WO2014209229A1 - Vasodilation peptides and uses thereof - Google Patents

Abstract

In one aspect, the invention is directed to a method of reducing blood pressure in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising a KNP peptide, a variant, and/or a biologically active portion thereof. In another aspect, the invention is directed to a method of treating high blood pressure (e.g., hypertension) in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising a KNP peptide, a variant, and/or a biologically active portion thereof. In another aspect, the invention is directed to a method of causing vasodilation without diuresis in an individual in need thereof comprising comprising administering to the individual an effective amount of a composition comprising a KNP peptide, a variant, and/or a biologically active portion thereof. The invention is also directed to compositions comprising a KNP peptide, a variant thereof and/or a biologically active portion thereof. In one aspect, the composition is a pharmaceutical composition.

Description

VASODILATION PEPTIDES AND USES THEREOF

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/839,040, filed on June 25, 2013.

[0002] The entire teachings of the above application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] Despite a thorough understanding of mammalian natriuretic peptides (NPs), an understanding of venom NPs is lacking. A greater understanding of venom NPs would open up newer avenues to understand the diversity of NPs.

SUMMARY OF THE INVENTION

[0004] In one aspect, the invention is directed to a method of reducing blood pressure in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising a peptide having an amino acid sequence of SEQ ID NO: 42, one or more variants thereof, and/or one or more biologically active portions thereof.

[0005] In another aspect, the invention is directed to a method of treating high blood pressure (e.g., hypertension) in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising a peptide having an amino acid sequence of SEQ ID NO: 42, one or more variants thereof, and/or one or more biologically active portions thereof.

[0006] In other aspects, the invention is directed to a method of treating hypertension in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising a peptide having an amino acid sequence of SEQ ID NO: 42, one or more variants thereof, and/or one or more biologically active portions thereof.

[0007] In yet another aspect, the invention is directed to a method of causing vasodilation without diuresis in an individual in need thereof comprising

administering to the individual an effective amount of a composition comprising a administering to the individual an effective amount of a composition comprising a peptide having an amino acid sequence of SEQ ID NO: 42, one or more variants thereof, and/or one or more biologically active portions thereof.

[0008] The invention is also directed to compositions comprising a KNP peptide (e.g., a KNP precursor; a mature KNP), a (one or more) variant thereof and/or a (one or more) biologically active portion thereof. In one aspect, the composition is a pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 : Schematic representation of the natriuretic peptides (NPs):

Mature NPs have a 17-residue ring held by a disulphide bond between 2 cysteines which are identified by #. The * represents evolutionarily conserved residues within the ring. The $ represents 2 D residues at position 8 and 14 of KNP. ANP (SEQ ID NO: 12), BNP (SEQ ID NO: 13), CNP (SEQ ID NO: 14), DNP (SEQ ID NO: 15) and KNP (SEQ ID NO: 7) have variable lengths of N-terminal and C-terminal segment with KNP having the longest (38 residues) which has propensity to form a- helix and CNP having no tail.

[0010] FIGs. 2A-2B: Sequence comparison of KNP with other NPs: (2A) Precursor NP found from the mRNA/transcriptome of the following species Na-NP (ADK12001) from Naja atra (SEQ ID NO: 16), KNP (ADF50041 from Bungarus flaviceps (SEQ ID NO: 11), Mf-NP (AAZ39879) from Micrurus fulvius (SEQ ID NO: 18) and Mc-NP (AAC60341) from Micrurus coralline (SEQ ID NO: 17) has been aligned. The signal peptide is shaded; the start of the mature protein is shaded and circled; the end of the mature protein is shaded and underlined. (2B) Mature NPs from elapid snakes and human are aligned. TNP-a (P83226) (SEQ ID NO: 19), TNP-b (P83229) (SEQ ID NO: 20), TNP-c (P83231) (SEQ ID NO: 21) identified from Oxyuranus species, PtNP-a (DQ116724) (SEQ ID NO: 22) from Pseudonaja textilis, PaNP-c (DQ116727) (SEQ ID NO: 23) from Pseudechis australis, Na-NP (ADK12001) (SEQ ID NO: 25) from Naja atra, Mc-NP (AAC60341) (SEQ ID NO: 24) from Micrurus coralline, DNP (SEQ ID NO: 15), BNP (AAH25785) (SEQ ID NO: 13) and human CNP (NP_077720) (SEQ ID NO: 14) have been compared. Identical residues are surrounded by a rectangle; similar residues are shaded. The arrows indicate the disulphide bond.

[0011] FIGs. 3A-3E: Heterologous expression, purification and refolding of KNP: (A) KNP was expressed as His-MBP-KNP fusion protein using pLIC vector in E. coli BL21 DE3. Expression was induced using 0.1 raM IPTG and grown overnight at 16°C. KNP fusion protein expressed in insoluble fraction, hence the insoluble fraction was partially purified with 50 mM Tris-Cl, 150 mM NaCl, 2 M urea pH 8 and then solubilized in 50 mM Tris-Cl, 150 mM NaCl, 2 M urea pH 8. Further, the protein in complete denaturation condition is dialyzed against 50 mM Tris-Cl, 0.2 M urea pH 8 at 0.50 mg/ml and then cleaved with TEV protease to obtain KNP. The samples were run of 15% Tris-glycine SDS PAGE for analysis. Lanes- UI: Uninduced whole cell lysate, I: Induced whole cell lysate, S: soluble fraction, PW: Pellet wash with 2 M urea, P: Pellet solubilized in 8 M urea, D:

Dialyzed fusion protein. (3B) TEV cleaved fusion protein was run on reversed-phase high performance chromatography (RP-HPLC) using Buffer A: 0.1% TFA and Buffer B: 0.1% TFA and 80% acetonitnle. The separation was performed on Jupiter CI 8, 5 μηι, 300 A (10 mm x 250 mm) with gradient between 37-43% B. The arrow indicates the protein of interest. (3C) Mass of KNP was determined using ESI-Ion Trap MS. 6604.35 ± 0.804Da corresponds to completely reduced KNP. (3D) KNP was refolded by DMSO mediated oxidation (100 mM Tris-Cl, 20% DMSO, 10% acetonitrile pH 8) and purified by RP-HPLC using Jupiter CI 8, 5 μιη, 300 A (4.6 mm x 250 mm) column, using 37-43% B as gradient. Arrow indicates the protein of interest. (3E) Mass of refolded KNP was determined using ESI-Ion Trap MS. 6602.8 ± 0.507 Da corresponds to mass of complete oxidized KNP.

[0012] FIGs. 4A-4C: Vasorelaxation assay: (4A) Data Acquisition of aortic strip relaxation - Thoracic aorta was isolated from 10 weeks Sprague Dawley rats and mounted at 2g tension resting tension. The tissue was equilibrated for an hour and then the presence endothelium was checked for the presence of endothelium by pre- contraction with 300 nM PE and the relaxation mediated by 100 μΜ ACh. The tissue was washed and allowed to rest before pre-contracting it again with 100 nM PE. A cumulative dose response of ANP (left) and KNP (right) are shown as a representation. (4B) Cumulative dose response of ANP and KNP in pre-contracted aortic strip with intact endothelium: Aortic rings are checked for the presence of endothelium and then pre-contracted with 100 nM PE. After the stabilization of response to PE, cumulative dose response is assessed. The tissue is incubated with each concentration for 10 min before the additive dosage is given. (4C) Cumulative dose response of ANP and KNP in pre-contracted aortic strip with denuded endothelium: The inner lining of the aortic strip was rubbed with cotton bud to remove endothelium. The absence of endothelium was checked with the response to 10 μΜ Ach after the pre-contraction with 300 nM PE. Tissues which showed no response to ACh were used to assess the dose response. The data is expressed as mean ± SD (n^).

[0013] FIGs. 5A-5C: Vasodilatory effect of KNP truncations: (5 A) Schematic representation of KNP deletion mutants. Last 23 residues of KNP tail putatively formed a helical segment. The 23 residues were excised to make Helix. KNP ring with 2 residue tail is designated as Ring and helix deleted constructed with 15 residue tail is Δ-Helix. (5B) Cumulative dose response of KNP deletion mutants in endothelium intact aortic strips - 100 nM PE contracted aortic strip and the dose response of different peptides has been evaluated. (5C) Cumulative dose response of KNP deletion mutants in endothelium denuded aortic strips - 100 nM PE contracted aortic strip and the dose response of different peptides has been evaluated. The data is represented as mean ± SD (n=3).

[0014] FIGs. 6A-6B: Downstream activators of KNP signaling: (6A) Schematic representation of endothelium dependent vasorelaxation pathway in response to agonists such as Acetyl choline, Bradykinin, receptor mediated activation of Phopholipase C which produces IP3 and DAG as secondary messengers. IP3 causing the intracellular calcium increase through release from sarcoplasmic reticulum. This increase in Ca2+, activates nitric oxide synthase (NOS) to synthesize nitric oxide (NO) and opens up Ca2+ activated K+ channel to cause hyperpolarization. Subsequently, NO binds to heme-core of cyclooxeygenase (COX). Further NO diffuses to smooth muscle to activate soluble guanylyl cyclase (sGC) thereby increasing cGMP. COX produces prostacyclins, which diffuses to smooth muscle to elevate cAMP levels. Hyperpolarization, NO and Prostacyclins are common intermediates identified in endothelium dependent vasodilation. (6B) Role of nitric oxide, prostacyclins and hyperpolarization factor in mediating KNP mediated vasorelaxation. The indicated inhibitors (L-NAME: 100 μΜ,

Indomethacin: 10 μΜ, Methylene blue: 20 μΜ) were incubated with aortic strip for

20 min prior to pre-contraction. Subsequently, the tissue was pre-contracted with 100 nM PE and the response of 300 nM of either ANP or KNP was tested. KC1 (40 mM) and BaC12 (30 mM were used for pre-contraction of the tissue and the response for 300 nM of ANP/KNP was recoreded. The data is represented as mean ± SD (n=3).

[0015] FIG. 7: Mechanism of action of KNP: A classical NP binds to its NPR on both endothelium and vascular smooth muscle to elevate cGMP to cause

vasodilation. Whereas, KNP which as the ring with NPR binding ability like a classical NP, is redirected to an unknown receptor on the endothelium by the putative segment in its tail. This helical segment elicits NO, prostacyclins and hyperpolarization to cause smooth muscle relaxation.

[0016] FIG. 8: Sequences of KNP, biologically active portions thereof and variants thereof (SEQ ID NOs: 1-11).

[0017] FIG. 9: Comparison of mammalian and venom NPs. A: Schematic representation showing conserved 17-residue ring held by a disulphide bond (^W^) in all NPs. Conserved residues ® , variable residues O. The NPs differ in their C- and N- terminal extensions; ANP (SEQ ID NO: 12), BNP (SEQ ID NO: 13), DNP (SEQ ID NO: 15) and KNP (SEQ ID NO: 7) have 5, 6, 15 and 38 residues in their C-terminal tail, respectively. CNP (SEQ ID NO: 14), however, lacks any tail. Last

21 residues of the KNP tail has propensity to form an a-helix. Two conserved G residues at positions 3 and 14 within the ring are replaced by D in KNP. B:

Alignment of KNP amino acid sequence (SEQ ID NO: 7) with other elapid venom NPs and mammalian NPs (SEQ ID NOs: 12, 13, 15, 19-23 and 25-29).

[0018] FIGs. 1 OA- IOC Figure 2. KNP mediates endothelium-dependent vasorelaxation. 10A: Vasodilatory ability of ANP and KNP on pre-contracted aortic rings. Rat thoracic aortic rings were isolated and mounted in an organ bath. The tissues were assessed for their viability by pre-contraction with 300 nM PE and relaxation response to 10 μΜ ACh (indicative of presence of endothelium). The tissues were washed and pre-contracted with 100 nM PE before assessing the ^■r e - relaxation ability of NPs. Representative traces showing cumulative dose-responses of ANP {left panel) and KNP {right panel) are depicted. 1 OB: Cumulative dose- response of ANP and KNP on pre-contracted aortic strip with intact endothelium. Each data point is an average of three independent trials and represented as mean ± SEM. The statistical analysis was performed using one way-ANOVA and P-value is 0.005. ANP and KNP induce vasorelaxation with EC50 of 16.3 ± 5.4 nM and 230.6 ± 37.2 nM, respectively. IOC: Cumulative dose-response of ANP and KNP in precontracted aortic strip with denuded endothelium. The inner lining of the aortic strip was rubbed with cotton bud to remove endothelium. The absence of endothelium was checked with the response to 10 μΜ Ach after the pre-contraction with 300 nM PE. Tissues which showed no response to ACh were used to assess the dose response. The data are expressed as mean ± SEM (n = 3, P-value: 0.004) only ANP retains its ability to relax endothelium-denuded aortic rings (EC50 = 23.4 ± 7.8 nM). KNP on the other hand failed to induce vasorelaxation indicating the requirement of endothelium for its function.

[0019] FIGs. 11 A- IE: KNP causes a prolonged reduction in MAP, PP and heart rate with no renal effects in anesthetized rats. 11 A: Femoral vein and artery, urinary blabber catheterization was performed for rats anesthetized with sodium pentobarbital. A fluid- filled pressure transducer was inserted into femoral artery and infusion saline/ peptide was given through a syringe pump at a constant flow rate of 2 ml/h through femoral vein. After the completion of surgery the animals were equilibrated for 20 min and the end of which the time is assumed to be t = 0.

