WO2012083385A1 - Serine protease inhibitors - Google Patents

Abstract

The invention relates to KLK protease inhibitors. In particular, the invention is directed to KLK14, KLK7 and/or KLK5 protease inhibitors and their uses in the diagnosis, prevention or treatment of a skin disease or pathology, or other undesirable skin condition.

Description

TITLE

SERINE PROTEASE INHIBITORS

FIELD OF THE INVENTION

The invention described herein relates generally to serine protease inhibitors. In particular, the invention is directed to kallikrein-related peptidase inhibitors and their use in the diagnosis, prevention and treatment of skin diseases, although the scope of the invention is not necessarily limited thereto.

BACKGROUND OF THE INVENTION

As the primary interface between the body and the external environment, the skin forms an essential protective frontier. This role demands considerable versatility. Thus, the skin provides a barrier that is normally impermeable to physical, chemical and microbial attack, able to regenerate and maintain water and electrolyte balance, and sufficiently resilient to endure mechanical stress. Cadherin function lies at the heart of the skin's unique ability to fulfil these functions. This is particularly significant within the epidermis, where networks of desmosomes and adherens junctions connect individual cellular subunits into a single cohesive unit via their cytoskeletal networks of actin and keratin filaments (Green and Gaudry, Nat. Rev. Mol. Cell. Biol. 1:208-16, 2000).

Cellular components of the epidermis originate from a population of self-renewing progenitors in its basal layers, and while this continual proliferation and maturation of cells imparts a capacity for self-renewal to the skin, it equally demands the presence of a balancing mechanism to maintain regular epidermal structure and thickness. As such, the skin routinely undergoes desquamation, a process of dynamic remodelling by which the outermost corneocytes are progressively shed and replaced by newly differentiated cornified cells.

The kallikrein-related peptidases (KLKs) are a highly conserved multi-gene family encoding serine proteases with either trypsin- or chymotrypsin-like activity. The KLK locus consists of fifteen homologous serine proteases clustered on chromosome 19ql3.3-13.4 (Borgono and Diamandis, Nat. Rev. Cancer 4:876-90, 2004; Clements et al, Crit. Rev. Clin. Lab. Sci. 41:265-312, 2004). A number of KLKs have been detected within the skin, including KLK1, 3-11 and 13-14 (Komatsu et al, J. Invest. Dermatol. 121 :542-49, 2003; Komatsu et al., J. Invest. Dermatol. 126:925-29, 2006). Evidence suggests that the significance of LK protease activity within the epidermis is two-fold: not only does it maintain the epidermal barrier by driving turnover of corneocytes through controlled proteolysis of desmosomal cadherins (Caubet et al, J. Invest. Dermatol. 122:1235-44, 2004; Stefansson et al, Biol Chem. 387:761-68, 2006), it is also at the forefront of a number of critical initial responses to compromised barrier integrity (Hachem et al, J. Invest. Dermatol. 126:2074-86, 2006; Cork et al, J. Invest. Dermatol. 129:1892-1908, 2009).

As with many physiological functions under the control of proteases, an imbalance between proteolysis and endogenous regulatory factors underlies several pathologies. Similarly, unrestrained proteolysis compromises the skin's protective ability, particularly KLK overactivity, which underpins the pathogenesis of a number of skin- based diseases.

Accordingly, there is a need for effective and selective inhibitors of KLKs expressed within the skin for use in therapeutic strategies for the treatment of skin pathologies.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a compound including a peptide or salt thereof according to Formula I:

Formula I wherein

R¹, R², R³, R⁴, R⁶, and R⁸ are each an amino acid residue, R⁵ is an amino acid residue other than Phe and R⁷ is an amino acid residue other than Asp or Glu. In one embodiment, R¹ is Tip or Tyr, R² is Val or He, R³ is Arg, R⁵ is Gin, and R⁷ is Asn.

In another embodiment, R¹ is Trp or Tyr, R² is Val or He, R³ is Arg, R⁴ is Pro, R⁶ is Pro, and R⁸ is Gly.

In yet another embodiment, R¹ is Trp or Tyr, R² is Val or lie, R³ is Arg, R⁴ is Pro,

R⁵ is Gin, R⁶ is Pro, R⁷ is Asn, and R⁸ is Gly.

In a further embodiment, R¹ is Trp or Tyr, R² is Ser, Thr or Asn, R³ is Arg, R⁵ is Gin, and R⁷ is Asn.

In yet a further embodiment, R¹ is Trp, R² is He, R³ is Arg, R⁵ is Asn, and R⁷ is Asn.

In another embodiment, R¹ is Tyr, R² is Ser, R³ is Arg, R⁵ is Asn, and R⁷ is Asn. In still another embodiment, R¹ is Thr, R² is Thr, R³ is Tyr, R⁵ is Asn, and R⁷ is

Asn.

In yet another embodiment, R¹ is Thr, R² is Thr, R³ is Arg, R⁵ is Asn, and R⁷ is Asn.

In a further embodiment, R¹ is Trp, R² is Val, R³ is Arg, R⁵ is Asn, and R⁷ is Asn. In yet a further embodiment, R¹ is Tyr R² is Leu, R³ is Phe, R⁵ is Asn, and R⁷ is

Asn.

In another embodiment, R^l is Thr, R² is Thr, R³ is Phe, R⁵ is Asn, and R⁷ is Asn. In still another embodiment, R¹ is Trp R² is Leu, R³ is Phe, R⁵ is Asn, and R⁷ is

Asn.

In some embodiments, a compound according to the first aspect of the invention is a KLK protease inhibitor, including a KLK14, KLK7 and/or KLK5 protease inhibitor.

According to a second aspect of the invention, there is provided a pharmaceutical or veterinary composition including an amount of a compound according to the first aspect of the invention effective to inhibit or decrease serine protease activity in a subject together with a pharmaceutically acceptable carrier or diluent.

In one embodiment, the pharmaceutical or veterinary composition is effective to inhibit or decrease the serine protease activity of a KLK protease (e.g., KLK14, KLK7 and/or KLK5) in the subject.

According to a third aspect of the invention, there is provided a pharmaceutical or veterinary composition for the diagnosis, prevention or treatment of a skin disease or pathology, or other undesirable skin condition in a subject, the composition including a compound according to the first aspect of the invention together with a pharmaceutically or veterinarially acceptable carrier or diluent.

In one embodiment, the skin disease is selected f om the group consisting of: Netherton syndrome, peeling skin syndrome, acne rosacea, psoriasis, eczema, and atopic dermatitis.

In another embodiment, the pharmaceutical or veterinary composition is effective to inhibit or decrease the serine protease activity of a L protease (e.g., KL 14, K.LK7 and/or KLKS) in the subject.

In yet another embodiment, the pharmaceutical or veterinary composition further comprises one or more additional active agents.

According to a fourth aspect of the invention, there is provided a compound according to the first aspect of the invention for the diagnosis, prevention or treatment of a skin disease or pathology, or other undesirable skin condition in a subject.

In one embodiment, the skin disease is selected from the group consisting of:

Netherton syndrome, peeling skin syndrome, acne rosacea, psoriasis, eczema, and atopic dermatitis.

According to a fifth aspect of the invention, there is provided a method for the diagnosis, prevention or treatment of a skin disease or pathology, or other undesirable skin condition in a subject, the method including administering to the subject a therapeutically effective amount of a compound according to the first aspect of the invention or a pharmaceutical or veterinary composition according to the second aspect of the invention.

In one embodiment, the skin disease is selected from the group consisting of: Netherton syndrome, peeling skin syndrome, acne rosacea, psoriasis, eczema, and atopic dermatitis.

According to a sixth aspect of the invention, there is provided a method for the diagnosis, prevention or treatment of a KLK-mediated disease in a subject, the method including administering to the subject a therapeutically effective amount of a compound according to the first aspect of the invention or a pharmaceutical or veterinary composition according to the second aspect of the invention.

In one embodiment, the KLK-mediated disease is a KLK14-mediated disease. In another embodiment, the L -mediated disease is a L 7-mediated disease. In still another embodiment, the KLK-mediated disease is a KLK5-mediated disease.

In yet another embodiment, the KLK-mediated disease is selected from the group S consisting of: Netherton syndrome, peeling skin syndrome, acne rosacea, psoriasis, eczema, and atopic dermatitis.

According to a seventh aspect of the invention, there is provided a method for disaggregating cells or tissue in vitro, the method including exposing the cells or tissue to a KLK protease (e.g., KLK14, KLK7 and/or KLK5), followed by exposure to a 0 compound according to the first aspect of the invention.

According to an eighth aspect of the invention, there is provided a compound according to the first aspect of the invention for use in treating a skin disease or pathology, or other undesirable skin condition.

In one embodiment, the skin disease is Netherton syndrome, peeling skin S syndrome, acne rosacea, psoriasis, eczema, or atopic dermatitis.

With reference to Formula I, R^l, R², R³, R⁴, R⁶, and R⁸ can be any amino acid residue, while R^s can be any amino acid residue other than Phe and R⁷ can be any amino acid residue other than Asp or Glu.

In order that the invention may be more readily understood and put into practice,0 one or more preferred embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Refined substrate specificity of KLK 14 determined by sparse matrix library screen. Thirty-six individually synthesised peptide pora-nitroanilide (pNA)5 substrates were assayed against a constant concentration of KLK 14. Amidolytic activity was measured by the change in absorbance at 405 nm using a Biorad Benchmark Plus multi-well spectrophotometer with readings taken every 10 s for 420 s. The rate of cleavage measured in

with the substrate identity labelled on the -axis. Data represents the mean ± standard0 error of the mean (S.E.M.) of three experiments performed in triplicate. Figure 2. Michaelis-Menten kinetic plots for KLK14 cleavage of preferred peptide-pNA substrates. Six peptide- ?NA substrates were purified and subjected to quantitative kinetic analysis: Ac-YAVR-/>NA (optimal substrate - panel A), Ac-WAVR- pNA (panel B), Ac-YASR- NA (second best substrate - panel C), Ac-YANR-pNA (reported optimal substrate iroin published positional scanning - panel D), Ac-YVSR- pNA (optimal substrate from positional scanning data - panel E), and Bz-FVR-pNA (panel F). Velocity of LK14 cleavage (nM sec^"1) was measured at six different substrate concentrations and fit to Michaelis-Menten kinetic plots using GraphPad Prism 5.01. The >-axes represent KLK14 velocity (nM sec^'1) and the x-axes show peptide-pNA substrate concentration (μΜ). Data represent the mean ± S.E.M. for three experiments performed in triplicate.

Figure 3. Inhibition of LK14 cleavage of YAVR JNA by engineered SFTI variants. Activity of KLK14 on 120 μΜ YAVRpNA across varying concentrations of engineered SFTI inhibitors (SFTI-YCVR N14 - panel A; SFTI-WCVR N14 - panel B; SFTI-WCVR Q12 N14 - panel C). Assays were performed as described for Figure 1, with activity measured by the change in absorbance at 405 nm for 300 sec with readings taken every 10 sec. Data were exported to GraphPad Prism 5.01 and fit with logio [inhibitor] vs response curves. Relative activity expressed as a percentage of activity in uninhibited KL 14 internal controls is plotted on the y-axis with logio [inhibitor (nM)] shown on the x-axis.

Figure 4. Engineered SFTI variants inhibit LK digestion of the high molecular weight protein substrate fibrinogen. Coomassie stained SDS-PAGE gels for inhibition of fibrinogen (FG) proteolysis by SFTI-WCVR F12 N14 (panel A), SFTI-YCVR F12 N14 (panel B), SFTI-YCSR F12 N14 (panel C), SFTI-YCNR F12 N14 (panel D), and several off-target proteases, including trypsin and matriptase. Undigested FG is loaded on the far left of each gel and resolves into three distinct subunits under reducing conditions. Loading for each protease is paired; FG digestion by uninhibited protease is shown on the left of each series and digestion in the presence of inhibitor on the right. Inhibitor concentrations are indicated above each lane. For off-target proteases (trypsin and matriptase), inhibitor concentrations reflect the highest concentration of inhibitor where no change in FG proteolysis was observed. For KL treatments, inhibitor concentration indicates the lowest concentration where complete inhibition was observed.

