Voltage-Gated Calcium Channel Auxilliary Subunit Alpha 2 Delta and Uses Thereof

Abstract

The Voltage-Gated Calcium Channel auxiliary subunit α2δ-1 is the target/receptor of gabapentinoid compounds known to exert therapeutic effects as for example in Epilepsy and Neuropathic pain. Gabapentinoids are known to exert their action via binding Arginine (R) within an RRR motif located at the N-terminal of the α2δ-1. The present invention describes a novel binding site for gabapentinoids which is located within the VGCC_a2 domain and within an IKAK aminoacid sequence of the α2δ-1. Such newly identified amino acid binding site finds utility in the identification and characterization of novel compounds with therapeutic properties in Neuropathic Pain and in other disorders and conditions in which α2δ-1 is involved in.

Claims

1. An isolated α2δ-1 peptide consisting of a fragment of an amino acid sequence substantially as set out in SEQ ID No. 5 and encompassing an amino acid sequence substantially as set out in SEQ ID No. 20.

2. A peptide according to claim 1, wherein the fragment comprises an amino acid sequence of SEQ ID No. 20, and further includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 N-terminal and/or C-terminal amino acids, which correspond to at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids that are located at the N-terminus and/or C-terminus of the fragment equivalent to SEQ ID No. 20 as disposed within SEQ ID No. 5.

3. A peptide according to claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, and SEQ ID No. 23.

4. A peptide according to claim 3, wherein the first four amino acids of SEQ ID Nos. 20 to 23 are not mutated, altered or substituted, preferably, wherein the fourth amino acid of SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No. 23 is not mutated, altered or substituted.

5. A peptide according to claim 1, wherein the peptide also encompasses a RRR motif or is conjugated to a separate peptide encompassing a RRR motif.

6. An antibody or antigen-binding fragment thereof capable of binding or interacting with a peptide according to claim 1.

7. An isolated nucleic acid encoding the peptide according to claim 1.

8. The isolated nucleic acid according to claim 7, wherein the nucleic acid encodes an amino acid sequence substantially as set out in SEQ ID No. 20.

9. A genetic construct comprising the nucleic acid according to claim 7.

10. A recombinant vector comprising the genetic construct according to claim 9.

11. A host cell comprising the genetic construct according to claim 9.

12. A method of preparing an isolated α2δ-1 recombinant peptide, the method comprising (i) culturing at least one cell according to claim 11; and (ii) isolating the peptide from the cell to create an isolated α2δ-1 recombinant peptide.

13. A membrane, micelle or liposome comprising the α2δ-1 peptide according to claim 1.

14. A membrane, micelle, liposome or α2δ-1 peptide according to claim 13, wherein the membrane, micelle, liposome or α2δ-1 peptide is recombinant.

15. A membrane, micelle, liposome or α2δ-1 peptide according to claim 14, wherein the membrane is a plasma membrane or an organelle membrane.

16. A binding assay test system for identifying an agent that binds to the α2δ-1 protein of a voltage-gated calcium channel, the system comprising the peptide according to claim 1 or a peptide which comprises an amino acid sequence substantially as set out in SEQ ID No. 20.

17. The test system according to claim 16, wherein the test system comprises a positive control that binds to the isolated peptide.

18. The test system according to claim 16, wherein the test system comprises a negative control that does not bind to the isolated peptide.

19. A method of identifying an agent that binds to the α2δ-1 protein of a voltage-gated calcium channel, the method comprising detecting for binding between the agent and the peptide according to claim 1 or a peptide comprising an amino acid sequence substantially as set out in SEQ ID No. 20.

20. The method according to claim 19, wherein the method comprises using Absorbance, Fluorescence intensity, Luminescence, Surface Plasmon Resonance (SPR), reverse Surface Plasmon Resonance (rSPR), Fluorescence Polarization, Fluorescence resonance energy transfer (FRET), Time resolved Fluorescence (TRF), Homogeneous Time Resolved Fluorescence (HTREF/TR-FRET), Alpha Screen Technology, Fluorescence lifetime, fragment complementation or FLIPR (for calcium readout), ELISA, Radioligand binding assays or Immunoprecipitation.

21. The method according to claim 19, wherein prior to detecting for binding between the agent and the peptide, the method comprises a step of contacting the agent and the peptide according to claim 1 or a peptide comprising an amino acid sequence substantially as set out in SEQ ID No. 20.

22. An agent identified by the method according to claim 19, for use in therapy or as a medicament, or in diagnosis.

23. An agent identified by the method according to claim 19 for use in the treatment of a medical condition in which the α2δ-1 subunit is a therapeutic target, the medical condition being selected from: pain, neuropathic pain, peripheral nervous system pain, central nervous system pain, hyperalgesia, tactile allodynia, fibromyalgia, restless legs syndrome, epilepsy, generalised anxiety disorder, migraine, social phobia, panic disorder, mania, bipolar disorder, and alcohol withdrawal, cancer, urinary tract infections, obstructive pulmonary disease, sexual dysfunction, Kawasaki disease, cardiovascular disorders, (such as angina, heart attacks, heart failure) and respiratory disorders (such as asthma and Chronic Obstructive Pulmonary Disease).

24. A method of treating, preventing or ameliorating a condition in which the α2δ-1 subunit is a therapeutic target, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an agent identified by the method according to claim 19.

25. A pharmaceutical composition of an agent, the composition comprising an agent identified by the method according to claim 19 and a pharmaceutically acceptable vehicle.

26. Use of a peptide comprising an isolated α2δ-1 peptide to identify an agent that binds thereto, wherein the peptide is the peptide according to claim 1 or a peptide comprising an amino acid sequence substantially as set out in SEQ ID No. 20.

27. Use of an isolated α2δ-1 peptide to identify an agent that can be used to treat a medical condition in which the α2δ-1 subunit is a therapeutic target, wherein the peptide is the peptide according to claim 1 or comprises an amino acid sequence substantially as set out in SEQ ID No. 20.

28. The use according to claim 27, wherein the medical condition is selected from: pain, neuropathic pain, peripheral nervous system pain, central nervous system pain, hyperalgesia, tactile allodynia, fibromyalgia, restless legs syndrome, epilepsy, generalised anxiety disorder, migraine, social phobia, panic disorder, mania, bipolar disorder, and alcohol withdrawal, cancer, urinary tract infections, obstructive pulmonary disease, sexual dysfunction, Kawasaki disease, cardiovascular disorders, (such as angina, heart attacks, heart failure) and respiratory disorders (such as asthma and Chronic Obstructive Pulmonary Disease).

29. A membrane, micelle or liposome comprising the α2δ-1 peptide obtained or obtainable by the method according to claim 12.

30. A membrane, micelle, liposome or α2δ-1 peptide according to claim 29, wherein the membrane, micelle, liposome or α2δ-1 peptide is recombinant.

31. A membrane, micelle, liposome or α2δ-1 peptide according to claim 30, wherein the membrane is a plasma membrane or an organelle membrane.

Description

[0111] FIG. 1 is a schematic showing the topological domains/features of the human α2δ-1 subunit, which is encoded by CACNA2D1.