Following a control period of 10 min, ANP (0.2 nmol/kg/min) or KNP (2

nmol/kg/min) was infused for 10 min. Subsequently, the animals were let to recover for 40 min. During control and recovery periods, 0.2% BSA saline was infused. Blood pressure/ heart rate/ urine flow rate value at t = 0 was assumed as baseline. Average values over a time period of 10 min is used for calculation. (ANP: n = 5, KNP: n = 5). 1 IB: Mean arterial pressure (MAP) was calculated as (½ systolic pressure + ¾ diastolic pressure). Change in MAP with reference to the baseline was plotted against time. 11C: Pulse pressure (PP) was calculated as difference between systolic and diastolic pressure. Change in PP with reference to the baseline was plotted against time. 11 D: Heart rate (HR) was calculated as number of beats per minute. Change in HR with reference to baseline was plotted against time. 1 IE: Urine output was represented as volume of urine collected per minute. Change in urine flow rate with reference to the baseline was plotted against time. Each group of peptide and control were tested on five independent animals and the data are represented as mean ± SEM. Statistical analysis between ANP and KNP were performed using one way student t-test and the data points which were significantly different (P-value < 0.05) are represented using *.

[0020] FIGs. 12A-12C: C-terminal helix mediates the vasodilatory effects of KNP. 12A: Schematic representation of KNP (SEQ ID NO: 7) deletion mutants designed based on the predicted structure and the potential processing sites. Δ Helix: C-terminal 26 residues of KNP which forms the putative helix was deleted to mimic other known elapid NPs (SEQ ID NO: 30); Helix: C-terminal 26 residues of KNP (SEQ ID NO: 31); Ring: KNP ring with 2-residue tail was designed to mimic mammalian NPs (SEQ ID NO: 32); R&H: KNP ring was fused with putative helix (SEQ ID NO: 33). 12B: Vasorelaxation of endothelium-intact rat aortic rings by various KNP deletion mutants. Aortic rings were pre-contracted with 100 nM PE and the dose response of different peptides have been evaluated. Cumulative dose- response of KNP deletion mutants was determined. 12C: Vasorelaxation of

- endothelium-intact rat aortic rings by various KNP deletion mutants. Endothelium- denuded aortic rings were pre-contracted with 100 nM PE and the dose response of different peptides have been evaluated. Cumulative dose response of KNP deletion mutants was determined. Every data point is an average of three independent trials for each concentration of the peptide and is represented as mean ± SEM. Statistical analysis was performed between the dose-response curves using one-way ANOVA. Dose-response curves of ANP and KNP were included for comparison. KNP ring shows similar dose-response in both endothelium-intact and -denuded aortic rings and thus acts like ANP. C-terminal extension in Δ-Helix leads to significant loss of potency. C-terminal helix shows similar properties as KNP; it induces

vasorelaxation of only endothelium-intact aortic rings.

[0021] FIGs. 13 A-l 3B: C-terminal tail of KNP redirects the ring away from NPR-A. cGMP response of ANP, KNP and KNP deletion mutants in CHO-Kl cells transfected with plasmid encoding NPR-A (13 A) and NPR-B (13B). Dose- dependent cGMP responses were measured after 30-min incubation of respective peptides at 37°C. Data points of 3 independent trials have been plotted as mean ± SEM. Statistical analysis has been performed using one-way ANOVA using one way t-test for comparing the different dose response curves with ANP or CNP. * represents curves with a P-value < 0.01

[0022] FIGs. 14A-14B: KNP requires NO, prostacyclins and hyperpolarization factor for vasodilation. 14A- Schematic representation of endothelium dependent vasorelaxation pathway. In response to agonists such as Acetylchloline/Bradykinin, receptor mediated activation of Phospholipase C (PLC) produces IP3 and DAG as secondary messengers. IP3 causes the intracellular Ca2+ to increase which through certain downstream targets activates nitric oxide synthase (NOS). Intracellular Ca2+ increase opens Ca2+activated K+ channels to cause hyperpolarization. Activation of NOS, stimulates production of nitric oxide (NO). Subsequently, NO binds to heme- core of cyclooxygenase (COX) and diffuses to smooth muscle to activate soluble guanylyl cyclase (sGC) thereby increasing cGMP. COX produces prostacyclins, which diffuses to smooth muscle to elevate cAMP levels. K+ ions from the endothelium cells open the K+ channels of smooth muscle, thus causing

hyperpolarization. NO, Prostacyclins and Hyperpolarization factors are common intermediates identified in endothelium dependent vasodilation. 14B- Role of nitric oxide, prostacyclins and hyperpolarization factor in mediating KNP mediated vasorelaxation. The indicated inhibitors (L-NAME: 100 μΜ, Indomethacin: 10 μΜ, ODQ: 20 μΜ) were incubated with aortic strip for 20 min prior to pre-contraction. Subsequently, the tissue was pre-contracted with 100 nM PE and the response of 300 nM of the peptides was tested. KCl (40 mM) was used for pre-contraction of the tissue and the response for 300 nM of ANP/KNP/ Ring/ Helix was recorded. Each data point is an average of three independent trials and is represented as mean ± SEM. The statistical analysis using one-tail student t-test has been performed to compare the significance of the response between control aortic rings and inhibitor treated aortic rings for each peptide. * represents the response with a P-value < 0.01.

[0023] FIGs. 15 A- 15D: Ring reduces MAP, PP and heart rate like ANP with no renal effects while ring and helix contribute to KNP's function. 15A: Mean arterial pressure (MAP) was calculated as (½ systolic pressure + ²/₃ diastolic pressure). Change in MAP with reference to baseline was plotted against time. 15B: Pulse pressure (PP) was calculated as difference between systolic and diastolic pressure. Change in PP with reference to baseline was plotted against time. 15C: Heart rate (HR) was calculated as number of beats per minute. Change in HR with reference to baseline was plotted against time. 15D: Urine output was represented as volume of urine collected per minute. Change in urine flow rate is plotted against time. Every data point is an average of five independent trials and represented as mean ± SEM. The statistically analysis for the comparison of ANP with the different KNP truncations was performed using one-way student t-test and P-value < 0.05 is considered significant.

[0024] FIG16: Sequence and structural analysis of KNP Precursor KNP was identified from transcriptome of the venom gland of Bungarus flaviceps. It encoded 148 residues protein along with a signal peptide (sequence represented in bold) (SEQ ID NO: 6). The possible maturation sites in the proteins are dibasic residues (indicated in red bold letters). The mature protein was assumed to be 60 residues protein with both Cys residues in a disulphide bond (SEQ ID NO: 1). M20 (*) was changed to I to prevent the protein from oxidation during the expression and purification (SEQ ID NO: 1). The secondary structure prediction using PSI-PRED, showed that the last 21 amino acid residues of KNP tail had propensity to form ot- helix.

[0025] FIGs. 17A-17F: Heterologous expression and purification of KNP A. 17A: 15 % SDS-PAGE analysis shows the expression of trx-KNP fusion protein in insoluble fraction. M- Precision Plus proteinTM Dual color standard, UI- Uninduced and I- Induced, E. coli whole cell lysate S- Supernatant containing soluble protein after cell lysis, P- Pellet containing insoluble proteins. 17B: The trx-fusion protein was purified by reversed-phase high performance liquid chromatography using . 0.05% formic acid (FA) as buffer- A and 100% ACN with 0.05% FA as buffer-B. The separation was performed on Jupiter CI 8 column (5μηι, 300 A 250 X 21.2 mm) with a linear gradient of 40- 70% B. The arrow indicates the protein of interest. 17C: Mass of the protein peak indicated by the arrow in panel B was determined by ESI Ion-Trap mass spectrometry. Peaks obtained were reconstructed to obtain a mass of 23734.9 ± 1.9 Da which corresponded to the mass of trx-KNP fusion protein. Pure fractions of the protein were freeze dried. 17D: The freeze dried fusion protein was dissolved in 50 mM Tris-HCl, 6 M urea pH 8 buffer. This protein was desalted with 50 mM Tris-HCl pH 8. TEV protease cleavage reaction was set with protein obtained at a ratio 1:40 (TEV: protein) at 4°C for 16-20 h. Cleaved protein, tag and uncut fusion protein were separated using cation exchange chromatography using 50 mM Tris-HCl pH 8 as buffer-A and 50 mM Tris-HCl, 500 mM NaCl pH 8 as buffer- B. The chromatographic separation was performed using Hi-Trap-Sulfopropyl (SP)- Sepharose column (34 μπι, 16 X 25 mm) with a linear gradient of 0-100% B. The arrow indicates the protein peak which was subsequently purified. 17E: Peak indicated in panel D was run on RP-HPLC using Jupiter CI 8 column (5 μπι, 300 A, 250 X 10 mm) with 0.05% FA as buffer-A and 100% ACN with 0.05% FA as buffer-B on a linear gradient of 15- 50% B. Arrow indicates is the protein of interest. 17F: Mass of the protein indicated from panel E was determined by ESI-ion trap mass spectrometer. The reconstructed mass of the mass spectrum indicated a protein of mass 6602.3 ± 0.9 Da, which corresponded to the calculated mass of oxidized KNP.

[0026] FIGs. 18 A- 18D: Purification of ANPA- ANP was synthesized by F-moc based manual SPPS and purified by RP-HPLC. Crude mixture of reduced peptides were separated on Jupiter CI 8 column (5μιη, 300 A, 250 X 21.2 mm) using 0.1% TFA as buffer A and 0.1% TFA with 80% ACN as buffer B. 18A: linear gradient 20- 30% B was used to obtain the purified peptide. Arrow indicates the peptide peak of interest. 18B- Mass of the peptide indicated in panel A was determined by ESI- ion trap mass spectrometry. The reconstructed mass spectrum indicated a protein of mass 3082.0 ± 0.5 Da, which corresponded to reduced mass of ANP. 18C- Purified ANP was folded in 100 mM Tris-HCl pH8 containing 10% ACN for 24 h. This folding mixture was purified by RP-HPLC using Jupiter CI 8 column (5μηι, 300 A, 250 X21.2 mm) using a linear gradient of 20- 30% B (0.1 % TFA as buffer A and 0.1% TFA with 80% ACN as buffer B). Arrow indicates the peptide peak of interest. 18D- Mass of the peptide indicated in panel C was determined by ESI-ion trap mass spectrometer. The reconstructed mass spectrum indicated a protein of mass 3080.6 ± 0.4 Da, which was 2 Da lesser than the mass of ANP before folding, suggesting the formation of disulphide linkage. [0027] FIGs. 19A-19F: Heterologous expression and purification of Δ HelixA. 19A: 15 % SDS-PAGE analysis shows the expression of trx-Δ Helix fusion protein in insoluble fraction. M- Precision Plus proteinTM Dual color standard, UI- Uninduced E. coli whole cell lysate, I- Induced E. coli whole cell lysate, S- Supernatant containing soluble protein after cell lysis, P- Pellet containing insoluble proteins. 19B: The trx-fusion protein was purified by reversed-phase high performance liquid chromatography using 0.05% formic acid (FA) as buffer-A and 100% ACN with 0.05% FA as buffer-B. The separation was performed on Jupiter CI 8 column (5μπι, 300 A 250 X 21.2 mm) with a linear gradient of 40- 70% B. The arrow indicates the protein of interest. 19C: Mass of the protein peak indicated by the arrow in panel B was determined by ESI Ion-Trap mass spectrometry. Peaks obtained were reconstructed to obtain a mass of 20985.51 ± 0.8 Da which corresponded to the mass of trx- AHelix fusion protein. Pure fractions of the protein were freeze dried. 19D: The freeze dried fusion protein was dissolved in 50 mM Tris-HCl, 6 M urea pH 8 buffer. This protein was desalted with 50 mM Tris-HCl pH 8. TEV protease cleavage reaction was set with protein obtained at a ratio 1 :40 (TEV: protein) at 4°C for 16-20 h. Cleaved protein, tag and uncut fusion protein were separated using cation exchange chromatography using 50 mM Tris-HCl pH 8 as buffer-A and 50 mM Tris-HCl, 500 mM NaCl pH 8 as buffer-B. The

chromatographic separation was performed using Hi-Trap-Sulfopropyl (SP)- Sepharose column (34 μηι, 16 X 25 mm) with a linear gradient of 0-100% B. The arrow indicates the protein peak which was subsequently purified. 19E: Peak indicated in panel D was run on RP-HPLC using Jupiter CI 8 column (5 μπι, 300 A, 250 X 10 mm) with 0.05% FA as buffer-A and 100% ACN with 0.05% FA as buffer-B on a linear gradient of 15- 50% B. Arrow indicates is the protein of interest. 19F: Mass of the protein indicated from panel E was determined by ESI-ion trap mass spectrometer. The reconstructed mass of the mass spectrum indicated a protein of mass 3855.6 ± 1.2 Da^ which corresponded to the calculated mass of oxidized Δ Helix.

