Thrombin inhibitors for treatment of stroke and related coagulative disorders
11091535 · 2021-08-17
Assignee
Inventors
- Richard J. Payne (Sydney, AU)
- Shaun Phillip Jackson (Sydney, AU)
- Pedro Jose Barbosa Pereira (Sydney, AU)
Cpc classification
A61P7/02
HUMAN NECESSITIES
International classification
Abstract
The treatment of stroke related diseases are described herein. In addition, fibrin clot formation and related activity is proposed for the disclosed compounds, in particular, peptides for inhibiting or modifying the cleavages of fibrinogen by thrombin. Methods for producing such compounds are also disclosed.
Claims
1. A peptide comprising the amino acid sequence ZXZYZXYZXXX (SEQ ID NO: 2), wherein Z is any amino acid selected from D, E, Q, S or P and X is any amino acid, wherein at least one residue of SEQ ID NO: 2 is a sulphated tyrosine wherein the peptide comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 28-38 or SEQ ID NOs: 40-44.
2. The peptide of claim 1, wherein the peptide binds to exosite II of thrombin.
3. The peptide of claim 1, wherein the peptide competitively inhibits the binding of clot-associated fibrin.
4. The peptide of claim 1, wherein the tyrosine at position 4 in SEQ ID NO: 2 is sulphated.
5. The peptide of claim 1, wherein the tyrosine at position 7 in SEQ ID NO: 2 is sulphated.
6. The peptide of claim 1, wherein the peptide consists of the amino acid sequence of as set forth in any one of SEQ ID Nos: 28-38 or SEQ ID Nos: 40-44.
7. The peptide of claim 1, wherein the peptide is no more than 35 amino acids in length.
8. The peptide of claim 3, wherein the peptide competitively inhibits the binding of fibrinogen γ′ to exosite II of thrombin.
9. The peptide of claim 1, wherein the tyrosine residues at positions 4 and 7 of SEQ ID NO: 2 are sulphated.
10. The peptide of claim 1, wherein the peptide comprises the amino acid sequence DADYDDYDEEG (SEQ ID NO: 31).
11. The peptide claim 1, wherein the peptide comprises the amino acid sequence DGDYDEYDNDE (SEQ ID NO: 29).
12. The peptide claim 1, wherein the peptide comprises 49-F the amino acid sequence DADYDEYEEDG (SEQ ID NO: 28).
13. The peptide of claim 1, wherein the peptide the amino acid sequence DADYDDYDEEG (SEQ ID NO: 31).
14. The peptide of claim 1, wherein the peptide the amino acid sequence DGDYDEYDNDE (SEQ ID NO: 29).
15. The peptide claim 1, wherein the peptide the amino acid sequence DADYDEYEEDG (SEQ ID NO: 28).
16. A thrombin inhibitor of Formula 1: wherein: A is the peptide of claim 1; B is a peptide having a sequence shown in any one of SEQ ID NOS: 15 to 24 for binding to the thrombin active site to prevent the thrombin active site from cleaving fibrinogen to form fibrin; and wherein A and B are linked so as to enable A to bind to exosite II of thrombin when B is bound to the thrombin active site.
17. The thrombin inhibitor of claim 16, wherein the thrombin inhibitor provides for a bleeding time or a clotting time that is at least 20% shorter than hirudin on a molar equivalent basis of the peptide and hirudin.
18. The peptide of claim 16, wherein the peptide comprises the amino acid sequence of DGDYDEYDNDE (SEQ ID NO: 29).
19. The peptide of claim 16, wherein the tyrosine residues at positions 4 and 7 of SEQ ID NO: 2 are sulphated.
20. A pharmaceutical composition including a peptide of claim 1, together with a pharmaceutically effective carrier, diluent or excipient.
21. A method of thrombolytic therapy comprising administering a therapeutically effective amount of a peptide of claim 1 to a subject in need thereof.
22. The method of claim 21, wherein the thrombolytic therapy includes tPA therapy.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
DETAILED DESCRIPTION OF THE EMBODIMENTS
(7) The invention is particularly concerned with inhibiting the binding of endogenous components of the coagulation cascade to exosite II of thrombin. Disclosed herein are peptides that bind to thrombin exosite II and that competitively inhibit some of these components.