[0112] FIG. 2 is a schematic showing domain organisation of the human α2δ-1 subunit. It specifically shows the topology of CACNA2D1 domains and the α2δ-1 recombinant peptide sequence within the VGCC_α2 domain.

[0113] FIG. 3 is a schematic representation of construct 1 (SEQ ID No. 2). It is a truncated α2δ-1 fragment containing the gabapentin/pregabalin binding site motif but lacking the VWA_N, VWF_A and carboxy-terminal regions downstream of VGGC-α2 domain.

[0114] FIG. 4 is a schematic representation of construct 2 (SEQ ID No. 3). It is a truncated α2δ-1 fragment containing the gabapentin/pregabalin binding site motif but lacking the VWA_N, VWF_A and carboxy-terminal regions downstream of VGGC-α2 domain and also lacking the Cache 1 domain.

[0115] FIG. 5 shows SPR sensorgrams for binding of NVA1309 to immobilized α2δ-1 construct 1.

[0116] FIG. 6 shows SPR sensorgrams for binding of NVA1309 to immobilized α2δ-1 construct 2.

[0117] FIG. 7 shows SPR sensorgrams for binding of pregabalin to immobilized α2δ-1 constructs 1 and 2.

[0118] FIGS. 8A and 8B show specific SPR binding of compound NVA1309 and pregabalin to truncated α2δ-1 Constructs 1 and 2.

[0119] FIG. 9A shows blank-subtracted sensorgram overlay. SPR sensorgrams showing binding of NVA1309 to immobilized peptide 1 (containing the gabapentin/pregabalin binding site).

[0120] FIG. 9B shows RU400 values of FIG. 9A.

[0121] FIG. 10 shows SPR sensorgrams for binding of pregabalin to immobilized peptide 1 (P1).

[0122] FIG. 11 shows SPR sensorgrams for binding of NVA1309 to immobilized an α2δ-1 recombinant peptide containing the VGGC_α2 domain.

[0123] FIG. 12 shows specific SPR binding of compound NVA1309 to immobilized an α2δ-1 recombinant peptide containing the VGGC_α2 domain.

[0124] FIG. 13A shows SPR sensorgrams for binding of NVA1309 to immobilized peptide 3 (carboxy terminal VGGC_α2 region (I)). Blank-subtracted sensorgram overlay. FIG. 13B shows RU400 values of FIG. 13A.

[0125] FIG. 14A shows SPR sensorgrams for binding of NVA1309 to immobilized peptide 4. Blank—subtracted sensorgram overlay. FIG. 14B shows RU400 values of FIG. 14A.

[0126] FIG. 15 is an RU400 plot for binding of NVA1309 to immobilized peptides P4 and P6.

[0127] FIG. 16A shows binding of NVA1309 to peptide P4: Sensorgram overlay. FIG. 16B shows binding of NVA1309 to peptide P13: Sensorgram overlay. FIG. 16C shows binding of NVA1309 to peptides P4 and P13: RU400 plot.

[0128] FIG. 17A shows binding of NVA1309 to peptide P14: Sensorgram overlay. FIG. 17B shows binding of NVA1309 to peptides P4 and P14: RU400 plot.

[0129] FIG. 18A shows binding of NVA1309 to peptide P4: Sensorgram overlay. FIG. 18B shows binding of NVA1309 to peptide P15: Sensorgram overlay. FIG. 18C shows binding of NVA1309 to peptides P4 and P15: RU400 plot.

[0130] FIG. 19 shows covalent immobilization of compound NVA1309 on the Biacore CM5 sensor chip surface.

[0131] FIG. 20 shows specific SPR binding of “an α2δ-1 recombinant peptide comprising the VGGC_α2 domain to NVA1309, covalently coupled to the surface of a Biacore CM5 optical sensor chip.

[0132] FIG. 21 shows mathematical sensorgram fitting (Langmuir 1:1 binding model) of the α2δ-1 recombinant peptide/NVA1309 binding curve.

[0133] FIG. 22(A to D) shows SPR sensorgram fitting of α2δ-1 constructs (P1-P4) encoding human recombinant α2δ-1 proteins (PR1-4)—NVA1309 binding curves.

EXAMPLES

[0134] The inventors decided to explore the significance of individual α2δ-1 domains in the binding profile of the gabapentinoid, NVA1309. Pregabalin and Gabapentin were used as comparative examples. Two truncated α2δ-1 proteins were generated by recombinant synthetic DNA cloning and transiently expressed as His-tagged GST fusion proteins in CHO cells. Also, a CACNA2D1 (human α2δ-1) recombinant peptide produced in yeast (CACNA2D1; CUSABIO, Cat. No. CSB-YP004407HU), lacking the predominant pregabalin binding region as established in the prior art [13] has also been used. FIG. 2 shows a schematic the topology of CACNA2D1 domains and the α2δ-1 recombinant peptide sequence comprising the VGCC_α2 domain.

[0135] Furthermore, the α2δ-1 recombinant peptide together with a set of synthetic peptides representing defined α2δ-1 regions and mutant derivatives thereof were used in target binding experiments. Using Surface Plasmon Resonance (SPR) technology for real time binding assessment, it was found that NVA1309 to bind to a recombinant full length α2δ-1 protein with high affinity but has also been unexpectedly shown to bind the α2δ-1 recombinant peptide comprising the VGCC_α2 domain but not the known binding site for gabapentinoids (R217). Compound NVA1309 was also specifically binding to a synthetic peptide comprising the pregabalin/gabapentin binding site described in prior art (R217) and also was found to bind independently to a site within the carboxy-terminal amino acid region of the α2δ-1VGCC_α2 domain, contained within the α2δ-1 recombinant peptide. This novel and unexpected finding postulates the existence of a novel and independent target binding site for gabapentinoid compounds on human CACNA2D1 which has not been identified in the prior art. The supporting data associated with these unexpected findings are discussed in detail in the following Examples.

Surface Plasmon Resonance (SPR)

[0136] Binding of the gabapentinoid, NVA1309, to recombinant fragments and synthetic peptides comprising different regions of the α2δ-1 target molecule was investigated using Surface Plasmon Resonance (SPR; Biacore) technology.

[0137] Biomolecular interaction analysis by the SPR technology applies optical sensor chips and allows to study in real time binding events in between molecules. Only μg amounts of non-labelled proteins (e.g. antibodies/antigens, recombinant receptors, ligands) or low molecular compounds (e.g. drug candidates) are sufficient to assess target binding strength and to determine accurate binding kinetics (on/off rates, affinities).

Example 1—Binding of Gabapentinoid NVA1309 and Pregabalin to Truncated Recombinant α2δ-1 Proteins, Construct 1 (SEQ ID No. 2) and Construct 2 (SEQ ID No. 3)

[0138] Binding experiments were carried out using Surface Plasmon Resonance (SPR) technology in a Biacore instrument. Recombinant construct 1 and construct 2 were immobilized on the surface of two different flow cells (FC2 and FC3, respectively) of a Biacore CM5 optical sensor chip by covalent amine coupling chemistry, using the Biacore amine coupling kit and following the Biacore amine coupling protocol. Human IgG was immobilized to the reference flow cell FC1 as intra-assay background binding control. Compound NVA1309 was injected as analyte at increasing concentrations and the binding reaction was monitored by generation of real time sensorgrams.

