Binding Compound and Uses Thereof

Abstract

Described are compounds for targeting proteases, e.g. serine proteases and their use in the diagnostic methods and methods for treatment of respiratory diseases such as cystic fibrosis. The compounds have the structure [H][B]-[A]; wherein [H] is a hydrophilic group, [B] is a subsite recognition group and [A] is a binding group; wherein A has the formula: C(0)CH.sub.2NR.sup.1COOR.sup.2 and wherein [B] has the structure: (i) [COCH.sub.2NR.sup.3]m-, or (ii) -[AA1-AA2]- or (iii) -(AA1-C0-CH.sub.2NR.sup.3) or (iv) (COCH.sub.2NR.sup.3-AA1)- or (v) (C0CH.sub.2NR.sup.4-AA1-AA3)-.

Claims

1. A compound which has the structural formula:
[H][B]-[A]; wherein [H] is a hydrophilic group, [B] is a subsite recognition group and [A] is a binding group; wherein [A] has the formula: ##STR00007## wherein R.sub.1 is H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl, and R.sub.2 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein said cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted; and wherein [B] has the structure: (i) [COCH.sub.2NR.sup.3].sub.m, (ii) -[AA1-AA2]-, (iii) -(AA1-COCH.sub.2NR.sup.3), (iv) (COCH.sub.2NR.sup.3-AA1)-, or (v) (COCH.sub.2NR.sup.4-AA1-AA3)-; wherein R.sup.3 is H or alkyl; m is 1-2; R.sup.4 is H, alkyl or a basic group; and AA1, AA2 and AA3 are amino acid residues, each of which, when linked to another of AA1, AA2 and AA3 are linked through a reverse amide bond.

2. The compound according to claim 1, wherein R.sub.1 is selected from the group consisting of CH(CH.sub.3).sub.2 benzyl, n-guanidino propyl, and n-amino butyl.

3.-5. (canceled)

6. The compound according to claim 1, wherein R.sub.2 is an optionally substituted heteroaryl group or a pentahalo substituted phenyl group.

7.-8. (canceled)

9. The compound according to claim 1, wherein the binding moiety [A] comprises an N-alkyl glycine carbamate moiety.

10. The compound according to claim 1, wherein [B] has the structure: (i) [COCH.sub.2NR.sup.3].sub.m- or (ii) -[AA1-AA2]-, wherein AA1 is joined to AA2 through a reverse amide bond and AA1 is an amino acid selected from Lys, Arg, Ser, Thr, and Gln, and AA2 is an amino acid selected from Lys and Arg.

11.-13. (canceled)

14. The compound according to claim 1, wherein the hydrophilic group comprises a biotin, glucoronyl, or morpholino carbamate group or comprises a moiety having the formula: NH-(CH.sub.2).sub.o(CH.sub.2CH.sub.2O).sub.p(CH.sub.2).sub.qNH or NH(CH.sub.2).sub.o(CH.sub.2CH.sub.2O).sub.p(CH.sub.2).sub.qCONH(CH.sub.2).sub.rNH, wherein o is 0, 1, 2 or 3; p is 1-10, q is 1, 2 or 3, and r is 1, 2 or 3.

15.-16. (canceled)

17. The compound according to claim 1, wherein the compound is selected from the group consisting of Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate, Biotin-PEG-NH-Gly-Gly-N-(benzyl)-glycine succinimidyl carbamate, Biotin-PEG-NH-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate, Biotin-PEG-NH-Gly-Gly-N-(n-amino-butyl)-glycine succinimidyl carbamate, Biotin-PEG-NHN-(n-guanidino-propyl)-glycine-Gln-Sar-N-(n-guanidino-propyl)-glycine succinimidyl carbamate, HO-Lys(Biotin)-Ahx-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP897), HO-Lys(Biotin)-Ahx-Gly-Gly-N-(n-guanidino-propyl)-glycine pentafluorophenyl carbamate (NAP966), Biotin-PEG-NHN-(n-guanidino-propyl)-glycine N(Val)-N(Lys)N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP1127), N--(1-acetylene-3-amino-phenyl)-di-carboxy-pentane-Lys-NH-Ahx-NH-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP884), Biotinyl-(PEG).sub.2-N(Arg)-Asn-N-(n-amino-butyl)-glycine succinimidyl carbamate (PRX1305), and Biotin-PEG-NArg-D-Gln-Sar-NArg-COOSu (NAP1099).

18.-29. (canceled)

30. A method of treating an inflammatory airway disease, said method comprising administration of a compound according to claim 1.

31. The method according to claim 30, wherein said inflammatory airway disease is selected from cystic fibrosis, chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis, emphysema, congenital alpha1-antitrypsin deficiency, asthma (for example, allergic asthma), and acute respiratory distress syndrome (ARDS).

32.-33. (canceled)

34. A method of treating a renal disease, said method comprising administration of a compound according to claim 1.

35. A pharmaceutical composition comprising a compound according to claim 1.

36. A method of inhibiting one or more channel activating proteases, said method comprising the administration of a compound according to claim 1.

37. A method of inhibiting activation of the epithelial sodium channel (ENaC), said method comprising the administration of a compound according to claim 1.

38. A method of inhibiting activation of protease-activated receptor (PAR-2), said method comprising the administration of a compound according to claim 1.

39. A method for the detection and/or inhibition of one or more proteases, comprising the steps of: mixing a biological sample, with the compound as claimed in claim 1, allowing the compound to stably bind a target protease in the sample to form a detectable complex, and detecting the detectable complex.