Figure 5. SFTI-WCIR N12 N14 is selective for L 14. Fibrinogen proteolysis assays were conducted using five off-target KLK proteases and four off-target non-KLK proteases. Proteases were treated ± inhibitor (indicated above each lane) before addition of fibrinogen substrate.

Figure 6. SFTI-YCSR N12 N14 was most effective against KLK5 and KLK14 (panel A), while SFTI-TCTR N12 N14 inhibited KLK5, KLK7 and KL 14 (panel B), and SFTI-TCTY N12 N14 inhibited KLK5 and KLK7 (panel C). Figure 7. Ex vivo desquamation assay. To quantify the effect of SFTI inhibitor variants in the most biologically relevant system available, skin flakes were harvested from healthy volunteers and incubated with/without exogenously added SFTI variants. Detached cells were collected by centrifugation and quantified by BCA assay.

Figure 8. SFTI-WCVR Q12 N14 reduces the desquamation-like activity of KLK 14 in simulated desquamation assays using HaCaT cell monolayers. A-E: Phase microscopy images of cell monolayers following treatment with (A) buffer (100 mM Tris-HCl pH 8.0, 0.5 mM EDTA) only, (B) thermolysin control (100 nM thermolysin - EDTA inhibited), (C) 35 nM KLK 14, (D) 35 nM KLK14 + 1.75 μΜ SFTI-WCVR Q12 N14, and (E) 35 nM KLK14 + 3.5 μΜ SFTI-WCVR Q12 N14. All treatments proceeded for 2 hours at 37°C. At the incubation end point, cells were gently agitated to assess biomechanical strength of the cell monolayer and visually assessed using a Nikon Eclipse TE2000-U microscope. Images are representative of three separate experiments (scale bar = 100 μιη).

Lower panel: To examine global changes across the monolayer, the number of cells detached from the culture surface was quantified by NucleoCount (Chemometec). After microscopy, detached cells in assay buffer were recovered and samples removed for cell count (carried out according to the manufacturer's instructions). The total number of cells detached was calculated and data were compiled in GraphPad Prism 5.01 as mean ± S.E.M. of three separate experiments. Figure 9. SFTI-WCVR Q12 N14 attenuates KLK14-mediated cell detachment by inhibiting proteolysis of desmoglein- 1. Western blot analysis for desmoglein 1, the extracellular link between cell-surface desmosomes which regulate tissue cohesion in the upper epidermis. Desmoglein 1 was equally present in negative control treatments (Buffer Only; Thermolysin Control). However, in KL 14 treated cells, the abundance of full length, monomelic desmoglein 1 was substantially reduced, along with dimeric desmoglein 1, an indicator of the number of intact desmosomes (35 nM KL 14). This suggested that desmoglein 1 was a target for proteolysis by LK14 in situ. KLK14- mediated proteolysis of desmoglein 1 was reversed in both treatments of SFTI-WCVR Q12 N14 (35 nM KLK14 + 1.75 μΜ SFTI; 35 nM KL 14 + 3.5 μΜ SFTI). Western blot analysis for desmoplakin I, the intracellular desmosome contact between desmoglein and the keratin cytoskeleton, confirmed equivalent protein loading.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following explanation of terms, abbreviations and methods is provided to better describe the present compounds, compositions and methods. It is understood that the terminology used in the disclosure is for the purpose of describing particular embodiments and examples only, and is not intended to be limiting.

Abbreviations:

SFTI: Sunflower trypsin inhibitor

SFTI-YCVRN14: SFTI variant with 2-Tyr, 3 -Cys, 4-Val, 5-Arg, and 14-Asn SFTI-WCVR N14: SFTI variant with 2-Trp, 3 -Cys, 4-Val, 5-Arg, and 14-Asn SFTI-YCIRN14: SFTI variant with 2-Tyr, 3-Cys, 4-Ile, 5-Arg, and 14-Asn SFTl-WCVR Q12 N14: SFTI variant with 2-Trp, 3-Cys, 4-Val, 5-Arg, 12-Gln, and 14-Asn

SFTI- WCIR N 12 N 14 : SFTI variant with 2-Trp, 3-Cys, 4-Ile, 5-Arg, 12-Asn, and

14-Asn

SFTI-YCSR N12 N14: SFTI variant with 2-Tyr, 3-Cys, 4-Ser, 5-Arg, 12-Asn, and

14-Asn

SFTI-TCTY N12 N14: SFTI variant with 2-Thr, 3-Cys, 4-Thr, 5-Tyr, 12-Asn, and

14-Asn

SFTI-TCTR N12 N14: SFTI variant with 2-Thr, 3-Cys, 4-Thr, 5-Arg, 12-Asn, and

14-Asn

SFTI-WC VR N 12 N 14 : SFTI variant with 2-Trp, 3-Cys, 4-Val, 5-Arg, 12-Asn, and

14-Asn

SFTI-YCLFN12 N14: SFTI variant with 2-Tyr, 3-Cys, 4-Leu, 5- Phe, 12-Asn, and

14-Asn

SFTI-TCTF N12 N14: SFTI variant with 2-Thr, 3-Cys, 4-Thr, 5- Phe, 12-Asn, and

14-Asn

SFTI-WCLF N12 N14: SFTI variant with 2-Trp, 3-Cys, 4-Leu, 5-Phe, 12-Asn, and

14-Asn

DMF: N,N-dimethylformamide

DBU: 1,8-Diazabicyclo [5.4.0]undec-7-ene

HBTU: 2-( 1 H-benzotriazole- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluoro-phosphate

HOBt: 1 -Hydroxybenzo-triazole

DEPEA: N,N-Diisopropylethylamine

FMOC: 9-fluorenylmethyl carbamate

SDS: Sodium dodecyl sulfate

PAGE: Polyacrylamide gel electrophoresis

KLK: Kallikrein-related peptidase

KL 4: Kallikrein-related peptidase 4

KLK5: Kallikrein-related peptidase 5

KL 7: Kallikrein-related peptidase 7

KL 14: Kallikrein-related peptidase 14

TFA: Trifluoroacetic acid DCM: Dichlormethane

MS: Mass spectroscopy

TOF: Time of flight

MALDI: Matrix assisted laser desorption ionization

pNA: /wra-Nitroanilide

Bz: Benzyl

The present invention relates to kallikrein-related peptidase inhibitors and their use in the diagnosis, prevention and treatment of skin diseases.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

In one aspect, the invention provides a compound including a peptide or salt thereof according to Formula I:

e

Formula I wherein

R 1 , R 2 , R 3 , R 4 , R 6 , and R 8 are each an amino acid residue, R 5 is an amino acid residue other than Phe and R⁷ is an amino acid residue other than Asp or Glu.

In one embodiment, R¹ is Trp or Tyr, R² is Val or He, R³ is Arg, R⁵ is Gin, and R⁷ is Asn.

In another embodiment, R¹ is Trp or Tyr, R² is Val or He, R³ is Arg, R⁴ is Pro, R⁶ is Pro, and R⁸ is Gly. In yet another embodiment, R is Trp or Tyr, R is Val or He, R is Arg, R is Pro, R⁵ is Gin, R⁶ is Pro, R⁷ is Asn, and R⁸ is Gly.

In a further embodiment, R^l is Tyr or Trp, R² is Ser, Thr or Asn, R³ is Arg, R⁵ is Gin, and R⁷ is Asn.

In yet a further embodiment, R¹ is Trp, R² is He, R³ is Arg, R⁵ is Asn, and R⁷ is

Asn.

In another embodiment, R¹ is Tyr, R² is Ser, R³ is Arg, R⁵ is Asn, and R⁷ is Asn. In still another embodiment, R is Thr, R is Thr, R is Tyr, R is Asn, and R is

Asn.

In yet another embodiment, R¹ is Thr, R² is Thr, R³ is Arg, R⁵ is Asn, and R⁷ is

Asn.

In a further embodiment, R¹ is Trp, R² is Val, R³ is Arg, R⁵ is Asn, and R⁷ is Asn. In yet a further embodiment, R¹ is Tyr R² is Leu, R³ is Phe, R⁵ is Asn, and R⁷ is

Asn.

In another embodiment, R¹ is Thr, R² is Thr, R³ is Phe, R⁵ is Asn, and R⁷ is Asn. In still another embodiment, R is Trp R is Leu, R is Phe, R is Asn, and R is

Asn.

In some embodiments, a compound according to the first aspect of the invention is a LK protease inhibitor, including a KLK14, LK7 and/or KL 5 protease inhibitor.

The term "amino acid" refers to both natural and unnatural amino acids in their D and L stereoisomers for chiral amino acids. Moreover, peptide compounds disclosed herein may contain asymmetric centers in addition to the chiral centers in the backbone of the peptide compound. These asymmetric centers may independently be in either the R or S configuration. It will also be apparent to those skilled in the art, that certain peptide compounds disclosed herein may exhibit geometrical isomerism. Geometrical isomers include the cis and trans forms of peptide compounds of the invention having alkenyl moieties. The present compounds comprise the individual geometrical isomers and stereoisomers and mixtures thereof.

The term "amino acid" is understood to refer to both amino acids and the corresponding amino acid residues, such as are present, for example, in peptides. Natural and unnatural amino acids are well known to those of ordinary skill in the art. Common natural amino acids include, without limitation, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Uncommon and unnatural amino acids include, without limitation, allyl glycine (AllylGly), biphenylalanine (Bip), citrulline (Cit), 4- guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2- napthylalanine (2-Nal), ornithine (Orn), and pentafluorophenylalanine (FsPhe).

Amino acids are typically classified in one or more categories, including polar, hydrophobic, acidic, basic, and aromatic, according to their side chains. Examples of polar amino acids include those having side chain functional groups such as hydroxyl, sulfhydryl, and amide, as well as the acidic and basic amino acids. Polar amino acids include, without limitation, asparagine, cysteine, glutamine, histidine, selenocysteine, serine, threonine, tryptophan, and tyrosine. Examples of hydrophobic or non-polar amino acids include those residues having nonpolar aliphatic side chains, such as, without limitation, leucine, isoleucine, valine, glycine, alanine, proline, methionine, and phenylalanine. Examples of basic amino acids include those having a basic side chain, such as an amino or guanidino group. Basic amino acids include, without limitation, arginine, homolysine and lysine. Examples of acidic amino acids include those having an acidic side chain functional group, such as carboxy group. Acidic amino acids include, without limitation aspartic acid and glutamic acid. Aromatic amino acids include those having and aromatic side chain group. Examples of aromatic amino acids include, without limitation, biphenylalanine, histidine, 2-napthylalananine, pentafluorophenylaline, phenylalanine, tryptophan, and tyrosine. It is noted that some amino acids are classified in more than one group. For example, histidine, tryptophan and tyrosine are classified as both polar and aromatic amino acids. Additional amino acids that are classified in each of the above groups are known to those of ordinary skill in the art.

"Equivalent amino acid" means an amino acid which may be substituted for another amino acid in the peptide compounds according to the invention without any appreciable loss of function. Equivalent amino acids will be recognized by those of ordinary skill in the art. Substitution of like amino acids is made on the basis of relative similarity of side chain substituents, for example regarding size, charge, hydrophilicity, and hydrophobicity as understood by those of ordinary skill in the art. The amino acid residues of Formula I may include N-alkyl and/or N-aralkyl amide bonds. Such substitutions can be made as is known to those of ordinary skill in the art to improve stability of a compound or to increase the affinity of a compound for the desired target.

