Patent Grant
11793864
This invention relates to therapeutic compounds which are inhibitors of serine proteases, to pharmaceutical compositions thereof and to their use in the treatment of the human or animal body. More specifically, the present invention relates to a method for the treatment, diagnosis or prognosis of skin diseases comprising the administration to a subject in need thereof of a therapeutically effective amount of a Serine protease inhibitor.
Proteases or proteolytic enzymes are essential in organisms, from bacteria and viruses to mammals Proteases digest and degrade proteins by hydrolyzing peptide bonds. Serine proteases (EC. 3.4.21) have common features in the active site, primarily an active serine residue. There are two main types of serine proteases; the chymotrypsin/trypsin/elastase-like and subtilisin-like, which have an identical spatial arrangement of catalytic His, Asp, and Ser but in quite different protein scaffolds. However, over twenty families (S1-S27) of serine proteases have been identified that are grouped into 6 clans on the basis of structural similarity and other functional evidence, SA, SB, SC, SE, SF & SG. The family of chymotrypsin/trypsin/elastase-like serine proteases have been subdivided into two classes. The “large” class (ca 230 residues) includes mostly mammalian enzymes such as trypsin, chymotrypsin, elastase, kallikrein, and thrombin. The “small” class (ca 190 residues) includes the bacterial enzymes.
The catalytic His, Asp and Ser are flanked by substrate amino acid side chain residue binding pockets termed S1′, S2′, S3′ etc on the C-terminal or ‘prime’ side of the substrate and S1, S2, S3 etc on the N-terminal side. This nomenclature is as described in Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding, Alan Fersht, 1999 (W.H. Freeman and Company) pages 40-43 and Brik et al, Org. Biomol. Chem., 2003, 1, 5-14. The chymotrypsin/trypsin/elastase-like serine proteases can also be further subdivided by the residues present in the 51 pocket as described in Introduction to Protein Structure, Carl Branden and John Tooze, 1991 (Garland Publishing Inc) pages 231-241. The subdivisions are chymotrypsin-like (Gly-226, Ser-189 and Gly-216 in S1 pocket), trypsin-like (Gly-226, Asp-189 and Gly-216 in S1) and elastase-like (Val-226 and Thr-216 in S1) where the residues numbering is taken from the standard chymotrypsin numbering. The trypsin-like serine proteases prefer substrates which place either Lys or Arg in the S1 pocket.
The serine proteases have a common catalytic mechanism characterized by a particularly reactive Ser residue at position 195 using the chymotrypsin numbering system. Examples of serine proteases include trypsin, tryptase, chymotrypsin, elastase, thrombin, plasmin, kallikrein, Complement Cl, acrosomal protease, lysosomal protease, cocoonase, α-lytic protease, protease A, protease B, serine carboxypeptidase 7E, subtilisin, urokinase (uPA), Factor Vila, Factor IXa, and Factor Xa. The serine proteases have been investigated extensively for many years and are a major focus of research as a drug target due to their role in regulating a wide variety of physiological processes.
Processes involving serine proteases include coagulation, fibrinolysis, fertilization, development, malignancy, neuromuscular patterning and inflammation. It is well known that these compounds inhibit a variety of circulating proteases as well as proteases that are activated or released in tissue. It is also known that serine protease inhibitors inhibit critical cellular processes, such as adhesion, migration, free radical production and apoptosis. In addition, animal experiments indicate that intravenously administered serine protease inhibitors, variants or cells expressing serine protease inhibitors, provide protection against tissue damage.
The serine proteases Kallikreins (KLK) are shown to play an essential role in the normal physiology of skin. KLK5 and 7 were originally isolated and cloned from the stratum corneum (Hansson et al., 1994; Brattsand and Egelrud, 1999) and were shown to be involved in skin desquamation through processing of extracellular adhesive proteins of the corneodesmosomes, i.e. corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) (Caubet et al., 2004; Descargues et al., 2005). KLK5 was shown to cleave all three components, while KLK7 was able to digest only CDSN and DSC1 (Caubet et al., 2004). Further IHC studies supported the proposed role of KLK7 in desquamation (Sondell et al., 1995). In-vitro studies demonstrated an potential activation mechanism of KLK7 through a proteolytic cascade, involving KLK5, and 14 (Brattsand et al., 2005). Also, varying levels of KLKs 1, 6, 8, 10, 11, and 13 have been reported in SC (Komatsu et al., 2005; Borgono et al., 2006) and KLK1, 5, 6, and 14 are believed to be involved in skin desquamation through DSG1 processing (Borgono et al., 2006). KLK14 is believed to play a major role in skin remodeling as it contributes to approximately half of the total trypsin-like proteolytic activity in the SC layer (Stefansson et al., 2006). KLK8 is suggested to play an overlapping function in skin desquamation processing DSG1 and CDSN (Kishibe et al., 2006). An additional antimicrobial function KLKs in skin through the regulation of cathelicidin peptides was shown in vitro and in vivo (Yamasaki et al., 2006).
