to the burden of non-communicable diseases (Chaudhari K., et al., Nat. Rev. Drug. Discov 13, 571-572 (2014)). Ischemic heart disease and stroke, both of which are pathological manifestations of thrombosis are the most common examples of cardiovascular disease and account for up to one in four deaths worldwide (Raskob G. Thromb Haem 112(5), 843-943 (2014)). Anticoagulants like direct thrombin inhibitors (DTIs), direct factor Xa (FXa) inhibitors and vitamin K antagonists (VKAs) comprise a significant fraction of the current therapeutic options as antithrombotic drugs. Some examples of DTIs used as therapeutic options are bivalirudin, the synthetic analogue of hirudin which is a bivalent inhibitor binding to the thrombin active site and exosite-I; argatroban and dabigatran, small molecule univalent DTIs that bind to the active site alone; and low-molecular-weight-heparins (LMWHs) which inhibit thrombin in an antithrombin-dependent manner (Michiel Coppens, et al., Circ Res 112, 920-931 (2012)). Despite being popular options of anticoagulant therapy, these classes are fraught with limitations like narrow therapeutic window, individual dosing, high bleeding risks, poor bioavailability and high food-drug interactions (Bauer K. A. Haem 464-470 (2013)). Therefore, novel, superior anticoagulants with greater benefits are being sought.
Hematophagous animals have adapted a blood feeding diet and have evolved an assortment of molecules that control host haemostasis to ensure a continuous blood flow for successful feeding. Among anticoagulants, thrombin inhibitors take a central stage in these blood sucking parasites (Koh C. Y. and Kini R. M. Expert Rev. Haematol 1(2), 135-139 (2008)). Hirudin, haemadin, triabin, ornithodorin and rhodniin are some of the most extensively studied examples of specific families of thrombin inhibitors from hematophagous animals (Huntington J. A. Thromb Haemost 111, 583-589 (2014)). We have previously isolated and characterized a novel thrombin inhibitor we dubbed variegin (PCT/IB2008/002109), a 32-residue long peptide which is a fast, tight binding, and competitive thrombin inhibitor from the salivary gland extracts of the hard tick-Amblyomma variegatum (Koh C. Y., J Biol Chem 282 (40), 29101-29113 (2007)).
There is a need to provide more effective peptides as therapeutic agents for the treatment of cardiovascular and cerebrovascular diseases. Examples of uses include the treatment and prophylaxis of arterial and venous thrombosis causing heart attacks, stroke and embolism; for anticoagulation during unstable angina, coronary angioplasty, percutaneous coronary intervention and heart surgery. In addition, these peptides can also be developed as reagents, as anticlotting agents in blood collection tubes and as surface coating materials on medical devices such as stents, catheters and medical tubing.
The present invention seeks to solve or ameliorate the problems described above and provide new peptides and variants thereof with improved affinity for thrombin. Although newly identified peptides ‘avathrin’ and ‘ultravariegin’ show limited sequence identity with variegin, they selectively inhibit thrombin in a similar fast, tight binding competitive mode with a Ki of 545 pM and 4.4 pM, respectively. These affinities are approximately 5 and 650 times higher, respectively, for target (thrombin) than a similar peptide-based thrombin inhibitor used in the clinic (Bivalirudin™), which suffered from a lack of clear efficacy and need for continuous infusion. We have identified their important functional sites, assisted by high resolution three-dimensional structures and structure-function relationships of a number of variants of these thrombin inhibitors. New variants of these peptides with improved affinity for thrombin were designed and synthesized. We have successfully demonstrated in vivo efficacy of these peptides through a murine arterial thrombosis model. Also identified and studied other novel peptides from Ixodid ticks.
A first aspect of the invention provides thrombin inhibitors comprising an amino acid sequence selected from the group comprising SGGHQTAVPKISKQGLGGDFEEIPSDEIIE (SEQ ID NO: 1), a variant or fragment thereof, SDEAVRAIPKMYSTAPPGDFEEIPDDAIEE (SEQ ID NO: 2), a variant or fragment thereof, SEQ ID NO: 22, a variant or fragment thereof, SEQ ID NO: 23, a variant or fragment thereof, SEQ ID NO: 24, a variant or fragment thereof and SEQ ID NO: 25, a variant or fragment thereof.
In a preferred embodiment the amino acid sequence is selected from the group comprising;
Another aspect of the invention provides an isolated thrombin inhibitor comprising an amino acid sequence according to any aspect of the invention for the prophylaxis or treatment of a disease associated with thrombus.
Another aspect of the invention provides a method of inhibiting thrombin activity, wherein the method comprises contacting thrombin with at least one thrombin inhibitor according to any aspect of the invention.
According to another aspect of the invention there is provided a use of a thrombin inhibitor according to any aspect of the invention for the preparation of a medicament for the prophylaxis and/or treatment of a disease associated with thrombus.
According to another aspect of the invention there is provided a method of prophylaxis and/or treatment of a disease associated with thrombus, comprising administering to a subject in need of such prophylaxis and/or treatment an efficacious amount of a thrombin inhibitor according to any aspect of the invention.
According to another aspect of the invention there is provided a method of detecting thrombin accumulation in a subject, comprising administering at least one inhibitor according to any aspect of the invention to a subject or to a tissue sample isolated from the subject, and detecting the presence of said at least one thrombin inhibitor bound to thrombin.
According to another aspect of the invention there is provided a pharmaceutical composition comprising an effective amount of at least one thrombin inhibitor according to any aspect of the invention.
According to another aspect of the invention there is provided an isolated nucleic acid molecule encoding a thrombin inhibitor according to any aspect of the invention. In a preferred embodiment the nucleic acid sequence encodes SEQ ID NO: 1 and is represented by TCGGGTGGCCATCAGACTGCTGTTCCGAAGATATCTAAGCAAGGCTTGGGTGGAGACTTT GAAGAAATTCCAAGTGATGAAATAATCGAG (SEQ ID NO: 26).
According to another aspect of the invention there is provided a kit comprising at least one thrombus inhibitor as herein defined.
Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.
For convenience, certain terms employed in the specification, examples and appended claims are collected here.