(8) It is proposed that these peptide inhibitors may be utilised to inhibit fibrin formation by clot-associated thrombin, thereby addressing some of the issues regarding re-canalisation associated with tPA therapy. The peptide inhibitors may also be used to modify the binding affinity and/or function of other peptides that bind to and block function of the thrombin active site, or the thrombin exosite I.
A. Definitions
(9) “Thrombin” is a serine protease having a central role in hemostasis through the conversion of fibrinogen to fibrin.
(10) “Thrombin active site” is a catalytic site that cleaves a range of substrates including fibrinogen, fibrinopeptides, Factor V, Factor VIII, protease activated receptors (PARs), glycoprotein V, Factor XI, Factor XIII, ADAMTS13, protein C.
(11) “Thrombin exosite II”, (also known as “heparin binding exosite”) is a positively charged recognition surface that influences substrate and cofactor binding, particularly fibrinogen, Gp1bα, and heparin. It may include residues R93, R101, R126, K236, K240, and R233.
(12) “Thrombin exosite I” (also known as “fibrinogen binding exosite”) is a positively charged recognition surface that influences substrate and cofactor binding, particularly fibrinogen, Factor V, Factor VIII, ADAMTS13, Factor XIII, PAR-1, Factor XI and thrombomodulin. It may include residues K36, H71 R73, R75, Y76, and R77.
(13) Thrombin, including the thrombin active site and exosites I and II are generally discussed in Lane D. et al. 2005 Blood J. 106:2605-2612.
(14) “sulphated tyrosine residue” and “tyrosine-O-sulfate” is a residue arising from the transfer of a sulfur-containing group to the hydroxyl side chain of tyrosine. The residue may arise from the action of a tyrosylprotein sulfotransferase (TPST).
(15) “bleeding time” generally refers to the time required for bleeding to stop. It has been utilised clinically to assess platelet function. A range of assays for assessing bleeding time are known in the art: See in general Greene T. K. et al. 2010 J. Thromb and Haem 8:2820-2822; Liu Y., et al. 2012 WJEM 2: 30-36; Broze G. J. et. al 2001 Thromb Haemost 85:747-748.
(16) “clotting time” generally refers to the time required for formation of a fibrin clot. Typically clotting time is assessed by the activated partial thromboplastin time (APTT) test.
(17) “Hirudin” is a low molecular weight peptide (7 kDa) comprised of 65 amino acids (Dodt et al., 1984 FEBS Lett., 165: 180-4) which prevents blood from coagulating by binding to exosite I and the thrombin active site (Stone and Hofsteenge, 1986 Biochem, 25:4622-28).
(18) “Comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.
B. Exosite II Inhibitors
(19) In one embodiment there is provided a peptide comprising, or consisting of an amino acid sequence shown in
(20) TABLE-US-00005 SEQ ID No: 1 PXYXXXZZPXYZZZ
(21) wherein
(22) Z is D or E and
(23) X is any amino acid.
(24) The peptide may have a sequence as shown in:
(25) TABLE-US-00006 SEQ ID No: 3 (PBYXXGZZPXYZZZ wherein Z is D/E and B is K/R/H and X is any amino acid.) SEQ ID No: 4 (PQYJXGZZPOYZZZ wherein Z is D/E and J is A/T/S or G and O is S or T and X is any amino acid.) SEQ ID No: 5 (PQYAXGZZPOYZZZ wherein Z is D/E and O is S/T and X is any amino acid.) SEQ ID No: 6 (PQYAXGZZPOYZZZXXXXZXX wherein Z is D/E and O is S/T and X is any amino acid.) SEQ ID No: 7 (PQYAXGZZPOYZZZ(D/T)(D/G/F)(D/A)(S/D/E)(D/E)(K/P/S) (L/V) wherein Z is D/E and O is S/T and X is any amino acid.) SEQ ID No: 8 (PQYAXGZZPOYZZZDDDDD(K/P/S)(L/V) wherein Z is D/E and O is S/T and X is any amino acid.) SEQ ID No: 9 (PQYAXGZZPOYZZZDDDEE(K/P/S)(L/V) wherein Z is D/E and O is S/T and X is any amino acid.) SEQ ID No: 10 (PQYAPGDEPSYDED) SEQ ID No: 11 (PQYAPGEEPSYDED) SEQ ID No: 12 (PQYTHGEEPEYDED) SEQ ID No: 13 (PQYAQGEEPTYDED) SEQ ID No: 14 (PQYARGDVPTYDEE)
(26) In another embodiment there is provided a peptide comprising, or consisting of an amino acid sequence shown in
(27) TABLE-US-00007 SEQ ID No: 2: ZXZYZXYZXXX
(28) wherein
(29) Z is D, E, Q, S or P and
(30) X is any amino acid.