Imbalances in the proteolytic activity of KLKs, through gene over-expression or dysregulation of activity is reported in a large number of skin disorders, including chronic itchy dermatitis, peeling skin syndrome, psoriasis, atopic dermatitis, and Netherton syndrome (Komatsu et al., 2005b; Descargues et al., 2005; Hachem et al., 2006; Komatsu et al., 2006; Hansson et al., 2002; Ekholm and Egelrud, 1999). The expression of multiple KLKs is significantly upregulated in psoriasis, atopic dermatitis, peeling skin syndrome type-B, and chronic lesions of atopic dermatitis (Komatsu et al., 2005b; Komatsu et al., 2006; Hansson et al., 2002). Patients with Netherton syndrome, an autosomal recessive skin disorder, have shown frame shifts and non-sense mutations in the SPINK5 gene encoding for LEKTI (Chavanas et al., 2000; Komatsu et al., 2002; Chavanas et al., 2000; Sprecher et al., 2001), LEKTI being a serine protease inhibitor with activity against several KLKs, including KLK5, 6, 7, 13, and 14 (Borgono et al., 2006; Egelrud et al., 2005; Deraison et al., 2007). Such genetic defects lead to loss of inhibitory domains (Chavanas et al., 2000; Sprecher et al., 2001).
Also of interest is the potential involvement of kallikreins in the skin inflammation aspect of desquamation type disorders through activation of protease activated receptors (PARs). PARs 1-4 are G protein-coupled receptors, activated by various proteases including kallikreins. PAR2 is of special interest, as it is activated by trypsin cleavage and is co-localized with tissue kallikreins in skin tissue. In skin lesions from atopic dermatitis and Netherton syndrome patients, PAR2 receptors were found overexpressed and co-localized with human tissue kallikreins (Descargues et al., 2006). This lead to the hypothesis that such a KLK-PAR pathway is involved in the pathogenesis of these diseases and that KLKs induce inflammation in these skin disorders via PAR2 activation.
Recent in vitro and in vivo work by Oikonomopoulou et al. (2006) has demonstrated that PAR activity may be targeted by active KLK5, 6, and 14. KLK5 and KLK6 were shown to activate PAR2, whereas KLK14 was reported to inactivate PAR1 and activate PAR2 and PAR4. Other reports showed activation of either PAR1 or PAR2 by KLK1, 2, 4, 5, 6 and 14 in different cell ltypes (Mize et al., 2008; Stefansson et al., 2008; Vandell et al., 2008)
PAR2 receptors are attractive research targets for dermatologists and cosmeticians due to implication in skin inflammation, cell proliferation, tumor suppression, skin pigmentation, and skin moisture. As activators of PAR2 receptors, kallikreins are of increasing interest to researchers investigating the above-mentioned skin processes. Natural non-denatured soybean-derived trypsin inhibitors are used as ingredients of cosmetic products targeting skin pigmentation, UV exposure, and skin moisture. Soybean-derived soy seeds and soymilk contain soybean trypsin inhibitor (STI) and Bowman-Birk inhibitor (BBI), respectively (Paine et al., 2001). The desired effects of these products are attributed to trypsin inhibition leading to blockade of PAR2 activation. KLK5 and KLK7 have been shown to be overexpressed under UVB irradiation concomitantly to a decrease of LEKTI expression, suggesting a contribution of these skin kallikreins in stratum corneum desquamation under UVB stress (Nin M et al., 2008).
It has been suggested that STI reduces UV light-induced skin cancer, as topical application of STI halts tumor progression in mice exposed to UVB for long periods (Huang et al., 2004). It is suggested that products containing natural soybean extracts block PAR2 activation by kallikrein inhibition. STI has been proven to inhibit trypsin-like KLK5 and 14 with high efficiency (Brattsand et al., 2005). Reduced KLK5 and 7 expression in the upper SC of dry skin and elevated KLK activity following UV radiation have been reported (Voegeli et al., 2007).