The term “comprising” is herein defined to be that where the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term “comprising” encompasses the more restrictive terms “consisting essentially of” and “consisting of.”
The term “isolated” is herein defined as a biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been isolated thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
The term “variant” is herein defined as one which has at least one nucleotide sequence that varies from a reference sequence via substitution, deletion or addition of at least one nucleic acid, but encodes an amino acid sequence that retains the ability to recognize, bind and inhibit thrombin. The term ‘variant’ also applies to an amino acid sequence that varies from at least one reference sequence via substitution, deletion or addition of at least one amino acid, but retains the ability to recognize, bind and inhibit thrombin. In particular, the variants may be naturally occurring or may be recombinant or synthetically produced. More in particular, the variant may be of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the reference sequences. For example, the AvathrinK10R amino acid sequence set forth in SEQ ID NO: 7 was created by substituting amino acids in the Avathrin amino acid sequence set forth in SEQ ID NO: 1 and may be considered a variant of SEQ ID NO: 1. The term “variant” may include peptide inhibitors of thrombin that have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “non-conservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNASTAR® software (DNASTAR, Inc. Madison, Wis., USA). Another form of variant includes substitution of an amino acid with a modified amino acid, such as beta-homoarginine in β-ultravariegin (SDEAVRAIPXMYSTAPPGDFEEIPDDAIEE) set forth as “X” in SEQ ID NO: 9. For the purpose of the sequence listing, the beta-homoarginine will be represented by “X” or “Xaa”.
The term “fragment” as used herein, refers to an amino acid sequence that is altered by one or more amino acids, but retains the ability to recognize and bind the same conformational epitope on thrombin as the reference sequence. For example, the QTAVPKISKQGLGGDFEEIPSDEIIE (QT26) amino acid sequence set forth in SEQ ID NO: 3 is shorter than the Avathrin sequence set forth in SEQ ID NO: 1 (SGGHQTAVPKISKQGLGGDFEEIPSDEIIE) and may be considered a fragment of SEQ ID NO: 1 because it retains the ability to recognize, bind and inhibit thrombin. Likewise, the IS20 (ISKQGLGGDFEEIPSDEIIE) amino acid sequence set forth in SEQ ID NO: 4 is shorter than the Avathrin sequence set forth in SEQ ID NO: 1 (SGGHQTAVPKISKQGLGGDFEEIPSDEIIE) and may be considered a fragment of SEQ ID NO: 1 because it retains the ability to recognize, bind and inhibit thrombin.
The term “sample,” as used herein, is used in its broadest sense. A biological sample suspected of containing thrombin may comprise a bodily fluid or a tissue.
The term “treatment”, as used in the context of the invention refers to prophylactic, ameliorating, therapeutic or curative treatment.
The term “subject” is herein defined as vertebrate, particularly mammal, more particularly human. For purposes of research, the subject may particularly be at least one animal model, e.g., a mouse, rat and the like. In particular, for treatment of thrombus and/or thrombus-linked diseases, the subject may be a human.
The present invention, in one aspect provides an isolated thrombin inhibitor comprising an amino acid sequence selected from the group comprising SGGHQTAVPKISKQGLGGDFEEIPSDEIIE (SEQ ID NO: 1), a variant or fragment thereof, SDEAVRAIPKMYSTAPPGDFEEIPDDAIEE (SEQ ID NO: 2), a variant or fragment thereof, SEQ ID NO: 22, a variant or fragment thereof, SEQ ID NO: 23, a variant or fragment thereof, SEQ ID NO: 24, a variant or fragment thereof and SEQ ID NO: 25, a variant or fragment thereof. It would be understood that these sequences, as herein disclosed, may be altered and still retain an ability to inhibit thrombin activity.
For example, it was found that residues 7(A), 9(P), 10(K), 19-21(DFE), 23(1) and 24(P) of SEQ ID NO: 1 and SEQ ID NO: 2 are important for thrombin inhibitory activity because they are the least tolerant to changes. If most of these key residues are retained, we have produced peptide variants with as low as 30% identity that retains thrombin inhibitory activity.
In a preferred embodiment the isolated thrombin inhibitor comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more preferably at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. More preferably, the isolated thrombin inhibitor comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, or more preferably at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.
In a preferred embodiment the isolated thrombin inhibitor amino acid sequence is selected from the group comprising;
Preferably said inhibitor has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
In another preferred embodiment, said thrombin inhibitor inhibits thrombin fibrinogenolytic activity and/or inhibits thrombin amidolytic activity.
In another preferred embodiment, the thrombin inhibitor has an IC50 of less than 400 nM, preferably less than 300 nM, less than 200 nM, less than 100 Nm, less than 50 nm, less than 10 nM, preferably less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM or less than 1 nM when assessed in an amidolytic assay. More preferably, the IC50 is less than 2 nM. It is intended that IC50 values between those specifically recited are within the scope of the invention.
In another preferred embodiment, the thrombin inhibitor has a Ki of less than 6000 nM, preferably less than 2000, less than 500, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 Nm, less than 50 nm, less than 10 nM, preferably less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM or less than 1 nM when assessed in an amidolytic assay. More preferably, the Ki is less than 2 nM. It is intended that Ki values between those specifically recited are within the scope of the invention.
Another aspect of the invention provides an isolated thrombin inhibitor comprising an amino acid sequence according to any aspect of the invention for the prophylaxis or treatment of a disease associated with thrombin activity.
Another aspect of the invention provides a method of inhibiting thrombin activity, wherein the method comprises contacting thrombin with at least one thrombin inhibitor according to any aspect of the invention. In a preferred embodiment, the at least one thrombin inhibitor is present as an anticlotting agent in blood collection tubes, or as a surface coating material on medical devices such as stents, catheters and other medical tubing.
According to another aspect of the invention there is provided a use of a thrombin inhibitor according to any aspect of the invention for the preparation of a medicament for the prophylaxis and/or treatment of a disease associated with thrombus. In a preferred embodiment, the disease associated with thrombus is selected from arterial and venous thrombosis causing heart attacks, stroke and embolism; for anticoagulation during unstable angina, coronary angioplasty, percutaneous coronary intervention and heart surgery.