[0139] Two recombinant truncated α2δ-1 fragments (constructs 1 and 2; see FIGS. 3 and 4), containing the reported pregabalin binding site were found to bind NVA1309 but lacked binding to pregabalin. These two truncated α2δ-1 proteins were generated by recombinant synthetic DNA cloning and transiently expressed as his-tagged GST fusion proteins in CHO cells.

Construct 1

[0140] Construct 1 (SEQ ID No. 2) lacks: [0141] a) the amino terminal VWA_N domain, which is found at the N-terminus of proteins containing von Willebrand factor type A (VWA) and Cache domains. It has been found in vertebrates, Drosophila and C. Elegans but has not yet been identified in other eukaryotes. It is probably involved in the function of some voltage-dependent calcium channel subunits; [0142] b) the complete Von Willebrand factor type A domain; and [0143] c) all carboxy-terminal regions downstream of VGGC-α2 domain, which includes (i) the gabapentin/pregabalin binding site motif, (ii) the Cache 1 domain, an extracellular protein domain predicted to play a role in small-molecule recognition in a wide range of proteins, including α2δ-1 and various bacterial chemotaxis receptors, and (iii) the VGGC_α2 domain, a specific domain present in various neuronal voltage-dependent calcium channel (VGCC) subunits to the N-terminus of a Cache domain.

Construct 2

[0144] Construct 2 (SEQ ID No. 3) is the same as construct 1 but also lacking the Cache 1 domain.

[0145] Sensorgram running conditions were: [0146] Compound stock solution: 20 mM, HBS-P buffer [0147] Compound working stock concentrations 0.66-3.62 mM [0148] Running buffer: HBS-P [0149] Flow rate: 30 μl/min

[0150] Results are presented as subtracted curves from the human IgG reference in FIG. 5 and FIG. 6.

[0151] Pregabalin was injected as analyte at a concentration of 500 μM, corresponding to the highest concentration applied for compound NVA1309 (see FIG. 7).

[0152] Subtractive binding sensorgrams clearly show lack of pregabalin binding to both the truncated α2δ-1 ligands although both truncated proteins contained an intact gabapentin/pregabalin binding motif (RRR) (see FIGS. 3 and 4). Similar data were obtained using gabapentin as analyte, with no binding observed to occur for both α2δ-1 Constructs 1 and 2.

[0153] Taking the relative SPR Response Units measured at 400 sec. after injection (RU400; early dissociation phase) as a reference report point, concentration-dependent binding of compound NVA1309 and lack of pregabalin binding to the truncated recombinant α2δ-1 constructs is clearly demonstrated. Specific SPR binding of NVA1309 and pregabalin to truncated α2δ-1 fragments is illustrated in FIGS. 8A & 8B.

Example 2—Binding of Gabapentinoid Compound NVA1309 to a Synthetic Peptide Comprising the Gabapentin/Pregabalin Binding Site of α2δ-1

[0154] A synthetic peptide was generated comprising the reported gabapentin/pregabalin binding site upstream of the VWF_A domain of α2δ-1, carboxy-terminally fused to a flexible spacer consisting of three glycines and a terminal cysteine (Peptide 1, P1; SEQ ID No. 4):

TABLE-US-00011 [SEQ ID No: 4] P1: RTPNKIDLYDVRRRPWYIQGAGGGC

[0155] This peptide was covalently coupled via the carboxy-terminal cysteine to the surface (FC2) of a Biacore CM5 optical sensor chip using the Biacore Thiol Coupling Kit, following the Biacore thiol coupling procedure. Thiol coupling allows for uniform surface presentation of the immobilized peptide molecules and provides freedom to adopt a steric conformation that is determined by the amino acid sequence of the peptide. Compound NVA1309 was injected as analyte at increasing concentrations and the binding reaction was monitored by generation of real time sensorgrams, presented as subtracted curves from the blank FC1 dextran reference surface.

[0156] Sensorgram running conditions were: [0157] Compound stock solution: 50 mM, DMSO [0158] Compound working stock dilution: 0.5-2 mM [0159] Running buffer: HBS-P [0160] Flow rate: 30 μl/min

[0161] Taking the relative SPR Response Units measured at 400 sec. after injection (RU400; early dissociation phase) as a reference report point, concentration-dependent binding of compound NVA1309 to the synthetic peptide comprising the reported gabapentin/pregabalin binding site is clearly demonstrated (see FIGS. 9A & 9B).

[0162] Pregabalin as analyte yielded no positive SPR signal for binding to the peptide P1 ligand and this has confirmed the data shown in Example 1 (see FIG. 10).

Example 3—Binding of Gabapentinoid Compound NVA1309 to the α2δ-1 Recombinant Peptide (SEQ ID No. 5) Comprising the VGCC_α2 Domain but not the Known Gabapentin/Pregabalin Binding Site

[0163] The α2δ-1 recombinant peptide comprising the VGGC_α2 domain and containing a single cysteine near the carboxy terminus, was immobilized on the surface of a Biacore CM5 optical sensor chip using the Biacore Thiol Coupling Kit following the covalent thiol coupling chemistry. This immobilization process provides a sterically more uniform attachment of the ligand molecules to the sensor chip surface than the randomized amine coupling procedure. Bovine serum albumin (BSA) was immobilized to the reference flow cell FC1 as intra-assay background binding control. Compound NVA1309 was injected as analyte at increasing concentrations and the binding reaction was monitored by generation of real time sensorgrams, presented as subtracted curves from the BSA reference (see FIG. ii).

[0164] Sensorgram running conditions were: [0165] Compound stock solution: 50 mM, Dimethyl sulfoxide (DMSO) DMSO [0166] Compound working dilution: 0.5-2 mM [0167] Running buffer: HBS-P [0168] Flow rate: 30 μl/min

[0169] The relative SPR Response Units measured at 400 sec. after injection (RU400; early dissociation phase) as reference report points, a concentration-dependent binding of compound NVA1309 to the α2δ-1 recombinant peptide is clearly demonstrated (see FIGS. 11 and 12).

Example 4—Specific Binding of Gabapentinoid Compound NVA1309 to a Synthetic Peptide Comprising the Carboxy Terminal Amino Acid Region of the VGGC_α2 Domain of α2δ-1

[0170] A synthetic peptide was constructed, representing the carboxy terminal of the VGGC_α2 domain of α2δ-1, carboxy-terminally fused to a flexible spacer consisting of three glycines and a terminal cysteine (Peptide 3; SEQ ID No. 6):

TABLE-US-00012 Peptide P.sub.3, carboxy-terminal VGGC_α2 region (I) + GGGC anchor [SEQ ID No. 6] P3: TYSFYIKAKLEETITQARYSETGGGC

[0171] This peptide was covalently coupled via the carboxy-terminal cysteine to the surface (FC2) of a Biacore CM5 optical sensor chip using the Biacore Thiol Coupling Kit following the Biacore thiol coupling procedure. This strategy allows for uniform surface presentation of the immobilized peptide molecules and provides freedom to adopt a steric conformation determined by the amino acid sequence of the peptide. Compound NVA1309 was injected as analyte at increasing concentrations and the binding reaction was monitored by generation of real time sensorgrams, presented as subtracted curves from the blank FC1 dextran reference surface (see FIG. 13A).