Serine protease inhibitors have also been predicted to have potential beneficial uses in the treatment of disease a wide variety of clinical areas such as oncology, neurology, hematology, pulmonary medicine, immunology, inflammation and infectious disease. Serine protease inhibitors may also be beneficial in the treatment of thrombotic diseases, asthma, emphysema, cirrhosis, arthritis, carcinoma, melanoma, restenosis, atheroma, trauma, shock and reperfusion injury. A useful review is found in Expert Opin. Ther. Patents (2002), 12(8). Serine protease inhibitors are disclosed in US published patent applications US 2003/0100089 and 2004/0180371 and in U.S. Pat. Nos. 6,784,182, 6,656,911, 6,656,910, 6,608,175, 6,534,495 and 6,472,393.
Skin diseases such as contact hypersensitivity, atopic dermatitis, rare genetic skin diseases (e.g. Netherton syndrome) and psoriasis are characterized by hyperproliferative and inflammatory skin reactions. A large population suffers from these diseases. For example, atopic dermatitis, a hereditary chronic disease of the skin, affects approximately 8 million adults and children in the United States. It is believed that a combination of multiple factors including genetic, environmental, and immunological factors may cause skin diseases. Although most skin diseases are not fatal, they significantly affect quality of life of those who suffer from the diseases.
Commonly used steroid-containing ointment or anti-histamine agents for treating skin diseases frequently cause considerable side effects. For example, steroids of external or oral application make the skin layer thin, cause osteoporosis, and inhibit growth in children upon long-term use. It was also observed that the termination of steroid application is often followed by lesion recurrence.
Therefore is a need to develop improved non-steroid agents for therapeutic, prophylactic or diagnostic approaches for the treatment of skin diseases. The present invention provides an improved and reliable method for the treatment, diagnosis or prophylaxis of skin diseases comprising the administration to a subject in need thereof of a therapeutically effective amount of a Serine protease inhibitor.
These and other objects as will be apparent from the foregoing have been achieved by the present invention.
The present invention concerns a method of treating or preventing as skin disease comprising administering to a mammal a pharmaceutical composition comprising a recombinant Serine protease inhibitor.
Also disclosed is the use of a Serine protease inhibitor in the preparation of a medicament for the treatment of a skin disease.
Another object of the invention is a kit for treating or preventing as skin disease comprising a pharmaceutical composition of a recombinant Serine protease inhibitor.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.
The present invention relates to the use of a Serine protease inhibitor in the preparation of a medicament for the treatment of a skin disease. Biologically active fragments of a Serine protease inhibitor are also useful in the preparation of said medicament.
Some of the serine proteases of the chymotrypsin superfamily, including t-PA, plasmin, u-PA and the proteases of the blood coagulation cascade are large molecules that contain, in addition to the serine protease catalytic domain, other structural domains responsible in part for regulation of their activity (Barrett, 1986; Gerard et al, 1986; Blasi et al., 1986). Among important serine proteases are trypsin-like enzymes, such as trypsin, tryptase, thrombin, kallikrein, and factor Xa. The serine protease targets are associated with processes such as blood clotting; complement mediated lysis, the immune response, glomerulonephritis, pain sensing, inflammation, pancreatitis, cancer, regulating fertilization, bacterial infection and viral maturation. By inhibiting serine proteases which have high specificity for a particular target, one can inhibit in vivo numerous biological processes, which may have dramatic effects on a host.
Serine proteinase inhibitors (serpins) comprise a diverse group of proteins that form a superfamily already including more than 100 members, from such diverse organisms as viruses, plants and humans Serpins have evolved over 500 million years and diverged phylogenetically into proteins with inhibitory function and non-inhibitory function (Hunt and Dayhoff, 1980). Non-inhibitory serpins such as ovalbumin lack protease inhibitory activity (Remold-O'Donnell, 1993). The primary function of serpin family members appears to be neutralizing overexpressed serine proteinase activity (Potempa et al., 1994). Serpins play a role in extracellular matrix remodeling, modulation of inflammatory response and cell migration (Potempa et al., 1994).