[0028] FIGs. 20A-20F: Heterologous expression and purification of R&HA. 20A: 15 % SDS-PAGE analysis shows the expression of trx-R&H fusion protein in insoluble fraction. M- Precision Plus proteinTM Dual color standard, UI- Uninduced E. coli whole cell lysate, I- Induced E. coli whole cell lysate, S- Supernatant containing soluble protein after cell lysis, P- Pellet containing insoluble proteins. 2 OB: The trx-fusion protein was purified by reversed-phase high performance liquid chromatography using 0.05% formic acid (FA) as buffer-A and 100% ACN with 0.05% FA as buffer-B. The separation was performed on Jupiter CI 8 column (5μιη, 300 A 250 X 21.2 mm) with a linear gradient of 40- 70% B. The arrow indicates the protein of interest. 20C: Mass of the protein peak indicated by the arrow in panel B was determined by ESI Ion-Trap mass spectrometry. Peaks obtained were reconstructed to obtain a mass of 22187.7 ± 0.9 Da which corresponded to the mass of trx-R&H fusion protein. Pure fractions of the protein were freeze dried. 20D: The freeze dried fusion protein was dissolved in 50 mM Tris-HCl, 6 M urea pH 8 buffer. This protein was desalted with 50 mM Tris-HCl pH 8. TEV protease cleavage reaction was set with protein obtained at a ratio 1 :40 (TEV: protein) at 4°C for 16-20 h. Cleaved protein, tag and uncut fusion protein were separated using cation exchange chromatography using 50 mM Tris-HCl pH 8 as buffer-A and 50 mM Tris-HCl, 500 mM NaCl pH 8 as buffer-B. The chromatographic separation was performed using Hi-Trap-Sulfopropyl (SP)-Sepharose column (34 μηι, 16 X 25 mm) with a linear gradient of 0-100% B. The arrow indicates the protein peak which was subsequently purified. 20E: Peak indicated in panel D was run on RP-HPLC using Jupiter C18 column (5 μηι, 300 A ,250 X 10 mm) with 0.05% FA as buffer-A and 100% ACN with 0.05% FA as buffer-B on a linear gradient of 15- 50% B. Arrow indicates is the protein of interest. 20F: Mass of the protein indicated from panel E was determined by ESI-ion trap mass spectrometer. The reconstructed mass of the mass spectrum indicated a protein of mass 5052.9 ± 0.6 Da, which corresponded to the calculated mass of oxidized R&H.

[0029] FIGs. 21A-21D: Purification and oxidation of Ring. 21A- Ring was synthesized by F-moc based manual SPPS and purified by RP-HPLC. Crude mixture of reduced peptides were separated on Jupiter C18column (5μηι, 300 A, 250 X 21.2 mm) using 0.1 % TFA as buffer A and 0.1 % TFA with 80% ACN as buffer B. A linear gradient 20- 30% B was used to obtain the purified peptide. Arrow indicates the peptide peak of interest. 2 IB- Mass of the peptide indicated in panel A was determined by ESI-ion trap mass spectrometer. The reconstructed mass spectrum indicated a protein of mass 2770.1 ± 0.5 Da, which corresponded to reduced mass of Ring. 21 C- Purified Ring was folded in 100 mM Tris-HCl pH8 containing 10% ACN for 24 h. This folding mixture was purified by RP-HPLC using Jupiter CI 8 column (5μπ , 300 A, 250 X21.2 mm) using a linear gradient of 20- 30% B (0.1 % TFA as buffer A and 0.1 % TFA with 80% ACN as buffer B). Arrow indicates the peptide peak of interest. 21 D- Mass of the peptide indicated in panel C was determined by ESI-ion trap mass spectrometer. The reconstructed mass spectrum indicated a protein of mass 2768.2 ± 0.7 Da, which was 2 Da lesser than the mass of Ring before folding, suggesting the formation of disulphide linkage

[0030] FIGs. 22 A-22B Purification of Helix. 22A- Helix was synthesized by F- moc based manual SPPS and purified by RP-HPLC. Crude mixture of peptides were separated on Jupiter C18 column (5μπι, 300 A, 250 X 10 mm) using 0.1% TFA as buffer A and 0.1% TFA with 80% Acn as buffer B. A linear gradient 20- 40% B was used to obtain the purified peptide. Arrow indicates the position of elution of Helix. 22B- Mass of the protein indicated in panel A was determined by ESI-ion trap mass spectrometer. The reconstructed indicated a protein of mass 2765.2 ± 0.5 Da, which corresponded to calculated mass of Helix.

[0031] FIG. 23: Vasodilatory abilities of Helix and Ring Representative traces of cumulative dose responses of Helix {left panel) and Ring {right panel) on precontracted aortic rings.

[0032] FIG. 24A-24E Changes in systolic and diastolic pressure induced by KNP deletion mutants. Change in systolic, diastolic and PP with reference to baseline was plotted against time for animals infused with 24A- ANP, 24B- Helix, 24C- KNP, 24D- AHelix and 24E- Ring. Each peptide was tested in five

independent animals. Data are represented as mean ± SEM.

[0033] FIG. 25: D3 and D14 within the ring may cause electrostatic repulsion on NPR-A binding Crystal structure of ANP- NPR-A shows that G3 and G20 are in the vicinity of El 69 residue of the receptor. Replacement of G to D might cause repulsion.

[0034] FIG. 26: Experimental setup for measurement of blood pressure and urine output

[0035] FIG. 27: Ex-vivo organ bath setup for vasorelaxation assay DETAILED DESCRIPTION OF THE INVENTION

[0036] Described herein is the study of the mechanism of action and structure- function relationship of a new class of NP from a krait (elapid), referred to herein KNP. Structurally, this NP is distinct compared to mammalian and other venom NPs. It has a 38 residues long, C-terminal tail in contrast to 4-6 residues in mammalian NPs. Further, this tail has the propensity to form a-helix, unlike the C- terminal extensions of other elapid venom NPs. The ex-vivo organ bath studies showed that the ability of the candidate NP to relax the pre-contracted aortic strip was weaker than ANP, and it does so via a different mechanism. It arbitrates vasodilation via endothelium-dependent pathways in contrast ANP which mediates via endothelium-independent mechanisms. Deletion of the C-terminal helical segment attenuates its activity suggesting its definitive role in vasorelaxation. Thus, the data shown herein establishes that KNP mediates vasodilation by a unique mechanism which is attributed to its C-terminal tail.

[0037] Accordingly, in some aspects, the invention is directed to a method of reducing blood pressure in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising, consisting essentially of or consisting of a KNP peptide, a variant, and/or a biologically active portion thereof.

[0038] In other aspects, the invention is directed to a method of treating high blood pressure (e.g., hypertension) in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising, consisting essentially of or consisting of a KNP peptide, a variant, and/or a biologically active portion thereof.

[0039] As used herein, high blood pressure is a blood pressure (BP) that is elevated, e.g., compared to a normal, physiologic blood pressure. As is apparent to those of skill in the art, a normal, physiologic blood pressure will vary from

, individual to individual. In some aspects, a normal, physiologic blood pressure is about 120 systolic pressure and/or about 80 diastolic pressure. In some aspects, the elevated blood pressure is more than about 130, 140, 150, 160, 170, 180, 190, 200 or greater systolic value and/or about 85, 90, 95, 100, 105, 110 or greater diastolic value. The reasons for the increase in BP can be attributed to several factors such as fat or cholesterol deposition along the blood vessel linings, the presence of diseases that affect the cardiovascular system, and also the weakening or loss of blood vessel elasticity. Fat deposits often block the normal passageway of blood. As a result, the lumen of the blood vessel narrows down thereby increasing the pressure of the passing blood. Similarly, renal and endocrine disorders often cause a sudden shift of BP due to hormonal abnormalities; even pregnancy can induce hypertension in mothers who are more at risk. Moreover, some medications are said to cause serious adverse effects relating to high BP. Lastly, the loss of blood vessel elasticity also influences BP as the blood vessels can no longer expand efficiently to accommodate the passage of blood. The increase in blood pressure can therefor occur for a limited time and/or for a short duration of time in an individual due to a variety of factors.

[0040] In some aspects, the high blood pressure is due to "hypertension", a medical condition of the cardiovascular system that is often chronic in nature. It is characterized by a persistent elevation of the BP. Hypertension can be classified as either essential (primary) or inessential (secondary) hypertension. The former is the most common form described as having no exact identifiable cause while the latter is mostly attributed to a secondary factor that is very easily identified. This condition is present in many people around the world and is regarded as the leading cause of more serious conditions like heart attacks, strokes, aneurysms, and heart failure, among other diseases.

[0041] Thus, in other aspects, the invention is directed to a method of treating hypertension in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising, consisting essentially of or consisting of a KNP peptide, a variant, and/or a biologically active portion thereof.

[0042] Further, as shown herein, the KNP peptides do not have a significant influence on diuresis. Thus, in another aspect, the invention is directed to a method of causing vasodilation without diuresis in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising, consisting essentially of, or consisting of a KNP peptide having an amino acid sequence of SEQ ID NO: 42, a (one or more) variant, and/or a (one or more) biologically active portion thereof. [0043] As used herein, KNP refers to a natriuretic peptide (natriuretic polypeptide) identified from red- headed kraits venom gland. Red headed krait (Bungarus flaviceps) is a highly venomous elapid snake found in South and

Southeast Asia. Transcriptomic analysis revealed the presence of a full length precursor NP (Bf-NP or KNP) [Siang et al, BMC Molec Biol, 11:24 (2010)]. As shown herein, KNP has a similar 17-residue ring as all known NPs, however, unlike other NPs, KNP has a 38-residue long C-tail (C -terminal tail) which has a propensity to form an a- helical segment in its C-tail (Figure 1). The C-tail of KNP has no sequence similarity to any known sequences in the database. As shown herein such distinct features, e.g., unique sequence, propensity to form a- helix and possessing a much longer C-tail than any known NP, conferred a distinct biological function. KNP showed an endothelium-dependent vasorelaxation, in contrast to classical NPs, and further, structure based functional studies revealed the putative helix region to be involved in conferring this function to KNP. This study has led to the

understanding of the action mechanism of KNP, and thereby, the therapeutic use of KNP.

[0044] In some aspects, the peptide comprises a (one or more) wild type

(naturally occurring) KNP peptide. In other aspects, the peptide can be a precursor KNP peptide. In some aspects, the precursor peptide comprises the sequence:

MVGPSRLAGGGLLLLLLLALLPLALDGKPAPPPQALPKDPAAASAAERIMR ALLPDSKSSRPATDRMVHPEHQAGGGDTRRLQEPAKKGLLISCFDRRIDRIS HTSDMGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRADSKQSRAA (SEQ ID NO: 45), where X at position 86 is a D or G; X at position 97 is a D or G; and/or X at position 98 is I, M, L or V. In one aspect, the precursor peptide is SEQ ID NO: 6; SEQ ID NO: 11 or a combination thereof.

[0045] In yet other aspects, the peptide comprises a (one or more) KNP variant peptide. A variant KNP peptide (KNP polypeptide) can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. Further, variant polypeptides can be fully or partially functional (e.g., ability to reduce blood pressure and/or treat hypertension; vasodilation; vasorelaxation) compared to the wild type KNP peptide. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[0046] Amino acids that are essential for function (e.g., vasodilation, vasorelaxation; ability to reduce blood pressure and/or treat hypertension) can be identified by methods known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (Cunningham et al, Science, 244: 1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each of the residues in the molecule (one mutation per molecule). The resulting mutant molecules are then tested for biological activity in vitro or in vivo. Sites that are critical for polypeptide activity can also be determined by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (See Smith et al, J. Mol. Biol., 224: 899-904 (1992); and de Vos et al. Science, 255: 306-312 (1992)).

[0047] In one aspect, the KNP peptide comprises an amino acid sequence of SEQ ID NO: 1. In another aspects the KNP variant peptide comprises an amino acid sequence of SEQ ID NO: 7. In other aspects, the KNP peptide comprises the following amino acid sequence:

GLLISCFXRRIDRISHTSXXGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRAD SKQSRAA (SEQ ID NO: 42), where X at position 8 is a D or G; X at position 19 is a D or G; and/or X at position 20 is I, M, L or V.

[0048] In some aspects, the peptide comprises a (one or more) biologically active portion (biologically active fragment) of a KNP peptide or variant thereof. As used herein, a biologically active portion of a KNP peptide or variant thereof includes a portion that retains at least one activity of KNP, e.g., vasodilation, vasorelaxation; ability to reduce blood pressure and/or treat hypertension.

Biologically active portions can be derived from a polypeptide comprising e.g., SEQ ID NO: 1, SEQ ID NO: 7 and/or SEQ ID NO: 42, or from a polypeptide encoded by a nucleic acid molecule that encodes KNP, and/or variant or portion thereof, complements thereof, or other variant thereof.

[0049] Biologically active fragments include peptides that are, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 more amino acid residues in length.

[0050] Specific examples of biologically active fragments include SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Other examples include Δ Helix: C-terminal 26 residues of KNP which forms the putative helix was deleted to mimic other known elapid NPs (SEQ ID NO: 30); Helix: C-terminal 26 residues of KNP (SEQ ID NO: 31); Ring: KNP ring with 2-residue tail was designed to mimic mammalian NPs (SEQ ID NO: 32). Yet another example is a KNP Ring comprising the amino acid sequence:

CFXRRIDRISHTSXIGC (SEQ ID NO: 44), where X at position 3 is D or G and/or X at position 14 is D or G. Other examples include variants of a KNP ring, e.g., a. G- Ring (CFGRRIDRISHTSGIGC (SEQ ID NO: 43).

[0051] Another example includes all or a functional portion of a KNP Ring {e.g., SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 44) fused to all or a functional portion of a KNP Helix {e.g., SEQ ID NO: 31), an example of which is referred to herein as R&H (SEQ ID NO: 33). As will be apparent to those of skill in the art, a fusion of all or a functional portion of a KNP Ring to all or a functional portion of a KNP Helix can include no intervening components {e.g., one or more additional amino acids, linkers and the like) or one or more such intervening components.

[0052] As indicated above, fragments can be discrete (not fused to other amino acid residues or polypeptides) or can be fused to one or more components {e.g., one or more amino acids {e.g., a polypeptide), linkers and the like). In one aspect, one of more fragments of the KNP peptide can be fused to one another {e.g., R&H peptide (SEQ ID NO: 33)). In some aspects, the KNP fragments that are fused to one another are contiguous fragments. In other aspect, the KNP fragments fused to one another are noncontiguous fragments {e.g., KNP Ring fused to KNP Helix (R&H) (SEQ ID NO: 33)). Further, several fragments can be comprised within a single larger polypeptide. In one embodiment, a fragment designed for expression in a host can have wild type and/or heterologous pre- and pro-polypeptide regions fused to the amino terminus and/or the carboxyl terminus of the KNP polypeptide and/or variant or fragment thereof.