(31) The peptide may have a sequence as shown in:
(32) TABLE-US-00008 SEQ ID No: 28 (DADYDEYEEDG) SEQ ID No: 29 (DGDYDEYDNDE) SEQ ID No: 30 (QGDYDEYDQDE) SEQ ID No: 31 (DADYDDYDEEG) SEQ ID No: 32 (DVSYDEYEDNG) SEQ ID No: 33 (QDDYDEYDADE) SEQ ID No: 34 (ETDYDEYEENE) SEQ ID No: 35 (ESDYDTYPDDN) SEQ ID No: 36 (DDEYDMYESDG) SEQ ID No: 37 (ETSYEEYPDDS) SEQ ID No: 38 (ESDYDTYPDDN) SEQ ID No: 39 (ERDDEDYDNSN) SEQ ID No: 40 (ZXZYZZYZZZZ wherein Z is D/E and X is T/M/E/S/D/ G/N/P) SEQ ID No: 41 ZSZYZTYPDDN wherein Z is D/E) SEQ ID No: 42 ZDZYZMYESDG wherein Z is D/E) SEQ ID No: 43 ZTSYZEYPDDS wherein Z is D/E) SEQ ID No: 44 ZSZYZTYPDDN wherein Z is D/E)
(33) In one embodiment the peptide consists of no more than about 35 amino acids, preferably about 30 to 35 amino acids, more preferably about 30, 31, 32, 33, 34 or 35 amino acids.
(34) Preferably the peptide binds to exosite II of thrombin.
(35) The peptide may competitively inhibit the binding of clot-associated fibrin, preferably fibrinogen γ′, to exosite II of thrombin. The peptide may have a higher affinity for exosite II of thrombin than does fibrinogen γ′. This would enable the peptide to ostensibly elute clot-associated thrombin from a clot the subject of tPA thrombolytic therapy, thereby minimising the incidence of reduced re-canalisation that is otherwise associated with tPA therapy.
(36) In one embodiment, the peptide may inhibit the binding of a compound selected from the group consisting of Factor V, Factor VIII, Gp1bα, chondroitin sulphate and heparin, to exosite II of thrombin.
(37) Typically at least one residue of the peptide is a sulphated tyrosine residue.
(38) Preferably the tyrosine at position 11 in SEQ ID No: 1 is sulphated. The tyrosine at position 3 in SEQ ID No: 1 may not be sulphated.
(39) In another embodiment, the tyrosine at position 11 in SEQ ID No: 1 is sulphated and the tyrosine at position 3 is sulphated.
(40) As described in the Examples herein, the sulfation of either or both tyrosine residues is associated with improvements in inhibition of thrombin activity.
(41) Preferably the tyrosine at position 4 in SEQ ID No: 2 is sulphated. The tyrosine at position 7 in SEQ ID No: 2 may not be sulphated.
(42) Preferably the tyrosine at position 7 in SEQ ID No: 2 is sulphated. The tyrosine at position 4 in SEQ ID No: 2 may not be sulphated.
(43) In another embodiment, the tyrosine at position 4 in SEQ ID No: 2 is sulphated and the tyrosine at position 7 is sulphated.