40. The method as claimed in claim 39 wherein the protease is an trypsin-like protease.

41. The method as claimed in claim 39, wherein the protease is kallikrein, neutrophil elastase or a plasminogen activator.

42. (canceled)

43. A method of detecting a pathological condition in a subject comprising the steps of: providing a sample from the subject, incubating the sample with a compound as claimed in claim 1 to form a detectable complex of said compound and protease and determining the amount of protease in the sample through comparison of the amount of the detectable complex present with a standard, wherein an elevated level of the protease compared to a normal level is indicative of a pathological condition.

44. (canceled)

45. The method as claimed in claim 43 wherein the pathological condition is a chronic or acute airways disease such as cystic fibrosis, chronic obstructive pulmonary disease, non-CF bronchiectasis, congenital alpha.sub.1 antitrypsin, emphysema, acute respiratory distress (ARDS); atherosclerosis; pancreatitis, acute periodontal disease, solid malignancy, disseminated intravascular dissemination, sepsis, aneurysm or chronic non-healing wound, or bacterial, viral or fungal infection.

46. An assay system for the detection of a protease, said system comprising a compound as claimed in claim 1.

47. The assay system of claim 46, wherein said system comprises an ELISA assay, a lateral flow device/dipstick or chip.

Description

[0227] The invention will now be described further in the following non-limiting examples with reference to the accompanying drawings in which:

[0228] FIG. 1 illustrates the structure of Biotin-PEG-NH-Gly-Gly-N-(benzyl)-glycine succinimidyl carbamate (NAP849);

[0229] FIG. 2 illustrates the structure of Biotin-PEG-NH-Gly-Gly-N-(n-guanidino propyl)-glycine succinimidyl carbamate (NAP858);

[0230] FIG. 3 illustrates the structure of Biotin-PEG-NH-Gly-Gly-N-(n-amino butyl)-glycine succinimidyl carbamate (NAP830);

[0231] FIG. 4 illustrates the structure of Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate (NAP800);

[0232] FIG. 5 illustrates the structure of Biotin-PEG-NHN-(n-guanidino-propyl)-glycine -Gln-Sar-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP1099);

[0233] FIG. 6 illustrates a synthetic scheme for the solid phase synthesis of Biotin-PEG-NH-Gly-Gly-N-(benzyl)-glycine succinimidyl carbamate, Biotin-PEG-NH-Gly-Gly-N-(n-amino butyl)-glycine succinimidyl carbamate and Biotin-PEG-NH-Gly-Gly-N-(n-guanidinopropyl)-glycine succinimidyl carbamate, illustrating the use of preformed Fomc-N-alkyl glycine monomers;

[0234] FIG. 7 illustrates a synthetic scheme for the solid phase synthesis of Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate, illustrating the sub-monomer approach for the on-resin construction of the N-alkyl glycine residue;

[0235] FIG. 8A illustrates progress curves for the inactivation of human neutrophil elastase by Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate in the presence of competing fluorogenic substrate Boc-Val-Pro-Val-NHMec;

[0236] FIG. 8B illustrates progress curves for the inhibition of trypsin by (NAP966); all assays were carried out in PBS, pH 7.4, at 37 C., in the presence of varying concentrations of inhibitor (0.25-50 M) and using a fixed substrate (Z-Gly-Gly-Arg-NHMec) concentration of 50 M;

[0237] FIG. 8C illustrates progress curves for the inhibition of trypsin by (NAP897); all assays were carried out in PBS, pH 7.4, at 37 C., in the presence of varying concentrations of inhibitor (0.25-50 M) and using a fixed substrate (Z-Gly-Gly-Arg-NHMec) concentration of 50 M;

[0238] FIG. 8D illustrates progress curves for the inhibition of trypsin by (NAP884); all assays were carried out in PBS, pH 7.4, at 37 C., in the presence of varying concentrations of inhibitor (0.25-1.50 M) and using a fixed substrate (Z-Gly-Gly-Arg-NHMec) concentration of 50 M;

[0239] FIG. 8E illustrates progress curves for the inhibition of tryptase by (PRX1305);

[0240] FIG. 8F illustrates progress curves for the inhibition of trypsin by (NAP858);

[0241] FIG. 9 shows a kinetic scheme for the irreversible inactivation of an enzyme by an inhibitor working through a complexing mechanism, in the presence of competing substrate. In this scheme, enzyme (E) and substrate (S) form a Michaelis complex (ES), prior to the formation of an acyl-enzyme intermediate (ES) with concomitant release of product (P.sub.1). The formation of ES is characterised by K.sub.m. Interconversion of ES into ES' is characterised by the first-order rate constant k.sub.1. Hydrolysis of ES' regenerates E, with the concomitant formation of the second product P.sub.2. This process is characterised by the rate constant k.sub.2. In the presence of competing inhibitor (I), formation of the reversible enzyme-inhibitor complex (EI) is characterised by the inhibitor constant K.sub.i. This is converted into the covalent/irreversible complex (E-I) characterised by a first-order rate constant k.sub.3. The overall second-order rate constant for the inactivation of E by I is given by the ratio k.sub.3/K.sub.i.