Suitable methods for administering a therapeutic composition in accordance with the methods of the present invention include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, intraarterial administration), oral delivery, topical administration, buccal delivery, rectal delivery, vaginal delivery, subcutaneous administration, intraperitoneal administration, surgical implantation, local injection, and hyper-velocity injection/bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site (see, e.g., U.S. Pat. No. 6,180,082).
Regardless of the route of administration, the peptides of the present invention are typically administered in amount effective to achieve the desired response. As used herein, the terms “effective amount” and “therapeutically effective amount” refer to an amount of the therapeutic composition (e.g., a composition comprising a thrombin inhibitor polypeptide, and a pharmaceutically vehicle, carrier, or excipient) sufficient to produce a measurable biological response (e.g., a decrease in the amount of a thrombus or thrombus-related disease). Actual dosage levels of active ingredients in a therapeutic composition of the present invention can be varied so as to administer an amount of the active polypeptide(s) that is effective to achieve the desired therapeutic response for a particular subject and/or application. Of course, the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. Preferably, a minimal dose is administered, and the dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.
For additional guidance regarding formulation and dose, see U.S. Pat. Nos. 5,326,902 and 5,234,933; PCT International Publication No. WO 93/25521; Berkow, et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, N.J.; Goodman, et al., (2006) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 11th ed. McGraw-Hill Health Professions Division, New York.
According to another aspect of the invention there is provided a method of prophylaxis and/or treatment of a disease associated with thrombin activity, comprising administering to a subject in need of such prophylaxis and/or treatment an efficacious amount of a thrombin inhibitor according to any aspect of the invention. In a preferred embodiment, the disease associated with thrombus is selected from arterial and venous thrombosis causing heart attacks, stroke and embolism; for anticoagulation during unstable angina, coronary angioplasty, percutaneous coronary intervention and heart surgery.
The thrombin inhibitors of the invention may also be used topically to treat, for example, bruises with or without hematoma. The peptide inhibitors of the invention may be administered in a cream in a similar regimen as previously trialed with Hirudin, in which 280 UI/100 g was applied to unilateral acute musculoskeletal injuries (bruises) in 3-4 daily applications for 5 days [Stamenova P K., et al, Eur Rev Med Pharmacol Sci. 5(2):37-42 (2001)]. Thrombin inhibitors have also been formulated as topical application (r-hirudin 1120 IU/40 g; MINAPHARM Pharmaceuticals, Cairo, Egypt) to treat AV shunt thrombosis, contusions, distortions, muscular tears, traumatic hematomas, edema, erythema, varicosities, periphlebitis and anal periphlebitis with haemorrhoids especially those associated with thrombo-embolic complications [see Thrombexx® at minapharm.com]. The molecules of the present invention are less than half the size of hirudin, which should increase the penetration of the active ingredient (ie. thrombin inhibitor) across the skin barrier when applied topically.
According to another aspect of the invention there is provided a method of detecting thrombin accumulation in a subject, comprising administering at least one inhibitor according to any aspect of the invention to a subject or to a tissue sample isolated from the subject, and detecting the presence of said at least one thrombin inhibitor bound to thrombin.
In a preferred embodiment there is provided a method of detecting thrombin accumulation in a subject, said method comprising:
a. obtaining a tissue sample from a patient; and
b. detecting whether thrombin has accumulated in the sample by contacting the sample with at least one inhibitor according to any aspect of the invention and detecting binding between thrombin and the at least one thrombin inhibitor.
Preferably, the thrombin inhibitor comprises an amino acid sequence selected from the group comprising SGGHQTAVPKISKQGLGGDFEEIPSDEIIE (SEQ ID NO: 1), a variant or fragment thereof, and SDEAVRAIPKMYSTAPPGDFEEIPDDAIEE (SEQ ID NO: 2), a variant or fragment thereof.
In another preferred embodiment of the method, the thrombin inhibitor comprises an amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.
Preferably said inhibitor has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
According to another aspect of the invention there is provided a method of diagnosing a disease or condition associated with thrombus, comprising administering at least one inhibitor according to any aspect of the invention to a subject or to a tissue sample isolated from said subject, and detecting the presence of said thrombin inhibitor bound to thrombin wherein the detection of an elevated level of said inhibitor bound to thrombin compared to a level of inhibitor bound to a normal thrombin level is indicative of said disease or condition.
In another preferred embodiment of the method, the thrombin inhibitor comprises an amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.
Preferably said inhibitor has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
According to another aspect of the invention there is provided a pharmaceutical composition comprising an effective amount of at least one thrombin inhibitor according to any aspect of the invention.
According to another aspect of the invention there is provided an isolated nucleic acid molecule encoding a thrombin inhibitor according to any aspect of the invention. In a preferred embodiment the nucleic acid sequence encodes SEQ ID NO: 1 and is represented by;
In another preferred embodiment, the isolated nucleic acid molecule encodes a thrombin inhibitor comprising an amino acid sequence selected from the group comprising SEQ ID NO: 1 and SEQ ID NO: 2, variants or fragments thereof.
In a preferred embodiment, the isolated nucleic molecule encodes SEQ ID NO: 2 a fragment or variant therof and may be obtained by modifying the nucleic acid sequence represented by;
TCAGACGAAGCTGTCAGGGCGATTCCCAAGATGTACTCGACTGCCCCACCGGGAG ATTTCGAAACAATCCCTGACGACGCTATTGAGGAG (SEQ ID NO: 27) a fragment or variant therof. Preferably the nucleotide sequence of SEQ ID NO: 27 is altered, by replacing the codon encoding Thr22 with a suitable codon to encode Glu22, to produce the peptide of SEQ ID NO: 2. It would be understood that the native nucleotide sequence (SEQ ID NO: 27) encodes a suitable thrombin inhibitor according to the invention.
In a preferred embodiment, the nucleic acid has a sequence represented by SEQ ID NO: 26 or SEQ ID NO: 27, a variant or derivative thereof.
According to another aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the invention as herein described.
According to another aspect of the invention there is provided a host cell comprising a nucleic acid molecule or a vector according to any aspect of the invention. The host cell may be prokaryotic or eukaryotic but is preferably eukaryotic.