(44) Sulfation of tyrosine residues can be achieved by expressing a nucleic acid encoding a peptide of SEQ ID No: 1 or SEQ ID No: 2 in a cell that contains a tyrosylprotein sulfotransferase (TPST). In more detail, inorganic sulfate may be actived in the forms of adenosine-5′-phosphosulfate (APS) and 3′-phospho-adenosine-5′-phosphosulfate (PAPS) by ATP sulfurylase and APS kinase respectively. The activated sulfate may then be transferred to tyrosine by TPST in the Golgi body.
(45) Another approach to obtain sulphated tyrosine residues in a peptide according to the invention is to utilise a recombinant expression system involving an amber codon suppression enabling sulfotyrosine to be incorporated into the peptide during the recombinant synthesis of the peptide.
(46) In the Examples herein, the inventors provide a synthetic method enabling the production of homogenous compositions of sulphated peptides (i.e. compositions that contain a peptide having only one sulfation profile).
(47) The exosite II inhibitors described herein may be tested for specificity for binding to thrombin exosite II by utilising a thrombin/γ′peptide binding assay as described in Lovely R S et al. 2002. Briefly, an exosite II inhibitor or binder, or putative exosite II inhibitor or binder is labelled, incubated with thrombin and fluorescence polarization is measured. The assay can be used in a competitive inhibitor model to enable the determination of inhibition constants for each exosite II binding peptide.
(48) As described above, at least 20-30% of patients who receive tPA therapy for ischaemic stroke will have complete artery re-canalisation following tPA therapy and of these, 20-30% will experience re-occlusion. Some consider that this arises when tPA therapy reveals clot-entrapped thrombin which is understood to be bound to the clot via exosite II, enabling the active site of the thrombin to cleave fibrinogen and fibrinopeptides to amplify and build the clot. Exosite II inhibitors described herein are provided to an individual receiving tPA therapy to enable the elution of clot-associated thrombin from a fibrin clot, thereby minimising the amount of thrombin at the clot which would otherwise cause fibrin production and clot expansion.
(49) Thus, in one embodiment there is provided, in a method of tPA therapy, the step of providing an exosite II inhibitor of thrombin described herein in a therapeutically effective amount.
(50) Typically the exosite II inhibitor of thrombin is provided in the form of a composition adapted for i.v. administration.
(51) Typically the exosite II inhibitor of thrombin is provided in an amount of about 100 μg/kg to 10 mg/kg of the recipient.
C. Thrombin Inhibitors
(52) In further embodiments the invention relates to utilising the exosite II binding peptides disclosed herein for design, modification and/or production of novel inhibitors of thrombin activity, in particular for inhibitors that prevent or at least minimise the cleavage of fibrinogen or fibrinopeptides by thrombin. These exosite II binding peptides may provide for inhibitors that have an improved affinity for the binding to the thrombin active site, and/or to exosite I.
(53) A thrombin inhibitor may be described according to Formula 1:
A-B wherein: A is an exosite II binding peptide having a sequence shown in SEQ ID No: 1 or SEQ ID No: 2 and as generally described above; B is a peptide having a sequence shown in one of SEQ ID No: 15 to 24 for binding to the thrombin active site to prevent the thrombin active site from cleaving fibrinogen to form fibrin; wherein A and B are linked so as to enable A to bind to exosite II of thrombin when B is bound to the thrombin active site.
(54) It will be understood that A may be located N terminal to B, or alternatively, B may be located N terminal to A.
(55) In a further embodiment, a thrombin inhibitor may be described according to Formula 2:
A-B—C wherein: A is an exosite II-binding peptide having a sequence shown in SEQ ID No: 1 or SEQ ID No: 2 and as generally described above; B is a peptide having a sequence shown in one of SEQ ID No: 15 to 24 for binding to the thrombin active site to prevent the thrombin active site from cleaving fibrinogen to form fibrin; C is a peptide having a sequence shown in one of SEQ ID No: 25 to 27 for binding to exosite I of thrombin; wherein A, B and C are linked so as to enable A to bind to exosite II of thrombin when B is bound to the thrombin active site and C is bound to exosite I of thrombin.