[0242] FIG. 10A illustrates the structure of QUB-TL1;

[0243] FIG. 10B shows a bar chart summarising the effect of serine protease inhibitors on mucociliary clearance (using primary cystic fibrosis airways epithelial cells grown at air-liquid interface); inhibitors (50 M) were added for 24 hours, prior to analysis (n=3);

[0244] FIG. 11 illustrates a bar chart showing inhibition of trypsin-like serine proteases in CF sol against the Cbz-Gly-Gly-Arg-AMC substrate using NAP858 and Cbz-Arg-DPP;

[0245] FIG. 12 illustrates the results of cytotoxicity assays on the CF human airway epithelial cell line CuFi using three inhibitors;

[0246] FIG. 13 are bar charts illustrating the ability of compounds of the invention to inhibit tryptic activity using primary human airway epithelial (HAE) cell cultures (A) and sputum sol samples collected from 8 individual CF patients (B);

[0247] FIG. 14 illustrates inhibition of ENac activity in primary CF cultures using QUB-TL1 and NAP858;

[0248] FIG. 15A shows a comparison of structures of HO-Lys(Biotin)-Ahx-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP897) and HO-Lys(Biotin)-Ahx-Gly-Gly-N-(n-guanidino-propyl)-glycine pentafluorophenyl carbamate (NAP966);

[0249] FIG. 15B shows a schematic of structure of Biotin-PEG-NHN-(n-guanidino-propyl)-glycine N(Val)-N(Lys)N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP1127);

[0250] FIG. 15C shows a schematic of structure of N-6-(1-acetylene-3-amino-phenyl)-di-carboxy-pentane-Lys-NH-Ahx-NH-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP884);

[0251] FIG. 15D shows a schematic of structure of Biotinyl-(PEG).sub.2-N(Arg)-Asn-N-(n-amino-butyl)-glycine succinimidyl carbamate (PRX1305); and

[0252] FIG. 15E shows a schematic of structure of Biotin-PEG-NArg-D-Gln-Sar-NArg-COOSu (NAP1099).

EXAMPLES

Examples 1-3: Synthesis of Biotin-PEG-NH-Gly-Gly-N-(benzyl)-glycine succinimidyl carbamate, Biotin-PEG-NH-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate and Biotin-PEG-NH-Gly-Gly-N-(n-amino-butyl)-glycine succinimidyl carbamate

[0253] Compounds of the invention, Biotin-PEG-NH-Gly-Gly-N-(benzyl)-glycine succinimidyl carbamate (FIG. 1), Biotin-PEG-NH-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (FIG. 2), and Biotin-PEG-NH-Gly-Gly-N-(n-amino-butyl)-glycine succinimidyl carbamate (FIG. 3) were prepared according to the scheme shown in FIG. 6. Briefly, these compounds were synthesised utilising a solid-phase approach, on Biotin-PEG NovaTag resin (Novabiochem). This resin contains a Biotin-PEG-amine moiety linked through a BAL linker, forming an immobilised secondary amine, which can be reacted with activated carboxylic acids and amino acids to furnish the desired compounds. To this resin was coupled two glycine residues (Fmoc-Gly-OH) through standard coupling procedures, prior to coupling of the appropriate N-alkyl glycine residue. The corresponding Fmoc-protected N-alkyl glycine monomers Fmoc-N-(benzyl)-Gly-OH, Fmoc-N-(n-guanidino-propyl)-Gly-OH and Fmoc-N-(n-amino-butyl)-Gly-OH were available commercially (Polypeptide Group), which enabled these residues to be incorporated directly into the common peptidic core sequence, using standard Fmoc-SPPS protocols. After the incorporation of each of these Fmoc-protected N-alkyl glycine monomers into the target sequence, removal of the Fmoc-group was achieved using piperidine (20% v/v solution in DMF). The on-resin synthesis of the corresponding succinimidyl carbamates was achieved essentially according to the method of Niphakis et al. (2013). Briefly, a solution containing N,N-disuccinimidyl carbonate (DSC) and 2,6-lutidine in anhydrous DCM/DMF (1:1) was added to each of the de-protected resins, in turn, until chloranil analysis indicated complete reaction of the terminal secondary N.sup. amine functions of each. The target peptides were then cleaved from the resin (and any side-chain-protecting group removed, simultaneously) using TFA/TIPS/DCM (95:2.5:2.5) and, following workup and precipitation in diethyl ether, the products were obtained in quantitative yield.

Example 4 Synthesis of Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate

[0254] A compound of the invention, Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate (FIG. 4), was prepared according to the scheme shown in FIG. 7. Briefly, since the N-alkyl glycine residue Fmoc-N-(isopropyl)-Gly-OH, was not commercially available, an on-resin sub-monomer approach was followed. In essence, Botin-PEG-amine derivatised BAL resin was subjected to two coupling cycles with Fmoc-Gly-OH. The terminal Fmoc-group was then removed from the resin-tethered peptide and the primary amino function was bromoacetylated in a di-isopropyl-carbodiimide-mediated reaction, using a ten-fold excess of bromoacetic acid, essentially according to a previously reported method (Tran et al., 2011). This acylation step was repeated (430 minutes) with fresh reagents until Kaiser analysis indicated complete acylation. This was followed by the addition of a solution of isopropylamine in N-methylpyrrolidone (NMP) to allow displacement of the bromine group to form the desired secondary amine. Formation of the target succinimidyl carbamate and its subsequent cleavage was carried out described above for the other three analogues, to provide the required compound in excellent yield.

Example 5: Synthesis of Biotin-PEG-NHN-(n-guanidino-propyl)-glycine -Gln-Sar-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP1099)

[0255] Another compound of the invention, Biotin-PEG-NHN-(n-guanidino-propyl)-glycine -Gln-Sar-N-(n-guanidino-propyl)-glycine succinimidyl carbamate (NAP1099) (FIG. 5) was synthesised according to the general Scheme illustrated in FIG. 6. The sarcosine, glutamine and N-(n-guanidino-propyl)-glycine residues were introduced as their N-Fmoc-protected derivatives, using standard coupling protocols.