The disclosed peptide compounds also encompass salts including, if several salt- forming groups are present, mixed salts and/or internal salts. The salts are generally pharmaceutically-acceptable salts that are non-toxic. Examples of salt-forming acidic groups include, but are not limited to, a carboxyl group, a phosphonic acid group or a boronic acid group, that can form salts with suitable bases. These salts can include, for example, nontoxic metal cations which are derived from metals of groups I A, IB, HA, and IIB of the periodic table of the elements. In one embodiment, alkali metal cations such as lithium, sodium or potassium ions, or alkaline earth metal cations such as magnesium or calcium ions can be used. The salt can also be a zinc or an ammonium cation. The salt can also be formed with suitable organic amines, such as unsubstituted or hydroxyl- substituted mono-, di- or tri-alkylamines, in particular mono-, di- or tri-alkylamines, or with quaternary ammonium compounds, for example with N-methyl-N-ethylamine, diethylamine, triethylamine, mono-, bis- or tris-(2-hydroxy-lower alkyl)amines, such as mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine or tris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-lower alkyl)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine, or N- methyl-D-glucamine, or quaternary ammonium compounds such as tetrabutylammonium salts.

Particular peptide compounds possess at least one basic group that can form acid- base salts with inorganic acids. Examples of basic groups include, but are not limited to, an amino group or imino group. Examples of inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid. Basic groups also can form salts with organic carboxylic acids, sulfonic acids, sulfo acids or phospho acids, or N-substituted sulfamic acid, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2- acetoxybenzoic acid, embonic acid, nicotinic acid, or isonicotinic acid, and, in addition, with amino acids, for example with a-amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonic acid, ethane- 1,2-disulfonic acid, benzenedisulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid (with formation of the cyclamates), or with other acidic organic compounds, such as ascorbic acid.

The peptide compounds of the disclosure can be prepared using virtually any technique known to one of ordinary skill in the art for the preparation of peptides. For example, the peptide compounds can be prepared using step-wise solution or solid phase peptide synthesis, recombinant DNA techniques or equivalents thereof.

Peptide compounds of the disclosure having either the D- or L-configuration can be readily synthesized by automated solid phase procedures well known in the art. Suitable syntheses can be performed by utilizing "T-boc" or "F-moc" procedures. Techniques and procedures for solid phase synthesis are described in Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the peptide compounds may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37:933-36, 1996; Baca et al., J. Am. Chem. Soc. 117:1881-87, 1995; Tam et al., Int. J. Peptide Protein Res. 45:209-16, 1995; Schnolzer and Kent, Science 256:221-25, 1992; Liu and Tam, J. Am. Chem. Soc. 116:4149-53, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91:6584-88, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31:322-34, 1988). This is particularly the case with glycine containing peptides. Other methods useful for synthesizing the peptide compounds of the disclosure are described in Nakagawa e/ a/., J. Am. Chem. Soc. 107:7087-92, 1985.

Additional exemplary techniques known to those of ordinary skill in the art of peptide and peptide analog synthesis are taught by Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis, Springer Verlag, New York, 1994; and by Jones, J., Amino Acid and Peptide Synthesis, 2nd ed., Oxford University Press, 2002. The Bodanszky and Jones references detail the parameters and techniques for activating and coupling amino acids and amino acid derivatives. Moreover, the references teach how to select, use and remove various useful functional and protecting groups. If a peptide compound is composed entirely of gene-encoded amino acids, or a portion of it is so composed, the peptide compound or the relevant portion can also be synthesized using conventional recombinant genetic engineering techniques. For recombinant production, a polynucleotide sequence encoding the peptide compound is inserted into an appropriate expression vehicle, that is, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation. The expression vehicle is then transfected into a suitable target cell which will express the peptide compound. Depending on the expression system used, the expressed peptide is then isolated by procedures well-established in the art. Methods for recombinant protein and peptide production are well known in the art (see, e.g., Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2^nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Ch. 17 and Ausubel et al. Short Protocols in Molecular Biology, 4^th ed., John Wiley & Sons, Inc., 1999).

To increase efficiency of production, the polynucleotide can be designed to encode multiple units of the peptide compound separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved {e.g., by treatment with the appropriate enzyme) in order to recover the peptide units. This can increase the yield of peptides driven by a single promoter. In one embodiment, a polycistronic polynucleotide can be designed so that a single mRNA is transcribed which encodes multiple peptides, each coding region operatively linked to a cap-independent translation control sequence, for example, an internal ribosome entry site (IRES). When used in appropriate viral expression systems, the translation of each peptide encoded by the mRNA is directed internally in the transcript, for example, by the IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual peptides. This approach eliminates the production and enzymatic processing of polyproteins and can significantly increase yield of peptide driven by a single promoter.

A variety of host-expression vector systems may be utilized to express the peptides described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems.

The expression elements of the expression systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. When cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter can be used. When cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters, the promoter for the small subunit of RUBISCO, the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV, the coat protein promoter of TMV) can be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. , the adenovirus late promoter, the vaccinia virus 7.5 K promoter) can be used.

The peptide compounds of the disclosure can be purified by many techniques well known in the art, such as reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, gel electrophoresis, and the like. The actual conditions used to purify a particular peptide will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those of ordinary skill in the art.

A detectable moiety can be linked to the peptide compounds disclosed herein, creating a peptide-detectable moiety conjugate. Thus, in another aspect, the invention provides a peptide compound as disclosed herein for the diagnosis of a skin disease or pathology, or other undesirable skin condition in a subject. Detectable moieties suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. The detectable moieties contemplated for the present disclosure can include, but are not limited to, an immunofluorescent moiety (e.g., fluorescein, rhodamine, Texas red, and the like), a radioactive moiety (e.g., ³H, ³²P, ¹²⁵I, and ³⁵S), an enzyme moiety (e.g., horseradish peroxidase and alkaline phosphatase), a colorimetric moiety (e.g., colloidal gold, biotin, colored glass or plastic, and the like). The detectable moiety can be linked to the peptides at either the N- and/or C-terminus. Optionally, a linker can be included between the peptide and the detectable moiety.

Means of detecting such moieties are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, while fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

The present inventors have found that compounds based on SFTI with specific protease inhibition properties can be identified and synthesised using the strategy illustrated herein below in the examples. These compounds have utility in the diagnosis, prevention and/or treatment in subjects of a skin disease or pathology, or other undesirable skin condition. This utility results from the ability of the compounds to modulate the activity of proteases involved in disease processes. Thus, these compounds have utility in inhibiting or decreasing the serine protease activity of a KLK protease in a subject, particularly KLK14, KLK7 and/or KLK5. One embodiment of the protease inhibitors disclosed herein includes compounds according to Formula I and their corresponding pharmaceutically acceptable salts.

With reference to Formula I, in one embodiment a cystine bridge, formed by cysteine residues linked through their side chains by a disulfide bond (cysteine-S-S- cysteine), stabilizes the protease inhibitor. In another embodiment, the protease inhibitor is stabilized by a crosslinking group. Examples of crosslinking groups include, but are not limited to, amides, esters, thioesters, ethers, sulfides, disulfides, diselenides, and aromatic and aliphatic groups, such as optionally substituted lower aliphatic carbon chains. In particular, the invention provides potent and specific KLK 14, KLK7 and/or KLK5 inhibitors that do not significantly inhibit off-target proteases (e.g., trypsin and matriptase).

As indicated herein above, the compounds according to the invention have utility in the diagnosis, prevention or treatment in subjects of a skin disease or pathology, or other undesirable skin condition. Skin diseases or pathologies, include, but are not limited to, Netherton syndrome, peeling skin syndrome, acne rosacea, psoriasis, eczema, and atopic dermatitis.

By "undesirable skin condition" is meant a problem affecting the skin or the appearance of the skin which may not necessarily be considered a disease or pathology of the skin. For example, skin blemishes and other imperfections of the skin may constitute an undesirable skin condition. Thus, the invention also contemplates methods of cosmetic treatment where peptide compounds of the disclosure, inclusive of salts thereof, are administered to improve or enhance skin quality or skin appearance.

Diagnosis of a skin disease or pathology, or undesirable skin condition using the disclosed peptide compounds can be achieved using methods well known in the art, including, for example, detecting the binding of a peptide of the invention with one or more KLK proteases (e.g., KLK14, KLK7 and or KLK5) in a sample from a subject. The basic principle of an assay used to identify a KLK protease that binds to a peptide of the invention involves contacting a peptide of the invention and a sample suspected of including one or more KLK proteases from a subject under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be detected.

The binding assays can be conducted in a variety of ways. For example, one method to conduct such an assay involves anchoring one or more labeled (either directly or indirectly) peptides of the invention to a solid support (such as, a microarray or in a microtitre plate), contacting the solid support with a sample suspected of including one or more KLK proteases and detecting peptide/protease complexes anchored to the solid support at the end of the reaction. Each of the peptides may be present on the solid support in one or more addressable positions. Alternatively, a sample suspected of including one or more KLK proteases is attached to a solid support and one or more detectable (i.e., labeled) peptides disclosed herein are applied to the solid support, followed by detection of peptide/protease complexes anchored to the solid support at the end of the reaction.

The peptide compounds of the invention have particular utility in the treatment of skin disorders in mammals, particularly humans. Therefore, in yet another aspect, the invention provides a pharmaceutical or veterinary composition including an amount of a peptide compound as disclosed herein together with a pharmaceutically acceptable carrier or diluent. Such compositions are effective to inhibit or decrease serine protease activity in a subject, for example, to inhibit or decrease the serine protease activity of a KLK protease, including, but not limited to, KLK 14, KLK7 and/or KLK5 in the subject. Such compositions are also useful for the diagnosis, prevention or treatment of a skin disease or pathology, or other undesirable skin condition in a subject, as discussed herein.

The peptide compounds of the invention are typically administered as a component of a pharmaceutical composition as described herein. Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatine or an adjuvant or an inert diluent. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, a mineral oil or a synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations will generally contain at least 0.1 wt% of the compound.

Parenteral administration includes administration by the following routes: intravenously, cutaneously or subcutaneously, nasally, intramuscularly, intraocularly, transepithelially, intraperitoneally, and topically. Topical administration includes dermal, ocular, rectal, nasal, as well as administration by inhalation or by aerosol means. For intravenous, cutaneous or subcutaneous injection, or injection at a site where treatment is desired, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of skill in the art will be well able to prepare suitable solutions using, for example, solutions of the subject peptide compounds or derivatives thereof.

In addition to a peptide compound and a pharmaceutically acceptable carrier or diluent, compositions according to the invention can further include a pharmaceutically or veterinarially acceptable excipient, buffer, stabiliser, isotonicising agent, preservative or antioxidant, or any other material known to those of skill in the art. It will be appreciated by the person of skill in the art that such materials should be non-toxic and should not interfere with the efficacy of the compound(s). The precise nature of any additive may depend on the route of administration of the composition, that is, whether the composition is to be administered orally or parenterally. With regard to buffers, aqueous compositions typically include such substances so as to maintain the composition at a close to physiological pH or at least within a range of about pH 5.0 to about pH 8.0.

Compositions according to the invention can also include additional active ingredients in addition to a peptide compound as disclosed herein. Such ingredients will principally be chosen for their efficacy as agents for preventing or treating a skin disease or pathology, or other undesirable skin condition in a subject, or for providing other therapeutic benefits to the skin, but can also be chosen for their efficacy against a non- skin associated condition involving a KL , such as KL 14, KLK7 and/or KLK.5.