(56) It will be understood that the A may be located N terminal to C, or alternatively, C may be located N terminal to A.
(57) In yet a further embodiment a thrombin inhibitor may be described according to Formula 3:
A-C—B wherein: A is an exosite II binding peptide having a sequence shown in SEQ ID No: 1 or SEQ ID No: 2 and as generally described above; B is a peptide having a sequence shown in one of SEQ ID No: 15 to 24 for binding to the thrombin active site to prevent the thrombin active site from cleaving fibrinogen to form fibrin; C is a peptide having a sequence shown in one of SEQ ID No: 25 to 27 for binding to exosite I of thrombin; wherein A, B and C are linked so as to enable A to bind to exosite II of thrombin when B is bound to the thrombin active site and C is bound to exosite I of thrombin.
(58) It will be understood that A may be located N terminal to B, or alternatively, B may be located N terminal to A.
(59) In yet a further embodiment, a thrombin inhibitor may be described according to Formula 4:
B-A-C wherein: A is an exosite II binding peptide having a sequence shown in SEQ ID No: 1 or SEQ ID No: 2 and as generally described above; B is a peptide having a sequence shown in one of SEQ ID No: 15 to 24 for binding to the thrombin active site to prevent the thrombin active site from cleaving fibrinogen to form fibrin; C is a peptide having a sequence shown in one of SEQ ID No: 25 to 27 for binding to exosite I of thrombin; wherein A, B and C are linked so as to enable A to bind to exosite II of thrombin when B is bound to the thrombin active site and C is bound to exosite I of thrombin.
(60) It will be understood that B may be located N terminal to C, or alternatively, C may be located N terminal to B.
(61) The thrombin inhibitors of Formula 1 to 4 may include a linker in the form of a peptide sequence (for example a peptide including 2 or more amino acid residues such as Gly and Ala) or other polymer (for example, a diethylene glycol linker) that links B (active site binding peptide) with A (exosite II binding peptide); or B (active site binding peptide) with C (exosite I binding peptide); or A (exosite II binding peptide) with C (exosite I binding peptide). Examples of peptide sequences include poly Ala or poly Gly peptides. The length of the linker peptides may be determined according to the molecular distance between the relevant thrombin sites. These molecular distances are as follows: (i) thrombin active site to exosite II: 38 to 60 angstroms; (ii) thrombin active site to exosite I: 27 to 40 angstroms; (iii) exosite II to exosite I: 65 to 100 angstroms.
(62) The above described inhibitors may be prepared by solid phase peptide synthesis. For example, an inhibitor of Formula 1 may be prepared by a method including the steps of solid phase synthesis of A, selective desulfurisation of A so as to retain a desired tyrosine sulfation profile, solid phase synthesis of B, and ligation of A to B, or as an alternative in the final step, ligation of A to a linker, and ligation of the A-linker conjugate to B to from an A linker-B conjugate.
(63) In other embodiments, the inhibitors may be synthesised by recombinant DNA technology. It is particularly preferred that the cell lines used in this technology are (i) capable of growing in the presence of inorganic sulfate and (ii) capable of assimilating inorganic sulfate into a biological system, in particular a system involving post translational modification of tyrosine residues. Such a cell line generally includes a tyrosylprotein sulfotransferase in the Golgi body, enabling the formation of one or more tyrosine-O-sulfate residues. In certain embodiments the expression products may be heterogeneous with respect to tyrosine sulfation pattern. A homogenous population of tyrosine-sulfated isoforms can be obtained by purifying the expression products on a variety of separation systems including a chromatographic system enabling differentiation of isoforms on the basis of tyrosine-sulfated phenotype.
(64) Inhibitors described herein may be investigated for thrombin selectivity by counter-screening against a panel of proteases including trypsin, chymotrypsin, elastase, papain, reptilase, and factor Xa and activated protein C from the blood coagulation cascade. Inhibitors are screened initially at a single concentration (5 μM) using a fluorescence polarisation assay as described above.