[0172] Sensorgram running conditions were: [0173] Compound stock solution: 50 mM, Dimethyl sulfoxide (DMSO) DMSO [0174] Compound working dilution: 0.5-2 mM [0175] Running buffer: HBS-P (Biacore) [0176] Flow rate: 30 μl/min

[0177] Taking the relative SPR Response Units measured at 400 sec. after injection (RU400; early dissociation phase) as reference report points, concentration dependent binding of compound NVA1309 to peptide P3, homologous to the carboxy-terminal end of the α2δ-1 VGGC_α2 region (I) domain is clearly demonstrated (see FIG. 13B).

Example 5—Mapping of the Gabapentinoid Compound NVA1309 Binding Site on the Carboxy Terminal Region of the VGGC_α2 Target Domain: Binding to Core Amino Acid Sequence IKAKLEETITQA

[0178] A synthetic peptide was constructed comprising the core amino acid sequence IKAKLEETITQAGGGC contained in the Peptide 3 (SEQ ID No. 6), carboxy-terminally fused to the flexible spacer consisting of three glycines and a terminal cysteine (Peptide 4; SEQ ID No. 7):

TABLE-US-00013 [SEQ ID No. 7] P4: IKAKLEETITQAGGGC

[0179] This peptide was covalently coupled via the carboxy-terminal cysteine to the surface (FC2) of a Biacore CM5 optical sensor chip as described in Example 4 following the Biacore thiol coupling procedure. Compound NVA1309 was injected as analyte at increasing concentrations and the binding reaction was monitored by generation of real time sensorgrams, presented as subtracted curves from the blank FC1 dextran reference surface (FIGS. 14A & 14B).

[0180] Sensorgram running conditions were: [0181] Compound stock solution: 50 mM, Dimethyl sulfoxide (DMSO) [0182] Compound working dilution: 0.5-2 mM [0183] Running buffer: HBS-P [0184] Flow rate: 30 μl/min

[0185] An additional peptide was synthesized starting with the carboxy-terminal half of peptide P4 extended to the final downstream VGGC_α2 amino acids, carboxy-terminally fused to the flexible spacer consisting of three glycines and a terminal cysteine: (Peptide P6; SEQ ID No. 8):

TABLE-US-00014 [SEQ ID No. 8] P6: ETITQARYSETLKPGGGC

[0186] This peptide was covalently coupled via the carboxy-terminal cysteine to the surface (FC3) of a new Biacore CM5 optical sensor chip using the Biacore thiol coupling procedure. Peptide P4 was immobilized on FC2 as a positive reference control, flow cell FC1 was left blank as negative background binding reference. Compound NVA1309 was injected as analyte at increasing concentrations and the binding reaction was monitored by generation of real time sensorgrams as described in the previous examples and presented as subtracted curves from the blank FC1 dextran reference surface.

[0187] The relative SPR R400 report point Response Units obtained from the blank subtracted sensorgrams for peptides P4 and P6 are shown in direct comparison in FIG. 15.

[0188] The results of the experiments disclosed in Example 5 clearly demonstrate that the amino acid sequence stretch IKAKLEETITQA at the carboxy-terminal end of the α2δ-1 VGGC_α2 domain defines the novel binding site for compound NVA1309. Deletion of the amino-terminal part of this sequence and further extension of the carboxy-terminal end of VGGC_α2 as represented in peptide P6 (SEQ ID No. 8) resulted in significant loss of NVA1309 binding capacity. Thus, the amino-terminal part of peptide P4 (SEQ ID No. 7), IKAKLE contributes to compound NVA1309 target binding.

Example 6: Molecular Characterisation of the Novel VGGC_α2 Gabapentinoid Binding Site (Peptide P4 Scanning)

[0189] A series of three concentration SPR binding experiments were carried out with peptides derived from the peptide P4 sequence (SEQ ID No. 7). These peptides were designed in order to determine the significance of single peptide 4-contained (P4) amino acid residues in binding compound NVA1309. The following peptides were prepared:

Peptide P8 (SEQ ID No. 9)

[0190] This is an inverse peptide P4+GGGC anchor.

TABLE-US-00015 [SEQ ID No. 9] AQTITEELKAKIGGGC

Peptide P9 (SEQ ID No. 10)

[0191] This is a P4 analogue all amino acids were substituted by most similar ones+GGGC anchor.

TABLE-US-00016 [SEQ ID No. 10] LRVRIDDSLSNVGGGC

Peptide P10 (SEQ ID No. 11)

[0192] This derives from P4, positively-charged side chains A-substituted+GGGC anchor.

TABLE-US-00017 [SEQ ID No. 11] IAAALEETITQAGGGC

Peptide P11 (SEQ ID No.)

[0193] This derives from P4, negatively-charged side chains A-substituted+GGGC anchor.

TABLE-US-00018 [SEQ ID No. 12] IKAKLAATITQAGGGC

Peptide P12 (SEQ ID No. 13)

[0194] This derives from P4, polar side chains A-substituted+GGGC anchor.

TABLE-US-00019 [SEQ ID No. 13] IKAKLEEAIAQAGGGC

[0195] Synthetic peptides were covalently coupled to optical sensor chips via the terminal cysteines following the Biacore thiol coupling procedure and SPR binding experiments were run exactly under the same conditions as described in the previous examples. Relative binding strengths of the single peptides were read from three concentration RU400 values and calculated as percentage binding of the peptide P4 reference (P4: RU400=100%). The comparative results of this Example are summarised in Table 1.

TABLE-US-00020 TABLE 1 Results of peptide P4 scanning binding experiments AA % BINDING (RU400) PEPTIDE DESCRIPTION SEQUENCE 0.5 mM 1 mM 2 mM mean P4 Positive control: 100 100 100 100 contains α2δ1 VGGC-α2 C-terminal region P6 C-terminal P4 RYSETLKP   9   7   9   8.3 extension P8 Inverse peptide P4   3   2   2   2.3 P9 P4 analogue-all aa LRVRIDDSLSNV   1   1   2   1.3 substituted by most similar ones P10 P4; positively IAAA   4   4   4   4 charged side chains A-substituted P11 P4; negatively AA  23  22  25  23.3 charged side chains A-substituted P12 P4; polar side AIA  14  15  17  15.3 chains A-substituted indicates data missing or illegible when filed

[0196] Supported by the lack of binding to the reverse peptide P8, the results of the peptide P4 scanning experiments confirm binding specificity of compound NVA1309 to peptide P4 and indicate that the intact N-terminal amino acid motif IKAKLE is of particular importance to NVA1309 binding.

Example 7: More Detailed Identification of the Novel Binding Site of Gabapentinoid NVA1309 within the VGCC_α2 Domain of Human α2δ-1 Voltage-Gated Calcium Channel Subunit

[0197] In order to identify functional amino acid residues within the binding sequence for NVA1309 within the VGCC_α2 domain of human α2δ-1, the inventors synthesized and used peptides P4 (SEQ ID No. 7), P13 (SEQ ID No. 14), P14 (SEQ ID No. 15) and P15 (SEQ ID No. 16). See below for details.