The International Cosmetic Ingredient Dictionary and Handbook, Twelfth Edition (2008) describes a wide variety of non-limiting cosmetic and pharmaceutical ingredients commonly used in the skin care industry, which are suitable for use in the compositions of the present invention. Examples of these ingredient classes include: abrasives; absorbents; aesthetic components, such as fragrances; pigments and colorings/colorants; essential oils; skin sensates; astringents, such as clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, and witch hazel distillate; anti-acne agents (e.g., resorcinol, sulfur, benzoyl peroxide, erythromycin, zinc, and the like); anti-caking agents; antifoaming agents; antimicrobial agents (i.e., agents capable of destroying microbes or preventing their development/pathogenic action); antioxidants (e.g., ascorbic acid and its salts, ascorbyl esters of fatty acids, ascorbic acid derivatives, tocopherol, tocopherol sorbate, tocopherol acetate, and other esters of tocopherol); binders; thickeners; biological additives; buffering agents; bulking agents; chelating agents (i.e., an agent capable of removing a metal ion from a system by forming a complex with it, such as, for example, firildioxime, furilmonoxime and derivatives thereof); denaturants; film formers; opacifying agents; pH adjusters; anti-inflammatory agents, such as steroidal anti- inflammatory agents, including but not limited to, corticosteroids (e.g., hydrocortisone) and nonsteroidal anti-inflammatory agents (e.g., ibuprofen, naproxen, flufenamic acid, etofenamate, aspirin, mefenamic acid, meclofenamic acid, piroxicam, and felbinac); reducing agents; sequestrants; skin bleaching and lightening agents (e.g., hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, and ascorbyl glucosamine); skin-conditioning agents (e.g., humectants); skin soothing and/or healing agents (e.g., panthenol and panthenol derivatives, aloe vera, pantothenic acid and pantothenic acid derivatives, allantoin, bisabolol, and dipotassium glycyrrhizinate); topical anesthetics, such as benzocaine, lidocaine, bupivacaine, chlorprocaine, dibucaine, etidocaine, mepivacaine, tetracaine, dyclonine, hexylcaine, procaine, cocaine, ketamine, pramoxine, phenol, and pharmaceutically acceptable salts thereof; retinoids (i.e., natural and or synthetic analogs of vitamin A or retinol-like compounds that possess the biological activity of vitamin A in the skin, as well as the geometric isomers and stereoisomers of these compounds); vitamins (e.g., vitamin B3 and its derivatives, such as, nicotinic acid esters, nicotinyl amino acids, nicotinyl alcohol esters of carboxylic acids, nicotinic acid N-oxide, and niacinamide N-oxide); sunscreen agents, such as metallic oxides (e.g., zinc oxide) and organic sunscreen compounds (e.g., p-aminobenzoic acid, its salts and derivatives); and propellants.

A pharmaceutical or veterinary composition according to the invention will be administered to a subject in either a prophylactically effective or a therapeutically effective amount as necessary for the particular situation under consideration. The actual amount of a peptide compound administered by way of a composition, and rate and time- course of administration, will depend on the nature and severity of the condition being treated or the prophylaxis required. Prescription of treatment such as decisions on dosage and the like will be within the skill of the medical practitioner or veterinarian responsible for the care of the subject. Typically however, compositions for administration to a human subject will include between about 0.01 and 100 mg of the compound per kg of body weight and more preferably between about 0.1 and 10 mg/kg of body weight.

The peptide compounds can be included in compositions as pharmaceutically or veterinarially acceptable derivatives thereof. As used herein "derivatives" of the compounds includes salts, coordination complexes with metal irons such as Mn and Zn²⁺, esters such as in vivo hydrolysable esters, free acids or bases, hydrates, or prodrugs. Compounds having acidic groups such as phosphates or sulfates can form salts with alkaline or alkaline earth metals such as Na⁺, K⁺, Mg²⁺, and Ca²⁺, and with organic amines such as triethylamine and Tris (2-hydroxyethyl) amine. Salts can also be formed between compounds with basic groups, such as amines, with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid. Compounds having both acidic and basic groups can form internal salts.

Esters can be formed between hydroxyl or carboxylic acid groups present in the peptide compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques that will be well known to those of skill in the art.

Prodrug derivatives of the peptide compounds of the invention can be transformed in vivo or in vitro into the parent compounds. Typically, at least one of the biological activities of a parent compound may be suppressed in the prodrug form of the compound, and can be activated by conversion of the prodrug to the parent compound or a metabolite thereof. Prodrugs of compounds of the invention include the use of protecting groups which may be removed in vivo to release the active compound or serve to inhibit clearance of the drug. Suitable protecting groups will be known to those of skill in the art.

The peptide compositions of the present invention are useful for protecting the skin by inhibiting protease activity and promoting healthy skin development. Thus, in a further aspect, the invention provides a method for preventing or treating a KL - mediated disease or pathology in a subject, for example, a KLK14-, KLK7- and/or L 5-mediated disease or pathology, the method including the step of administering to the subject a therapeutically effective amount of a peptide compound as disclosed herein, or a pharmaceutical or veterinary composition including a therapeutically effective amount of a peptide compound as disclosed herein.

Also provided is a method of preventing or treating a skin disease or pathology, or other undesirable skin condition in a subject, the method including the step of administering to the subject a therapeutically effective amount of a peptide compound as disclosed herein, or a pharmaceutical or veterinary composition including a therapeutically effective amount of a peptide compound as disclosed herein.

The peptide compositions of the invention can be used for both prophylactic and therapeutic treatment of a skin disease or pathology, or other undesirable skin condition in a subject. For example, compositions of the invention may be used for preventing skin irritations, preventing rashes, promoting healing of skin tissue after a rash or irritation has occurred (i.e., building the epidermis and/or dermis layers of the skin), preventing and/or retarding atrophy of the skin, preventing and/or retarding the appearance of spider vessels and/or red blotchiness on the skin, preventing and/or relieving itching in the skin, regulating skin texture (e.g., ameliorating roughness, swelling and soreness), and improving skin color (e.g., reducing redness).

Treating skin tissues involves topically applying to the skin tissue a safe and effective amount of a composition of the invention. The amount of the composition that is applied, the frequency of application and the period of use will vary widely depending upon the amount of the peptide compound and other ingredients in a given composition, and the level of regulation desired, for example, in light of the level of skin tissue damage present or expected to occur.

In some embodiments, a composition of the invention is chronically applied topically to the skin. By "chronic topical application" is meant continued topical application of the composition over an extended period during the subject's lifetime, for example, for a period of at least about one week, or for a period of at least about one month, or for at least about three months, or for at least about six months, or for at least about one year. While benefits are obtainable after various periods of use (e.g., two, five, ten, twenty, or more days), chronic application can continue for a year or more. Typically applications would be on the order of about once per day over such extended periods, however application rates can vary from about once per week up to about three times per day, or more.

A wide range of quantities of the peptide compounds and compositions thereof of the invention can be employed to provide a beneficial effect upon the skin. Quantities that are typically applied per application are, in mg composition/cm² skin, from about 0.01 mg cm to about 10 mg/cm . A desirable and useful application amount is about 1 mg/cm to about 2 mg/cm².

Prophylactic or therapeutic treatment of a skin disease or pathology, or other undesirable skin condition in a subject can be practiced by applying a peptide compound or composition thereof in the form of a skin lotion, cream, gel, foam, ointment, paste, emulsion, spray, conditioner, tonic, or the like that is preferably intended to be left on the skin. After applying the composition to the skin, it can be left on the skin for a period of at least about 15 minutes, or at least about 30 minutes, or at least about 1 hour, or for at least several hours, for example, at least about 2 hours, 3 hours, 6 hours, 12 hours, or 24 hours.

The peptide compositions of the present invention are also useful for regulating in vitro desquamation by inhibiting the serine protease activity of a KLK protease (e.g., KLK 14, KLK7 and/or KLKS) in cell/tissue culture procedures. Thus, in an additional aspect, the invention provides a method for disaggregating cells or tissue in vitro, the method including the steps of exposing the cells or tissue to a KLK protease (e.g., KLK14, KLK7 and/or KLK5) followed by exposure to a peptide compound of the disclosure, inclusive of salts thereof.

It will be appreciated that this method has application to cell culture procedures generally, where the method can be used to dissociate and detach cells for passage. Furthermore, this method can be used in the culturing of artificial skin, where the selective cleavage of cell-cell adhesion proteins by a KLK protease (e.g., KLK14, KLK7 and/or KLK5) in conjunction with regulation of such cleavage by a peptide compound of the disclosure, inclusive of salts thereof, results in cells/tissue better suited for subsequent therapeutic uses.

Peptide compounds according to the invention have utility for use in preventing or treating a skin disease or pathology, or other undesirable skin condition in a subject, as well as in the manufacture of a medicament for the diagnosis, prevention or treatment in a subject of a skin disease or pathology, or other undesirable skin condition. Processes for the manufacture of such medicaments will be known to those of skill in the art and include the processes used to manufacture the pharmaceutical compositions described herein.

Having broadly described the invention, non-limiting examples of the compounds, their synthesis, and their biological activities, will now be given.

EXAMPLES EXAMPLE 1 - KLK14 protease inhibitors

The inventors used substrate-based design principles to engineer potent and selective inhibitors to KLK 14. This approach probes the target enzyme active site with a library of molecules to determine the optimal substrate for the protease. This substrate is then used as the basis for engineering SFTI through substituting that portion of the inhibitor that directly contacts the enzyme's active site. Experimental Procedures

Recombinant KLK14 Production

Recombinant KLK14 was produced in Spodoptera frugiperda (Sf9) insect cells (Invitrogen, Mount Waverly, Australia) using an expression construct produced by ligating the KLK14 human open reading frame into the pIB/V5-His vector (Invitrogen) (Swedberg et al., Chem. Biol. 16:633-43, 2009). This allows production of full length pro- L 14 with a C-terminal V5 epitope for identification by Western analysis and a poly- His tag for facile purification. Sf9 cells were transfected using Cellfectin (Invitrogen) and cells carrying the expression vector were selected with 50μg/ml blasticidin (InvivoGen, San Diego, CA, USA). Conditioned media containing secreted pro-KLK14 was dialysed against PBS, pH 7.4 at 4°C overnight then purified by Ni-NTA superflow resin (Qiagen, Doncaster, Australia) according to the manufacturer's instructions. Eluant fractions were analysed by Coomassie stained SDS-PAGE, with pro-KLK14 containing fractions aliquoted and stored at -80°C until use. Pro-KLKl 4 Activation

When required for experiments, pro- L 14 was activated with thermolysin (Calbiochem, San Diego, CA, USA) at 37°C with the proportion of thermolysin required optimised for each protease preparation. To monitor activation progress, 1 iL samples were removed at 5 min intervals and assayed for amidolytic activity against Bz-FVR- /?NA in KLK14 assay buffer (100 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.005% Triton- X) as described by Swedberg et al. {Chem. Biol. 16:633-43, 2009). Thermolysin activity was inhibited by the addition of 5% (v/v) 0.5 M EDTA pH 8.0.

Synthesis of Peptide Substrates

Peptide-/?NA substrates were produced by Fmoc solid phase synthesis methods (Abbenante et al., Lett. Pept. Sci. 7:347-351, 2000), optimised further within our laboratory for high efficiency synthesis (Swedberg et al., Chem. Biol. 16:633-43, 2009).

Chemicals were from Sigma-Aldrich and solvents from AusPep unless otherwise stated.