According to another aspect of the invention there is provided a kit to prevent or treat a disease or condition associated with thrombus comprising at least one thrombus inhibitor or medicament according to any aspect of the invention. The kit may comprise a medical device, such as a stent, catheter or other form of tube coated in the thrombus inhibitor of the invention.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books.
Materials and Methods
Kallikrein, human fibrinogen, and bovine trypsin were purchased from Merck. Chemicals Ltd. (Nottingham, UK). Bovine chymotrypsin, ferric chloride hexahydrate and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, Mo., USA). All other serine proteases were from Hematologic Technologies, Inc. (Essex Junction, Vt., USA). Recombinant thrombin was a gift from the Chemo-Sero-Therapeutic Research Institute (Kaketsuken, Japan). Chromogenic substrates were purchased from Chromogenix and Spectrozyme FIXa was from American Diagnostica. All other chemicals and reagents used were of analytical grade.
Peptide Synthesis and Purification
All peptides were synthesized using solid-phase peptide synthesis on an Intavis MultiPep RSi peptide synthesizer. Intavis Bioanalytical Instruments, Cologne, Germany) and cleaved from resin as described previously [[Koh C Y, et al., J Biol Chem 282: 29101-13 (2007)]. Crude peptides were purified using reverse-phase HPLC on an AKTA purifier from GE Healthcare (Uppsala, Sweden) with a Jupiter Proteo (5 μm, 250 mm×10 mm, 90 Å) column. The purity and mass of all peptides were determined by ESI-MS using an LCQ Fleet Ion Trap MS from Thermo Fisher Scientific (Waltham, Mass., USA).
CD Spectroscopy
Far-UV CD spectra (260-190 nm) of avathrin, QT26 and IS20 dissolved in 10 mM sodium phosphate buffer (pH 7.4) were measured using a Jasco™ J-810 spectropolarimeter (Easton, Md.). All measurements were carried out at room temperature using a 0.1-cm path length cuvette with a scan speed of 50 nm/min, a bandwidth of 2 nm and a resolution of 0.2 nm.
Inhibition of Amidolytic and Fibrinogenolytic Activities of Thrombin and Protease Activities
Thrombin amidolytic activity assays for all peptides were performed in 96-wells microtiter plates in 50 mM Tris buffer (pH 7.4) containing 100 mM NaCl and 1 mg/ml bovine serum albumin. Typically, 100 μl of peptide and 100 μl of thrombin were pre-incubated for different durations before 100 μl of S2238 was added to the reaction wells. The rates of formation of p-nitroaniline were followed by measuring the absorbance at 405 nm for 10 min with a Tecan InfinitePro M200 microplate reader from Tecan (Mannedorf, Switzerland). Dose-response curves were fitted using the GraphPad Prizm software (San Diego, Calif., USA) to calculate IC50 values and Hill coefficients. To measure the inhibitory constants, different concentrations of S2238 were used and the residual velocities were determined using the Morrison's tight binding equation. Inhibition of thrombin fibrinogenolytic activity of peptides were tested by measuring absorbance at 650 nm using a Sunrise microplate reader from Tecan (Mannedorf, Switzerland) as described previously [Koh C Y, et al., J Biol Chem 282: 29101-13 (2007)]. The selectivity of avathrin was examined against 13 serine proteases (
X-Ray Crystallography
Recombinant α-thrombin (in 150 mM NaCl) was desalted using 3000 MWCO spin filters in 20 mM NH4HCO3 and lyophilized before crystallization. The crystallization conditions for other inhibitor complexes, thrombin with variegin, hirugen and hirulog, were used and optimized further [Koh C Y, et al., PLoS One 2011; 6; Skrzypczak-Jankun E, et al., J Mol Biol 221: 1379-93 (1991)]. Avathrin (81.7 μM) was dissolved in 50 mM HEPES buffer (pH 7.4) containing 375 mM NaCl. Thrombin was dissolved in the avathrin solution to a final concentration of 54.5 μM. Crystallization was achieved using the hanging drop vapor diffusion method. Typically, 1 μl of mixture containing avathrin and thrombin was mixed with 1 μl of precipitant (100 mM HEPES, pH 7.4, 20 to 25% (w/v) PEG 8000) and left at 4° C. Crystals appeared after approximately six weeks. Crystals were soaked in a cryoprotectant solution containing the mother liquor, supplemented with 25% (v/v) glycerol, and flash cooled at 100 K in cold nitrogen gas stream (Oxford Cryosystem, Oxford, UK). A data set of 180 frames was collected (180° oscillation) using a CCD mounted on a rotating anode Rigaku X-ray generator. The data set was processed and scaled using Mosflm [Battye T G G, et al., Acta Crystallogr Sect D Biol Crystallogr 67: 271-81 (2011)] and AIMLESS [Evans P R, and Murshudov G N. Acta Crystallogr Sect D Biol Crystallogr 69: 1204-14 (2013)] respectively [Leslie A G W, and Powell H R. Evolving methods for macromolecular Crystallography. 2007]. The structure of the complex was determined by molecular replacement, using the Phaser program and the thrombin-variegin crystal structure (PDB:3B23) as the template [McCoy A J. Acta Crystallogr Sect D Biol Crystallogr International Union of Crystallography 63: 32-41 (2006)]. Model building and refinement was performed using COOT [Emsley P, and Cowtan K. Acta Crystallogr Sect D Biol Crystallogr International Union of Crystallography 60: 2126-32 (2004)].
Clot-Bound Thrombin Inhibition
Clot-bound thrombin activity was tested using S2238. Briefly, fibrin clots were prepared by incubating 100 μL of 2 mg/mL fibrinogen (in 50 mM HEPES buffer, pH 7.5, 150 mM NaCl, 10 mg/mL CaCl2)) with 100 μL 30 nM thrombin. After 2 h at 37° C., the clots were extensively washed with the same buffer. This washing was repeated after every three hours for a period of 24 hr. Different concentrations of avathrin were then added to the clots and incubated for 60 min. Chromogenic substrate, S2238 (final concentration 200 μM) was then added and the reaction mixture was incubated for 90 min at 37° C. Aliquots were taken, and substrate hydrolysis was estimated by end point reading at 405 nm, using a Tecan Infinite®Pro microplate reader. Experiments were performed in quadruplicate and percentage inhibition was plotted.