[0053] Standard molecular biology methods for generating polypeptide fragments are known in the art. Once the fragments are generated, they can be tested for biological activity, using, for example, any of the methods described herein.

[0054] The invention thus provides chimeric or fusion polypeptides. These can comprise a KNP polypeptide variant and/or biologically active portion thereof operatively linked to a heterologous protein or polypeptide having an amino acid sequence not substantially homologous to the polypeptide. "Operatively linked" indicates that the polypeptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide. In one embodiment, the fusion polypeptide does not affect the function of the polypeptide per se. For example, the fusion polypeptide can be a GST-fusion polypeptide in which the polypeptide sequences are fused to the C-terminus of the GST sequences. Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example, β-galactosidase fusions, yeast two- hybrid GAL fusions, poly-His fusions, FLAG-tagged fusions and Ig fusions. Such fusion polypeptides can facilitate the purification of recombinant polypeptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a polypeptide can be increased by using a heterologous signal sequence. Therefore, in another embodiment, the fusion polypeptide contains a heterologous signal sequence at its N-terminus.

[0055] EP-A 0464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. The Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). Thus, this invention also encompasses soluble fusion polypeptides containing a polypeptide of the invention and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE).

[0056] A chimeric or fusion polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al., "Current Protocols in Molecular Biology " John Wiley & Sons, (1998), the entire teachings of which are incorporated by reference herein). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A nucleic acid molecule encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide.

[0057] Useful biologically active portions include those that retain one or more of the biological activities of the polypeptide {e.g., vasodilation, vasorelaxation; ability to reduce blood pressure and/or treat hypertension). In particular

embodiments, the biologically active portion of KNP or variant thereof comprises SEQ ID NOs: 2, 3, 4, 5, 8, 9, 10, 30, 31, 32, 33, 42, 43, 44 and combinations thereof.

[0058] In some aspects, the KNP peptide, variant and/or biologically active portion thereof is isolated. As used herein, a polypeptide is said to be "isolated," "substantially pure," or "substantially pure and isolated" when it is substantially free of cellular material, when it is isolated from recombinant or non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized. In addition, a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell (e.g., in a "fusion protein") and still be "isolated," "substantially pure," or "substantially pure and isolated." An isolated, substantially pure, or substantially pure and isolated polypeptide may be obtained, for example, using affinity purification techniques described herein, as well as other techniques described herein and known to those skilled in the art.

[0059] The substantially pure, isolated, or substantially pure and isolated KNP polypeptide, variant and/or biologically active can be purified from cells that naturally express it, purified from cells that have been altered to express it

(recombinant), or synthesized using known protein synthesis methods. In one embodiment, the polypeptide is produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector is introduced into a host cell, and the polypeptide is expressed in the host cell.

[0060] As will be appreciated by those of skill in the art, the methods can comprise administering nucleic acids that encode the KNP peptide(s), variants and/or biologically active fragments thereof. As will also be appreciated by those of skill in the art, a variety of methods for introducing nucleic acid encoding a peptide for expression in an individual in need thereof is known in the art. For example, naked nucleic acid encoding the KNP peptide(s), variants and/or biologically active fragments thereof can be introduced. In addition, or alternatively, nucleic acid encoding the KNP peptide(s), variants and/or biologically active fragments thereof can be introduced using any of a variety of delivery vehicles routinely used to deliver nucleic acids in vivo such as gene guns, vectors {e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, etc.) and the like.

[0061] Any suitable route of administration can be used to administer, either systemically or locally, a composition comprising, consisting essentially of, or consisting of a KNP peptide(s), variant and/or biologically active portion thereof, and/or nucleic acid nucleic acid encoding the KNP peptide(s), variants and/or biologically active fragments thereof. As will be apparent to those of skill in the art, the KNP peptide, variant and/or biologically active peptide thereof can be administered as a nucleic acid encoding the KNP peptide, variant and/or biologically active peptide thereof.

[0062] Examples of suitable routes of administration include oral, dietary, topical, transdermal, rectal, parenteral, intraarterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), ocular, pulmonary, nasal, gene gun and the like. Administration can be local or systemic as indicated. The preferred mode of administration can vary depending on the particular agent chosen. Suitable dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. The mode of administration will vary depending on the particular agent chosen.

[0063] The KNP peptide, variant and/or biologically active portion thereof can be administered in a single dose (e.g., in a day) or in multiple doses. In addition, the KNP peptide, variant and/or biologically active portion thereof can be administered in one or more days (e.g. over several consecutive days or non-consecutive days).

[0064] The KNP peptide, variant and/or biologically active portion thereof used in the methods described herein can be administered to a subject as part of a pharmaceutical composition. Formulations will vary according to the route of administration selected (e.g., solution, emulsion or capsule). A "pharmaceutical composition" comprises a (one or more) composition or compound described herein as the active ingredient and inert ingredient(s), such as pharmaceutically acceptable excipients, that make up the carrier. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's

Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's lactate and the like. Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying, solubilizing, pH buffering, wetting agents). Methods of encapsulation compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art. For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer or nebulizer or pressurized aerosol dispenser).

[0065] In one aspect, the therapy or treatment ameliorates the symptoms associated with the condition and/or disease in an individual. In other aspect, the therapy arrests and/or delays onset of the condition and/or disease in the individual. In yet other aspects, the therapy eradicates the condition and/or disease in an individual. In yet other aspects, the treatment lessens the severity or frequency of symptoms of the disease.

[0066] As used herein an "individual" refers to an animal, and in a particular aspect, a mammal. Examples of mammals include primates, a canine, a feline, a rodent, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats and mice.

[0067] The term "individual in need thereof refers to an individual who is in need of treatment or prophylaxis as determined by a researcher, veterinarian, medical doctor or other clinician. In one embodiment, an individual in need thereof is a mammal, such as a human.

[0068] The need or desire for administration according to the methods of the present invention is determined via the use of well kriown risk factors. The effective amount of a (one or more) particular compound is determined, in the final analysis, by the physician in charge of the case, but depends on factors such as the exact condition and/or disease to be treated, the se verity of the condition and/or disease from which the patient suffers, the chosen route of administration, other drugs and treatments which the patient may concomitantly require, and other factors in the physician' s judgment.

[0069] An effective amount of KNP peptide, variant and/or biologically active portion thereof is delivered to an individual in need thereof. As used herein, "effective amount" or "therapeutically effective amount" means an amount of the active compound that will elicit the desired biological or medical response in a tissue, system, subject, or human, which includes alleviation of the symptoms, in whole or in part, of the condition and/or disease being treated.

[0070] The composition can be administered in a single dose (e.g., in a day) or in multiple doses. In addition, the composition can be administered in one or more days (e.g. over several consecutive days or non-consecutive days).

[0071] In other aspects, the invention is directed to pharmaceutical compositions comprising one or more KNP peptides, variants and/or biologically active portions thereof described herein and/or nucleic acids that encode one or more KNP peptides, variants and/or biologically active portions thereof described herein. The

pharmaceutical compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. The formulation should suit the mode of administration.

[0072] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.

[0073] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose, magnesium carbonate, etc.

[0074] The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other compounds. The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active compound. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. [0075] For topical application, norisprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., that are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The compound may be incorporated into a cosmetic formulation. For topical

application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air. In addition, or alternatively, long-term use of continuous infusion e.g., using Alzet pumps, dermal patches and slow release formulations can be used.

[0076] Compounds described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0077] The compounds are administered in a therapeutically effective amount. The amount of compounds that will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms of an angiogenic disease, a vascular disease, a heart disease, or a circulatory disease, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. [0078] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, that notice reflects approval by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the compounds can be separated, mixed together in any combination, present in a single vial or tablet. Compounds assembled in a blister pack or other dispensing means is preferred. For the purpose of this invention, unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each compound and administered in FDA approved dosages in standard time courses.

[0079] The present invention also encompasses all (e.g., a KNP precursor; a mature KNP), a (one or more) variant thereof and/or a (one or more) biologically active portion thereof. In one aspect, the invention is directed to a composition comprising, consisting essentially of, or consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 30, 31, 32, 33, 42, 43 and/or 44. In particular aspects, the invention is directed to a pharmaceutical composition comprising, consisting essentially of, or consisting of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 30, 31, 32, 33, 42, 43 and/or 44..

[0080] Exemplification

[0081] Example 1

[0082] Materials and Methods

[0083] Cloning, expression and purification of KNP and Δ Helix

[0084] The DNA sequence encoding KNP sequence was optimized for expression in Escherichia coli, and cloned in pLIC vector (Kindly donated by) into Kpnl and Sacl (Fermentas fast digest) restriction site. KNP was expressed as a 6X- histidine (His) maltose binding protein (MBP) fusion protein with a Tobacco-ecth virus (TEV) protease cleavage site (His-MBP-TEV-KNP). Expression was carried out in BL21 DE3 E. coli strain using 0.1 mM IPTG induction at 16 °C overnight. The expression of KNP fusion protein was observed in the insoluble fraction. To purify the expressed protein, the cells were harvested and re-suspended in native lysis buffer (50 mM Tris-Cl, 150 mM NaCl pH 8) and sonicated. The lysed cells were spun at 12,000g and pellet was washed with wash buffer (50 mM Tris-HCl, 150 mM NaCl, 2 M urea pH 8). The slurry was spun at 12,000 g and the pellet was solubilized in denaturation buffer (50 mM Tris-Cl, 150 mM NaCl, 8 M urea pH 8). The purity of the sample was assessed by running the samples on 15% Tris-Glycine SDS-PAGE. The KNP fusion at a concentration of 0.5 mg/ml was then dialysed against 50 mM Tris-HCl, 0.2 M Urea pH 8, to cleave the protein. The dialysed fusion protein was cleaved using recombinantly expressed and purified TEV in the ratio 25 : 1 (fusion protein: TEV) in 50 mM Tris-HCl, 0.2 M Urea, 1 mM DTT, 0.5 mM EDTA pH 8. Reduced cleaved KNP was purified by Reversed phase chromatography (RP-HPLC) with Jupiter C 18, 5 μηι, 300 A ( 10 X 250 mm

Phenomenox) column with Buffer A - 0.1 % Tri-fluro-acetic acid (TFA) and Buffer B- 0.1% TFA with 80% Acetonitrile, on Akta purifier system (GE Healthcare). Purity and homogeneity of KNP was checked using Electrospray- ionization (ESI) - Mass Spectrometer (MS) (LCQ fleet Ion trap, Thermoscientific) and freeze dried. The freeze dried protein was quantified after reconstituting it in 1 OOmM Tris-HCl pH 8 by absorbance at 280 nm and set for folding at 5 μΜ final concentration. The folding was carried out for 16-20 h in buffer containing 100 mM Tris-HCl pH8, 20% DMSO, 10% Acetonitrile. Further the folded protein was purified by RP-HPLC in the same gradient as the reduced protein and checked on ESI-MS for its purity and homogeneity. The folded protein was freeze dried and reconstituted in phosphate buffer saline before the assay. Δ-Helix was designed based on the predicted secondary structure of KNP. The last 26 residues which putatively formed helix in KNP were deleted to make Δ-Helix. The DNA encoding Δ Helix was amplified from KNP. Further cloning, expression, purification and folding were done in the same as full length KNP. [0085] Peptide Synthesis

[0086] ANP, Helix and Ring were synthesized using manual Fmoc- based peptide synthesis. ANP was synthesized using Tyr-preloaded Wang resin, Helix using Novasyn TGA resin and Ring using Novasyn TGR resin. 5 times excess of Fmoc- Amino acid derivatives were activated using 4.9 times HATU and 10 times DIPEA and the coupling reaction was performed in the ratio 2:1 DMF: NMP. Following coupling, the F-moc group was removed using 20% (v/v) piperidine in DMF. The peptides were cleaved from the resin using TFA:EDT:TIS:water (94:2.5:1:2.5) and precipitated using ice cold 1,2 diethyl ether. The crude peptides were purified using Jupiter CI 8 , 5 μπι, 300 A (10 X 250 mm Phenomenox) column with Buffer A - 0.1 % Tri-fluro-acetic acid (TFA) and Buffer B - 0.1% TFA with 80% acetonitrile, on Akta purifier system (GE Healthcare). The purity and homogeneity of the peptides were assessed using ESI-MS and pure fractions were freeze fried.

[0087] ANP was subjected to air oxidation in 100 mM Tris pH 8 with 10% acetonitrile while Ring was oxidized using the same conditions as KNP using 100 mM Tris-HCl pH 8, 20% DMSO and 10% Acetonitrile. The oxidized peptides were purified by RP-HPLC and the mass of the peptides determined by ESI-MS.

[0088] Vasorelaxation assay

[0089] Male Sprague Dawley (SD) rats (10 weeks old) were euthanized and the descending thoracic aorta was isolated and flushed with physiological solution (Krebs Buffer: 115 mM NaCl, 5.9 mM KC1, 1.2 mM MgC12, 1.2 mM NaH2P04, 1.2 mM Na2S04, 2.5 mM CaC12, 25 mM NaHC03, 10 mM glucose, pH 7.4). The fat tissues and connective tissues on aorta were removed, cut into 2-3mm rings and mounted under 2 g tension into the organ bath chamber using two stainless steel hooks. The aortic strips were fixed to MLT0201/RAD Force transducer (AD Instruments). The tissues were equilibrated in chambers at 37Χ^ containing carbogenated (95% 0₂ and 5% C0₂) Krebs buffer for an hour. The presence of endothelium in the aortic strip was checked by pre-contracting the tissue with 300 nM phenylephrine (PE) and relaxing with 10 μΜ Acetylcholine (ACh). The aortic rings were pre-contracted with 100 nM PE above a cumulative dose response for different peptides/ protein was obtained. Further, the aorta was denuded of endothelium by rubbing the inner surface of the aortic ring with a cotton bud and then pre-contracted with 100 nM PE to construct the cumulative dose-response of the protein/peptides.