(65) Molecular details of thrombin recognition and inhibition by the inhibitors described herein may be determined by solving the three-dimensional structures of their complexes with thrombin. Briefly, thrombin-inhibitor complexes are prepared in vitro and subjected to extensive sub-microlitre scale screenings for crystallisation conditions. Preliminary conditions are refined and optimised using custom grid screens. Determination of cryoprotection conditions and initial sample characterisation is performed using a X-ray diffractometer. High resolution X-ray diffraction data is collected at high brilliance synchrotron sources, ensuring an adequate level of detail in the resulting models. The structures are solved by molecular replacement techniques using the coordinates of unliganded human thrombin as search model and refined and interpreted using a computational platform. These data provide detail on the binding mode of the inhibitors and unveil key interactions with thrombin.
(66) The anticoagulant activity of the thrombin inhibitors disclosed herein is determined by measuring their ability to prolong clotting of human plasma in vitro using a clinical TT assay. Briefly, human plasma from healthy donors (800 μL) is mixed with a concentration range of inhibitors, clotting initiated by addition of thrombin, and clotting time measured using a coagulometer. Compounds which prolong clotting time to 30 sec at a concentration of 50 nM may be further investigated in vitro/ex vivo for APTT. In brief, pooled citrated plasma from C57BL6/J mice is pre-incubated with various concentrations (0-12 μg/mL) of inhibitors. APTT of each plasma sample is quantified following addition of a coagulation activator and CaCl.sub.2). In an ex vivo assay, mice are injected i.v. with inhibitors (fixed concentration determined from in vitro APTT) and whole blood collected into sodium citrate (˜130 μL) 0, 5, 30 and 60 min post-administration. APTT is quantified on isolated plasma using a RANDOX APTT kit, with fibrin generation monitored to measure clotting time.
(67) The inventors show in the Examples that polypeptides having an exosite II binding peptide described above possess potent in vivo antithrombotic activity with reduced clotting time and reduced bleeding time compared to known anticoagulants. This is advantageous because many anti-thrombotic drugs are limited in their use due to risk of bleeding (including intracranial bleeding in stroke therapy).
(68) Bleeding time is an important issue clinically, as bleeding risk is markedly increased in patients receiving anticoagulant therapy. Specifically, there is no effective treatment available against bleeding caused by known anticoagulants including hirudin. There is therefore a need for agents that reduce bleeding time.
(69) The inventors demonstrate the surprising effect that the sulfated polypeptides according to the present invention display significantly less bleeding compared to the known coagulant hirudin.
(70) In one embodiment, the sulfated polypeptides according to the present invention reduce bleeding by more than 2-fold when compared to hirudin. In another embodiment, the sulfated polypeptides according to the present invention reduce bleeding time by more than 3-fold when compared to hirudin. In another embodiment, the bleeding time in the presence of sulfated polypeptides according to the present invention occurs for no longer than 10 minutes.
(71) In another embodiment, the bleeding time in the presence of sulfated polypeptides according to the present invention is reduced by more than 20%, by more than 30%, by more than 40%, by more than 50%, or by more than 60% when compared to hirudin.
(72) The disclosed peptides and compositions can be used for inhibiting thrombin activity, for example in anti-thrombotic amounts sufficient to inhibit thrombin activity in a subject, such as a human, in whom pathological thrombosis is not desired. The compositions can be used in subjects who suffer from a condition such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, disseminated intravascular coagulation, cardiovascular and cerebrovascular thrombosis, thrombosis associated with post-operative trauma, obesity, pregnancy, side effects of oral contraceptives, prolonged immobilization, and hypercoaguable states associated with hematologic, immunologic or rheumatological disorders. Alternatively, the subject may suffer from unstable angina, arteriosclerosis, a reblockage of vessels after angioplasty with a balloon catheter, or blood clotting in hemodialysis.
EXAMPLES
Example 1—Synthesis of Sulfated Exosite II Binders
(73) Fragments of full length thrombin inhibitors that bind only exosite II were synthesised by Fmoc-strategy SPPS.
(74) Resin Loading: 2-Chlorotrityl chloride resin, Rink amide resin or Wang resin was loaded with the C-terminal amino acid of the target sulfopeptide as per the manufacturer's instructions.