Briefly, 2-chlorotrityl chloride resin (Cross-Link 100-200 mesh, 1.3 mmol equiv per g) was derivatised with four-fold excess of />-phenylenediamine and 0.25M N,N- diisopropylethylamine (DIPEA) in N,N-dimethylformamide (DMF) overnight. Peptide elongation was carried out using four-fold excess of Fmoc-protected amino acids with 0.25M each of 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU); 1-hydroxybenzo-triazole (HOBt); and DIPEA dissolved in DMF. Coupling proceeded for 60 min. Rapid deprotection of the Fmoc protecting group was achieved using 50% (v/v) piperidine and 5% (v/v) l,8-diazabicyclo[5.4.0]undec-7- ene (DBU) in DMF (twice, 5 min). Following synthesis of complete peptides, the N- terminus was acetylated using 50% (v/v) acetic anhydride in DMF for 120 min. Mild cleavage from the resin was carried out using three changes each of 1, 2 and 3% (v/v) trifluoroacetic acid (TFA) in dichloromethane (DCM) followed by precipitation in 10 volumes of ice-cold diethyl ether. The C-terminal 7-phenylenediamine group was oxidised in 6 molar equivalents of Oxone dissolved in 50% (v/v) acetonitrile / H₂0 overnight. Oxidised peptides were recovered by separation with 50% (v/v) ethyl acetate in DCM, dried down with nitrogen gas, and side chain protecting groups removed by cleavage in 95% (v/v) TFA, with scavengers: 2.5% (v/v) thioanisole, 1.25% triisopropyl silane (TIS) and 1.25% ¾0. Again, peptides were collected by ether precipitation, re- solubilised in 40% (v/v) 2-propanol / ¾0 and lyophilised overnight. Peptides were stored at -80°C until the complete series had been synthesised.

Production of a Positional Scanning-Combinatorial Library

Using the method described above, a fully degenerate P1-P4 peptide- NA positional scanning library was produced within our laboratory (for binding site nomenclature, see, Schechter and Berger, Biochemical and Biophysical Research Communications 27:157-62, 1967). As the first phase of determining the optimal substrate specificity for LK14, positional scanning-combinatorial library (PS-SCL) analysis was carried out. Substrates were prepared by solubilising a small mass of each sub-library in 45% (v/v) EtOH, 45% (v/v) H₂0, 10% (v/v) glycerol, adjusted such that the concentration of each substrate sub-library was 3.75 mM. Triplicate assays for each of the eighty sub-libraries were carried out by adding 15μ1 of substrate to 285μ1 KLK14 assay buffer (as above) containing 10% 2-propanol to aid with substrate solubility. Thermolysin-activated LK14 (2 μΐ) was added just prior to assay, with cleavage of the p K reporter group measured by an increase in absorbance at 405 nm over 300 sec using a Benchmark Plus Microplate Spectrophotometer (Biorad). Data was compiled in GraphPad Prism 5.01 and expressed as mean activity ± standard error of the mean (SEM). Sparse Matrix Library Screen

A sparse matrix library was designed based on high scoring residues from our PS- SCL results and the previously reported peptide- ACC PS-SCL data using Arg fixed at PI (Borgono et al., Biol. Chem. 388:1215-25, 2007). The four top scoring residues at P4 and three top scoring residues at P3 and P2 were taken, while Arg showed a definite preference at PI. The resulting 36 peptides were individually synthesised as peptide- pNAs as described herein.

Prior to assay, crude peptides were solubilised in 500 μΐ 40% (v/v) 2-propanol and adjusted to equal molarity according to total cleavage of the />NA group. Triplicate assays were prepared in 96 well non-binding transparent assay plates (Corning) by adding 5 μΐ of each substrate to 245 μΐ assay buffer (100 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.005% Triton-X) containing 1.5 μΐ thermolysin-activated KLK14. Single point absorbance readings were taken at 405 nm prior to addition of KL 14 and following the overnight incubation. The change in total absorbance was used to calculate the volume of substrate required for each peptide such that equal loading was achieved.

Sparse matrix library (SML) analysis was carried out across three triplicate assays. Each substrate was added to three separate wells of a 96 well assay plate (2.5 μΐ per well adjusted to equal molarity) along with 1 μΐ thermolysin-activated KLK14, with volume made up to 250 μΐ with LK14 assay buffer (as above). Enzyme velocity was measured by the change in absorbance at 405nm over 420 sec (as above). Data were compiled in GraphPad Prism 5.01 and expressed as mean activity ± standard error of the mean (S.E.M.).

Synthesis ofSFTI Variants

Inhibitors were synthesised as linear peptides on 2-chlorotrityl resin using a Discover SPS Microwave System (CEM Corporation). All steps were performed using 20 W power and ΔΤ = 5°C. Resin was firstly derivatised with 2 eq. Fmoc-Ser (10 min, 75°C). The following nine residues were coupled according to the manufacturer's recommendations (7 min, 75°C) using 5 eq. Fmoc-protected amino acids and 125 mM HBTU dissolved in DMF containing 5% DIPEA. Cysteine residues were coupled at 50°C to reduce racemisation. After each coupling, the Fmoc protecting group was removed by incubation in 50% piperidine in DMF (3 min, 75°C) except for aspartate residues which were deprotected using 20% piperidine containing 0.1 M HOBt to suppress aspartimide formation. The final four residues were coupled for 10 min and deprotected for 4 min to reduce aggregation-associated losses.

Assembled linear peptides were liberated from the resin by successive changes of 0.5% TFA in DCM at room temperature and collected in 10 volumes of diethyl ether. Microwave-assisted head to tail cyclisation (30 min, 20 W, 65°C) occurred in solution using 125 mM each of l-hydroxy-7-aza-benzotriazole (HO At) and benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) dissolved in DMF containing 5% DIPEA.

Peptides were extracted by exploiting the hydrophobic nature of the side chain protecting groups. DMF solutions containing peptide and activators were mixed with an equal volume of DCM, followed by an equal volume of H₂0. The DCM phase (containing peptide) was recovered and washed a further three times with an excess of H₂0 to remove coupling reagents. Finally, the DCM was removed by rotary evaporation. Side chain protecting groups were removed by cleavage in TFA for 2 hours containing 1.25% (v/v) TIS, 1.25% (v/v) H₂0 and 3.75% (v/v) thioanisole to reduce modification of cysteine residues. Peptides were recovered by precipitation in diethyl ether (as previous) and pelleted by centrifugation.

To extract cyclic peptides from synthesis by-products and reagents, peptides were solubilised in 10% (v/v) isopropanol / H₂0 containing 0.1% TFA and purified by reverse phase high performance liquid chromatography using a C-18 column. Fractions containing correct product were identified by SELDI-TOF/MS and pooled. Cysteine residues were oxidised to complete the internal disulphide bond using a redox regeneration buffer containing 150 mM Tris-HCl pH 8.0, 10 mM reduced glutathione, 1 mM oxidised glutathione and 1 mM EDTA. Oxidation proceeded for 24 hours at room temperature with gentle stirring. Completed inhibitors were purified from glutathione by reverse phase high performance chromatography (as above) and lyophilised overnight.

SFTI variants are indicated by the nomenclature SFTI-X'X^X⁴, where X'-X⁴ are amino acid residues at positions 2-5 of SFTI. For example SFTI-WCVR indicates an SFTI variant with 2-Trp, 3-Cys, 4-Val and 5-Arg. Variants of the aforementioned are then indicated by the substitution and position of the substitution. For example, SFTI-WCVR Q12 N14 indicates an SFTI variant with 2-Trp, 3-Cys, 4-Val, 5-Arg, 12-Gln, and 14-Asn. Kinetic Studies

Selected peptides from the SML screen (Ac-YAVR-pNA, Ac-WAVR-/>NA, Ac- YASR-pNA, Ac-YANR- NA, Ac-YVSR-pNA) were purified by reverse phase high performance liquid chromatography (HPLC) using a C-18 column across a 20-100% isopropanol gradient. Fractions containing pure product were identified by MALDI- TOF/MS and freeze dried by overnight lyophilisation. Dry peptides were weighed out and reconstituted to give a 6 mM substrate solution by dissolving peptide in 40% isopropanol in ¾0. Kinetic constants were determined across three triplicate assays. For each substrate, enzyme activity was measured by the change in absorbance at 405 nm over 420 sec (as above) using six substrate concentrations and a constant concentration of KLK14 (0.7 nM). Raw enzyme rates were converted from mOD min^"1 to nM sec^"1 according to total pNA cleavage. Data were compiled in GraphPad Prism 5.01 as mean velocity ± S.E.M. and fit to Michaelis-Menten kinetic plots.

Inhibition Assays

Serial dilutions of SFTI variants were assayed against 1 nM KLK14 and 120 μΜ

Ac-YANR- NA in 250 μΐ assay buffer with activity of uninhibited enzyme measured by the change in absorbance at 405 nm over 7 min. K\ values were determined in GraphPad Prism 5 using the Morrison equation for tight binding inhibitors which has previously been shown to compare well with experimentally determined inhibition constants for SFTI variants (Swedberg et al. , PLoS One 6:e 19302, 2011 ).

Protein Proteolysis

Fibrinogen was incubated with active proteases, KLK14 (20 ng), KLK4 (50 ng), KLK5 (80 ng), trypsin (10 ng) and matriptase (50 ng) and various concentrations of inhibitor. Assays proceeded at 37°C for an incubation time dependent on the rate of fibrinogen proteolysis for each protease: 20 min - trypsin; 90 min - KLK4 and KLK14; 180 min - KLK5 and matriptase. Assay buffers for each protease were 0.1 M Tris-HCl pH 8.0, 0.1 M NaCl, 25 mM EDTA, 0.005% Triton-X (KLK14, KLK.5 and matriptase); 0.1 M Tris-HCl pH 7.4, 0.1 M NaCl, 25 mM EDTA, 0.005% Triton-X (KLK4); 0.1 M Tris-HCl pH 8.0, 0.1 M NaCl, 10 mM CaCl₂, 0.005% Triton-X (trypsin).

Protease activity was quenched at the assay end point by the addition of reducing

SDS-PAGE buffer and heating to 95°C for 5 min. Digestion products were resolved by SDS-PAGE and stained with colloidal Coomassie blue. Gels were visualised using the LI- COR Odyssey infrared imaging system (Millennium Science).

Cell Culture

HaCaT cells were maintained in Dulbecco's Modified Eagle's Medium (D-MEM) supplemented with 10% fetal calf serum (In vitro Technologies, Noble Park, Australia), 100 U/ml penicillin (Invitrogen) and 100 μg/ml streptomycin (Invitrogen). Cultures were propagated at 37°C in a humidified atmosphere containing 5% CO₂.

Results

Positional Scanning-Combinatorial Library Analysis

The first phase in our approach to substrate specificity mapping involved positional scanning; a combinatorial method which uses pools of similar peptide substrates to identify general amino acid preferences at individual sub-sites (Harris et ah, Proc. Natl. Acad. Sci. USA 97:7754-59, 2000). KLK14 PS-SCL analysis was carried out using a fully degenerate P1-P4 peptide-pNA library. Interestingly, the most preferred residues at each sub-site have common physical or chemical properties. Within the P4 sub-library, large residues able to mediate polar interactions (such as hydrogen bonding) were preferred; particularly aromatic residues Trp and Tyr, while His and Lys were preferred to a lesser extent. Surprisingly, Met was also a high scoring residue at P4. P3 preferences fit two distinct profiles. Small, aliphatic residues such as Val, Leu and Ala were most preferred, although large, positive residues such as Arg and Lys were also tolerated. Similarly, P2 was typified by a preference for small residues such as Ser, Val, Asn, and Pro, while the strict requirement for Arg at PI was expected given that L 14 is a trypsin-like kallikrein serine protease.

Sparse Matrix Library Analysis

Although positional scanning gave valuable information regarding general amino acid preferences, degenerate residues occupy three of the four positions within the peptide. Consequently, the preferences indicated within each sub-library must be considered independently, since the contribution of adjacent residues cannot be determined due to the lack of definition for the remainder of the peptide. Additionally, assurance that every sequence is equally represented is challenging since differences in coupling efficiency when the library is synthesised cannot be considered. Therefore, the results from PS-SCL analysis were validated and further analysis carried out to determine the optimal tetrapeptide substrate for the KL 14 protease.

To achieve this, high scoring residues at each position were selected and synthesised in all combinations as individual peptides in a refined sparse matrix library (Swedberg et al., Chem. Biol. 16:633-43, 2009). The SML for LK14 was constructed using both our PS-SCL data and previously reported data of Borgono et al. {Biol. Chem. 388:1215-25, 2007), as well as taking into consideration the restrictions of the SFTI bioscaffold and the likely impact of certain amino acid residues.