[0090] To study the effect of different inhibitors, the tissue was incubated with inhibitor; L-NAME- 100 μΜ, Indomethacin- 10 μΜ, Methylene blue- 20 μΜ, for 20 min prior to pre-contraction of tissue. The inhibition was confirmed with Ach relaxation ability. To understand the role of hyperpolarization in mediating KNP signalling, tissues were either pre-contracted with 40 mM KC1 or 30 mM BaC12. The pre-contracted tissue in the presence of a particular inhibitor was used to assess the activity of 300 nM of either ANP/KNP.

[0091] Results

[0092] Sequence and structural analysis of KNP

[0093] The transcriptome of B. flaviceps revealed the presence of KNP as precursor protein which was assumed to be processed at the C-terminus of dibasic residue at position 87. This assumption was validated by comparing the length of N- terminal extension which varied between 5-7 residues in all known NPs (Figs. 2A- 2B). While the possible processing sites along the C-terminus could be either after dibasic residues at position 111 and 113 or at position 124. Processing at SI 24 rendered KNP to have 15 residues tail similar to other known elapid NPs. Since B. flaviceps is a rare snake, the identification of KNP from venom was impossible. Hence, to avoid ambiguity KNP with 60 amino acid residues was deduced to be the full length. Mature KNP has a much longer C-terminal tail; hence secondary structure prediction was done using PSI-PRED. The last 26 residues of KNP C- terminus tail had propensity to form a- helix; a unique feature of NPs. Another distinct feature of KNP is the two D residue in position 8 and 19 (in mature KNP) in place of G within the ring. Thus, KNP had to be recombinantly expressed and tested for its function.

[0094] Heterologous expression and purification of KNP

[0095] KNP was heterologously expressed in E. coli as a His-MBP fusion protein. This protein was obtained from the insoluble fraction after a 2 M urea wash. Partially purified pellet was solubilized in complete denaturation condition with 8 M urea, which was dialyzed to be cleaved using TEV protease. The cut protein was purified by RP-HPLC and the mass was determined to be 6604.35 ± 0.804 Da, which matched to the calculated mass completely reduced protein (6604.5 Da). Hence, the protein was refolded. The folded protein was purified and the mass was determined to be 6602.8 ± 0.507 Da. The loss of 2 Da, indicates the removal of 2 hydrogen atoms from the free sulfhydryl group from the two cysteines that has formed a disulphide bond. The purified and homogenously folded KNP was used to assay its function. See Figs. 3 A-3E.

[0096] Ability of KNP and ANP to relax pre-contracted aortic strip

[0097] Vasodilation is one of the key mechanisms through which NPs cause reduction of blood pressure. NPs bind to their NPR and increase intracellular increase in cGMP levels which eventually leads to myosin light chain

dephosphorylation leading to relax the smooth muscle [Brenner, B.M., et al., Physiol Rev, 1990. 70(3): p. 665-99]. The aortic strip isolated from rat thoracic aorta was mounted on an organ bath chamber and pre-contracted with 100 nM PE to test the dilation caused by KNP and ANP. ANP induced vasorelaxation had an EC50 of 16.3 ± 5.4 nM was comparable to EC50 values reported by other groups. This peptide caused a quick vasodilatory response for 10 nM or higher concentration. In contrast KNP induced relaxation was slower at high concentrations. KNP's potency (EC50 230.6 ± 37.2 nM) to cause vasorelaxation was 15 folds lower than ANP.

[0098] The expression of NPRs is observed both in endothelium as well as vascular smooth muscle. Hence, a NP can elicit relaxation directly on the smooth muscle, independent of endothelium. Thus, the ability of these two peptides to relax endothelium denuded aortic strip was evaluated. ANP showed an equipotent response in the presence and the absence of endothelium while the activity of KNP was abolished with endothelium was denuded. This observation indicated that, KNP is not binding to NPR to evoke vasodilation. Although, KNP shows weak vasodilatory property in comparison to ANP, but it does so via a different mechanism. See Figs. 4A-4C.

[0099] Design and Synthesis of KNP deletion mutants

[00100] KNP was truncated into different segments based on the structure and possible maturation sites to understand its non-classical function. Secondary structure prediction showed residues between position 40 and 58 had propensity to form α-helix. Further, KNP ring has all crucial residues for NPR binding, except for D residues replacing the well conserved G within the ring at position 3 and 14. To understand the functional role of these segments, Ring (considering the maturation at dibasic residue of precursor protein at position 111) and Helix (last 23 residues of the C-terminus tail) were designed. Subsequently, Δ-Helix construct was designed in which the putative helix was deleted, to have ring with 15 residue tail. This truncation also encompasses the possible maturation of KNP at this location.

[00101] Ring and Helix were manually synthesized, while Δ-Helix was cloned from the full length KNP and heterologously expressed and purified. Ring and Δ- Helix were folded exactly in the same conditions as full length KNP. The masses of Ring, Helix and Δ-Helix was assessed using ESI-MS showed 2767.975 ± 1.153 Da (theoretical mass: 2769.12 Da), 2765.11 ± 1.05 Da (theoretical mass: 2765.7 Da) and 3855.6 ± 1.23 Da (theoretical mass: 3855.8 Da) respectively which matched the calculated mass.

[00102] Dilation of aortic strip by KNP truncations

[00103] The truncated segments of KNP were assessed for their ability to relax pre-contracted aortic strip (Figs. 5A-5C). The Ring evoked equipotent relaxation in aortic strips with and without endothelium with an EC50 of 228.6 ± 43.1 nM and 279.5 ± 47.2 nM respectively. Although Ring's ability to relax was comparable to full length KNP, it elicited an endothelium independent relaxation as a classical NP.

[00104] Helix relaxed aortic strip with endothelium with an EC50 of 326.7 ± 64.3 nM, while failed to evoke relaxation when endothelium was removed. This observation was identical to that of the full length KNP in terms of potency as well as mechanism. Δ-Helix had lower vasodilatory property compared to full length KNP in endothelium intact aortic strip, while its activity was not hampered on endothelium removal. Thus, suggesting classical NP like function for Δ-Helix with much lower potency. Although the EC50 of full length KNP, Ring and Helix are comparable, Helix attributes function to full length KNP.

[00105] This experiment indicated that KNP has two pharmacologically active segments, the Ring and Helix. Helix with equivalent activity of Ring, seems to take the full length away from NPR. Also, the lower potency of Δ-Helix that remained unaffected in the absence of endothelium, reiterates the fact; Helix readdresses KNP to endothelium and mediate its function.

[00106] Downstream activators of KNP signaling

[00107] KNP requires endothelium to function and hence, an investigation to understand the molecular players in KNP mediated vasodilation was studied.

Endothelial cells respond to different vasoactive factors by synthesizing nitric oxide (NO), prostacyclins and factor that stimulate hyperpolarization of smooth muscle [Mitchell, J.A., et a!., Exp Physiol, 2008. 93(1): p. 141-147].

[00108] Vasoactive mediators produced by the endothelial cells diffuse into the vascular smooth muscle to mediate relaxation. Hence, the requirement these endothelium derived factors were studied using various inhibitors against the enzymes. L-NAME, Indomethacin were used to inhibit nitric oxide synthase (NOS) which is responsible for NO synthesis and cyclooxygenase (COX-1 and COX2) involved in prostacyclin production respectively. Further, the aortic rings were pre- contacted with KC1 and BaC12 to understand the role of potassium channels mediated hyperpolarization in mediating KNP function. As shown in Figs. 6A-6B, KNP' s activity was completely abrogated when L-NAME was used to inhibit NO synthesis. NO diffuses to vascular smooth muscle and interacts with the heme- core of soluble guanylyl cyclase (sGC) to produce cGMP. On inhibiting sGC with methylene blue, KNP function was abolished, showing that NO is a play mediator of KNP signaling. COX-1 and COX-2 inhibition rendered KNP to lose about 60% of its activity; signifying prostacyclins are as well contributing to KNP function.

[00109] KNP evoked 32.1% relaxation when pre-contracted with KC1 which was 50% lower when compared to PE contracted rings. These experiments show the definitive role of potassium channel in KNP signaling.

[00110] Discussion

[00111] Natriuretic peptides are vital components of venom, as they have the ability to offset homeostasis by drastically reducing blood pressure, thereby rendering the prey incapacitated. Like mammalian NPs, venom NPs are synthesized as precursors and processed to their active form. These mature counterparts have the conserved 17- residue ring with variable C-terminal extensions. Despite their overall structural similarity, NPs from reptilian venom have distinct biological activity compared to mammalian NPs [Vink, S., et al, Toxicon, 2012. 59(4): p. 434-45; Rockwell, N.C., et al, Chem Rev, 2002. 102(12): p. 4525-48]. Though this has been attributed to subtle changes in the sequence, no reports on structure- activity relationship of venom NPs have been described.

[00112] Described herein is the investigation of the influence of structure on activity using KNP, a novel NP from Bungarus flaviceps. The precursor encoding KNP was found from the transcriptome analysis of venom gland of B.flaviceps, which encoded for a 147 amino acid residues long precursor with signal peptide. The precursor was speculated to be processed at position 87 after dibasic (KK) residue by a common pre-hormone processing enzyme Kexin [Rockwell, N.C., et al, Chem Rev, 2002. 102(12): p. 4525-48], to produce the mature KNP (60 a.a residues). Mature KNP has 5 residues in the N-terminal segment, 17-residue ring and 38 residues long C- terminal tail. Comparing KNP with well studied ANP, BNP, CNP and DNP, one may speculate KNP to have specificity to NPR-A as ANP, BNP and DNP as it has a long tail which is absent in CNP. Ring of KNP has all the evolutionarily conserved residues except D residues at position 8 and 19 in mature KNP, which are G residues in all known NPs. In ANP, F8 and R14 within the ring, N24 and R27 in the tail, are pivotal for receptor binding. Mature KNP has F7, R13 and K26 in equivalent positions [He, X., et al, Science, 2001. 293(5535): p. 1657- 62; Li, B., et al, Science, 1995. 270(5242): p. 1657-60; Bovy, P.R., Med Res Rev, 1990. 10(1): p. 115-42].

[00113] Although ring of KNP has striking similarity to known NPs, the distinct feature is its tail. The 38 residues long tail has no similarity to any sequence known and is predicted to have the ability to have an a-helical structure, which is not reported for any NP. With KNP's structural features; a possible NPR-A binding ring and a longer C-tail, one would hypothesize KNP may elicit vasorelaxation by binding to NPR-A with lesser potency due to the steric hindrance imposed by the tail. In contrast to the assumption, KNP evoked aortic strip relaxation in an endothelium dependent manner, otherwise a NPR-A independent mechanism with lower potency compared to ANP. This observation shakes the understanding of NPs, as KNP with necessary residues for NPR binding fails to induce activity similar to ANP, in an endothelium independent mechanism [Winquist, R.J., et al.., Proc Natl Acad Sci USA, 1984. 81(23): p. 7661-4].

[00114] Endothelium is a monolayer of cells lining the vascular smooth muscle in a blood vessel, which dynamically produces vasoactive mediators in response to several agonists and shear stress [Furchgott, R.F., Annual Review of Pharmacology and Toxicology, 1984. 24: p. 175-197; Ignarro, L.J., Circ Res, 1989. 65(1): p. 1-21]. Among the various factors, NO, prostacyclins, and Endothelium Derived

Hyperpolarization Factor (EDHF; presently debated to be potassium ions) are major players which diffuse from endothelium to the vascular smooth muscle to arouse cGMP/cAMP to mediate relaxation. Endothelial responses, response to agonist stimulated G-protein couple reactor is the increase of intracellular Ca2+, which activates two important target; NOS and Ca2+ activated potassium channels. NOS produces NO, which activates COX-1 and sGC by binding to the heme-core and thereby activates prostacyclin and cGMP synthesis [Sautebin, L., et al., Br J Pharmacol, 1995. 114(2): p. 323-8]. Further, opening of Ca2+ activated potassium channels, causes efflux of K+ which causes hyperpolarization of endothelial cells. This increase in K+ ions in the vascular smooth muscle gap junction, causes opening of K+ channels in smooth muscle, hence mediates hyperpolarization [Busse, R., et al., Trends Pharmacol Sci, 2002. 23(8): p. 374-80]. Different inhibitors were used to understand the necessity of these vasoactive factors for KNP to show endothelium dependent activity. Inhibition of NOS and sGC completely abrogated the activity, while inhibition of COX-1 and COX-2 reduced the activity of KNP to one third. Hence, when NO synthesis is shut down, the activity of KNP is completely lost, whereas inhibition of prostacyclin synthesis retains activity as available NO may activate sGC to produce cGMP. Pre-contraction of aortic strip with KC1, causes the disruption of hyperpolarization and causes inward rectified potassium channels to open. KNP's activity was lowered when pre-contraction was done using KCl, while it was restored back when BaCl₂ was used. These observation suggest a definitive role of hyperpolarization in mediating KNP's function.

[00115] With the explicit difference in the function of KNP observed, it was necessary to understand why KNP displays a non-classical activity. Structure based activity assessment revealed the answer to this unique mechanism. Deletion mutants of KNP designed based on the structure were namely, Ring (2 residue C-tail), Δ- Helix (Ring with 12 residue C-tail, putative helix region deleted) and Helix (last 26 residues, predicted to form helix). KNP ring functioned as a classical NP, with a lesser potency compared to ANP. Interestingly, helix relaxed aortic strip in an endothelium dependent manner with comparable activity of the full length. This showed the KNP has two functional segments, which induce vasorelaxation in dissimilar pathway. Although both the segments have equipotent activity, it is comparable to that of full length, suggesting that the function of full length KNP is conferred by helix. Further, Δ-Helix showed lower aortic strip dilation abilities compared to full length which was not influenced by endothelium.