Example 6: Inhibition Studies

[0256] The ability of compounds of the invention to inhibit the activities of a range of commercially sourced proteases was assessed using steady state fluorogenic substrate assays. In essence, samples of each protease were added to PBS buffer, pH 7.4, maintained at 37 C., containing a fixed concentration of appropriate substrate (Suc-Ala-Ala-Pro-Phe-NHMec, chymotrypsin; MeO-Suc-Ala-Ala-Pro-Val-NHMec, Neutrophil Elastase; Cbz-Gly-Gly-Arg-NHMec, trypsin; Boc-Val-Pro-Arg-NHMec, thrombin; Boc-Val-Leu-Lys-NHMec, plasmin; all used at 50 M) and varying concentrations (0.25-50 M) of the N-alkyl glycine carbamate inhibitors. The final concentration of protease ranged from 0.05-0.3 g/ml, depending on the activity of each. Progress curves were then generated by recording the increase in fluorescence as a function of time, for a period of between 30-60 minutes, using an excitation wavelength of 360 nm and emission wavelength of 485. FIG. 8A shows exemplar progress curves for neutrophil elastase-catalysed hydrolysis of MeO-Suc-Ala-Ala-Pro-Val-NHMec in the presence of varying concentrations of Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate. Assays were carried out in PBS, pH 7.4, at 37 C., in the presence of varying concentrations of inhibitor (10-50 M) and a fixed concentration of substrate (50 M). Similar progress curves were obtained for other proteases and other inhibitors used in the study, (FIGS. 8B-8F). These progress curves are indicative of the action of an irreversible inhibitor working through a complexing mechanism shown in FIG. 9. In this mechanism, the inhibitor I, binds the target protease to form an initial reversible complex EI, characterised by the inhibitor constant K.sub.i. This is followed by the formation of an irreversible complex E-I from EI, characterised by the first-order rate constant k.sub.3. The overall second-order rate constant for the inactivation of the protease by the inhibitor is then given by the ratio k.sub.3/K.sub.i (Walker and Elmore, 1984; Tian and Tsou, 1982).

[0257] The results of some of these inhibition studies are shown in Table 1. This records the kinetic parameters (K.sub.1, k.sub.3 and k.sub.3/K.sub.i) for the inactivation of the series of proteases utilised in the study with the various N-alkyl glycine carbamates.

TABLE-US-00001 TABLE 1 Inhibitory Kinetic Data Inhibitor Protease K.sub.i (M) k.sub.3 (min.sup.1) k.sub.3/K.sub.i (M.sup.1 min.sup.1) NAP858 Biotin-PEG-NH-Gly-Gly- Trypsin (n = 3) 4.86 (3.76) 10.sup.7 0.299 (0.037) 1.95 (2.62) 10.sup.6 N-(n-guanidino-propyl)- glycine succinimidyl Matriptase (n = 3) 7.33 (0.10) 10.sup.7 0.314 (0.084) 1.30 (0.943) 10.sup.6 carbamate Human Airways 6.59 (4.72) 10.sup.7 0.464 (0.164) 1.50 (1.76) 10.sup.6 Trypsin-like protease (HAT) (n = 3) NKP830 Biotin-PEG-NH-Gly-Gly- Trypsin (n = 3) 2.2 (0.8) 10.sup.7 0.5 (0.1) 2.7 (0.5) 10.sup.6 N-(n-amino-butyl)- glycine succinimidyl Plasmin (n = 2) 7.2 (0.3) 10.sup.6 0.4 (0.05) 6.2 (0.9) 10.sup.4 carbamate NFP849 Biotin-PEG-NH-Gly-Gly- Chymotrypsin (n = 3) 8.9 (6.7) 10.sup.7 0.8 (0.3) 1.3 (0.4) 10.sup.6 N-(benzyl)-glycine succinimidyl carbamate NVP800 Biotin-PEG-NH-Gly-Gly- Neutrophil 6.0 (1.4) 10.sup.6 0.4 (0.05) 6.4 (0.6) 10.sup.4 N-(iso-propyl)-glycine Elastase (n = 2) succinimidyl carbamate NAT988 Biotin-PEG-NH(N-benzyl- Thrombin (n = 2) 8.8 (2.5) 10.sup.7 0.4 ((0 02) .sup.4.7 ( 1.1) 10.sup.5 glycyl)-(D)-phenylalanyl- N-(n-guanidino-propyl)- glycine succinimidyl carbamate CG877 Biotin-PEG-NH(N- Chymotrypsin (n = 3) 2.8 ((n = 6) 10.sup.6 0.7 ((11) 2.5 ((922) 10.sup.5 methyl glycyl)- (N-methyl glycyl)- N-(benzyl)-glycine succinimidyl carbamate NAP1099 Biotin-PEG-NHN-(n- Trypsin (n = 3) 1.50 (1.26) 10.sup.7 0.354 (0.09) 3.80 (3.16) 10.sup.6 guanidino-propyl)- glycine-Gln-Sar-N-(n- Matriptase (n = 3) 8.33 (7.84) 10.sup.8 0.302 (0.03) 8.52 (9.05) 10.sup.6 guanidino-propyl)- glycine succinimidyl Human Airways 3.22 (4.23) 10.sup.7 0.474 (0.16) 3.71 (2.89) 10.sup.6 carbamate Trypsin-like protease (HAT) (n = 3) NAP966 Trypsin (n = 3) 2.1 (0.3) 10.sup.6 0.4 (0.007) 1.9 (0.25) 10.sup.5 NAP897 Trypsin (n = 3) 6.6 (4.1) 10.sup.7 0.7 (0.2) 1.3 (0.4) 10.sup.6 NAP884 Trypsin (n = 3) 4.20 (1.11) 10.sup.7 0.528 (0.064) 1.34 (0.351) 10.sup.6 Mean (SD); n is the number of determinations