Serine protease inhibitors are divided into the following families: the bovine pancreatic trypsin inhibitor (Kunitz) family, also known as basic protease inhibitor (Ketcham et al., 1978); the Kazal family; the Streptomyces subtilisin inhibitor family; the serpin family; the soybean trypsin inhibitor (Kunitz) family; the potato inhibitor family; and the Bowman-Birk family (Laskowski et al., 1980; Read et al., 1986; Laskowski et al., 1987). Serine protease inhibitors belonging to the serpin family include the plasminogen activator inhibitors PAI-1, PAI-2 and PAI-3, Cl esterase inhibitor, alpha-2-antiplasmin, contrapsin, alpha-1-antitrypsin, antithrombin III, protease nexin I, alpha-1-antichymotrypsin, protein C inhibitor, heparin cofactor II and growth hormone regulated protein (Carrell et al., 1987; Sommer et al., 1987; Suzuki et al., 1987; Stump et al., 1986).
Many of the serine protease inhibitors have a broad specificity and are able to inhibit both the chymotrypsin superfamily of proteases, including the blood coagulation serine proteases, and the Streptomyces subtilisin superfamily of serine proteases (Laskowski et al., 1980). The inhibition of serine proteases by serpins has been reviewed in Travis et al. (1983); Carrell et al. (1985); and Sprengers et al. (1987). Crystallographic data are available for a number of intact inhibitors including members of the BPTI, Kazal, SSI, soybean trypsin and potato inhibitor families, and for a cleaved form of the serpin alpha-1-antitrypsin (Read et al., 1986). Despite the fact that these serine protease inhibitors are proteins of diverse size and sequence, the intact inhibitors studied to date all have in common a characteristic loop, termed the reactive site loop, extending from the surface of the molecule that contains the recognition sequence for the active site of the cognate serine protease (Levin et al., 1983). The structural similarity of the loops in the different serine protease inhibitors is remarkable (Papamokos et al., 1982). The specificity of each inhibitor is thought to be determined primarily by the identity of the amino acid that is immediately amino-terminal to the site of potential cleavage of the inhibitor by the serine protease. This amino acid, known as the Pi site residue, is thought to form an acyl bond with the serine in the active site of the serine protease (Laskowski et al., 1980). Whether or not a serpin possesses inhibitory function depends strongly on the consensus sequence located in the hinge region of the reactive site loop near the carboxy-terminus of the coding region. Outside of the reactive site loop, the serine protease inhibitors of different families are generally unrelated structurally, although the Kazal family and Streptomyces subtilisin family of inhibitors display some structural and sequence similarity.
As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.
“A” or “an” means “at least one” or “one or more.”
The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components.
As used herein, the terms “protein”, “polypeptide”, “polypeptidic”, “peptide” and “peptidic” or “peptidic chain” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
Preferably, the Serine protease inhibitor is a recombinant Serine protease inhibitor and is selected from the group comprising the SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14 or a biologically active fragment thereof having a Serine protease inhibitor activity.
Preferably also the recombinant Serine protease inhibitor is selected from the group comprising the SEQ ID NOS:39 to 59 or a biologically active fragment thereof having a Serine protease inhibitor activity.
“Amino acid residue” means any amino acid residue known to those skilled in the art. This encompasses naturally occurring amino acids (including for instance, using the three-letter code, Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Be, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), as well as rare and/or synthetic amino acids and derivatives thereof (including for instance Aad, Abu, Acp, Ahe, Aib, Apm, Dbu, Des, Dpm, Hyl, McLys, McVal, Nva, and the like).
Said amino acid residue or derivative thereof can be any isomer, especially any chiral isomer, e.g. the L- or D-isoform.
By amino acid derivative, we hereby mean any amino acid derivative as known in the art. For instance, amino acid derivatives include residues derivable from natural amino acids bearing additional side chains, e.g. alkyl side chains, and/or heteroatom substitutions.
“Biologically active fragments” refer to sequences sharing at least 40% amino acids in length with the respective sequence of the substrate active site. These sequences can be used as long as they exhibit the same properties as the native sequence from which they derive. Preferably these sequences share more than 70%, preferably more than 80%, in particular more than 90% amino acids in length with the respective sequence the substrate active site.