[00116] The structure based activity distinctly showed that, although KNP has a ring with NPR-A binding ability, it is the tail of KNP which confers function for the entire molecule. Thus, the tail wags the dog; the tail of KNP redirects the full length away from NPR-A to that as yet unknown receptor on the endothelium to cause vasorelaxation using NO, prostacylins and hyperpolarization.

[00117] Example 2

[00118] This example includes data presented in Example 1 _> as well as new data.

[00119] Sequence and structural analysis of KNP

[00120] The transcriptome of B. flaviceps revealed the transcript which encodes 148-residue precursor protein (Fig. 16). B. flaviceps is an uncommon snake which is hard to obtain and difficult to keep in captivity. Further, venom yields per milking are low. There are three dibasic processing sites in the precursor protein. Processing at the second dibasic site at position 87 leads to 5-residue N-terminal segment ahead of the conserved 17-membered NP ring; such short N-terminal extensions are found in all known NPs (Figure IB). Processing at the third dibasic site at position 113 leads to two-residue C-terminal tail; such short tails have not found in elapid NPs and hence, we assumed that the processing may not occur at this site. Therefore, it was hypothesized that mature KNP is 60-residue long with a 38-residue tail. The C- terminal tail did not show similarity to any protein in the database and interestingly, the last 21 residues showed propensity to form a- helix (Fig. 16). Despite the presumption, the functions of the shorter version of KNP with a short two-residue tail were also tested (see below).

[00121] Further, in KNP two conserved G residues in the ring are replaced by D residues. Described herein the unique structural features and the functional properties of KNP.

[00122] Heterologous expression and purification of KNP

[00123] As there was no natural venom source, KNP was recombinantly expressed and purified. Mature protein was heterologously expressed in E. coli as Trx-His-fusion protein. This protein was obtained from the insoluble fraction.

Following a 2 M urea wash, the protein pellet was solubilized protein in 8 M urea containing buffer and was further purified by reversed phase high performance liquid chromatography (RP-HPLC), and the purity of the protein was assessed using ESl-ion trap mass spectrometry. Fusion protein showed 23734.9 ± 1.9 Da which matched with the calculated mass. The fusion protein was cut by recombinant TEV protease and purified by cation exchange chromatography (CIEX) followed by RP- HPLC (Figs. 17A-17F). The protein peak showed mass of 6602.3 ± 0.9 Da which corresponded to the calculated mass of the oxidized KNP. Thus completely oxidized KNP was obtained and was used to assess its function.

[00124] Synthesis and folding of ANP

[00125] ANP was synthesized by manual Fmoc-based peptide synthesis. The cleaved and purified peptide showed a mass of 3082.0 ± 0.5 Da which corresponded to the reduced mass of the peptide. It was folded by air-oxidation and the peptide after folding showed mass of 3080.6 ± 0.5 Da indicating the formation of the disulphide bond (Figs. 18A-18D)

[00126] KNP induced an endothelium-dependent relaxation of aortic strip

[00127] ANP and KNP induced vasorelaxation in the pre-contracted rat aortic rings (Fig. 10A). ANP induced a dose-dependent vasorelaxation with an EC₅₀ of 16.3 ± 5.4 nM was comparable to EC50 values reported in the literature. KNP also induced a dose-dependent vasorelaxation, but with ~13-fold lower potency (EC50 230.6 ± 37.2 nM) (Fig. 10B). ANP caused a quick vasodilatory response at > 10 nM, while KNP induced slow relaxation for all the concentrations tested (Figure 1 OA). The effects of KNP on vasorelaxation in endothelium-denuded aortic rings was also examined. ANP induced equipotent vasorelaxation with an EC₅₀ of 23.4 ± 7.8 nM, as NPRs are expressed in both endothelium as well as vascular smooth muscle. Interestingly, the activity of KNP was significantly (P- value: 0.0043) reduced when endothelium was denuded (Figure IOC). Thus, KNP mediates endothelium- dependent vasodilatory response, which is not NPR NPR-dependent. These results indicate that KNP induces vasodilation through a different mechanism.

[00128] Effect of KNP and ANP on hemodynamic and renal parameters

[00129] The effects of KNP on blood pressure (BP), heart rate and urine output were examined in experimental animals (Fig. 11 A). ANP and KNP were

intravenously infused into two groups of anesthetized rats for 10 min. The baseline mean arterial pressure (MAP) of the animals used were 114.5 ± 2.4 mmHg. In comparison to control animals (0.2% BS A saline infused n = 5) infusion of ANP (0.2 nmol/kg/min in 0.2% BSA saline n = 5) induced 6.5 ± 0.9 mmHg drop in MAP at the end of the infusion period which dropped reaching 8.5 ± 0.5 mmHg during the first 10 min recovery. By the end of 30 min the MAP was restored to the baseline. A 10-fold higher dosage was chosen for KNP based on the difference in activity on the aortic strip. KNP (2 nmol/kg/min in 0.2% BSA saline n = 5) showed a similar reduction in MAP at the end of the infusion period (6.1 ± 3 mmHg) (Fig. 1 IB). MAP continued to drop further reaching a difference of 11.9 ± 1.9 mmHg after the second recovery period. Although the MAP of animals infused with KNP started recovering, it did not recover back to the baseline within the experimental recovery period (40 min); at the end of 40 min, MAP was 10 ± 3 mmHg below the baseline (Fig. 1 IB). Thus, KNP induced a sustained drop in MAP compared to ANP.

[00130] ANP caused a steep drop in pulse pressure (PP), calculated as the difference of systolic BP and diastolic BP (Baseline PP: 47.4 ± 2.4 mmHg), by 7.2 ± 0.9 mmHg during infusion. PP began to restore back to baseline by the end of the experiment (Fig. 11C). In the case of KNP, although there was a milder drop in PP (3.7 ± 1 mmHg), it did not restore back during recovery period. PP is dependent on stroke volume and compliance of arteries. ANP is shown to reduce venous return of blood to the heart, which decreases stroke volume. In the case of KNP, change in systolic BP was comparable to ANP, while decrease in diastolic BP was more profound (ANP: 6.5 ± 1 mm Hg, KNP: 11.1 ± 1.8 mmHg). Due to similar differences in systolic and diastolic BP, the net PP was not greatly altered for KNP (Figs. 24A-24E).

[00131] Baseline heart rates of the animals were 371.9 ± 10.9 BPM. When ANP was infused, the heart rate was reduced by 39.9 ± 23.6 BPM during infusion which further dropped to a difference of 58.1 ± 19 BPM within the first recovery period (Fig. 1 ID). The reduced heart rate recovered back to baseline within the next 30 min. In the case of animals infused with KNP, the reduction in heart rate was not as profound (10.6 ± 21 BPM). But during the recovery period, heart rate persistently dropped reaching a difference of 42.1 ± 16 BPM at the end of experimental period (Fig. 1 ID).

[00132] In the same experimental animals, urine was collected throughout the experimental period by bladder catheterization. The baseline urine flow rate was 3.7 ± 0.5 μΐ/min. ANP showed marked diuresis with an increased flow rate of 8.8 ± 1.4 μΐ/min. The urine flow rate returned back to baseline within 20 min of recovery. Similar changes in renal output were observed previously. In contrast, the infusion of KNP showed only a minor increase in urine flow rate (2 ± 1.2 μΐ/min) which was sustained all throughout the experimental period (Fig. 1 IE).

[00133] These results indicated that KNP influences blood pressure, heart rate and renal output differently than ANP and further supports on contention that KNP exerts its biological effects through a distinct mechanism.

[00134] Design and Synthesis of KNP deletion mutants

[00135] To understand the role of distinct regions in KNP, several truncated and deletion mutants were designed (Fig. 12 A). To examine the role of C-terminal helix, KNP without this a-helix (AHelix, residues 1-34), C-terminal a-helix (Helix, residues 3 -60) and the helix directly attached to KNP ring (R&H fusion of residues 1-22 with residues 39-60) were designed. The shorter version of KNP (Ring, residues 1-24 that could be formed during maturation as described above) was also designed. Ring and Helix peptides were manually synthesized by SPPS, while AHelix and R&H peptides were obtained by heterologous expression. The masses of purified Ring, Helix, AHelix and R&H peptides, determined by ESI-MS, matched the theoretical masses (Table 2, Figs. 19A-19F, 20A-20F, 21 A-21D, 22A-22B)).

[00136] Vasodilatory properties of KNP deletion mutants [00137] Various deletion mutants of KNP were assessed for their ability to relax pre-contracted aortic strip (Figs 12B and 12C) and the EC50 values were determined (Table 1). Ring peptide evoked equipotent relaxation in aortic rings with and without endothelium (P- value: 0.33 no significant difference). Although its ability to relax was comparable to full length KNP, it elicited an endothelium-independent relaxation similar to a Classical NP (P-value: 0.25 no significant difference).

[00138] AHelix had much lower potency compared to KNP (P-value: 0.0002), but it showed equivalent response in both endothelium-intact and endothelium-denuded aortic rings. Helix, on the other hand, showed similar vasorelaxation ability in both types of aortic rings (P-value of helix with and without endothelium: 0.015). Also Helix induced slow relaxation as KNP (Fig. 23). These results indicate that the extension of the C-terminal tail decreases the potency of KNP ring (Ring vs.

AHelix) and C-terminal putative helix, independent of the NP ring, induces equipotency vasorelaxation as KNP. Vasorelaxation ability of R&H is similar to KNP (P-value: 0.25) indicating addition of the C-terminal helix to the KNP ring (with or without a spacer segment) switches the mechanism from endothelium- independent to endothelium-dependent mechanism (P-value R&H of with and without endothelium: 0.02). These results indicated that there are two vasoactive parts in KNP, namely, NP ring and the C-terminal helix; the former most-likely functions through NPR-A, while the latter functions through unknown mechanism. Further, it was hypothesized that the C-terminal helix redirects the KNP ring away from NPR-A-dependent vasorelaxation.

[00139] Activation of NPR-A and NPR-B by KNP and its mutants

[00140] To understand the ability of the KNP and its mutants to activate NPR-A and NPR-B, cells endogenous lacking these receptors (CHO-K1) were transfected with plasmids encoding rat NPR-A and NPR-B separately. The abilities of KNP and its deletion mutants to induce signal transduction and evoke cGMP response in these cells were measured and compared with those of ANP on NPR-A and CNP on NPR-

B.

[00141] On cells expressing NPR-A, ANP evoked a dose-dependent cGMP response (Fig. 13 A). Ring elicited a 10-fold less potency compared to ANP.

Interestingly, C-terminal extensions in KNP, AHelix and R&H led to further loss in cGMP response. Helix did not cause any elevation of cGMP indicating that it does not interact with NPR-A. These observations were in agreement to the vasodilatory properties of these peptides.

[00142] On cells expressing NPR-B, CNP evoked highly potent cGMP response. However, as expected because of the presence of C-terminal residues, KNP and its deletion mutants were not able to evoke any cGMP response in cells expressing NPR-B.

[00143] These results showed that Ring is a specific ligand to NPR-A, but with a lower potency compared to ANP and the presence of the C-terminal tail redirects the

[00144] Downstream signaling of KNP and its vasoactive segments

[00145] As KNP and Helix require endothelium to function, the role of various intermediates involved in endothelium-mediated vasorelaxation was examined. Endothelial cells respond to different vasoactive factors by synthesizing nitric oxide (NO), prostacyclins and factor that stimulate hyperpolarization of smooth muscle (Fig. 14A). These factors diffuse from endothelium to the vascular smooth muscle to arouse cGMP/cAMP levels to mediate vasorelaxation. In a NPR mediated signaling, the cGMP is aroused by the GC domain linked to the receptor. Hence, the above mentioned vasoactive factors have negligible role in NP-NPR-signaling. Therefore, specific inhibitors, namely L-NAME for NOS, indomethacin for COX, ODQ for sGC and KCl-induced precontraction, were used to subdue the hyperpolarization process (Fig. 14 A), to investigate the role of the endothelium-derived vasoactive factors in KNP signaling.

[00146] ANP's ability to relax the pre-contracted aortic strip was unmodulated in the presence of any of the inhibitors (Fig. 14B). With KCl-pre-contracted rings there was a slight decrease in the activity. Similar to ANP, Ring also had no influence of L-NAME, ODQ and indomethacin on its activity, but lost about 30% of its activity which pre-contracted with KC1. Thus, Ring acts similar to classical NPs and does not require any endothelial factors to cause vasodilation. A NPR-mediated vasodilation involves cGMP-dependent activation of PKG which (a) phosphorylates myosin light chain and (b) opens big conductance potassium channel leading to hyperpolarization. These two processes cause the smooth muscle relaxation. Thus, the activity of ANP and Ring are lowered when KC1 was used for pre-contraction.

[00147] K P's activity was completely abrogated in the presence of L-NAME. NO either activates COX or diffuses to vascular smooth muscle and activates soluble guanylyl cyclase (sGC) (Fig. 14A). The inhibition of COX-1 and COX-2 by indomethacin and that of sGC by ODQ leads to 60% and 90% loss of KNP's activity, respectively. Similarly, inhibition by L-NAME and ODQ completely abolished the ability of Helix to cause vasodilation, while COX inhibition by indomethacin resulted in 70% reduction in its activity. These results indicated that NO plays a key role in KNP-induced vasorelaxation. KNP evoked 25% relaxation in KCl-contracted aortic rings; this is 50% lower when compared to PE-contracted rings. Helix failed to evoke relaxation of KCl-contracted aortic strip. These results can be explained in two alternate ways: (a) hyperpolarization plays an important role in KNP signaling, but not in Helix signaling; and (b) high salt concentration breaks the interaction of Helix with its receptor. The second possibility is supported by high density of positively (4) and negatively (2) residues in 26 residues. In this scenario, high salt would also interfere with binding of C-terminal helix to its receptor and the observed partial activity could be due to the binding of the KNP ring.