[0258] It can be appreciated from Table 1, that the series of N-alkyl glycine succinimidyl carbamates function as irreversible inhibitors of the serine proteases studied. For example, Biotin-PEG-NH-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate and Biotin-PEG-NH-Gly-Gly-N-(n-amino-butyl)-glycine succinimidyl carbamate, both of which contain a basic N-alkyl glycine carbamate moiety, function as potent inhibitors of trypsin, exhibiting virtually identical over all second-order rate constants of 2.60 (0.884)10.sup.6M.sup.1.Math.min.sup.1 and 2.74 (0.547)10.sup.6M.sup.1.Math.min.sup.1, respectively. Similarly, potent inhibition of chymotrypsin was obtained using Biotin-PEG-NH-Gly-Gly-N-(benzyl)-glycine succinimidyl carbamate, which inactivated this protease with a second-order rate constant of 1.28 (0.442)10.sup.6 M.sup.1.Math.min.sup.1. The replacement of the two glycine residues with two N-methyl-glycine residues to give Biotin-PEG-NH-(N-methyl glycyl)-(N-methyl glycyl)-N-(benzyl)-glycine succinimidyl carbamate, still provided an effective inhibitor of chymotrpsin, albeit with a reduced second-order rate constant of 2.46 (0.214)10.sup.5 M.sup.1.Math.min.sup.1, in comparison to that obtained for Biotin-PEG-NH-Gly-Gly-N-(benzyl)-glycine succinimidyl carbamate. This drop in effectiveness can be attributed to poorer K.sub.i and k.sub.3 values for the former in comparison to the latter. The elastase-directed sequence Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate functioned as an inhibitor of this protease exhibiting a second-order rate constant of 6.41 (0.572)10.sup.4M.sup.1.Math.min.sup.1. Although not as effective as the chymotrypsin or trypsin directed sequences against their respective targets, Biotin-PEG-NH-Gly-Gly-N-(iso-propyl)-glycine succinimidyl carbamate is an efficient inhibitor of elastase, since, for example, a 5 M concentration will inactivate this protease with a t.sub.1/2 of less than 2 minutes. Finally, the plasma trypsin-like serine proteases plasmin and thrombin were efficiently inhibited by the basic N-alkyl glycine carbamate sequences Biotin-PEG-NH-Gly-Gly-N-(n-amino-butyl)-glycine succinimidyl carbamate and Biotin-PEG-NH-(N-benzyl-glycyl)-(D)-phenylalanyl-N-(n-guanidino-propyl)-glycine succinimidyl carbamate, respectively.

Example 7Biotin-PEG-NH-Gly-Gly-N-(n-guanidino-propyl)-glycine succinimidyl carbamate Enhances Mucociliary Clearance

[0259] Mucociliary clearance was quantified by measuring the velocity of microbeads apically applied to differentiated primary CF Airway Epithelial Cells (AECs). Compounds (50 M) were added to the apical surface of primary CF AECs for a period of 24 hours. Mucociliary flow was then determined by tracking and measuring the velocity of apically applied microbeads across the CF AEC surface. The effect of the compounds of the invention were compared to a vehicle only negative control and a positive control, QUB-TL1. The inventors have previously shown that QUB-TL1, the structure of which is shown in FIG. 10A, increases mucociliary clearance using primary CF AECs (Reihill et al, 2016). As shown in FIG. 10B, treatment of CF AECs by NAP858 (50 M) for 24 hours resulted in markedly improved mucociliary clearance (from 3.5 (vehicle control) to 6.83 m/s; n=3).

BIOLOGICAL EXAMPLES

Example 8 NAP858 Inhibits Trypsin-Like Serine Proteases in CF Sputum Sol

[0260] The inventors investigated the effect of compounds of the invention on biological samples using sol from patients with cystic fibrosis and cystic fibrosis cell lines.

[0261] FIG. 11 illustrates that NAP858 causes complete inhibition of the hydrolytic action of trypsin-like serine proteases in CF Sol as determined by inhibition of hydrolysis of the substrate, Cbz-Gly-Gly-Arg-AMC. Briefly, the spectrofluorimetic analysis was conducted on a sample of pooled supernatant recovered by centrifugation from expectorated cystic fibrosis sputum, which had been diluted 4 with phosphate-buffered saline (sputum sol). Samples were treated in duplicate with NAP858 (10 M) and a control compound, Cbz-Arg-diphenylphosphonate (Cbz-Arg-DPP) (10 M). The rate of hydrolysis of the substrate at 37 C. was monitored and inhibition as a % of an untreated control calculated.

Example 9 Cytotoxicity

[0262] The inventors performed cytotoxicity assays on cells of the human CuFi cell line. Briefly, CuFi cells (a CF human epithelial cell line) were treated for 24 hours with NAP858, NAP1099, and NAP1127 over a ranged of concentrations (1-100 M), before the addition of MTS tetrazolium compound (Abcam). MIS Cell Proliferation Assay Kit is a colorimetric method for sensitive quantification of viable cells in proliferation and cytotoxicity assay. The assay is based on the reduction of MIS tetrazolium compound by viable cells to generate a colored formazan product that is soluble in cell culture media. This conversion is thought to be carried out by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells. The formazan dye produced by viable cells can be quantified by measuring the absorbance at 490-500 nm. As shown in FIG. 12, each of the inhibitors tested were well tolerated.