Ferric Chloride-Induced Carotid Artery Thrombosis Model
All animal experiments were carried out under protocol 041/12 approved by Institutional Animal Care and Use Committee, National University of Singapore. The ferric chloride induced carotid artery thrombosis model was performed as described previously [Eckly A, et al., J Thromb Haemost 9: 779-89 (2011)] with minor modifications. Typically, C57BL/6 male mice (9-11 weeks old, 24.5-27.5 g) were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and medetomidine (1 mg/kg). 100 μL of different doses of avathrin were injected into the mice via tail vein. The right carotid artery was dissected using blunt dissection and a vascular injury was caused applying filter paper of 2 mm×2 mm saturated with FeCl3 on top of the carotid artery. After 3 min, the filter paper was removed and the vessel was washed with sterile normal saline. To determine the time to occlusion, a miniature Doppler flow probe (Transonic Systems Inc., Ithaca, N.Y., USA) was placed around the carotid artery and blood flow was recorded using a Transonic® flow meter from ADlnstruments (Dunedin, New Zealand). The maximal time for monitoring the blood flow after injury was 30 min. Mice were euthanized by cervical dislocation immediately after the completion of the experiment and prior to recovery from anesthesia.
Collection of Blood into Blood Tubes with Peptides
Individual peptides are dissolved in phosphate buffered saline to prepare 10× peptide solutions (ie. concentrations of peptides are 10 times higher than the final concentrations at which they are to be tested). 0.3 ml of the 10× peptide solutions are placed in blood tubes without any additives. Blood was drawn from healthy volunteers using syringes and immediately transferred to a 50 ml conical centrifuge tube (Falcon tube) before pipetting 2.7 ml of the blood into blood tubes with peptide solutions. Final concentrations of peptides are as stated in Table 5 and 6.
Anti-Coagulation Effect of Peptides in Blood Tubes
Blood tubes with various peptides at different concentrations were left standing at room temperature until tested at designated times. At different time points, tubes were inverted several times and visually inspected for insoluble materials as indications of clot formation.
Preservation of Platelet Function in Blood Tubes Added with Peptides
Blood tubes with various peptides at different concentrations were left standing at room temperature until tested at designated times. At different time points, tubes were inverted several times and blood samples were taken out for platelet aggregation tests using a Multiplate® Platelet aggregometer with adenosine diphosphate (ADP) as agonist according to manufacturer's recommended protocols.
Results
Detection of Variegin-Like Transcripts in Salivary Glands of Amblyomma variegatum
Degenerate primers based on variegin sequence amplified a transcript (AB183707) encoding a 219-residue precursor protein (BAD29729) from the salivary gland cDNA of 9-days fed female Amblyomma variegatum. This precursor contains a putative secretion signal and five identical 30-residue repeats with putative cleavage sites in between, enabling post-translational cleavage of the precursor to five active peptides. Expression of this precursor protein in the salivary glands was confirmed by in situ hybridization. The probe hybridized in the cytoplasm of the large basal granular cells of type II salivary gland acini. Localization of the transcripts in salivary glands of nymphs and adult female and male ticks demonstrated differences in expression during the course of feeding as well as individual variability in onset of expression. The strongest expression was detected in 2-4 days fed nymphs, 5 days fed females and in 12 day fed males, i.e. at the time when females were engorged and started to detach (
The active peptides encoded by this transcript showed ˜40% sequence identity to variegin. These peptides also showed several differences in key functional residues compared to variegin:
(i) Variegin has an acidic N-terminus which was postulated to be important for fast binding kinetics [Koh C Y, et al., J Biol Chem 282: 29101-13 (2007)]. In contrast, acidic residues are generally absent in these variegin-like peptides;
(ii) Variegin inhibits thrombin by disrupting the charge relay system of the active site catalytic triad by its His12 possibly hydrogen bonding with Ser195 of thrombin [Koh C Y, et al., PLoS One 2011; 6]. In these variegin-like peptides, this functional histidine is replaced by serine;
(iii) Thr14 in native variegin is glycosylated and it showed 14-fold higher affinity than synthetic variegin [Koh C Y, et al., J Biol Chem 282: 29101-13 (2007)]. In variegin-like peptides, glutamine, which cannot be glycosylated was present at this position;
(iv) Pro16 and Pro17 in variegin introduce kinks in the backbone, possibly limiting conformational flexibility in the linkage between the active site and the exosite-I binding segment [Koh C Y, et al., PLoS One 2011; 6]. The similar region in variegin-like peptides contains three glycines, imparting a lot more flexibility to the peptide.
All these differences provided the impetus to evaluate the structure-function relationships of these variegin-like peptides. Hence, we proceeded to synthesize the active peptide from the precursor protein (BAD29729) to further characterize its inhibitory effect on human α-thrombin.