The present invention also includes variants of a Serine protease inhibitor sequence. The term “variants” refer to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide that is amino acid sequences that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:
I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly
II. Polar, positively charged residues: His, Arg, Lys
III. Polar, negatively charged residues: and their amides: Asp, Asn, Glu, Gln
IV. Large, aromatic residues: Phe, Tyr, Trp
V. Large, aliphatic, nonpolar residues: Met, Leu, He, Val, Cys.
“Administering”, as it applies in the present invention, refers to contact of a pharmaceutical, therapeutic, diagnostic agent or composition, to the subject, preferably a human.
The term “kallikrein” relates to glandular or tissue kallikreins. Glandular or tissue kallikreins are a sub-family of serine proteases, with a high degree of substrate specificity and diverse expression in various tissues and biological fluids. The term “kallikrein” appeared in the literature for the first time in the 1930s, when large amounts of protease enzymes were found in pancreas isolates (pancreas is “Kallikreas” in Greek) (Kraut et al. 1930, Werle 1934). Nowadays kallikrein enzymes are divided into two groups, plasma and tissue kallikreins, which differ significantly in their molecular weight, substrate specificity, immunological characteristics, gene structure, and type of the kinin released.
Kallikreins comprise a family of 15 homologous single chain, secreted serine endopeptidases of ˜25-30 kDa, with orthologues present in species from at least six mammalian orders. These kallikreins are hK2, hK3, hK2, hK5, hK6, hK7, hK8, hK9 hK10, hK11, hK12, hK13, hK14 and hK15. Preferably, kallikreins to be inhibited are selected from the group comprising hK2, hK5, hK7, and hK14.
“Disease”, as used herein, refers to a pathological condition of a part, organ, or system of an organism resulting from various causes, such as infection, genetic defect, or environmental stress, and characterized by an identifiable group of signs or symptoms.
The epidermis has been shown to express several serine proteases including kallikrein, urokinase, plasmin, typtase-like and neutrophile elastase enzymes. These serine proteases are involved in multiple activities in the skin including epidermal cell proliferation, cell differentiation, skin and lipid barrier homeostasis and tissue remodelling. Most importantly, proteolysis of stratum corneum (SC) corneodesmosomes by serine proteases together with other enzymes is a crucial event prior to shedding of the outermost skin layer, called desquamation. Furthermore, increased protease activity, including kallikrein, plasmin and urokinase enzymes are implicated in inflammatory reactions of the skin. A list with inflammatory skin diseases is shown in TABLE XX.
Increased protease activity was also observed as stress response to various stimuli including environmental factors as ultraviolet radiation exposure and temperature changes or as reaction to different surfactants.
Several kallikreins, notably hK5, hK7 and hK14 have been implicated in the proteolytic cascade in skin desquamation. This proteolytic process is controlled through a complex inhibition and activation process and its deregulation can cause serious skin disorders. Rare genetic diseases (Netherton Syndrome, peeling skin syndrome) as well as more common skin diseases like atopic dermatitis or psoriasis are characterized by increased desquamation of the skin caused at least in part by an increased kallikrein activity.
The present invention also relates to the use of a Serine protease in the preparation of a cosmetic or cosmeceutical agent for the treatment or improvement of an undesirable skin condition. Biologically active fragments of a Serine protease inhibitor are also useful in the preparation of said cosmetic or cosmeceutical agent.
“An undesirable skin condition” refers, in the present invention, to a problem affecting the skin or the appearance of the skin which might not always be considered as a disease.
As used herein “Cosmetics” are compositions used to enhance or protect the appearance of the human skin. Cosmetics include skin-care creams, lotions, powders, perfumes, lipsticks, fingernail and toenail polishes, eye and facial makeup, permanent waves, hair colors, hair sprays and gels, deodorants, baby products, bath oils, bubble baths, bath salts, butters and many other types of products.
“Cosmeceuticals” are cosmetic products that are thought to have drug-like benefits. Examples of products typically labeled as cosmeceuticals include anti-aging creams and moisturizers. Cosmeceuticals may contain purported active ingredients such as vitamins, phytochemicals, enzymes, antioxidants, and essential oils.
As used herein, “Skin disease” relates to conditions affecting the skin. Usually, the skin disease is selected from Table XX. Preferably, the invention is suitable for treatment of skin diseases, such as atopic dermatitis, contact dermatitis (allergy), contact dermatitis (irritant), eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation or pruritus, rosacea, netherton syndrome, peeling skin syndrome type A and B, hereditary ichtyosis, hidradenitis suppurativa and erythroderma (generalized exfoliative dermatitis). Most preferably, the skin disease is selected from the group comprising Netherton syndrome, Atopic dermatitis, Psoriasis and Peeling Skin Syndrome.