Example 10 Inhibition of Endogenous Protease Activity

[0263] The ability of compounds of the invention to inhibit extracellular (apical surface) tryptic activity was investigated using primary human airway epithelial (HAE) cell cultures (A) and sputum sol samples collected from 8 individual CF patients (B). Briefly, HAE cells grown to confluence on 96-well plates were treated with a range of compounds (NAP858, NAP1099, NAP 1127 and QUB-TL1; all at 10 M) and the rate of hydrolysis of Boc-QAR-AMC (50 M) determined at 37 C. Inhibition of endogenous protease activities in CF sputum sol, was conducted as for Example 8 but in the presence of Boc-Gln-Ala-Arg-AMC (50 M) as substrate. The rate of hydrolysis of the substrate at 37 C. was monitored and residual activity determined. In primary HAE NAP858 and NAP1099 reduced extracellular tryptic activity to a similar degree as that of QUB-TL1 (FIG. 13A). Further FIG. 13B shows that NAP858, and NAP1099 were superior to QUB-TL1 when incubated with CF sputum sol.

Example 11 Effect of NAP858 on ENac Activity

[0264] The effect of compounds of the invention on ENaC current was investigated. ENaC-expressing FRT cells were treated with QUB-TL1 or NAP858 (50 M) for 45 mins and Ieq measured. The experiment was terminated by the addition of amiloride (10 M) which inhibited >95% of the observable current. All determinations were conducted using a TECC24 semiautomated system. Unlike the traditional Ussing chamber assays, where the short circuit current (I.sub.SC) is measured as an index of CFTR correction, the TECC-24 assay measures the equivalent current (I.sub.EQ). The TECC-24 assay format offers some advantages over the Ussing chamber assay, one of which is the higher throughput; the other involves the ability to measure the I.sub.EQ with the test compounds continuously present in the recording medium.

[0265] As shown in FIG. 14, NAP-858 reduced ENaC activity in primary CF cultures to a similar degree as QUB-TL1 (36 vs 33 A/cm2) over a 90 minute period.