Peptide P4

[0198] Positive binding reference (100% relative binding): homologous to the carboxy-terminal region of α2δ-1 VGCC-2 domain (MP3)+GGGC anchor.

TABLE-US-00021 [SEQ ID No. 7] IKAKLEETITQAGGGC

Peptide P13

[0199] P4 analogue; K2 A-substituted+GGGC anchor.

TABLE-US-00022 [SEQ ID No. 14] IAAKLEETITQAGGGC

Peptide P14

[0200] P4 analogue; K4 A-substituted+GGGC anchor.

TABLE-US-00023 [SEQ ID No. 15] IKAALEETITQAGGGC

Peptide P15

[0201] amino-terminal part of P4+GGGC anchor.

TABLE-US-00024 [SEQ ID No. 16] IKAKGGGC

[0202] Peptide P4 was covalently coupled via the carboxy terminal cysteine to the surface of flow cell 2 (FC2) of a Biacore CM5 optical sensor chip as reference target-ligand using the thiol coupling chemistry. Peptide P13 was covalently thiol-coupled to the surface of flow cell 3 (FC3) of the same sensor chip. Flow cell FC1, representing a blank carboxyl-dextran surface was used as negative (blank) binding reference.

Binding Analysis of NVA1309 to Peptides P4 and P13

[0203] Binding of NVA1309 to peptides P4 and P13 was analysed by generation of concentration dependent sensorgrams. (Background subtracted sensorgrams FC2-FC1 and FC3-FC1)(FIGS. 16A-C).

Binding Analysis of NVA1309 to Peptides P4 and P14

[0204] Binding of NVA1309 to peptides P4 and P14 was analysed by generation of concentration dependent sensorgrams for peptide P14 (see FIG. 17A, background subtracted sensorgrams FC4-FC1). RU400 plots comparing P14 data with the corresponding values obtained for peptide P4 (FC2-FC1; FIG. 16A) is shown in FIG. 17B.

Binding Analysis of NVA1309 to Peptides P4 and P15

[0205] Binding of NVA1309 to peptides P4 and P15 was analysed on a new Biacore CM5 sensor chip by generation of concentration dependent sensorgrams shown below in FIGS. 18A-C (Background subtracted sensorgrams FC4-FC1 and FC3-FC1).

[0206] The data illustrated in FIGS. 16 to 18 confirm the specificity of binding of compound NVA1309 to the newly discovered VGCC_α2 target. Using peptide P4 as on-chip reference, relative RU400 values and binding scores presented in Table 2 are consistent and underline the importance of the IKAK sequence and in particular of Lysine (K) in position 4 for compound NVA1309 binding on target. The carboxy-terminal half of P4 (ETITQA) per se was found not to be sufficient to interact with NVA1309.

TABLE-US-00025 TABLE 2 Summary of relative peptide binding data for NVA1309 using P4, P13, P14 and P15 % BINDING (RU400) Peptide Description AA Sequence 0.5 mM 1 mM 2 mM Mean SCORE P4 Positive control, IKAKLEETITQAGGGC 100 100 100 100 ++++ contains the α2δ-1 VGCC_a2 C-Terminal region P13 P4; K2 A-substituted IAAKLEETITQAGGGC 133  88  90 104 ++++ P14 P4; K4 A-substituted IKAALEETITQAGGGC   0  11   6   6 +/− P15 P4; IKAK core  IKAKGGGC  19  25  26  23 ++ sequence

Example 8—Binding of α2δ-1 Recombinant Protein (SEQ ID No. 5) Containing the VGCC_α2 Domain on the Gabapentinoid Compound NVA1309 which is Covalently Immobilized on the Surface of a Biacore CM5 Optical Sensor Chip

[0207] In order to confirm data presented in the previous examples and to allow for Biacore SPR experiments with large proteins like recombinant full length α2δ1 constructs (SEQ ID No. 1), an SPR protocol was established which allowed to use compound NVA1309 as ligand by covalently linking the single primary amino group of compound NVA1309 to the carboxyl groups of the dextran matrix as schematically shown in FIG. 19.

[0208] Chemical coupling of compound NVA1309 to the sensor chip was achieved by applying compound NVA1309 in a 50 mM stock solution diluted to 2 mM in phosphate buffered saline (PBS) and subsequently following the protocol recommended in the Biacore amine coupling Kit.

[0209] The following ligands were coupled to the surface of a Biacore CM5 sensor chip: [0210] Flow cell 1 (FC1): Amine (ethanolamin) activated blank surface [0211] Flow cell 2 (FC2): Non related protein 1 ( . . . RU) [0212] Flow cell 3 (FC3): Non related protein 2 ( . . . RU) [0213] Flow cell 4 (FC4): NVA1309 (44 RU)

[0214] α2δ-1 recombinant peptide (101) was passed over all flow cells as analyte. FC1 subtracted sensorgrams are shown in FIG. 20.

[0215] In order to estimate the affinity of α2δ-1 recombinant peptide binding to immobilized NVA1309, the binding sensorgram was analyzed by mathematical curve fitting applying a Langmuir 1:1 interaction algorithm (BiaEvaluation 4.1 software) (see FIG. 21).

[0216] Kinetics of the α2δ-1 recombinant peptide VGGC_α2-NVA1309 interaction were assessed by determination of kinetic binding constants (k-on; k.sub.a, k-off; k.sub.d) based on curve fitting shown in FIG. 21. The equilibrium dissociation constant K.sub.D=k.sub.d/k.sub.a was calculated and provides a single concentration estimate of the compound target binding affinity.

TABLE-US-00026 TABLE 3 Kinetic constants and binding affinity of α2δ-1 recombinant peptide - NVA1309 target interaction (single concentration data) compound k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (nM) NVA1309 1.8 × 10.sup.4 6.1 × 10.sup.−4 33.8

Example 9—Binding of Human Recombinant Full Length α2δ-1 Protein (SEQ ID No. 1) and Derivatives Thereof (SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19) to Compound NVA1309, Covalently Immobilized on the Surface of a Biacore CM5 Optical Sensor Chip

[0217] Recombinant full length α2δ-1 protein and mutants containing a single amino acid residue replaced by alanin were generated by standard DNA cloning and expression techniques and used as analytes for SPR binding to amino-coupled compound NVA1309. The analyte proteins shown on Table 4 were investigated:

TABLE-US-00027 TABLE 4 Human recombinant full length α2δ-1 proteins and alanine mutants thereof human α2δ-1 recombinant protein Mutant PR1 (SEQ ID No. 1), Construct P1 wt α2δ-1 PR2 (SEQ ID No. 17), Construct P2 α2δ-1-R217A PR3 (SEQ ID No. 18), Construct P3 α2δ-1-K634A PR4 (SEQ ID No. 19), Construct P4 α2δ-1-R217A + K634A

[0218] PR1 represents native wild type human α2δ-1 protein, PR2 contains a single arginine (R) to alanine (A) substitution described as the binding site for gabapentin/pregabalin within the RRR motif as reported in prior art and literature. PR3 contains a single lysine (K) to alanine (A) substitution representing to the novel binding site for gabapentinoid NVA1309 on the α2δ-1 target, as disclosed in example 7. PR4 protein contains both point mutations as in PR2 and PR3.