[00148] In-vivo effect of KNP truncations on MAP, PP, heart rate and urine output

[00149] To understand the contribution of different pharmacophores of KNP to its in-vivo activity, Ring, AHelix and Helix (2 nmol kg/min) were intravenously infused in experimental rats. Ring decreased MAP by 10.7 ± 2.5 mmHg during infusion which recovered back within the experimental period while AHelix and Helix caused a mild drop (4.3 ± 1.4 mmHg and 4.7 ± 1.1 mmHg ) during infusion period which was sustained during the recovery period (Fig. 15 A). Alteration in MAP profile of Ring was similar to that of ANP (P-value > 0.05; no significant difference) while the additive effect of Helix and AHelix seemed to reflect KNP's profile.

[00150] Ring and AHelix caused a meek reduction in heart rate (22.2 ± 12 BPM) which quickly returned back to baseline in case of ring while was sustained in case of AHelix (Fig. 15B). Heart rate remained unperturbed in animals infused with Helix similar to control. Although Ring and AHelix had lowered potencies, the overall heart rate profile of Ring matched that of ANP while AHelix was similar to KNP.

[00151] Ring reduced PP to similar extent as that of KNP (4.5 ± 1 mmHg) but restored within 20 min after the infusion was stopped (Fig. 15C). AHelix and Helix showed a mild decrease (2 ± 1.3 mmHg) which restored back in case of AHelix while sustained in case of Helix. Like the heart rate profile, Ring evoked similar changes in PP like ANP with lesser effects (at t =30, 40, 50 and 60 min, P-value < 0.05 compared to ANP) while Helix caused comparable effects to that of KNP (P- value > 0.05, no significant difference).

[00152] None of the KNP truncation had influence on urine volume like the full length molecule (Fig. 15D). The Ring has two substitutions in position 3 and 14 of the Ring where conserved G residue is replaced by D. These residues come in proximity to negatively charged pocket on the receptor. Hence, to understand the influence of these substitutions, Ring with both D residues replaced by G called G- Ring (CFGRRIDRISHTSGIGC (SEQ ID NO: 43) was synthesized and evaluated for its in- vivo activity. G-Ring evoked effects on hemodynamic parameters similar to Ring with restored renal effects. Thus, the overall effect on hemodynamic parameters evoked by the Ring was like a classical NP while both the

pharmacophores seems to contribute to KNP's function.

[00153] Discussion

[00154] Natriuretic peptides are vital components of venom. They are thought to cause potent hypotension that would lead to rapid unconsciousness. Like

mammalian NPs, venom NPs are synthesized as precursors and processed to their active forms. These mature counterparts have the conserved 17-residue ring with variable C-terminal extensions. Despite their overall structural similarity, NPs from reptilian venom have distinct biological activity compared to mammalian NPs (Fig. 9). Though this has been attributed to subtle changes in the sequence, no reports on structure-activity relationship of venom NPs have been described.

[00155] The study described herein focuses on the mode of action and structure- activity of KNP, a novel NP from Bungarus flaviceps. The precursor encoding KNP was found from the transcriptome analysis of venom gland of B. flaviceps, which encoded for a 148 amino acid residues long precursor with a signal peptide. The precursor was speculated to be processed at position 87 after dibasic (KK) residue by a common prehormone processing enzyme Kexin, to produce the mature KNP (60 amino acid residues). Mature KNP has 5 residues in the N-terminal segment, 17- residue ring and 38 residues long C- terminal tail. This tail has no similarity to any sequence known and is predicted to have the ability to form an a-helical structure, which is not reported for any NP. Ring of KNP has all the evolutionarily conserved residues except D residues at position 8 and 19 of mature KNP, which are G residues in all known NPs. In ANP, F8 and R14 within the ring, N24 and R27 in the tail, are pivotal for receptor binding. KNP has F7, R13 and K26 in equivalent positions. Hence, with these structural features it is likely that KNP with a long C- terminal tail interacts with NPR-A with lower affinity due to the steric hindrance that the tail might impose.

[00156] In contrast to the assumption, KNP evoked aortic strip relaxation in an endothelium dependent manner or otherwise NPR-A independent mechanism, unlike ANP. Thus, initial characterization of KNP showed it was a weak vasodilator compared to ANP but required endothelium derived vasoactive factors to do so.

[00157] Previous studies have shown that infusion of supraphysiological doses of ANP results in marked reduction of BP. This lowering of BP is due to decreased, stroke volume, heart rate, peripheral resistance and increased excretion of water. The experiments on effect of KNP on vascular and renal parameters described herein indicate that KNP does not seem to follow the paradigm of NPs. It was evident that KNP infusion resulted in reduction in cardiac output as it lessened heart rate and decreased stroke volume indicated by decreased diastolic pressure. But KNP seems to affect these parameters in a sustained manner in contrast to the prompt recovery which seen in case of ANP. Further, KNP did not have any significant renal effect which was a striking difference between the two peptides.

[00158] To understand the differences between KNP and ANP, a structure-based activity assessment revealed the answer to this non-classical activity. KNP ring functioned as a classical NP, with 10 fold lower potency compared to ANP. The lower activity of Ring could be attributed to two important features of the ring. The two D substitution in place of G at positions 8 and 19 of mature KNP and the lack of C-terminal NSFRY sequence similar to ANP. The crystal structure of ANP with NPR-A indicates that G residues (9, 20) at equivalent positions fall in the vicinity of E169A and E169B of the receptor (Fig. 25). On replacement of this G with D as in case of Ring might cause an electrostatic repulsion, which could result in lower potency. Further, receptor binding studies have shown that ANP loses its affinity to NPR-A on deletion of the C-terminal extension. This fact is supported by the information from the crystal structure. Residues N24, S25, F26 and R27 of the C- terminal extension of ANP form a β-sheet with Q186 B and F188 B of the receptor along with a hydrogen bond between N24 of ANP with El 87 B. KNP ring lacks these interactions with two basic residues as its tail. These structural changes may explain the lower potency of Ring.

[00159] Interestingly, Helix relaxed aortic strip in an endothelium dependent manner with comparable activity to that of full length. This showed that KNP has two pharmacologically active segments, which induced vasorelaxation by distinct mechanisms. Although both the segments have equipotent activity comparable to that of full length, putative helical segment of the tail seemed to contribute to the function of KNP. The vasorelaxation ability of R&H was in agreement with what was observed for KNP. The presence of the helical segment attributed function to R&H as well as KNP in an endothelium dependent manner. Further, Δ Helix showed much lower aortic strip dilation abilities compared to full length which was not influenced by endothelium.

[00160] Similar observations were obtained when intracellular cGMP levels were measured in response to activation NPR-A by different ligands. Ring elicited a 10 fold lower activity compared to ANP. In comparison to ANP, KNP, AHelix and R&H showed a 300 fold lower potency to elevate cGMP levels in the cells. This showed that the presence of any segment of tail of KNP lower the binding ability of the ring. In other words, tail segment of KNP was redirecting the otherwise functional ring away from the NPR. These results were conclusive to show that despite the presence of a functional NP ring, KNP lacks classical NP like functions due to its C-terminal tail.

[00161] In an investigation to obtain insights into the molecular players of KNP evoked vasorelaxation, endothelium derived vasoactive factors namely NO, prostacyclins and hyperpolarization seem to influence KNP's vasodilatory properties. KNP and Helix did have alteration in their vasodilatory properties in the presence of different inhibitors. Although all three factors seem to influence the activity of the peptides, inhibition of NO production lead to complete loss of function. Hence, looking at the possible mechanisms through which NOS is activated would likely lead to the molecular target. Endothelial NOS is activated by pathways that increase intracellular calcium. This increase in Ca²⁺ is dependent either on IP3 and DAG or Gq protein when different receptors are activated. Further, direct potassium channel openers have shown to increase intracellular calcium. Hence, sequential inhibition phospholipase C (enzyme that produces IP3 and DAG), Gq protein activation, different class of potassium channel (Calcium activated-, voltage gated-, ATP dependent- potassium channels) and monitoring the

vasodilatory properties of Helix and KNP will further narrow down the possible molecular target through which these peptides function.

[00162] Thus, the study described herein provides a mechanism of action of KNP. A classical NP like ANP will bind to NPR on both endothelium and vascular smooth muscle to elevate cGMP to cause vasodilation. While KNP, a non-classical NP, binds to a unknown target receptor or ion- channel on the endothelium cells and produces NO, prostacyclins and K+ hyperpolarization to cause relaxation of the underlying smooth muscle cells using its tail, despite the presence of a function ring. Thus, KNP's tail wags its ring (Fig. 7).

[00163] In-vivo the activity of KNP seems to be the contribution of both the pharmacophores. It may be speculated that KNP may be proteolytically cleaved to produce both the functional segments and hence an additive effect of both the segments is observed as KNP's activity. Further, the presence of a long tail may increase the PK of KNP.

[00164] Neither KNP nor its truncations had any renal effects. Despite being able to bind to NPR-A like ANP, Ring did not cause diuresis which was an intriguing observation. In the past, alternately spliced variant of BNP was shown to have renal effects while lacking vascular functions. The presence of the D residues in place of conserved G at positions 3 and 14 of the KNP ring may alter the conformational plasticity of the receptor which dictates the physiological response. Thus, it is likely that the conformational changes for the receptor activation may be altered in the case of Ring which dictates the tissue specific response.

[00165] Experiments

[00166] Materials

[00167] Codon optimized Synthetic DNA of KNP cloned in pUC-57 from Genescript corporation (Piscataway, NJ, USA), pET-32a vector (Novagen, Merck biosciences), RP- Jupiter CI 8 columns ( 300 A, 5 μιη, 250 mm X 21.2 mm and 250 mm X 10 mm) from Phenomenex (Torrance, CA, USA), Hiprep 26/10 desalting ^" column and Hi-Trap-Sulfopropyl (SP)-Sepharose column (GE healthcare, Little chatfont, UK), TGA, TGR, pre-loaded Wang resin (Novabiochem, Merck chemicals, NY,USA), CHO-K1 cell line (American Type Culture Collection, Manassas, VA, USA), L-NAME, Indomethacin, Dulbecco's Modified Eagle's Medium (DMEM), IBMX, BSA (Sigma-Aldrich ,St. Louis, MO, USA), Penicillin- Streptomycin, Trypsin and trypsin neutrahzer (GIBCO, Invitrogen, CA, USA), ODQ (Cayman Chemical, Ann Arbor, MI, USA), cGMP complete ELISA kit (Enzo lifesciences, NY,USA).

[00168] Animals

[00169] Animals (Sprague Dawley rats) were obtained from the Invivos and acclimatized in Comparitive medicine, Animal Holding Unit for a minimum of one day before the experiments. The animals were kept under standard conditions with food and water available ad libitum in a light-controlled room (12 h light/dark cycle, light on 07:00 h) at 23°C and 60% relative humidity

[00170] Cloning of Synthetic gene

[00171] The required DNA fragments were amplified using primers as listed in Table 3. The forward primers included Kpnl restriction site and tobacco-etch virus protease (TEV) recognition site and reverse primers included Sacl restriction site. The DNA fragment encoding R&H was amplified by sequential amplification of two independent fragment followed by fusion of the two products. Synthetic DNA (0.1 ig) containing plasmid was mixed with 1 μΐ of 10 μΜ primer stock, 1 μΐ of 10 mM dNTPs, 1 unit of Kapa HiFi polymerase and 10 μΐ of 5X PCR buffer containing MgC12 in a 50 μΐ reaction. The cycling parameters were set as one cycle of 98°C for 2 min, 35 cycles of 98°C for 10 s, 60°C for 30 s and 72°C for 45 s, and a final extension at 72°C for 7 min.

[00172] Expression of KNP, AHelix and R&H

[00173] Protein of interest was expressed as a Thioredoxin (Trx)- 6X histidine (His) fusion protein with a Tobacco-etch virus (TEV) protease cleavage site (Trx- His-TEV-protein).The amplified fragments were cloned into the multiple cloning site of pET 32a vector transformed into E.coli DE3 plys expression strain. Single colony was chosen and was used to inoculate 100 ml of LB Broth containing 100 μg/ml ampicillin and grown for 16 h at 37°C at 200 rev/min. This culture was added to 1 1 of LB broth containing 100 g/ml ampicillin and was growth at 37°C, 200 rev./min until the optical density at 600 nm reached ~ 0.6. The cells were induced to produce the fusion protein with 0.1 mM IPTG and further incubated for 16 - 20 h at 16°C, 200 rev/min. Subsequently, the cells were harvested and sonicated after resuspension in native lysis buffer (50 mM Tris-Cl, 150 mM NaCl pH 8). The lysed cells were spun at 14,000 g and pellet was washed with wash buffer (50 mM Tris- Cl, 150mM NaCl, 2 M urea pH 8). The slurry was spun at 14,000 g and the pellet was solubilized in denaturation buffer (50 mM Tris-Cl, 150 mM NaCl, 8 M urea pH 8). The expression of the fusion protein was analyzed on SDS-PAGE using 15% polyacrylamide gel.