[0266] All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

REFERENCE LIST

[0267] 1. Gibson, R. L., Burns, J. L., & Ramsey, B. W. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am. J. Respir. Crit Care Med. 168, 918-951 (2003). [0268] 2. Donaldson, S. H. & Boucher, R. C. Sodium channels and cystic fibrosis. Chest 132, 1631-1636 (2007). [0269] 3. Boucher, R. C., Cotton, C. U., Gatzy, J. T., Knowles, M. R., & Yankaskas, J. R. Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures. J. Physiol 405, 77-103 (1988). [0270] 4. Stutts, M. J. et al. CFTR as a cAMP-dependent regulator of sodium channels. Science 269, 847-850 (1995). [0271] 5. Boucher, R. C. Airway surface dehydration in cystic fibrosis: pathogenesis and therapy. Annu. Rev. Med. 58, 157-170 (2007). [0272] 6. Sheng, S., Carattino, M. D., Bruns, J. B., Hughey, R. P., & Kleyman, T. R. Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition. Am. J. Physiol Renal Physiol 290, F1488-F1496 (2006). [0273] 7. Kleyman, T. R., Carattino, M. D., & Hughey, R. P. ENaC at the cutting edge: regulation of epithelial sodium channels by proteases. J. Biol. Chem. 284, 20447-20451 (2009). [0274] 8. Bruns, J. B. et al. Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit. J. Biol. Chem. 282, 6153-6160 (2007). [0275] 9. Nimishakavi, S. et al. Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells. Am. J. Physiol Lung Cell Mol. Physiol 303, L97-106 (2012). [0276] 10. Myerburg, M. M. et al. Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis. Am. J. Physiol Lung Cell Mol. Physiol 294, L932-L941 (2008). [0277] 11. Ornatowski, W., Poschet, J. F., Perkett, E., Taylor-Cousar, J. L., & Deretic, V. Elevated furin levels in human cystic fibrosis cells result in hypersusceptibility to exotoxin A-induced cytotoxicity. J. Clin. Invest 117, 3489-3497 (2007). [0278] 12. Tong, Z., Illek, B., Bhagwandin, V. J., Verghese, G. M., & Caughey, G. H. Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line. Am. J. Physiol Lung Cell Mol. Physiol 287, L928-L935 (2004). [0279] 13. Coote, K. et al. Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease. J. Pharmacol. Exp. Ther. 329, 764-774 (2009). [0280] 14. Rowe, S. M. et al. Reduced sodium transport with nasal administration of the prostasin inhibitor camostat in subjects with cystic fibrosis. Chest 144, 200-207 (2013). [0281] 15. Myerburg, M. M., Harvey, P. R., Heidrich, E. M., Pilewski, J. M., & Butterworth, M. B. Acute regulation of the epithelial sodium channel in airway epithelia by proteases and trafficking. Am. J. Respir. Cell Mol. Biol. 43, 712-719 (2010). [0282] 16. Dubois, C. M. et al. Evidence that furin is an authentic transforming growth factor-beta1-converting enzyme. Am. J. Pathol. 158, 305-316 (2001). [0283] 17. Arkwright, P. D. et al. TGF-beta(1) genotype and accelerated decline in lung function of patients with cystic fibrosis. Thorax 55, 459-462 (2000). [0284] 18. Gaggar, A. et al. The role of matrix metalloproteinases in cystic fibrosis lung disease. Eur. Respir. J. 38, 721-727 (2011). [0285] 19. Ra, H. J. & Parks, W. C. Control of matrix metalloproteinase catalytic activity. Matrix Biol. 26, 587-596 (2007). [0286] 20. Stieneke-Grober, A. et al. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J. 11, 2407-2414 (1992). [0287] 21. Ortiz, J. R. et al. Influenza-associated cystic fibrosis pulmonary exacerbations. Chest 137, 852-860 (2010). [0288] 22. Inocencio, N. M., Moehring, J. M., & Moehring, T. J. Furin activates Pseudomonas exotoxin A by specific cleavage in vivo and in vitro. J. Biol. Chem. 269, 31831-31835 (1994). [0289] 23. Ogata, M., Chaudhary, V. K., Pastan, I., & FitzGerald, D. J. Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol. J. Biol. Chem. 265, 20678-20685 (1990). [0290] 24. Jaffar-Bandjee, M. C. et al. Production of elastase, exotoxin A, and alkaline protease in sputa during pulmonary exacerbation of cystic fibrosis in patients chronically infected by Pseudomonas aeruginosa. J. Clin. Microbiol. 33, 924-929 (1995). [0291] 25. Yasuoka, S. et al. Purification, characterization, and localization of a novel trypsin-like protease found in the human airway. Am. J. Respir. Cell Mol. Biol. 16, 300-308 (1997). [0292] 26. Chokki, M. et al. Human airway trypsin-like protease increases mucin gene expression in airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 30, 470-478 (2004). [0293] 27. Matsushima, R. et al. Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway. Am. J. Physiol Lung Cell Mol. Physiol 290, L385-L395 (2006). [0294] 28. Liu, C., Li, Q., Zhou, X., Kolosov, V. P., & Perelman, J. M. Human airway trypsin-like protease induces mucinSAC hypersecretion via a protease-activated receptor 2-mediated pathway in human airway epithelial cells. Arch. Biochem. Biophys. 535, 234-240 (2013). [0295] 29. Donaldson, S. H. et al. Regulation of the epithelial sodium channel by serine proteases in human airways. J. Biol. Chem. 277, 8338-8345 (2002). [0296] 30. Adebamiro, A., Cheng, Y., Johnson, J. P., & Bridges, R. J. Endogenous protease activation of ENaC: effect of serine protease inhibition on ENaC single channel properties. J. Gen. Physiol 126, 339-352 (2005). [0297] 31. Vallet, V., Chraibi, A., Gaeggeler, H. P., Horisberger, J. D., & Rossier, B. C. An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature 389, 607-610 (1997). [0298] 32. Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., & Thomas, G. Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J. Biol. Chem. 267, 16396-16402 (1992). [0299] 33. Cravatt, B. F., Wright, A. T., & Kozarich, J. W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383-414 (2008). [0300] 34. Kay, G., Bailie, J. R., Halliday, I. M., Nelson, J., & Walker, B. The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases. Biochem. J. 283 (Pt 2), 455-459 (1992). [0301] 35. Cullen, B. M., Halliday, I. M., Kay, G., Nelson, J., & Walker, B. The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture. Biochem. J. 283 (Pt 2), 461-465 (1992). [0302] 36. McGinty, A., Moore, M., Halton, D. W., & Walker, B. Characterization of the cysteine proteinases of the common liver fluke Fasciola hepatica using novel, active-site directed affinity labels. Parasitology 106 (Pt 5), 487-493 (1993). [0303] 37. Gilmore, B. F. et al. Synthesis, kinetic evaluation, and utilization of a biotinylated dipeptide proline diphenyl phosphonate for the disclosure of dipeptidyl peptidase IV-like serine proteases. Biochem. Biophys. Res. Commun. 