Avathrin is a Potent and Selective Inhibitor of Thrombin
The active peptide, named avathrin (Amblyomma variegatum thrombin inhibitor) was synthesized by fmoc-based solid phase peptide synthesis and purified to homogeneity (
The selectivity of avathrin was examined by screening it against 13 serine proteases. At 10 nM, avathrin inhibited 65% of thrombin activity. However, even at 100 μM, inhibition of other proteases was <30% (
Avathrin Exhibits Prolonged Thrombin Inhibition
Competitive inhibition of thrombin peptidyl substrate S2238 indicates that avathrin binds to the active site, and hence it may be susceptible to proteolytic cleavage by thrombin, similar to all other macromolecular substrates or inhibitors such as variegin and bivalirudin. We investigated the cleavage of avathrin by thrombin by incubating it with the enzyme (30:1 ratio) for increasing amounts of time and analysed the reaction by reverse-phase chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS). With incubations, two new peaks corresponding to SGGHQTAVPK (981.3 Da) and ISKQGLGGDFEEIPSDEIIE (2176.3 Da) were identified, indicating cleavage at Lys10-Ile11 scissile bond (
Thrombin Binding Segments on Avathrin
We synthesized two additional truncated variants of avathrin, namely QT26 and GL16, to localize thrombin binding segments on avathrin. Four and 15 N-terminal residues of avathrin were deleted in QT26 and GL16, respectively. Both peptides were tested for their ability to inhibit thrombin amidolytic and fibrinogenolytic activity. QT26 inhibited thrombin amidolytic activity (IC50=8.94±0.64 nM and Ki=760.32±0.91 μM) (
Structure-Function Relationships of Thrombin-Avathrin Interactions
Despite low overall sequence identity between variegin and avathrin, and the changes in several key functional residues as outlined above, functionally avathrin showed a high degree of similarity to variegin in its thrombin inhibitory activity. To further investigate the significance of differences in the two sequences, we evaluated a series of avathrin substitution mutants informed by previous structure-function studies with variegin [Koh C Y, et al., PLoS One 2011; 6]:
(i) The key functional residue-VHis12 of variegin that most likely disrupts the catalytic triad of thrombin is replaced with ASer12 in avathrin. We synthesized two mutants replacing ASer12 with Ala (S12A) or His (S12H). S12A showed a similar drop in potency (>10-fold) to that observed in the analogous variegin mutant (
(ii) Variegin contains two Glu residues in its N-terminus and these two acidic residues were suspected to steer variegin towards the thrombin exosite-II and confer a fast binding inhibitory mode [Koh C Y, et al., J Biol Chem 282: 29101-13 (2007)]. Although avathrin exhibited fast binding kinetics without an acidic N-terminus, we were interested in investigating the role on an acidic N-terminus on the thrombin inhibitory activity of avathrin. In one double mutant peptide G2D, Q5D, acidic residues were introduced into avathrin N-terminus to emulate the possible role of electrostatic steering in conferring fast binding kinetics to variegin [Koh C Y, et al., J Biol Chem 282: 29101-13 (2007)]. This mutant showed slightly weaker inhibition (<2-folds) than avathrin and QT26 (
(iii) To test the hypothesis that a more rigid, proline-rich linker between the active site and exosite-I binding segments in variegin (15APPF18) is beneficial compared to the flexible glycine-rich linker in avathrin (15GLGG18), the double mutant peptide L16P, G17P was synthesized and tested. This peptide registered a drop of >25-fold in activity (IC50, 181.32±3.76 nM) compared with avathrin (
(iv) Thrombin is known to prefer an arginine residue at P1 [Berliner L J. Journal of Chemical Information and Modeling. (1992)] and the substitution of P1 Arg by Lys causes a drop of 10-fold in activity [Gallwitz M, et al., PLoS One 2012; 7]. Both variegin and avathrin possess a lysine at P1 and the mutation of the P1 Lys to Arg in variegin resulted in a small gain in activity (<3-fold) [Koh C Y, et al., PLoS One 2011; 6]. Therefore, we substituted the P1 Lys in avathrin by Arg (K10R), and observed a similar 3- to 4-fold gain in activity (IC50, 1.15±0.45 nM) (
(v) Next, we synthesized a variant named β-avathrin in which the scissile peptide bond (VLys10-VIle11) was substituted by a proteolytically stable bond (β-homoArg10-Ile11). β-avathrin was >100-fold less potent than avathrin and inhibited thrombin with an IC50 of 332±1.32 nM (
Crystal Structure of Thrombin-Avathrin Complex
Thrombin-avathrin complex was crystallized in C2 space group and refined to a resolution of 2.09 Å (Table 3). The electron density for most thrombin residues is well-defined except for residues in the termini of the light chain; and in the autolysis loop and C-terminus of the heavy chain. Unfortunately, not all residues in the bound avathrin have density clear enough for unambiguous building of the model. No density for the first four (1SGGH4) and last two residues (29IE30) are observed. Electron density for avathrin is also discontinuous C-terminal to the scissile bond between AIle11 and AGly18. Thus, we built two segments of peptide, representing the active site binding segment N-terminus to the scissile bond (5QTAVPK10) and exosite-I binding segment (19DFEEIPSDEI28) (
#Ramachandran Plot statistics are as reported by the Molprobity server
Thrombin inhibition by avathrin appears to be through blocking of the active site as expected from amidolytic assays showing competitive inhibition with small peptidyl substrates. The model displayed the state of the active site after cleavage, in which thrombin charge relay system appears to be in place. Oγ of TSer195 is 2.7 Å away from Nε of THis57, and the Nδ of THis57 is in turn 2.7 Å away from 00 of TAsp102. The Ca of ALys10 (P1) appears to shift further away from the nucleophile (Oγ of TSer195) at 3.2 Å since the cleavage has occurred. The ALys10 carbonyl oxygen is still stabilized in the oxyanion hole, positioned at a distance of 3.1 Å from the backbone nitrogen of TGly193. P1 ALys10 binds in the S1 subsite as expected, with its side chain amine forming a hydrogen bond with TAsp189 at the bottom of the specificity pocket. P2 APro9 pyrrolidine ring appears to interact with aromatic side chains of THis57 and TTyr60A perpendicularly, resembling typical edge-to-face pi interactions. P3 AVal8 side chain is solvent exposed and without specific interactions. The methyl group of P4 AAla7 side chain in contrast is completely buried in a hydrophobic pocket formed by TAsn98, TLeu99, TIle174 and TTrp216. P5 AThr6 and P6 AGln5 are both solvent exposed as the peptide approaches exosite-II but there is lack of electron density for avathrin beyond this point such that it is not possible to determine if the peptide extends towards exosite-II (