[00174] Purification of KNP, AHelix and R&H

[00175] The crude protein mixture from the solubilized pellet was syringe filtered and purified by reversed-phase chromatography using Jupiter C18 (5 μηι, 300 A, 250 X 21.2 mm ) preparative grade HPLC column using 0.05 % formic acid (FA) as buffer A and 100 % acetonitrile with 0.05 % FA as buffer B on Akta purifier system (GE Healthcare Life Sciences, Little Chatfont, UK). A linear gradient of 40-70 % B was used to achieve required separation of the protein peaks and the quality of the fusion protein was analyzed using Electrospray-ionization (ESI) - Mass

Spectrometer (MS) (LCQ Fleet Ion trap, Thermoscientific, Massachusettes, USA). The fractions which showed pure protein mass were freeze dried.

[00176] The freeze dried fusion protein was dissolved in 50 mM Tris-HCl, 150 mM NaCl, 6 M urea pH 8 and desalted using 50 mM Tris-HCl pH 8 buffer using a Hiprep 26/10 desalting column (26 mm X 100 mm). The desalted fusion protein was cleaved using recombinantly expressed and purified TEV protease in the ratio 40: 1 (fusion protein:TEV) in 50 mM Tris-HCl containing 0.5 mM EDTA pH 8, overnight at 4°C.

[00177] Following cleavage, cation exchange chromatography was performed with 50 mM Tris-HCl pH 8 as buffer A and 50 mM Tris-HCl, 500 mM NaCl pH 8 as buffer B, on a Hi-Trap-Sulfopropyl (SP)-Sepharose column (34 μιη, 16 X 25 mm) to separate the protein of interest from the tag. Further the cleaved protein peak was purified to homogeneity using RP-HPLC with Jupiter CI 8 column (5 μηι, 300 A, 250 mm X 10 mm) with buffer A- 0.05 % FA and buffer B- 0.05% FA with 100% Acetonitrile on a linear gradient of 20-40% B. Purified protein peak was checked on ESI-MS for its purity and freeze dried. The protein was reconstituted in phosphate buffer saline and quantified by absorbance at 280 nm before the assay.

[00178] Synthesis and purification of ANP, Ring and Helix

[00179] ANP, Helix and Ring were synthesized using manual Fmoc- based peptide synthesis. ANP was synthesized using Tyr-preloaded Wang resin, Helix using Novasyn TGA resin and Ring using Novasyn TGR resin. 5 times excess of Fmoc- Amino acid derivatives were activated using 4.9 times HATU and 10 times DIPEA and the coupling reaction was performed in the ratio 2:1 DMF: NMP.

Following coupling, the F-moc group was removed using 20% (v/v) piperidine in DMF. After the completion of synthesis, peptides were cleaved from the resin using TFA:EDT:TIS: water (94:2.5:1 :2.5) and precipitated using ice cold 1,2 diethyl ether. The crude peptides were purified using Jupiter CI 8 , 5 μηι, 300 A (10 X 250 mm Phenomenox) column with Buffer A- 0.1 % Tri-fluro-acetic acid (TFA) and Buffer B- 0.1% TFA with 80% acetonitrile. The purity and homogeneity of the peptides were assessed using ESI-MS and pure fractions were freeze fried.

[00180] ANP was subjected to air oxidation in 100 mM Tris pH 8 with 10% acetonitrile while Ring was oxidized using 100 mM Tris-Cl pH 8, 20% DMSO and 10% Acetonitrile. The oxidized peptides were purified by RP-HPLC and the mass of the peptides determined by ESI-MS.

[00181] Vasorelaxation assay

[00182] Male Sprague Dawley (SD) rats (10 weeks old) were euthanized and the descending thoracic aorta was isolated and flushed with physiological solution (Krebs Buffer: 118 mM NaCl, 4.7 mM KC1, 1.2 mM KH₂P0₄, 1.2 mM MgS0₄, 1.25 mM CaCl₂, 25 mM NaHC0₃, 11 mM glucose, pH 7.4). The fat tissues and connective tissues on aorta were removed, cut into 2-3 mm rings and mounted under 2.5 g tension into the organ bath chamber using two stainless steel hooks and fixed to MLT0201/RAD Force transducer (AD Instruments). The ssues were e uilibrated in chambers at 37 C containing carbogenated (95% 0₂ and 5% C0₂) Krebs buffer for an hour. The presence of endothelium in the aortic strip was checked by precontracting the tissue with 300 nM phenylephrine (PE) and relaxing with 10 μΜ Acetylcholine (ACh). The aortic rings were pre-contracted with 100 nM PE and a cumulative dose response for different peptides/ protein was obtained. Further, in experiments involving endothelium denuded aortic rings; the inner surface of the aortic ring was rubbed with a cotton bud to remove the endothelial lining.

[00183] To study the effect of different inhibitors, the tissue was incubated with individual inhibitors such as 100 μΜ L-NAME (L- arginine methyl ester

hydrochloride, inhibitor of NO synthesis), 10 μΜ Indomethacin (cycloxygenase 1 and 2 inhibitor) or 20 μΜ ODQ (lH-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one, soluble guanylyl cyclase inhibitor) for 20 min prior to pre-contraction of tissue. The inhibition was confirmed with attenuated Ach relaxation ability. To understand the role of hyperpolarization in mediating KNP signalling, tissues were pre-contracted with 40 mM KC1. The pre-contracted tissue in the presence of a particular inhibitor was used to assess the activity of 300 nM of the peptides.

[00184] Three independent trials for each concentration of every peptide has been evaluated and statistical analytical analysis was done using one-way ANOVA and a P-value < 0.05 is considered significant difference.

[00185] Measurement of Mean arterial pressure and urine volume

[00186] Experiments were performed on male Sprague Dawley (SD) rats (220- 300 g) in accordance with protocol 041/12 approved by Institutional Animal Care & Use Committee, NUS. Rats were anesthetized with 60-70 mg/kg Pentobarbital (IP) and placed on heating pad at 37°C to maintain body temperature. Urinary bladder catheterization was performed as illustrated in 36 with certain modifications as described. A midline incision was made to expose the urinary bladder. The bladder was retracted and an 18 G needle was used to puncture the bladder neck. A 5 cm long PE 50 catheter was cut at one end to have a pointed and elliptical end, which was introduced into the bladder through the puncture. A purse-string suture was placed in the bladder surface at the site of entry of the catheter to secure it. 0.9% saline was injected into the bladder through the catheter to check if the bladder was leaking.

[00187] Subsequently, a left oblique groin incision was made to expose the femoral artery and vein which were isolated and cannulated. After the surgery 1 ml of 0.2% BSA containing saline was injected intravenous to compensate for surgical loss. A fluid filled physiological pressure transducer (MLT 844, AD Instruments) filled with 50 IU/ml heparinized saline was connected to the femoral artery catheter, while saline containing 0.2% BSA was infused through the catheter inserted into femoral vein at 2 ml/h for equilibration period of 20 min. Mean Arterial Pressure (MAP), heart rate and urine volumes were measured continuously. The experiment begun with 1 control periods (C- 10 min) followed by the 2 experimental periods (El and E2, 5 min each) during which 0.2 nmol/kg/min ANP or 2 nmol/kg/min KNP/Ring /Helix/AHelix in 0.2% BSA saline was infused. The animal was left to recover for 40 min (R1-R4, four recovery periods of 10 min each) during which 0.2% BSA saline was infused.

[00188] Five independent trials has been performed for each group and the statistical comparison for each time point was done using student t-test and a P- value < 0.05 is considered significant.

[00189] Cell culture

[00190] CHO-Kl cells were maintained in high-glucose Dulbeco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin and 2 mM glutamine in a humidified incubator at 37°C with 5% C0₂. The cells were sub-cultured by trypsinization every three days. The cells were counted using Bio-rad TC20™ automated cell counter.

[00191] NPR-A and NPR-B plasmid preparation

[00192] Plasmid encoding NPR-A and NPR-B were generously gifted by Dr. Ruey-Bing Yang (Academia Sinica) and Prof. Micheala Kuhn (University of Wuerzburg). The plasmids were transformed into JM109 E.coli strain. Single colony of the transformant was inoculated in 100 ml of LB broth containing 100 μg/ml ampicillin and was grown at 37°C, 200 rev./min for 22-24 h. The cells were harvested by spinning at 5,000 g for 20 min and the plasmids were extracted using Pure yield™ plasmid maxiprep kit (Promega) using manufacturer's protocol.

[00193] Transfection of CHO-K1 cells

[00194] 1X10⁵ cells were seeded per well in 24- well plate. The cells were allowed to growth for 20-22 h before transfection. The used media was aspirated from the wells and replenished with 400 μΐ of DMEM containing 10% FBS without any antibiotics. Each well was provided with 0.8 μg of plasmid and 2 μΐ of lipofectamineTM 2000 transfection reagent. The cells were incubated with the DNA-lipid complex for 6 h at 37°C with 5% C02 after which DMEM containing 10% FBS and antibiotics. These cells were incubated at 37°C with 5% C0₂ for 16- 20 h before it was treated with the peptides.

[00195] Whole cell cGMP elevation assay

[00196] CHO-K1 cells transfected with either NPR-A/ NPR-B/ empty-vector pCMV4.0 was used for the study. Post transfection, the media was aspirated and the cells were washed with 500 μΐ of PBS. The cells were incubated for 30min after the addition of 150 μΐ of 0.5 mM IBMX containing vascular growth media. Meanwhile peptides are reconstituted in 4X concentrations. After 30 min of incubation, 50 μΐ of the peptide was added to the cells and further incubated for 30 min at 37°C with 5% C0₂. Subsequently peptide containing basal media was removed and 150 μΐ of 0.1 M HC1 was added and incubated at room temperature for 30 min with shaking at 50 rpm. The cell lysates were collected and assayed for cGMP levels using Enzo life sciences cGMP ELISA kit, using manufacturer's protocol. Total protein content of the cell lysates of a few wells was determined by BCA (bichinchonic acid) assay and this value was used to normalize across different 24 well plates used. Three independent trials were performed for each peptide and the statistical analysis was perfor med using one-way ANOVA and a p-value < 0.05 is considered significant. Table 1. Comparison of vasorelaxation properties of ANP, KNP and KNP mutants

Peptide ECs, (n )

Endothelium + Endothelium -

ANP 16.3 ± 5.4 23.4 ± 7.8

KNP 230.6 ± 37.2 -

Ring 228.6 * 43.1 279.5 ± 47.2

Helix 326.7 ± 64.3 -

R&H 267.13 ± 45.7 -

ΔΗβΗχ > 1000 > 1000

Table 2 '■ Primers used for cloning KNP, Λ Helix and R&H

Construct Forward primer Reverse primer

S'GGTACCS AAAACCTGTACTTGCAATCG G S'GATAGTAAAGAGAGCCSTGCCGCATTAA GCCTGCTGATTT3' CSEQ Π) NO. 34) TAAGAGCTC3' (SEQ ED NO. 35)

S'GGTAC BAAAACCTGTACnrXAATCG G S'CTTG AGCTCTTATTATTATGCCGGCGGTG GC TGCTGATTT3" (SEQ ID NO. 35) CACGCGG3' (SEQ ID NO. 37)

5'GGTACCGAAAAC TGTACTTCCAATCGG 5 CACCGCCAG03GCGCAC3GACAACCGATA GCCTGCTGATTT3' (SEQ ID NO. 38) T? (SEQ Π NO. 39)

PCR2 5'CACCGCCAGCGGCGCACGACAACCGATA 5'GATAGTAAACAGAGCCGTGCCGCATTA

T3' (SEQ ID NO. 40) ATAAGAGCra' (SEQ ID NO. 41)

Table 3 : List of observed and theoretical masses of different KMP and its truncations

Construct Observed mass (Da) Theoretical mass (Da) NP 6602.3 ±0.9 6602.5*

AMP 3080.6 ±0.4 3080.2*

Ring 2768.7 ±0.7 2769.1*

Helix 2765.1 ± 1.0 2765.7

AHeltx 3855.6 ± 1.2 3855.8*

R&H 5052.5 ±0.6 5052.4*

* Represents oxidized mass

[00197] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[00198] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of reducing blood pressure in an individual in need thereof

comprising administering to the individual an effective amount of a composition comprising a peptide having an amino acid sequence of SEQ ID NO: 42, one or more variants thereof, and/or one or more biologically active portions.

2. The method of claim 1 wherein the amino acid sequence of the peptide comprises SEQ ID NO: 1, SEQ ID NO: 7 or a combination thereof.

3. The method of claim 1 wherein the amino acid sequence of the biologically active portion of the peptide comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 44 or a combination thereof.

4. A method of treating high blood pressure in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising a peptide having an amino acid sequence of SEQ ID NO: 42, one or more variants thereof, and/or one or more biologically active portions thereof.

5. The method of claim 4 wherein the amino acid sequence of the peptide

comprises SEQ ID NO: 1 , SEQ ID NO: 7 or a combination thereof.

6. The method of claim 4 wherein the amino acid sequence of the biologically active portion of the peptide comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 44 or a combination thereof.

7. The method of any one of claims 4-6 wherein the individual has

hypertension.

8. A method of causing vasodilation without diuresis in an individual in need thereof comprising administering to the individual an effective amount of a composition comprising a peptide having an amino acid sequence of SEQ ID NO: 42, one or more variants thereof, and/or one or more biologically active portions thereof.

9. The method of claim 8 wherein the amino acid sequence of the peptide comprises SEQ ID NO: 1, SEQ ID NO: 7 or a combination thereof.

10. The method of claim 8 wherein the amino acid sequence of the biologically active portion of the peptide comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 44 or a combination thereof.

11. The method of any one of claims 1-10 wherein the individual is a human.

12. A pharmaceutical composition comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 30, 31, 32, 33, 42, 43, 44 or a combination thereof.