347, 373-379 (2006). [0304] 38. Gilmore, B. F. et al. Expedited solid-phase synthesis of fluorescently labeled and biotinylated aminoalkane diphenyl phosphonate affinity probes for chymotrypsin- and elastase-like serine proteases. Bioconjug. Chem. 20, 2098-2105 (2009). [0305] 39. Walker B & McGinty A Detection and labelling of proteases using biotinylated active site-directed affinity labels (ed. Celis J. E.) 351-357 (Academic Press, 1998). [0306] 40. Tian, W. X. & Tsou, C. L. Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier. Biochemistry 21, 1028-1032 (1982). [0307] 41. Walker, B. & Elmore, D. T. The irreversible inhibition of urokinase, kidney-cell plasminogen activator, plasmin and beta-trypsin by 1-(N-6-amino-n-hexyl)carbamoylimidazole. Biochem. J. 221, 277-280 (1984). [0308] 42. Zabner, J. et al. Development of cystic fibrosis and noncystic fibrosis airway cell lines. Am. J. Physiol Lung Cell Mol. Physiol 284, L844-L854 (2003). [0309] 43. Hallenberger, S. et al. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature 360, 358-361 (1992). [0310] 44. Kohler, D., App, E., Schmitz-Schumann, M., Wurtemberger, G., & Matthys, H. Inhalation of amiloride improves the mucociliary and the cough clearance in patients with cystic fibroses. Eur. J. Respir. Dis. Supp1146, 319-326 (1986). [0311] 45. App, E. M., King, M., Helfesrieder, R., Kohler, D., & Matthys, H. Acute and long-term amiloride inhalation in cystic fibrosis lung disease. A rational approach to cystic fibrosis therapy. Am. Rev. Respir. Dis. 141, 605-612 (1990). [0312] 46. Hirsh, A. J. et al. Evaluation of second generation amiloride analogs as therapy for cystic fibrosis lung disease. J. Pharmacol. Exp. Ther. 311, 929-938 (2004). [0313] 47. Coakley, R. D. et al. Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium. Proc. Natl. Acad. Sci. U. S. A 100, 16083-16088 (2003). [0314] 48. Pezzulo, A. A. et al. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 487, 109-113 (2012). [0315] 49. Verghese, G. M., Gutknecht, M. F., & Caughey, G. H. Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor. Am. J. Physiol Cell Physiol 291, C1258-C1270 (2006). [0316] 50. Oberst, M. D. et al. Characterization of matriptase expression in normal human tissues. J. Histochem. Cytochem. 51, 1017-1025 (2003). [0317] 51. Wang, J. K. et al. Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition. Am. J. Physiol Cell Physiol 297, C459-C470 (2009). [0318] 52. Friis, S. et al. A matriptase-prostasin reciprocal zymogen activation complex with unique features: prostasin as a non-enzymatic co-factor for matriptase activation. J. Biol. Chem. 288, 19028-19039 (2013). [0319] 53. Vallet, V., Pfister, C., Loffing, J., & Rossier, B. C. Cell-surface expression of the channel activating protease xCAP-1 is required for activation of ENaC in the Xenopus oocyte. J. Am. Soc. Nephrol. 13, 588-594 (2002). [0320] 54. Iwakiri, K. et al. Human airway trypsin-like protease induces PAR-2-mediated IL-8 release in psoriasis vulgaris. J. Invest Dermatol. 122, 937-944 (2004). [0321] 55. Beaulieu, A. et al. Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium. J. Virol. 87, 4237-4251 (2013). [0322] 56. Johansen, H. K. & Hoiby, N. Seasonal onset of initial colonisation and chronic infection with Pseudomonas aeruginosa in patients with cystic fibrosis in Denmark. Thorax 47, 109-111 (1992). [0323] 57. Hughey, R. P. et al. Epithelial sodium channels are activated by furin-dependent proteolysis. J. Biol. Chem. 279, 18111-18114 (2004). [0324] 58. Hughey, R. P., Carattino, M. D., & Kleyman, T. R. Role of proteolysis in the activation of epithelial sodium channels. Curr. Opin. Nephrol. Hypertens. 16, 444-450 (2007). [0325] 59. Liu, P. V. Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130 Suppl, S94-S99 (1974). [0326] 60. Miyazaki, S., Matsumoto, T., Tateda, K., Ohno, A., & Yamaguchi, K. Role of exotoxin A in inducing severe Pseudomonas aeruginosa infections in mice. J. Med. Microbiol. 43, 169-175 (1995). [0327] 61. Woods, D. E., Cryz, S. J., Friedman, R. L., & Iglewski, B. H. Contribution of toxin A and elastase to virulence of Pseudomonas aeruginosa in chronic lung infections of rats. Infect. Immun. 36, 1223-1228 (1982). [0328] 62. Klinger, J. D., Straus, D. C., Hilton, C. B., & Bass, J. A. Antibodies to proteases and exotoxin A of Pseudomonas aeruginosa in patients with cystic fibrosis: Demonstration by radioimmunoassay. J. Infect. Dis. 138, 49-8 (1978). [0329] 63. Cross, A. S., Sadoff, J. C., Iglewski, B. H., & Sokol, P. A. Evidence for the role of toxin A in the pathogenesis of infection with Pseudomonas aeruginosa in humans. J. Infect. Dis. 142, 538-546 (1980). [0330] 64. Kounnas, M. Z. et al. The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J. Biol. Chem. 267, 12420-12423 (1992). [0331] 65. Gu, M., Gordon, V. M., FitzGerald, D. J., & Leppla, S. H. Furin regulates both the activation of Pseudomonas exotoxin A and the Quantity of the toxin receptor expressed on target cells. Infect. Immun. 64, 524-527 (1996). [0332] 66. Schidlow, D. V., Taussig, L. M., & Knowles, M. R. Cystic Fibrosis Foundation consensus conference report on pulmonary complications of cystic fibrosis. Pediatr. Pulmonol. 15, 187-198 (1993). [0333] 67. Mentz, W. M. et al. Deposition, clearance, and effects of aerosolized amiloride in sheep airways. Am. Rev. Respir. Dis. 134, 938-943 (1986). [0334] 68. Hirsh, A. J. Altering airway surface liquid volume: inhalation therapy with amiloride and hyperosmotic agents. Adv. Drug Deliv. Rev. 54, 1445-1462 (2002). [0335] 69. Noone, P. G. et al. Airway deposition and clearance and systemic pharmacokinetics of amiloride following aerosolization with an ultrasonic nebulizer to normal airways. Chest 112, 1283-1290 (1997). [0336] 70. Niphakis, M J., Cognetta, A B., Chang J W., Matthew W. Buczynski, M W., Parsons, L H., Byrne, F., Burston, Victoria Chapman, V., Cravatt, B F. Evaluation of NHS Carbamates as a Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors. ACS Chem. Neurosci., 4, 1322-1332 (2013). [0337] 71. Tran, H., Gael, S. L., Connolly, M. D., Zuckermann, R. N. Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets. J. Vis. Exp. 57, e3373, doi:10.3791/3373 (2011). [0338] 72. Reihill, J A, Walker B, Hamilton R A, Ferguson T E G, Maye J R, Elborn J S, Stutts M J, Harvey B J, Saint-Criq V, Hendrick S and Martin S L. (2016a) Inhibition of Protease-Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways. Am J Respir Crit Care Med; 94 (6): 701-710.