When the individually synthesised substrates of the SML were assayed, a number of similarities with the positional scanning data could be observed. However, a number of important differences were also identified. At P4, Tyr and Trp were clearly preferred compared to His, as suggested by PS-SCL. Interestingly, selection between Tyr and Trp was dependent on the P3 residue. Tyrosine performed better when adjacent to Ala or Leu, while Trp scored higher with P3 Val. These trends extended across all P2 positions. Surprisingly, Met performed poorly in every substrate combination, despite ranking third in the P4 positional scanning analysis. In a further deviation, the PS-SCL preferred residue at P3 (Val) was consistently outperformed by both Ala and Leu (Ala > Leu > Val). This trend was independent of the adjacent residue at P2 or P4.

At P2, Ser was generally the preferred residue, ranking highest when Leu or Val were present at P3. However, while it also scored well with Ala at P3, it was outperformed by Val when Tyr or Trp were present at P4. Further, Asn at P2 performed relatively poorly despite ranking highly in two independent PS-SCL screens. PI was not varied considering KLK14's preference for Arg at this site was almost exclusive. These findings highlight the potential limitations of using PS-SCL alone to determine a single, optimal substrate for a target protease.

To reveal the optimal substrate for KL 14, the thirty-six individual substrates were ranked by mOD (405nm) min^"1 and plotted in descending order (Figure 1). The clear optimal substrate for KLK14 was YAVR. Interestingly, the differences in rate between higher ranking substrates were consistently larger than differences between poorer substrates, and suggests an additive effect on rate of cleavage by increasing the number of "most preferred" residues in a substrate. Substrate ranking in SML was based on crude peptides that had been adjusted to apparent equal molarity. Therefore, the data was further validated by purifying several substrates by HPLC and calculating kinetic constants for cleavage by a known concentration of KLK.14. After converting enzyme activity from mOD min^"1 to nM s^"1 according to total j?NA cleavage results, kinetic constants were determined in GraphPad Prism 5.01 by fitting data to Michaelis-Menten plots (Figure 2). This confirmed YAVRpNA as the preferred substrate, closely followed by YASRpNA (Table 1); the two highest ranking substrates in the SML screen.

Substrate-Guided Engineering of the SFTI Bioscaffold Using a High Scoring KLK14 Substrate

Following SML analysis, key features of optimal L 14 substrates were grafted onto the SFTI bioscaffold to produce a potent LK14 inhibitor. The contact β-sheet of SFTI overlays with the β-sheet formed by peptide-/?NA/protease binding, hence these residues can be modified to re-direct the SFTI scaffold towards a target protease {e.g., KLK14). However, P3 Cys cannot be modified since it forms an internal disulphide bond which is essential for inhibitor function. From our KLK.14 SML analysis, YAVR and YASR were tested, however YAVR was selected since it was the optimal substrate. Also, Asp 14 was changed to Asn since this modification added an additional internal hydrogen bond, increasing inhibitor potency. Further, replacing Asp with Asn increases the success of synthesis by an Fmoc strategy since the tendency for conversion of aspartate to aspartimide is removed.

In addition to SFTI-YCVR N14, two further SFTI variants were designed using PS-SCL data available for KLK4 and KLK5 (Debela et al._t J. Biol. Chem. 281:25678-88, 2006), and matriptase (Takeuchi et al, J. Biol. Chem. 275:26333-42, 2000) to improve potency and selectivity of KL 14 inhibitors: P4 Tyr was substituted to Trp (SFTI-WCVR N14) and P2 Val was substituted to lie (SFTI-YCIR N14), since this residue had a similar chemistry to Val but seemed to be not preferred by either off-target KLK.

Structure-Based Refinement of Lead KLK14-SFTI Inhibitors

Although a SML screen of individually synthesised peptide-pNA substrates enabled the contact β-sheet of the KLK14 inhibitor to be successfully re-engineered, it gave little insight into how the remainder of the inhibitor interacts with the KLK 14 protease (including prime site residues). These contacts must be optimised by other methods. Accordingly, an in silico approach involving molecular dynamics simulations was utilised to improve contacts between KLK14 and SFTI-WCVR N14 to improve the inhibitor's potency and selectivity.

Analysis of the energy-minimised L 14/SFTI- WC VR N 14 complex revealed an unusually polar region on LK14 (Argl06) in the vicinity of SFTI residues 12-14. This was not seen on KL 4, the closest relative to KLK14, nor on KLK5, the most prominent off-target skin-expressed KLK. This region was closest to the Phel2 and Asnl4 residues on SFTI-WCVR N14 and already seemed to make a favourable contact with the protease. Phel2 seemed to stack with the short hydrocarbon chain on the Argl06 side-chain, while the carbonyl group on the Asnl4 side-chain formed transient hydrogen bonds with the amide and amine groups on Argl06. Despite this, it was hypothesised that Phel2 could be substituted with a polar residue to better exploit the Argl06 residue on KLK14 which possessed both a hydrogen bond acceptor (peptide bond carbonyl group), as well hydrogen bond donors on the amine and amide groups on the amino acid side-chain.

Of the natural amino acids examined to optimise interaction with Argl06, Gin seemed the best fit both in terms of size and orientation of hydrogen bond donors and acceptors. Therefore, Phel2 was substituted with Gin and the new inhibitor-protease complex was subjected to energy minimisation. As predicted, the amine group of the Gin side chain formed a hydrogen bond with the peptide bond carbonyl group, while the Gin side chain carbonyl group formed transient hydrogen bonds with the amide groups on the Argl06 side chain. Further, there was no detrimental effect on the Asnl4 interaction as it still maintained hydrogen bonds with both the protease and inhibitor backbone. Consequently, the SFTI-WCVR Q12 N14 variant was synthesised to confirm an improved interaction with KLK14 as predicted by molecular modelling.

Affinity of KLK14 Inhibitors

Competitive kinetic assays were carried out to assess the affinity of the SFTI- YCVR N14, SFTI-WCVR N14 and SFTI-WCVR Q12 N14 variants for KLK14 (Figure 3). This confirmed SFTI-WCVR Q12 N14 as a more potent KLK14 inhibitor (Table 2). Specificity of KLK14 Inhibitors

The selectivity of SFTI-WCVR F12 N14, SFTI-YCVR F12 N14, SFTI-YCSR F12 N14, and SFTI-YCNR F12 N14 variants was assessed by proteolysis assays using a common substrate digested by several serine proteases, fibrinogen (FG) (Figure 4). Under reducing conditions, FG separates into three chains (alpha, beta and gamma) with inhibition of proteolysis identified by the persistence of all three chains at the digest end point.

SFTI-WCVR F12 N14 is the most selective variant: complete inhibition of KLK14 was achieved with 2.5 μΜ inhibitor, while no effect was seen on KLK.5, trypsin and matriptase with higher concentrations. SFTI-YCVR F12 N14 was also relatively selective: complete inhibitor of KLK14 was achieved with 1.75 μΜ inhibitor, with no effect seen on KLK5, trypsin and matriptase. However, higher inhibition of KLK4 was seen with this variant.

SFTI-YCSR F12 N14 was the most potent L 14 inhibitor and also reduced activity of L 4, KLK5 and, to a lesser extent, trypsin. No inhibiton of matriptase was observed. SFTI-YCNR F12 N14 displayed a similar inhibiton profile, inhibiting LK4, KLK5, LK14 and, to a lesser extent, trypsin.

EXAMPLE 2 - Improved KLK protease inhibitors

The inventors used structural/computational techniques to refine an inhibitor initially designed using substrate guided engineering principals. Systematic analysis of hydrogen bonds in the wild type and variant SFTI inhibitors revealed that the molecule's potency was critically dependent on the frequency of hydrogen bonds within a prescribed portion of the SFTI scaffold. Accordingly, hydrogen bond frequency was used as a method for scoring and selection of SFTI variants.

Experimental Procedures

Molecular Dynamics Simulations for in silico Refinement of SFTI Variants

Since the structure of KLK 14 remains to be solved, a homology model was created with SWISS-MODEL (Guex et al., Electrophoresis 30 Suppl l:S162-73, 2009) using KLK5 as a template (PDB ID 2PSY). SFTI variant KLK14 complexes were generated by overlay of the KLK 14 model and the trypsin/SFTI-1 complex (PDB ID 1SFI) in SPDBV v4.01 (Guex et al, Electrophoresis 30 Suppl l :S162-73, 2009) (average Ca RSMD: 0.96 A) before substitutions were carried out in YASARA Dynamics 9.12.13 (Krieger et al, Proteins 47:393-402, 2002). Complexes were solvated, neutralized, equilibrated and simulated as described by Swedberg et al. (PLoS One 6:el9302, 2011), with the exception that the three independent production runs of 1 ns were carried out in NAMD 2.6 (Phillips et al., J. Comput. Chem. 26:1781-1802, 2005). Coordinates were saved every 100 simulation steps producing 5000 frames per trajectory. Hydrogen bonds within SFTI were analyzed using VMD 1.8.7 (Humphrey et al., J. Mol. Graph. 14:33-38, 1996) with hydrogen bond lengths and angles set to 3.3 A and 40° respectively.

Ex vivo Skin Desquamation Assay

The procedure of Lundstrom and Egelrud was followed (see, J. Invest. Dermatol.

91:340-43, 1988). Briefly, skin flakes were harvested from the heels of healthy volunteers (0.5 mm thick). Defined cylinders were then cut from these flakes using a 3 mm biopsy punch. Skin cylinders were then soaked in 0.5 mL 0.01 M sodium phosphate pH 7.4 + 140 mM NaCl + 0.1% sodium azide + 0.5% Triton X-100 (4 hr / 22°C) either with or without inhibitors. Cells that were only partially attached to the skin cylinder were removed by vortexing for 5 seconds followed by a further incubation of the skin cylinder in 0.75 mL 0.1 M Tris-HCl pH 8.0, 5 mM EDTA, 0.1 % sodium azide for 18 hr at 37°C (+/- inhibitors). Cells detached during this second incubation were harvested by a second round of vortexing followed by removal of the skin cylinder from the incubation and centrifugation (10,000 x g for 10 min). Cells were then washed by resuspension with PBS and re-pelleted (10,000 x g for 10 min) prior to solubilisation in 250 uL 1 M NaOH at 60°C for 90 min. 25 μΐ, alkali soluble protein was then neutralised with 25 1 M HC1 and total protein quantified using bicinchoninic acid assay.

Results

SFTI Variants with Affinity for KLK14

Inhibition constants (A¾ for LK14 with SFTI-WCVR variants were determined using the Morrison equation for tight binding inhibitors by fitting data to [inhibitor] vs response curves using GraphPad Prism 5.01 (Table 3). Molecular modelling analyses were carried out on SFTI-WCVR N14 variants (different amino acids at residue 12) in complex with KLK14 and hydrogen bonds within SFTI were calculated (Table 4). Substitutions at residue 12 aimed at increasing hydrogen bonds within SFTI produced more potent KLK14 inhibitors. To confirm that Asnl4 remained highly preferred with the new composition (Asnl2), similar modelling analyses were carried out except that residue 14 was modified. Serl4 produced a slightly more potent inhibitor, however Asnl4 is beneficial for biosynthesis in plants.

Inhibition constants (Κ) for KLK 14 with SFTI-YCSR variants were determined using the Morrison equation for tight binding inhibitors by fitting data to [inhibitor] vs response curves using GraphPad Prism 5.01 (Table 5). Replacing Phel2 with Asn also considerably improved the potency of SFTI-YCSR N14 which was consistent with molecular modelling analyses (Table 6).