[0219] Each α2δ-1 construct was run as analyte at three concentrations across the NVA1309 sensor chip surface. (NVA1309 surface density=204RU).

[0220] Kinetic binding constants (k-on; k.sub.a, k-off; k.sub.d) were determined from blank amine activated subtracted sensorgrams by mathematical sensorgram fitting. The fitted curves are shown in FIG. 22(A-D).

[0221] For calculation of the kinetic constants a Langmuir 1:1 interaction algorithm supplemented with mass transport limitation correction (where applicable) was applied by using the BiaEvaluation 4.1 software. The equilibrium dissociation constant K.sub.D=k.sub.d/k.sub.a was calculated. Kinetic binding constants and calculated affinities obtained for all recombinant proteins are summarized in Table 5.

TABLE-US-00028 TABLE 5 Kinetic constants and binding affinity of the recombinant α2δ-1 proteins-NVA1309 target interaction human α2δ-1 recombinant protein Mutant K.sub.a (1/Ms); e.sup.−4 K.sub.d (1/s); e.sup.−4 K.sub.D (nM) PR1 (Seq. ID NO. 1), wt α2δ-1 4.2 1.7 3.9 Construct 1 PR2 (SEQ ID NO. 17), α2δ-1-R217A 2.8 2.1 7.5 Construct 2 PR3 (SEQ ID NO. 18), α2δ-1-K634A 2.5 3.1 12.4 Construct 3 PR4 (SEQ ID No. 19), α2δ-1-R217A + 0.3 2.4 83.4 Construct 4 K634A

Conclusion

[0222] I-NVA1309 Displays a Novel Mechanism of Interaction with Target

[0223] NVA1309 is a gabapentinoid which binds to α2δ-1 by a molecular interaction mechanism different from that of gabapentinoids (Pregabalin/Gabapentin) described in the prior art. NVA1309 was able to bind to both recombinant truncated α2δ-1 proteins whereas no binding signals were obtained for pregabalin. Binding of pregabalin on these two truncated α2δ-1 fragments would have been expected as claimed in prior art and published literature [13]. This surprising finding clearly demonstrates a fundamental difference between compound NVA1309 and pregabalin in respect to their mode of target interaction: It has been shown that intact three dimensional folding of the full length α2δ-1 protein is necessary for pregabalin to interact with its known binding region (RRR motif) as postulated in literature [18], no such structural constraint is mandatory for NVA1309.

[0224] This unexpected finding was further extended by showing that compound NVA1309 was also able to bind to a short synthetic peptide exclusively comprising the reported PGB binding region upstream of the VWA domain of α2δ-1 (Peptide P1; Sequence Id 4). In contrast, pregabalin was unable to bind this peptide.

II-NVA1309 Interacts with a Novel Target Region within α2δ-1

[0225] Compound NVA1309 was found to independently bind to a region within the VGGC_α2 domain of the α2δ-1 protein, which is located downstream of the reported pregabalin binding sequence. An α2δ-1 recombinant peptide, expressed in yeast and comprising the VGGC_α2 domain but not the known in the prior art pregabalin/gabapentin binding site (RRR) (Sequence Id5), was used for SPR binding experiments.

[0226] Unexpectedly, compound NVA1309 was capable to specifically bind to the recombinant α2δ-1 recombinant peptide lacking the α2δ-1 region upstream of the VWF_A domain, in literature referred to as binding site for pregabalin and gabapentin. Binding proved to be specific (Bovine Serum Albumin, BSA as negative binding control) and also was concentration-dependent.

[0227] SPR binding analysis using synthetic peptides within the VGGC_α2 binding region were carried out with compound NVA1309 and the specific binding region was further narrowed down to the carboxy-terminal amino acid part of the VGGC_α2 domain. This was achieved by demonstrating NVA1309 binding to a synthetic peptide (peptide P3; SEQ ID No. 6) comprising this carboxy terminal VGGC_α2 region. Additional SPR binding experiments with shorter peptides (peptides P4 (SEQ ID No. 7), and peptide P6 (SEQ ID No. 8) as well as P4 peptide-derivatives containing alanine substituted amino acid residues, P9 (SEQ ID No. 10), peptide P10 (SEQ ID No. 11), peptide P11 (SEQ ID No. and peptide P12 (SEQ ID NO. 13) allowed the identification of a stretch of amino acids comprising the sequence IKAKLEETITQA as a novel binding region involved in target-compound NVA1309 interaction.

[0228] Specific interaction of compound NVA1309 with recombinant full length α2δ-1 protein (SEQ ID No. 1) as well as with α2δ-1 recombinant peptide (SEQ ID No. 5) was confirmed by SPR analysis using compound NVA1309 as ligand, after its covalent coupling to optical sensor chip surface via its single primary amino group and by applying the recombinant proteins as analytes in solution. Single recombinant full length α2δ-1 mutated protein containing alanine substitutions for the respective amino acid residues contained within the pregabalin binding site, as described in prior art (SEQ ID No. 14) and/or amino acid residues contained within the newly detected carboxy terminal VGGC_α2 region IKAKLEETITQA (SEQ ID No. 15, SEQ ID No. 16) was also shown to bind to covalently immobilized compound NVA1309.

[0229] Determination of kinetic binding constants obtained from the SPR sensorgrams allowed calculation of binding affinities (K.sub.D values) of the various analytes to the chemically immobilized compound NVA1309 and were found to lie in the range of 5-80 nM. These novel and unexpected findings postulate a unique mode of target interaction with gabapentinoid compound NVA1309 which is different from the target interaction binding properties of gabapentinoids gabapentin and pregabalin, as reported in the prior art. Furthermore, these findings constitute a novel and inventive basis to be used for the identification of novel voltage-gated calcium channel modulators with potentially unique biological/pharmacological properties.

[0230] The results disclosed in Examples 1 to 9, therefore, describe the identification and molecular characterisation of a novel binding site for gabapentinoid compounds within the VGGC_α2 domain of the auxiliary α2δ-1 subunit of voltage-gated calcium (CaV) channels. This target site may operate synergistically with the gabapentin/pregabalin binding site reported in the prior art located upstream of the VWA domain in order to generate a high affinity binding pocket. However, the newly identified novel VGGC_α2-located binding site may also act as stand-alone target for identification and development of novel therapeutically active calcium channel modulators.