(75) Iterative Peptide Assembly (Fmoc-SPPS)
(76) Deprotection: The resin was treated with piperidine/DMF (1:4, v/v, 3 mL, 3×5 min), filtered and then washed with DMF (5×3 mL), CH.sub.2Cl.sub.2 (5×3 mL) and DMF (5×3 mL).
(77) Coupling (standard Fmoc-protected amino acids): A solution of a standard Fmoc-protected amino acid (4 equiv.), PyBOP (4 equiv.) and NMM (8 equiv.) in DMF (final concentration 0.1 M) was added to the resin. After 1 h, the resin was filtered and washed with DMF (5×3 mL), CH.sub.2Cl.sub.2 (5×3 mL) and DMF (5×3 mL).
(78) Site specific incorporation of tyrosine sulfate: Tyrosine sulfate was incorporated into the desired peptide fragments by one of two methods: 1) Coupling of an amino acid cassette [Fmoc-Tyr(SO.sub.2OCH.sub.2C(CH.sub.3).sub.3)—OH]: A solution of Fmoc-Tyr(SO.sub.2OCH.sub.2C(CH.sub.3).sub.3)—OH (1.2 equiv.), HATU (1.2 equiv.) and NMM (2.4 equiv.) in DMF (final concentration 0.1 M) was added to the resin. After 18 h, the resin was filtered and washed with DMF (5×3 mL), CH.sub.2Cl.sub.2 (5×3 mL) and DMF (5×3 mL). 2) Solid phase sulfation following incorporation of Fmoc-TyrOAll-OH: Solid phase sulfation was carried out with a imidazolium sulfating reagent using the method described in: Taleski, D et al. 2011 or Hsieh, Y. S. et al 2014.
(79) Capping: Acetic anhydride/pyridine (1:9, v/v, 3 mL) was added to the resin. After 3 min the resin was filtered and washed with DMF (5×3 mL), CH.sub.2Cl.sub.2 (5×3 mL) and DMF (5×3 mL).
(80) Cleavage: The sulfated peptide was cleaved from the resin with concomitant side chain deprotection using an acidic cleavage cocktail, e.g. 90:5:5 v/v/v trifluoroacetic acid/triisopropylsilane/water.
(81) Deprotection of Sulfate Ester Protecting Groups: 1) Deprotection of the neopentyl sulfate ester (from cassette strategy) was performed by treatment with sodium azide solution or aqueous ammonium acetate as described in Ziarek, J. J et al 2013. 2) Deprotection of the dichlorovinylsulfate esters and trichloroethylsulfate esters (from the solid-phase sulfation strategy) was performed using hydrogenation as described in Taleski, D. et al 2011. Deprotection of trifluoroethylsulfate esters was achieved using aqueous ammonium acetate as described in Hsieh, Y. S. et al. 2014.
(82) Purification: Purification of site specifically sulfated exosite II binding peptide fragments was achieved using reversed-phase HPLC purification.