Selectivity analysis for SFTI-WCIR N12 N14 was assessed. Inhibition constants

(Ki) were determined (as herein described) for proteases where greater than 50% inhibition was observed with 10,000 nM inhibitor (Table 7). Otherwise, IC50 > 10,000 nM is listed. Inhibition values for SFTI-1 are shown for reference. (P2 Val was replaced with He to improve selectivity over KLK4: SFTI-WCVR N12 N14 KLK4 K = 1.49 ± 0.20 nM compared to KLK14 ^i = 1.46 ± 0.13 nM.)

Fibrinogen proteolysis assays were also conducted to assess selectivity of SFTI- WCIR N12 N14 using five off-target KLK proteases and four off-target non-KLK proteases (Figure 5).

The data for SFTI-WCVR and SFTI-YCSR variants, as well as existing data for SFTI-FCQR variants for KLK4 and wild type SFTI (SFTI- 1 ), highlight the importance of hydrogen bonding within SFTI to inhibitor function. Accordingly, the inventors reasoned that placing residues which promoted hydrogen bonding within the inhibitor at as many positions as possible would produce an inhibitor which was highly potent. Additionally, this would reduce the number of side chain contacts from the inhibitor with the target, resulting in a compound that can target a broader range of proteases.

SFTI Variants With Affinity for KLKs in Addition to or Other Than KLK 14

Observations on hydrogen bonding within SFTI led to the design of SFTI-TCTR N12 N14 (to target trypsin-like KLK proteases, for example, KLK5 and KLK14) as well as SFTI-TCTY N12 N14 and SFTI-TCTF N12 N14 variants (more suited to chymotrypsin-like KLK proteases, for example, KLK7). Competitive inhibition assays were undertaken to determine inhibition constants for these new variants with trypsin and chymotrypsin. The K_{ for SFTI-TCTR N12 N14 and trypsin was determined to be 0.366 ± 0.019 nM, which is an improvement on SFTI-1 (wild-type) when the same assay and conditions were used (Ki = 0.793 ± 0.033 nM, Table 8). This was consistent with molecular modelling analyses which indicated hydrogen bonds were more prevalent within SFTI- TCTR N12 N14. Assays with chymotrypsin demonstrated SFTI-TCTY N12 N14 and SFTI-TCTF N12 N14 were both effective inhibitors. K values were determined to be 0.414 ± 0.034 nM and 0.488 ± 0.078 (Table 8) respectively which represent over a 2,500 fold improvement on SFTI-1 which has Lys at PI (IC₅₀ = 2,300 nM).

Collectively, these findings validate the design strategy of engineering residue 2 (P4: Thr), residue 4 (P2: Thr), residue 12 (Asn) and residue 14 (Asn) to focus on hydrogen bonding, while modifying the PI residue to inhibit different types of serine proteases.

Fibrinogen proteolysis assays were also conducted to assess selectivity of the SFTI-YCSR N12 N14, SFTI-TCTR N12 N14 and SFTI-TCTY N12 N14 variants (Figure 6).

In addition to the in vitro digestion of fibrinogen, the inventors undertook a more biologically relevant assay of inhibitor potency based on skin flakes. Previously, it has been shown that broad range serine protease inhibitors are able to reduce desquamation- like activity in a simple ex vivo model using flakes of human stratum corneum (see, Lundstrom and Egelrud, J. Invest. Dermatol. 91 :340-43, 1988). Accordingly, this method has been applied to examine the ability of the KLK14-selective and LK5/7/14 inhibitors to modulate desquamation in non-diseased tissue (Figure 7). This data shows that the SFTI-based inhibitors can bring about almost complete inhibition of desquamation in this assay, that the process is driven by kallikrein activity and that desquamation can be selectively modulated by the application of combinations of SFTI variants with discrete selecti vities.

The Ki for SFTI-TCTY N12 N14 and chymotrypsin was determined to be 0.414 ± 0.034 nM, compared to the recorded K of 2.3 μΜ for SFTI-1 with chymotrypsin. Given the potency of TCTY N12 N14 against chymotryptic proteases, the inventors investigated the possibility of developing a potent selective KLK7 inhibitor. Due to solubility issues inherent for tyrosine and phenylalanine-based ^ara-nitroanilide substrates, it was not possible to carry out a sparse matrix scan as for KL 14, instead a small focused library of SFTI variants was constructed with positions Rl, R2 and R3 diversified. Deconvolution of this library in complex with LK7 showed that both YCLF and WCLF variants of SETI had strong affinity for this protease. Accordingly, these variants were synthesised in bulk and characterised (Table 8).

As a result of the inventors' refinements, inhibitor target profiles for a number of variants of SFTI were determined, as shown in Table 9.

EXAMPLE 3 - Dissociation of adherent cells in culture The inventors combined the ability of KLK14 to selectively cleave cellular junctions with the potency of SFTI-based inhibition to demonstrate the utility of these reagents in tissue culture based activities. This approach has broad applicability in tissue culture procedures and is particularly useful in processes where non-selective proteolytic activity is undesirable, including cell culture as a means of producing artificial skin.

Trypsin (± EDTA) is commonly used to dissociate and detach cells for passage. However, this approach has several drawbacks, most notably the unwanted proteolytic removal of cell-surface proteins due to trypsin's broad substrate specificity. Consequently, it may be more effective to utilise a protease that selectively cleaves proteins required for cell-cell adhesion in vivo. Whereas trypsin is a digestive enzyme intended to completely degrade many proteins, LK14/KLK5 are potentially able to cleave desmosomal cadherins and detach cells without causing collateral damage to cell surface proteins. Cells harvested in this fashion will be better suited for subsequent therapeutic uses.

To use LK14/KLK5 to passage cells in vitro, a semi-confluent cell monolayer would be rinsed with PBS, then incubated with active KLK14 KL 5 in 0.1 Tris-HCl pH 8.0 + 0.5 mM EDTA at 37°C. Previously, 25 nM KLK14 for 2 hours has been sufficient to detach and dissociate the cell monolayer of HaCaT cells. Higher concentrations of L 14 KLK5 and/or EDTA would decrease the incubation time, for example, commercially available lx trypsin-EDTA solutions typically contain 22 μΜ trypsin and 0.5 mM EDTA. LK14/KLK5 would be inhibited by addition of selective KLK14/ LK5 inhibitors, either in solution or attached to a solid support. Cells would be harvested by adding 5-10 volumes of fresh serum-containing (10%) media followed by centrifugation. Experimental Procedures

Cell Culture

HaCaT cells were maintained in Dulbecco's Modified Eagle's Medium (D-MEM) supplemented with 10% fetal calf serum (In vitro Technologies, Noble Park, Australia), 100 U/ml penicillin (Invitrogen) and 100 μg/ml streptomycin (Invitrogen). Cultures were propagated at 37°C in a humidified atmosphere containing 5% CO₂.

Cell monolayer desquamation Assays

Confluent monolayers of HaCaT cells were established to mimic an epidermal- like barrier. After reaching confluence (2 days), media was replaced with fresh serum- containing media and cells were cultured for a further 4 days. At time = 0, media was aspirated, monolayers were rinsed with 2 ml warm PBS and triplicate cell monolayers were treated with proteolysis buffer (100 mM Tris-HCl pH 8.0 containing 0.5 mM EDTA) containing either 100 nM EDTA-inhibited thermolysin, 35 nM LK14, 35 nM KLK14 + 1.75 μΜ SFTI-WCVR Ql 2 N14, 35 nM KL 14 + 3.5 μΜ SFTI-WCVR Q12 N14, or buffer only controls. Incubation proceeded for 2 hours at 37°C, after which cells were gently agitated to assess monolayer biomechanical strength. Each culture was visually inspected by phase contrast microscopy to examine local changes in monolayer integrity using a Nikon Eclipse TE2000-U microscope.

Global changes across the monolayer were determined by quantifying the number of detached cells by NucleoCount (ChemoMetec, carried out according to the manufacturer's instructions). The total number of cells detached was calculated and data were compiled in GraphPad Prism 5.01 as mean ± S.E.M. of three separate experiments.

Cell Lysate Preparation

Following microscopy in the desquamation assays, detached cells were recovered and pelleted by centrifugation at 1,000 rpm for 5 min. Cell pellets were resuspended in 150 μΐ ice-cold 0.1% SDS, added to remaining attached cells and lysis was completed mechanically using a cell scraper. Lysates were clarified by shearing DNA using short duration, low power sonication. To allow equal loading for subsequent Western blot analysis, samples for each lysate were adjusted to equivalent protein content by bicinchoninic acid (BCA) assay (Pierce) carried out according to the manufacturer's instructions. SDS-PAGE and Western Blot Analysis

Following denaturation by heating to 95°C for 5 min, lysate samples were separated by SDS-PAGE using 7.5% and 10% acrylamide gels for desmoglein-1 and desmoplakin respectively. Protein was transferred to nitrocellulose membranes under a constant current of 280 mA at 4°C for 70 min. Unoccupied binding sites were blocked by overnight incubation with 5% (m/v) skin milk powder in TBS containing 0.005% (v/v) Tween-20. Desmoglein-1 was detected using mouse anti-human desmoglein 1 monoclonal IgG (R&D Systems) while desmoplakin I + II was detected using rabbit anti- human desmoplakin I + II polyclonal IgG. Secondary incubations were goat anti-mouse horse radish peroxidise (HRP)-conjugated antibody (Pierce) and goat anti-rabbit HRP- conjugated antibody respectively. Signal was developed using West FEMTO (Pierce) according to the manufacturer's instruction and blots were visualised by exposure to film.

Results

SFTI-WCVR Q12 N14 Inhibits KLK14 Mediated Desmosome Remodelling in Desquamation Assays

Previously, the inventors had established that KLK14 could contribute to a desquamation-like event by cleaving cell-cell adhesion proteins such as desmoglein 1. Therefore, this assay was used to assess the ability of SFTI-WCVR Q12 N14 to modulate the proteolytic activity of KL 14 in a cell-based environment. Negative control treatments of assay buffer (100 mM Tris-HCl pH 8.0, 0.5 mM EDTA) and thermolysin (used to activate KL 14) had relatively little effect on the confluent HaCaT monolayer at the incubation end point (Figure 8A and Figure 8B, respectively). However, when treated with 35 nM active KLK14, a majority of the monolayer was lifted from the culture surface and detached cells were dissociated (Figure 8C). The cell detachment and dissociation are thought to result from different factors. Initially, KL 14 cleaves cell-cell adhesion proteins, such as desmosomal cadherins, to begin to break apart the monolayer integrity. Subsequently, EDTA affects cell-culture surface contacts (integrins). Treatment with SFTI-WCVR Q12 N14 markedly reduced this effect (Figure 8D and Figure 8E).

To analyse global changes across the monolayer, the detached cells at the treatment end point were collected and quantified by cell count using a Chemometec NucleoCounter™. This confirmed a significant increase in the number of cells detached by treatment with KLK14 compared to negative control treatments with assay buffer and thermolysin (Figure 8, lower panel). Further, treatment with SFTI-WCVR Q12 N14 at two different concentrations (1.75 μΜ and 3.5 μΜ) reversed this effect, returning the number of detached cells to that seen in control treatments (Figure 8, lower panel).

SFTI-WCVR Q12 N14 Attenuates KLK14-Mediated Cell Detachment by Inhibiting Proteolysis of Desmoglein- 1

To explore the molecular mechanism underlying KLK14's role in desquamation and examine changes in desmosome integrity and abundance, lysate samples from each treatment were balanced for protein loading by BCA and separated by SDS-PAGE for western blot analysis. Samples were probed for two components of the desmosomal complex: desmoglein 1, a desmosomal cadherin which forms the extracellular link between adjacent cells, and desmoplakin, an intracellular adaptor which facilitates connection between the keratin cytoskeleton and the cell-cell contact architecture (Figure 9). This suggested that desmoglein 1 (165 kDa) and desmoplakin I (230 kDa) were abundantly expressed in HaCaT cells cultured at confluence for several days. Further, there was some indication that dimers of desmoglein 1 may be SDS-stable as seen by detection of a product above 250 kDa in control treatments (Figure 9). In samples treated with KLK14, the level of monomelic desmoglein 1 was noticeably reduced while there was no indication of the product above 250 kDa which the inventors propose may be a desmoglein 1 dimer (Figure 9). This effect was completely prevented by treatment with SFTI-WCVR Q12 N14, which displayed a desmoglein 1 profile similar to that seen in negative controls (Figure 9). Desmoplakin I, which is not a substrate for proteolysis by KLK14 confirmed equal protein loading (Figure 9).