Residues located immediately C-terminal to the scissile bond did not show good electron density until around exosite-I. Overall, this part of avathrin binds in the exosite-I groove like hirugen, hirulog-1 and variegin [Koh C Y, et al., PLoS One 2011; 6; Skrzypczak-Jankun E, et al., J Mol Biol 221: 1379-93 (1991); Qiu X, et al., Biochemistry 31: 11689-97 (1992)]. Both electrostatic and hydrophobic interactions appear to be important for this binding. Three electrostatic interactions (AAsp19-TArg73, AGlu21-TArg75 and AGlu27-TArg77A) between the avathrin C-terminus and exosite-I are observed (
Clot-Bound Thrombin Inhibition
Haemostatic fibrin clots trap active thrombin, limiting its circulation [Francischetti I M B, et al., Biochemistry 38: 16678-85 (1999)]. This clot-bound thrombin is protected from inhibition by the heparin-antithrombin III complex and acts as a reservoir of active thrombin, which is thought to be instrumental in re-thrombosis [Francischetti I M B, et al., Biochemistry 38: 16678-85 (1999); Bridge K I, et al., Thromb Haemost 112: 1-8 (2014)]. Thus, the inhibition of clot-bound thrombin may prevent re-thrombosis. Therefore, we evaluated the ability of avathrin to inhibit clot-bound thrombin. Avathrin inhibited clot-bound thrombin in a dose-dependent manner with an IC50 of 1.74±0.35 μM (
FeCl3-Induced Carotid Artery Thrombosis Model
In order to evaluate the in-vivo antithrombotic efficacy of avathrin, we used the FeCl3-induced carotid artery thrombosis model in mice [Wan C, et al., J Thromb Haemost 13: 248-61 (2015); Eckly a, et al., J Thromb Haemost 2011; 9: 779-89]. The average time to occlusion (TTO) increased in a dose-dependent manner in mice intravenously injected with avathrin. From 7.24±1.46 min in control animals the TTO increased to 15.03±3.23 and 22.51±4.19 min in animals injected with 3 and 10 mg/kg, respectively. The efficacy of avathrin was compared to hirulog-1 as a comparator drug. The TTO in mice injected with 3 and 10 mg/kg of hirulog-1 were 9.70±3.15 and 15.22±3.39 min, respectively. Thus, avathrin showed a better antithrombotic efficacy compared to hirulog-1 (
Identification of Peptide Sequences from Ixodid Tick Transcriptomes
Peptide sequences which were similar to variegin and avathrin were identified by performing a standalone BLAST analysis of published transcriptomes of Amblyomma variegatum, Rhipicephalus puichellus, Amblyomma americanum, Amblyomma cajenesse, Amblyomma maculatum and Hyalomma marginatum rufipes. These sequences were manually aligned with variegin and avathrin and one peptide from each tick was selected for further analysis.
Inhibition of Amidolytic Activity and Selectivity of Other Peptides
A few more protein sequences with similarity to the avathrin transcript from the A. variagatum and other hard ticks can be found in NCBI database. Some of these sequences were synthesized and tested for their thrombin inhibitory activity (
Amblyomma americanum
Amblyomma maculatum
Rhipicephalus sanguineus
Hyalomma marginatum
rufipes
Ultravariegin is a 30-residue peptide represented by SEQ ID NO: 2 and is based on 30 residues stretch of a 212 amino acids protein sequence derived from a database transcript. However, we substituted one amino acid residue in ultravariegin from the 30 residues stretch sequence found in the 212 amino acids protein sequence (Thr22Glu to arrive at SEQ ID NO: 2). The peptide of SEQ ID NO: 2 was found to inhibit thrombin with a Ki of 4.4 μM. Compared to variegin, which has a Ki of 342 μM, ultravariegin is more than 70-fold more potent. To further understand structure-function relationships of ultravariegin in the inhibition of thrombin, a few more variants of ultravariegin were synthesized as follows. UV003, UV004 and UV005 are ultravariegin-variegin hybrid peptides. Based on ultravariegin sequence, the first 7 residues on the N-terminal were replaced by variegin sequence in UV003. The subsequent 7 residues were replaced in the same manner in UV004. In UV005, the last 6 residues in ultravariegin were replaced by last 8 residues of variegin (variegin has two extra residues). The Ki of UV003 and UV004 were found to be similar to that of ultravariegin, showing that replacement of ultravariegin sequences with that of variegin in the first 14 residues on the N-terminal is largely inconsequential for its activity (
To ascertain the role of ultravariegin C-terminal, the C-terminal cleavage product of ultravariegin by thrombin was synthesized and tested as UV011 (
Four peptides with cysteine residue(s) either at the N- or C-terminal are synthesized and tested for activities. Peptide variants with cysteine residue(s) provide a mean to covalently immobilize the peptides on surfaces for coating. So far, addition of cysteines at the termini of peptides generally cause a slight but acceptable drop in the activity compared to analogous sequences without cysteine(s). IC50 and Ki values of the peptides are as listed in Table 5.
Inhibition of thrombin amidolytic activity and selectivity of peptides from other ticks reported above was tested. All peptides were found to selectively inhibit thrombin. Kinetics and selectivity of these peptides are shown in detail in
Thrombin Inhibitors as Stabilizing Agent in Blood Collection Devices
We tested variegin, ultravariegin and avathrin for anticoagulant effect in blood tubes, at room temperature, at three concentrations: 75 μM, 150 μM and 300 μM. Variegin, but not avathrin and ultravariegin, was previously included as an additive in blood tubes in a patent application (WO2012075407A2). Avathrin (Ki=545 μM) has similar affinity as variegin (Ki=318 μM) and it showed comparable anticoagulation effect as variegin (Table 6).
The time for clot formation to be observed is identical for both avathrin and variegin at the same concentration (eg. 52-66 h at 150 μM). As expected, ultravariegin outperformed avathrin and variegin (Table 3). This is consistent with the >200-fold stronger affinity of ultravariegin (Ki=1.5 μM) compared to the latter two peptides. Even at the lowest concentration tested, the anticoagulation effect of ultravariegin (91-101 h at 75 μM) lasted longer than the highest concentration of variegin/avathrin tested (52-66 h at 300 μM). In contrast, another variant (β-variegin) has Ki>30 nM (ie. >100-fold weaker than variegin) and is only able to prevent clot formation for 3-4 h. The data showed that affinity of peptides for thrombin correlated well with their anticoagulation effect in blood tubes.