Example 2—Synthesis of Thrombin Inhibitor of Formula 2 (AAa)
(83) ##STR00001## ##STR00002##
(84) Fmoc-Pro-OH (84 mg, 250 μmop was loaded onto CTC resin using standard loading procedure. Iterative Fmoc-SPPS was then carried out as outlined above to give the fully assembled resin bound A.sup.Aa (32-61). The peptide on resin was split and 25 μmol peptide was used in the following treatment. The peptide was Fmoc-deprotected with 20% piperidine in DMF (2×5 mL) and Boc-Asp(STmb, OtBu)-OH (25 mg, 50 μmol, 2.0 eq.) was subsequently coupled using PyBOP (26 mg, 50 μmol, 2.0 eq.) and NMM (0.11 mL, 101 mg, 0.10 mmol, 4.0 eq.) in DMF (1.5 mL) at room temperature for 16 h. The resin was washed with DMF (5×5 mL), CH.sub.2Cl.sub.2 (5×5 mL) and DMF (5×5 mL). The peptide was deprotected and cleaved from resin using TFA/iPr.sub.3SiH/H.sub.2O (4 mL, 90:5:5 v/v/v) and the resulting mixture was agitated for 2 h. The crude peptide was precipitated from ice-cold Et.sub.2O (20 mL) and purified by reversed-phase preparative HPLC (20 to 80% B over 60 min, Eluent A) to afford the desired peptide 88 as a TFA salt (8.8 mg, 9.0%) after lyophilisation. See
(85) ##STR00003## ##STR00004## ##STR00005##
(86) Fmoc-Thr(OtBu)-OH (100 mg, 250 μmop was loaded onto CTC resin using standard loading procedure. Iterative Fmoc-SPPS was then carried out as outlined above to give the fully assembled resin bound A.sup.Aa (1-30). The peptide on resin was split and 25 μmol peptide was used in the following treatment. The protected peptide was liberated from resin using HFIP/CH.sub.2Cl.sub.2 (4 mL, 7:3 v/v). The resulting product was subsequently treated with PyBOP (65 mg, 0.13 mmol, 5.0 eq.), iPr.sub.2NEt (22 μL, 16 mg, 0.13 mmol, 5.0 eq.), and ethyl 3-mercaptopropionate (160 μL, 170 mg, 1.3 mmol, 50 eq.) in DMF (2 mL) at −40° C. overnight. At this point the reaction mixture was concentrated in vacuo. The peptide was then deprotected with TFA/iPr.sub.3SiH/H.sub.2O (4 mL, 90:5:5 v/v/v) and the reaction mixture was agitated for 2 h. The resulting crude product was precipitated from ice-cold Et.sub.2O (20 mL) and purified by preparative HPLC (20 to 80% B over 60 min, Eluent A) to afford peptide thioester 87 as a TFA salt (11 mg, 12%) after lyophilisation. See
(87) General Procedure for One-Pot Synthesis of Disulfated A.sup.Aa
(88) ##STR00006##
(89) Scheme 3 One-Pot Synthesis of Doubly Sulfated A.sup.Aa (86)
(90) A solution of peptide 88 (6.0 mg, 1.6 μmol, 1.2 eq.) in ligation buffer (6 M Gn.HCl, 100 mM Na.sub.2HPO.sub.4, 25 mM TCEP, pH 6.8, 320 μL) was added to peptide thioester 87 (5 mg, 1.3 μmol, 1.0 eq.) to give a final 5 mM solution of peptide thioester 87. The resulting solution was carefully readjusted to pH 7.4 with 2 M NaOH followed by addition of TFET (10 vol. %) and incubated at 30° C. for 2 h. UPLC-MS analysis indicated complete conversion to the ligated protein 95. A neutral solution of TCEP (0.5 M) and glutathione (100 mM) in buffer (6 M Gn.HCl, 100 mM Na.sub.2PO.sub.4, 320 μL) was then added to give a 2.5 mM final concentration of the ligation product. The resulting solution was adjusted to pH 6.5 and then degassed by sparging with Ar for 10 min which also removed the excess TFET from the reaction mixture. VA-044 (10 mM) was then added in solid form and the reaction mixture was gently agitated and incubated at 37° C. for 16 h. After this time, UPLC-MS analysis showed a complete conversion to the doubly sulfated A.sup.Aa 86. The crude reaction mixture was subjected to preparative HPLC purification (0 to 60% B over 60 min, 0.1 M NH.sub.4OAc) to afford disulfated A.sup.Aa 86 as an ammonium acetate salt (3.9 mg, 43%) after lyophilisation. See
(91) ##STR00007##
Example 3—Clotting and Bleeding Time of A.SUP.Aa .Compared with Hirudin
(92) We investigated the ability of the inhibitor of Example 2 (A.sup.Aa) to prolong clotting time in an in vitro activated partial thromboplastin time (APTT) assay (
(93) We have additionally shown that whilst A.sup.Aa possesses very similar antithrombotic activity to hirudin in an in vivo needle injury model (
Example 4—Inhibition of Thrombin by Sulphated Forms of A.SUP.Aa
(94) The inventors demonstrate, as shown in
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