The foregoing embodiments are illustrative only of the principles of the invention, and various modifications and changes will readily occur to those skilled in the art. The invention is capable of being practiced and carried out in various ways and in other embodiments. It is also to be understood that the terminology employed herein is for the purpose of description and should not be regarded as limiting.

All computer programs, algorithms, patent and scientific literature referred to in this specification is incorporated herein by reference in their entirety. Table 1. Kinetic constants for L 14 cleavage of six peptide-pNA substrates

Theoretical Determined

Substrate )

Mass Mass *. (μΜ) v.., (nM s ') *«.,/_¥,,, (M ' s ¹

Ac-YAVR-pNA 669.83 670.93 45.01 ± 4.95 65.33 93.33 ± 2.75 2.074 x 10⁶

Ac-WAVR-pNA 692.87 693.81 51.54 ± 3.10 68.12 97.31 ± 1.63 1.888 x I0⁶

Ac-YASR-pNA 657.78 658.87 22.03 ± 2.88 58.70 83.85 ± 2.29 3.806 x 10⁶

Ac-YANR-pNA 684.80 685.77 16.78 ± 1.96 37.13 53.04 ± 1.15 3.161 x 10⁶

Ac-YVSR-pNA 685.83 686.88 25.23 ± 3.11 24.20 34.57 ± 0.96 1.370 x lO⁶

Bz-FVR-pNA 644.73 645.71 15.81 ± 2.07 28.63 40.90 ± 1.36 2.587 x 10⁶

Kinetic constants (K_m and were determined by fitting enzyme velocity data vs substrate concentration (Figure 2) to Michaelis-Menten kinetics using GraphPad Prism 5.01. Substrate identity is shown down the first column.

Table 2. Inhibition constants (K) for engineered SFTI variant inhibition of KLK14- YAVRpNA cleavage

Protease Inhibitor K, (nM) Calculated Mass Determined Mass LK14 SFTI-1 16.82 ± 1.68 1513.81 1515.07

SFTI-WCVR F12 N14 19.05 ± 0.98 1568.89 1570.86

SFTI-YCNR F12 N14 16.56 ± 0.99 1560.83 1562.77

SFTI-YCVR F12 N14 9.60 ± 0.74 1545.86 1547.47

SFTI-YCSR F12 N14 1.41 ± 0.19 1533.80 1535.31

SFTI-WCVR Q12 N14 9.02 ± 0.66 1549.85 1550.86

Table 3. Inhibition constants (Kji for KLK14 with SFTI-WCVR variants

Fold

Protease Inhibitor K, (nM) Calculated Mass Determined Mass

change

KLK14 SFTI-WCVR F12 N14 19.05 ± 0.98 1568.89 1570.86 -

SFTI-WCVR E12 N14 18.11 ± 1.14 1550.83 1551.67 1.05

SFTI-WCVR Q12 N14 9.02 ± 0.66 1549.85 1550.86 2.11

SFTI-WCVR T12 N14 3.46 ± 0.28 1522.82 1524.04 5.51

SFTI-WCVR D12 N14 2.48 ± 0.23 1536.81 1537.60 7.68

SFTI-WCVR N12 N14 1.46 ± 0.13 1535.82 1537.02 13.05

SFTI-WCVR N 12 S14 1.00 ± 0.1 1 1508.80 1509.81 - Table 4. Molecular modelling analysis of SFTI-WCVR N14 variants

Table 5. Inhibition constants (Kj) for KLK14 with SFTI-YCSR variants

Fold

Protease . Inhibitor Kt (nM) Calculated Mass Determined Mass

change L 14 SFTI-YCSR F12 N14 1.41 ± 0.19 1533.80 1535.31 -

SFTI-YCSR N12 N14 0.11 ± 0.01 1500.73 1501.73 12.82

Table 6. Molecular modelling analysis of SFTI-YCSR N14 variants

Increase

Residue Average SFTI

from

12 hydrogen bonds

Phe

Asn (N) 5.37 ± 0.11 7.6%

Phe (F) 4.99 ± 0.07 Table 7. Selectivity analysis for SFTI-WCIR N12 N14

Fold

Protease Inhibitor Ki (nM) ICso (nM) Substrate

selectivity

SFTI-WCIR

KLK4 8.99 ± 0.88

N12 N14 FVQR-pNA 6.71 SFTI-WCIR

LK5 93.01 ± 14.01 Ac-YASR-

N12 N14 69.4 pNA

SFTI-WCIR

KLK7 Ac-GRPY-

N12 N14 > 5,000 NI pNA

KL 8 SFTI-WCIR

602.1 ± 89.2 Ac-YASR-

N12 N14 449 pNA

SFTI-WCIR

LK14 1.34 ± 0.12 Ac-YANR-

N12 N14 pNA

Plasma KLK SFTI-WCIR Ac-RQFR-

N12 N14 > 10,000 NI pNA

β-trypsin SFTI-1 0.1^a BAPNA

SFTI-WCIR

306.4 ± 17.3

N12 N14 Bz-FVR-pNA 229

Matriptase SFTI-1 0.92^b N-t-Boc-LRR- AMC

SFTI-YCSR Ac-KQSR- N12 N14 > 10,000 NI pNA

SFTI-WCIR Ac-KQSR- N12 N14 > 10,000 NI pNA

Chymotrypsin SFTI-1 2,300 ± N-succinyl- 100° AAPP-pNA

SFTI-WCIR

> 10,000 Ac-GRPY- N12 N14 NI pNA

SFTI-WCIR Ac-

Plasmin

N12 N14 > 10,000 RM(0₂)YR- NI pNA

Thrombin SFTI-1 136^» Not reported

SFTI-1 5,050^b N-t-Boc-LRR- AMC

SFTI-WCIR

N12 N14 > 10,000 Bz-FVR-pNA NI

Luckett et al, J. Mol. Biol. 290:525- 33, 1999, Long et al., Bioorg. Med Chem. Lett. 1 1:2515-9, 2001, ⁰ Descours et al., ChemBioChem 3:318^■23, 2002

NI denotes no substantial inhibition Table 8. Inhibition constants for engineered KLK.7 variants and improved broad-range inhibitors

Protease Inhibitor A^", (nM) IC(nM)

KLK7 SFTI- YCLF N 12 N 14 8.14 ±0.55 - L 5 SFTI-YCLF N12 N14 - > 5,000

KLK14 SFTI- YCLF N 12 14 - > 5,000

KLK7 SFTI-WCLFN12N14 12.30 ±0.68 -

Trypsin SFTI-1 0.793 ±0.033 -

Trypsin SFTI-TCTRN12N14 0.366 ±0.019 -

Chymotrypsin SFTI-1 - 2,300 ±100"

Chymotrypsin SFTI-TCTY N12 N14 0.414 ±0.034 -

Chymotrypsin SFTI-TCTF N12 N14 0.488 ±0.078 -

* Descours etal., ChemBioChem 3:318-23, 2002

Table 9. Inhibitor target profiles

High affinity

Non-target skin

Inhibitor target skin

proteases

proteases

Aprotinin Universal serine protease inhibitor

SFTI-WCIR N12 N14 KLK14 KL 5, KLK7,

LK8, matriptase

SFTI-YCLF N12N14 KLK7 LK5, KLK7

SFTI-YCSR N12 N14 KLK5, LK14 Matriptase

SFTI-TCTY N12N14 KLK7 Matriptase

SFTI-TCTF N12N14 KLK7 -

SFTI-TCTR N12 N14 LK5, L 7, Matriptase

LK14

SFTI-FCQRN14 KLK4 (non-skin) KLK5, KLK14,

matriptase

SFTI-1 Broad range

Claims

. A compound comprising a peptide or salt thereof according to Formula I:

Formula I wherein

R¹, R², R³, R⁴, R⁶, and R⁸ are each an amino acid residue, R⁵ is an amino acid residue other than Phe and R is an amino acid residue other than Asp or Glu.

2. The compound or salt according to claim 1, wherein R¹ is Trp or Tyr, R² is Val or He, R³ is Arg, R⁵ is Gin, and R⁷ is Asn.

3. The compound or salt according to claim 1, wherein R is Trp, R is Val, R is Arg, R⁵ is Gin, and R⁷ is Asn.

4. The compound or salt according to claim 1 , wherein R¹ is Trp, R² is Val, R³ is Arg, R⁴ is Pro, R⁶ is Pro, and R⁸ is Gly.

5. The compound or salt according to claim 1 , wherein R¹ is Trp or Tyr, R² is Val or He, R³ is Arg, R⁴ is Pro, R⁵ is Gin, R⁶ is Pro, R⁷ is Asn, and R⁸ is Gly.

6. The compound or salt according to claim 1 , wherein R¹ is Trp or Tyr, R² is Ser, Thr or Asn, R³ is Arg, R⁵ is Gin, and R⁷ is Asn.

7. The compound or salt according to claim 1, wherein R¹ is Trp, R² is lie, R³ is Arg, R⁵ is Asn, and R⁷ is Asn.

8. The compound or salt according to claim 1, wherein R is Tyr, R is Ser, R is Arg, R⁵ is Asn, and R⁷ is Asn.

9. The compound or salt according to claim 1, wherein R¹ is Thr, R² is Thr, R³ is Tyr, R⁵ is Asn, and R⁷ is Asn.

10. The compound or salt according to claim 1, wherein R is Thr, R is Thr, R is Arg, R⁵ is Asn, and R⁷ is Asn.

11. The compound or salt according to claim 1, wherein R is Trp, R is Val, R is Arg, R⁵ is Asn, and R⁷ is Asn.

12. The compound or salt according to claim 1, wherein R¹ is Tyr R² is Leu, R³ is Phe, R⁵ is Asn, and R⁷ is Asn.

13. The compound or salt according to claim 1, wherein R¹ is Thr, R² is Thr, R³ is Phe, R⁵ is Asn, and R⁷ is Asn.

14. The compound or salt according to claim 1, wherein R¹ is Trp R² is Leu, R³ is Phe, R⁵ is Asn, and R⁷ is Asn.

15. The compound according to any one of claims 1-8, 10 and 11, wherein the compound is a KLK14 protease inhibitor.

16. The compound according to any one of claims 1-6 and 8-10, wherein the compound is a KLK5 protease inhibitor.

17. The compound according to any one of claims 9, 10, 12, and 13, wherein the compound is a KLK7 protease inhibitor.

18. The compound according to claim 8, wherein the compound is a L 5 protease inhibitor or a KLK14 protease inhibitor.

19. The compound according to claim 9, wherein the compound is a KLK5 protease inhibitor or a KLK7 protease inhibitor.

20. The compound according to claim 10, wherein the compound is a KLK5 protease inhibitor, a LK7 protease inhibitor, or a KL 14 protease inhibitor.

21. A method for treating a skin disease in a subject, the method including the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound according to any one of claims 1-14.

22. The method according to claim 21, wherein the skin disease is selected from the group consisting of: Netherton syndrome, peeling skin syndrome, acne rosacea, psoriasis, eczema, and atopic dermatitis.

23. A compound according to any one of claims 1-14 for use in treating a skin disease.

24. The compound according to claim 23, wherein the skin disease is Netherton syndrome, peeling skin syndrome, acne rosacea, psoriasis, eczema, or atopic dermatitis.