Extended Stability of Blood for Platelet Function Test
We also tested the platelet aggregation responses of blood anticoagulated with ultravariegin, avathrin and variegin at 150 μM and 300 μM using Multiplate® analyzer. We used ADP (6.5 μM) as the agonist. We used citrate and hirudin as two controls. Results obtained are shown in Table 7 and
Since there is no consensus in the literature on the lower limit of response which can be
$Concentration of hirudin is as defined in the hirudin Vacuette ® tube product information
considered as reliable reading, we decided on using a cut-off of 32 U (
Blood tubes with ultravariegin, avathrin and variegin all showed as least 6-fold increase in stabilization window compared to the gold standard, citrate. In the first 24 h, blood in hirudin (
Despite the low overall sequence identity between avathrin and variegin (40%) and variations in key functional residues as detailed above, avathrin appears to function in a similar manner to variegin. Both inhibit thrombin as fast, tight-binding bivalent inhibitors targeting the active site and exosite-I with similar inhibitory constants (Ki of variegin=342 μM, Ki of avathrin=545 μM). Both variegin and avathrin bind to thrombin canonically, and are hence cleaved by thrombin upon binding. Cleavage of variegin (4 h to completion) by thrombin proceeds faster than avathrin (10 h to completion). The slower cleavage of avathrin could be due to the overall flexibility conferred by AGly15, AGly17 and AGly18. The cleavage products for both peptides inhibited thrombin non-competitively with respect to small peptidyl substrates, with the affinity of variegin cleavage product (MH22) 2-fold lower than that of avathrin's (IS20) [Koh C Y, et al., ChemBioChem 10: 2155-8 (2009)]. The slower cleavage rate of avathrin could likely be due to the higher affinity of IS20, reducing the availability of free thrombin for cleavage of the peptide at equilibrium. We have also shown that serine at position 12 appeared to confer slightly better affinity than histidine, while acidic residues at the N-terminus do not determine the fast binding kinetics. Comparison between sequences of variegin and avathrin allowed these focused structure-function studies and helped in understanding thrombin-inhibitor interactions.
The structure of the thrombin-avathrin complex is best compared to that of thrombin-hirulog-1 complex (RMSD of 0.41 Å for 279 residues) as both are peptidyl bivalent thrombin inhibitors crystallized as cleaved peptides [Skrzypczak-Jankun E, et al., J Mol Biol 221: 1379-93 (1991); Bourdon P, et al., FEBS Lett 294: 163-6 (1991)]. Hirulog-1 is a shorter peptide compared to avathrin but the two peptides show a significant degree of identity in their thrombin active site and exosite-I binding sequences. After cleavage, only the segment N-terminal to the scissile bond and the segment in exosite-I can be built in their respective structures. Both crystals have similar unit cell dimensions (C2; a/b/c≈70/72/72 Å; β=100°). In contrast, the thrombin-variegin complex crystallized in a different crystal form (C2; a/b/c=125/51/62 Å; β=99°), and appears to have only the cleaved C-terminal peptide bound.
Both avathrin and hirulog-1 have their P1 residues binding to the same S1 pocket on thrombin. Although the P1 Lys of hirulog-1 binds to TAsp189 at the bottom of the S1 pocket through a water molecule [Bode W. Blood Cells, Mol Dis 36: 122-30 (2006)], we observed direct interaction between ALys10 and TAsp189. The same study with hirulog-1 showed a 10-fold decrease in affinity when Arg is replaced by Lys but we see a smaller change (˜3-fold) with ALys10 to Arg mutation, in agreement with the direct interaction observed in the structure. Hirulog-1 and avathrin share the same P2 amino acid (Pro) and hence interact in a similar way to the S2 subsite. Hirulog-1 P3 is a D-Phe, occupying the same hydrophobic pocket that avathrin P4 AAla7 occupies [Skrzypczak-Jankun E, et al., J Mol Biol 221: 1379-93 (1991)]. The chromogenic substrate S2238 used in all our enzymatic assays is almost identical to hirulog-1 at P3 to P1 (D-Phe-Pipecolic acid-Arg). Considering that avathrin and hirulog-1 bind to the same sites on thrombin at these positions, the competitive mechanism of inhibition is in agreement with the structure.
The first six residues of exosite-I binding segments of avathrin (DFEEIPSDEIIE), hirulog-1 (DFEEIPEEYL) and variegin (DFEAIPEEYLDDES) are almost identical (underlined). These residues are also aligned well in the crystal structures (
Despite the use of variegin sequence to design primers for amplification, we did not manage to amplify a variegin gene, suggesting a high degree of variability in the peptides produced by a multitude of genes in these ticks at different points of feeding. More surprisingly, both avathrin and variegin appear to be synthesized as larger precursor proteins containing multiple repeats, and processed into shorter active peptides which possess thrombin inhibitory activity. Despite the low overall sequence identity and variation in some key functional residues, both peptides have similar inhibitory mechanisms and effects on thrombin. A search of the database uncovered more similar sequences of such precursor proteins containing repeats of variegin-like peptides in Amblyomma variegatum (BM291228: 3 peptides, incomplete transcript; BM293052: five peptides; BM289492: five peptides), Amblyomma americanum (ACG76173: 5 peptides, incomplete transcript), and Amblyomma cajennense (ACAJ0085C_1: 4 peptides) [Nene V, et al., Int J Parasitol 32: 1447-56 (2002); Batista I F C, et al., Toxicon 51: 823-34 (2008)] (
In conclusion, we have demonstrated that hard ticks disable thrombin, which is a crucial enzyme in coagulation by employing a multitude of diversified sequences while maintaining a largely similar overall scaffold and function. We have demonstrated that avathrin and several other peptides prevented thrombosis better than hirulog-1, despite similarity in sequences, in the FeCl3-induced carotid artery thrombosis model. This family of molecules could produce anticoagulants useful for several clinical indications and cardiovascular procedures such as prevention of arterial thrombosis and reocclusion during invasive procedures, venous thrombosis prophylaxis after an orthopaedic surgery and management of myocardial infarction after detailed evaluation of safety and efficacy profiles [Bauer K A. Hematology Am Soc Hematology Educ Program 2013: 464-70 (2013)].
This application is a U.S. National Phase Application of International Patent Application No. PCT/SG2016/050278 having an international filing date of Jun. 17, 2016, published as WO 2016/204696 A1 on Dec. 22, 2016, which claims priority to U.S. Provisional Patent Application No. 62/230,923 filed Jun. 18, 2015, the disclosures of which are herein incorporated by reference in their entireties for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2016/050278 | 6/17/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62230923 | Jun 2015 | US |