SEQUENCE LISTING

Peptide Amino Acid Sequences

SEQ ID NO. 1 (Human Full Length α2δ-1, Numbering Includes the Signal Peptide):

[0231] MAAGCLLALTLTLFQSLLIGPSSEEPFPSAVTIKSWVDKMQEDLVTLAKTASGVNQLVDIYEKYQDLY TVEPNNARQLVEIAARDIEKLLSNRSKALVRLALEAEKVQAAHQWREDFASNEVVYYNAKDDLDPEKN DSEPGSQRIKPVFIEDANFGRQISYQHAAVHIPTDIYEGSTIVLNELNWTSALDEVFKKNREEDPSLL WQVFGSATGLARYYPASPWVDNSRTPNKIDLYDVRRRPWYIQGAASPKDMLILVDVSGSVSGLTLKLI RTSVSEMLETLSDDDFVNVASFNSNAQDVSCFQHLVQANVRNKKVLKDAVNNITAKGITDYKKGFSFA FEQLLNYNVSRANCNKIIMLFTDGGEERAQEIFNKYNKDKKVRVFTFSVGQHNYDRGPIQWMACENKG YYYEIPSIGAIRINTQEYLDVLGRPMVLAGDKAKQVQWTNVYLDALELGLVITGTLPVFNITGQFENK TNLKNQLILGVMGVDVSLEDIKRLTPRFTLCPNGYYFAIDPNGYVLLHPNLQPKPIGVGIPTINLRKR RPNIQNPKSQEPVTLDFLDAELENDIKVEIRNKMIDGESGEKTFRTLVKSQDERYIDKGNRTYTWTPV NGTDYSLALVLPTYSFYYIKAKLEETITQARYSETLKPDNFEESGYTFIAPRDYCNDLKISDNNTEFL LNFNEFIDRKTPNNPSCNADLINRVLLDAGFTNELVQNYWSKQKNIKGVKARFVVTDGGITRVYPKEA GENWQENPETYEDSFYKRSLDNDNYVFTAPYFNKSGPGAYESGIMVSKAVEIYIQGKLLKPAVVGIKI DVNSWIENFTKTSIRDPCAGPVCDCKRNSDVMDCVILDDGGFLLMANHDDYTNQIGRFFGEIDPSLMR HLVNISVYAFNKSYDYQSVCEPGAAPKQGAGHRSAYVPSVADILQIGWWATAAAWSILQQFLLSLTFP RLLEAVEMEDDDFTASLSKQSCITEQTQYFFDNDSKSFSGVLDCGNCSRIFHGEKLMNTNLIFIMVES KGTCPCDTRLLIQAEQTSDGPNPCDMVKQPRYRKGPDVCFDNNVLEDYTDCGGVSGLNPSLWYIIGIQ FLLLWLVSGSTHRLL

SEQ ID NO. 2 (Recombinant α2δ-1 Truncation 1, Construct 1):

[0232] ATGLARYYPASPWVDNSRTPNKIDLYDVRRRPWYIQGAASPKGRPMVLAGDKAKQVQWTNVYLDALEL GLVITGTLPVFNITGQFENKTNLKNQLILGVMGVDVSLEDIKRLTPRFTLCPNGYYFAIDPNGYVLLH PNLQPKPIGVGIPTINLRKRRPNIQNPKSQEPVTLDFLDAELENDIKVEIRNKMIDGESGEKTFRTLV KSQDERYIDKGNRTYTWTPVNGTDYSLALVLPTYSFYYIKAKLEETITQARYSET

SEQ ID No. 3 (Recombinant α2δ-1 Truncation 2, Construct 2):

ATGLARYYPASPWVDNSRTPNKIDLYDVRRRPWYIQGAASPKGRPMVLAGDKAKQVQKRRPNIQNPKS QEPVTLDFLDAELENDIKVEIRNKMIDGESGEKTFRTLVKSQDERYIDKGNRTYTWTPVNGTDYSLAL VLPTYSFYYIKAKLEETITQARYSET

SEQ ID NO. 4 (Peptide P1, Containing the Pregabalin Binding Site):

RTPNKIDLYDVRRRPWYIQGAGGGC

SEQ ID NO. 5 (Recombinant CACNA2D1 (Cusabio) Fragment):

QPKPIGVGIPTINLRKRRPNIQNPKSQEPVTLDFLDAELENDIKVEIRNKMIDGESGEKTFRTLVKSQ DERYIDKGNRTYTWTPVNGTDYSLALVLPTYSFYYIKAKLEETITQARYSETLKPDNFEESGYTFIAP RDYCN

SEQ ID NO. 6 (Peptide P3, Carboxy-Terminal VGGC_α2 Region (I)+GGGC Anchor):

TYSFYIKAKLEETITQARYSETGGGC

SEQ ID NO. 7 (Peptide P4, Carboxy-Terminal VGGC_α2 Region (II)+GGGC Anchor):

IKAKLEETITQAGGGC

SEQ ID No. 8 (Peptide P6, Carboxy-Terminal VGGC_α2 Region (IV)+GGGC Anchor):

ETITQARYSETLKPGGGC

SEQ ID NO. 9 (Peptide P8, Inverse Peptide P4+GGGC Anchor):

AQTITEELKAKIGGGC

SEQ ID NO. 10 (Peptide P9, P4 Analogue—all aa Substituted by Most Similar Ones+GGGC Anchor):

LRVRIDDSLSNVGGGC

SEQ ID NO. 11 (Peptide P10, P4 Analogue—Positively Charged Side Chains A-Substituted+GGGC Anchor):

IAAALEETITQAGGGC

SEQ ID NO. 12 (Peptide P11, P4 Analogue—Negatively Charged Side Chains A-Substituted+GGGC Anchor):

IKAKLAATITQAGGGC

SEQ ID NO. 13 (Peptide P12, P4 Analogue—Polar Side Chains A-Substituted+GGGC Anchor):

IKAKLEEAIAQAGGGC

[0233] SEQ ID NO. 14 (Peptide P13, P4 Analogue with Lysine in Position 2 (K) Substituted by Alanine (A)+GGGC Anchor):

IAAKLEETITQAGGGC

[0234] SEQ ID NO. 15 (Peptide P14, P4 Analogue with Lysine in Position 4 (K) Substituted by Alanine (A)+GGGC Anchor):

IKAALEETITQAGGGC

[0235] SEQ ID NO. 16 (Peptide P15, P4 Analogue Containing Only the Amino-Terminal Part of P4+GGGC anchor):

IKAKGGGC

[0236] SEQ ID NO. 17 (Full Length Human α2δ-1 Recombinant Protein PR2 with R217A Mutation):
EPFPSAVTIKSWVDKMQEDLVTLAKTASGVNQLVDIYEKYQDLYTVEPNNARQLVEIAARDIEKLLSNRSKALVR LALEAEKVQAAHQWREDFASNEVVYYNAKDDLDPEKNDSEPGSQRIKPVFIEDANFGRQISYQHAAVHIPTDIYE GSTIVLNELNWTSALDEVFKKNREEDPSLLWQVFGSATGLARYYPASPWVDNSRTPNKIDLYDVRRPWYIQGAA SPKDMLILVDVSGSVSGLTLKLIRTSVSEMLETLSDDDFVNVASFNSNAQDVSCFQHLVQANVRNKKVLKDAVNN ITAKGITDYKKGFSFAFEQLLNYNVSRANCNKIIMLFTDGGEERAQEIFNKYNKDKKVRVFTFSVGQHNYDRGPI QWMACENKGYYYEIPSIGAIRINTQEYLDVLGRPMVLAGDKAKQVQWTNVYLDALELGLVITGTLPVFNITGQFE NKTNLKNQLILGVMGVDVSLEDIKRLTPRFTLCPNGYYFAIDPNGYVLLHPNLQPKPIGVGIPTINLRKRRPNIQ NPKSQEPVTLDFLDAELENDIKVEIRNKMIDGESGEKTFRTLVKSQDERYIDKGNRTYTWTPVNGTDYSLALVLP TYSFYYIKAKLEETITQARYSETLKPDNFEESGYTFIAPRDYCNDLKISDNNTEFLLNFNEFIDRKTPNNPSCNA DLINRVLLDAGFTNELVQNYWSKQKNIKGVKARFVVTDGGITRVYPKEAGENWQENPETYEDSFYKRSLDNDNYV FTAPYFNKSGPGAYESGIMVSKAVEIYIQGKLLKPAVVGIKIDVNSWIENFTKTSIRDPCAGPVCDCKRNSDVMD CVILDDGGFLLMANHDDYTNQIGRFFGEIDPSLMRHLVNISVYAFNKSYDYQSVCEPGAAPKQGAGHRSAYVPSV ADILQIGWWATAAAWSILQQFLLSLTFPRLLEAVEMEDDDFTASLSKQSCITEQTQYFFDNDSKSFSGVLDCGNC SRIFHGEKLMNTNLIFIMVESKGTCPCDTRLLIQAEQTSDGPNPCDMVKQPRYRKGPDVCFDNNVLEDYTDCGGV SGLNPSLWYIIGIQFLLLWLVSGSTHRLL
SEQ ID NO. 18 (Full Length Human α2δ-1 Recombinant Protein PR3 with K634A Mutation):
EPFPSAVTIKSWVDKMQEDLVTLAKTASGVNQLVDIYEKYQDLYTVEPNNARQLVEIAARDIEKLLSNRSKALVR LALEAEKVQAAHQWREDFASNEVVYYNAKDDLDPEKNDSEPGSQRIKPVFIEDANFGRQISYQHAAVHIPTDIYE GSTIVLNELNWTSALDEVFKKNREEDPSLLWQVFGSATGLARYYPASPWVDNSRTPNKIDLYDVRRRPWYIQGAA SPKDMLILVDVSGSVSGLTLKLIRTSVSEMLETLSDDDFVNVASFNSNAQDVSCFQHLVQANVRNKKVLKDAVNN ITAKGITDYKKGFSFAFEQLLNYNVSRANCNKIIMLFTDGGEERAQEIFNKYNKDKKVRVFTFSVGQHNYDRGPI QWMACENKGYYYEIPSIGAIRINTQEYLDVLGRPMVLAGDKAKQVQWTNVYLDALELGLVITGTLPVFNITGQFE NKTNLKNQLILGVMGVDVSLEDIKRLTPRFTLCPNGYYFAIDPNGYVLLHPNLQPKPIGVGIPTINLRKRRPNIQ NPKSQEPVTLDFLDAELENDIKVEIRNKMIDGESGEKTFRTLVKSQDERYIDKGNRTYTWTPVNGTDYSLALVLP TYSFYYIKALEETITQARYSETLKPDNFEESGYTFIAPRDYCNDLKISDNNTEFLLNFNEFIDRKTPNNPSCNA DLINRVLLDAGFTNELVQNYWSKQKNIKGVKARFVVTDGGITRVYPKEAGENWQENPETYEDSFYKRSLDNDNYV FTAPYFNKSGPGAYESGIMVSKAVEIYIQGKLLKPAVVGIKIDVNSWIENFTKTSIRDPCAGPVCDCKRNSDVMD CVILDDGGFLLMANHDDYTNQIGRFFGEIDPSLMRHLVNISVYAFNKSYDYQSVCEPGAAPKQGAGHRSAYVPSV ADILQIGWWATAAAWSILQQFLLSLTFPRLLEAVEMEDDDFTASLSKQSCITEQTQYFFDNDSKSFSGVLDCGNC SRIFHGEKLMNTNLIFIMVESKGTCPCDTRLLIQAEQTSDGPNPCDMVKQPRYRKGPDVCFDNNVLEDYTDCGGV SGLNPSLWYIIGIQFLLLWLVSGSTHRLL
SEQ ID NO. 19 (Full Length Human α2δ-1 Recombinant Protein PR4 with R217A+K634A Mutations)):
EPFPSAVTIKSWVDKMQEDLVTLAKTASGVNQLVDIYEKYQDLYTVEPNNARQLVEIAARDIEKLLSNRSKALVR LALEAEKVQAAHQWREDFASNEVVYYNAKDDLDPEKNDSEPGSQRIKPVFIEDANFGRQISYQHAAVHIPTDIYE GSTIVLNELNWTSALDEVFKKNREEDPSLLWQVFGSATGLARYYPASPWVDNSRTPNKIDLYDVRRAPWYIQGAA SPKDMLILVDVSGSVSGLTLKLIRTSVSEMLETLSDDDFVNVASFNSNAQDVSCFQHLVQANVRNKKVLKDAVNN ITAKGITDYKKGFSFAFEQLLNYNVSRANCNKIIMLFTDGGEERAQEIFNKYNKDKKVRVFTFSVGQHNYDRGPI QWMACENKGYYYEIPSIGAIRINTQEYLDVLGRPMVLAGDKAKQVQWTNVYLDALELGLVITGTLPVFNITGQFE NKTNLKNQLILGVMGVDVSLEDIKRLTPRFTLCPNGYYFAIDPNGYVLLHPNLQPKPIGVGIPTINLRKRRPNIQ NPKSQEPVTLDFLDAELENDIKVEIRNKMIDGESGEKTFRTLVKSQDERYIDKGNRTYTWTPVNGTDYSLALVLP TYSFYYIKAZLEETITQARYSETLKPDNFEESGYTFIAPRDYCNDLKISDNNTEFLLNFNEFIDRKTPNNPSCNA DLINRVLLDAGFTNELVQNYWSKQKNIKGVKARFVVTDGGITRVYPKEAGENWQENPETYEDSFYKRSLDNDNYV FTAPYFNKSGPGAYESGIMVSKAVEIYIQGKLLKPAVVGIKIDVNSWIENFTKTSIRDPCAGPVCDCKRNSDVMD CVILDDGGFLLMANHDDYTNQIGRFFGEIDPSLMRHLVNISVYAFNKSYDYQSVCEPGAAPKQGAGHRSAYVPSV ADILQIGWWATAAAWSILQQFLLSLTFPRLLEAVEMEDDDFTASLSKQSCITEQTQYFFDNDSKSFSGVLDCGNC SRIFHGEKLMNTNLIFIMVESKGTCPCDTRLLIQAEQTSDGPNPCDMVKQPRYRKGPDVCFDNNVLEDYTDCGGV SGLNPSLWYIIGIQFLLLWLVSGSTHRLL

SEQ ID NO. 20

IKAK

SEQ ID NO. 21

IKAKLE

SEQ ID NO. 22

IKAKLEET

SEQ ID NO. 23

IKAKLEETITQA

SEQ ID NO. 24 (PGB Binding Site)

VRRRPWYIQGAAS

SEQ ID NO. 25 (RRR Motif)

RRR

[0237] A CRF ASCI text file of the Sequence Listing is submitted with the specification and is herein incorporated by reference; the ACI text file (i) is named SEQLTXT_1_16616752. txt, (ii) was created on 10 May 2021, and (iii) is 47,825 bytes in size.

NON PATENT CITATIONS

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