Netherton syndrome (NS) is a rare autosomal recessive genodermatosis caused by mutations in SPINK5 (LEKTI) one of the major inhibitor of the skin kallikrein cascade. Increased kallikrein activities have been shown to be causative for its clinical symptoms.
NS, a multisystem ichthyosiform syndrome, is characterized by ichthyosis, erythroderma, hair shaft defects and atopic features. Multiple infections due to the seriously impaired barrier function of the skin are very common.
NS is very rare, but little data on frequency is available, probably in part due to the difficulty to identify NS. Currently, less than 10 cases per million are diagnosed.
Treatment options are very limited and non-curative. They concentrate mainly on management of the various cutaneous infections and reduction of itching and pain (e.g. corticosteroid).
Excessive kallikrein activity (hK5, hK7, hK14) was proven causative for symptoms of the skin disorder. Decreased activity of the natural kallikrein inhibitor (LEKTI) could be replaced by alternative kallikrein inhibitors.
Surprisingly, Applicants have shown, e.g. in example 4, that the application of Serine protease inhibitors including MD67 (SEQ ID NO:8) mouse model (orthotopic hK5 overexpressing) considerably decreased the severity of the symptoms, which were observed in the untreated skin disease (e.g. NS) models. The symptoms are characterized by severe peeling of the skin, due to premature desmosomal protein degradation resulting in splitting of corneodesmosomes and stratum corneum detachment. This causes a severe loss of skin barrier functions leading to severe dehydratation, erythema and intense scratching.
Atopic dermatitis (AD) is a pruritic disease of not well defined origin that usually starts in early infancy and is typified by itching, eczematous lesions and dry, thick skin. AD is associated with other atopic diseases (eg, asthma, allergic reactions in about 30% of patients) and cutaneous infections are common.
The pathophysiology of AD is poorly understood. There appears to be a genetic component. An immune defect involving an abnormality of TH2 cells is suggested and a dysregulation of protease activity was found to be involved in the disease. This dysregulation is believed to cause a defective barrier function in the stratum corneum leading to the entry of antigens, which results in the production of various inflammatory cytokines. The prevalence rate in US is 10-12% in children and 0.9% in adults. In other developed countries the prevalence rate is as high as 18% and is rising, especially in developed countries. The disease is chronic, but the majority of patients improve from childhood to adult age.
No curative treatment is available yet. Depending on the severity of the symptoms topical steroids, antihistamines and immunomodulators or antibiotics, antiviral and antifungal agents are usually prescribed.
Psoriasis is a chronic disease, it is noncontagious and commonly appears as inflamed, edematous skin lesions, but also occurs on the oral mucosa. Joints (arthritis) also are affected in 10% of patients. Flares are related to various systemic and environmental factors including stress events or infections. There is a genetic predisposition for psoriasis and there is mounting evidence for signs of an autoimmune disorder. Increased protease (e.g. kallikrein) activity is involved in the typical excessive desquamation of the skin. In the US 2 to 3% of the population are affected and over 200,000 new cases occur annually. Approximately 1.5 million people with psoriatic arthritis seek medical care each year and 400 hundred people die annually from psoriasis-related causes. Incidence of psoriasis in other countries is similar but dependent on the climate and genetic heritage of the population. It is less common in the tropics and in dark-skinned persons.
Currently, there is no curative treatment. Depending on severity of symptoms topical corticosteroids, coal tar, keratolytic agents or retinoids are prescribed.
“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is human.
“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
The term “subject” refers to patients of human or other mammal and includes any individual it is desired to examine or treat using the methods according to the present invention. However, it will be understood that “patient” does not automatically imply that symptoms or diseases are present.
The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
As used herein, the term “protease” refers to a class of enzymes which recognizes a molecule and cleaves an activation sequence in the molecule. The protease can be an endopeptidase which cleaves internal peptide bonds. Alternatively, the protease can be an exopeptidase which hydrolyzes the peptide bonds from the N-terminal end or the C-terminal end of the polypeptide or protein molecule. The protease folds into a conformation to form a catalytic site which receives and cleaves the activation sequence.
“Inhibitors” refer to a polypeptide, or a chemical compound, that specifically inhibit the function of a kallikrein or serine protease by, preferably, binding to said kallikrein or serine protease.
“Reactive Serpin Loop” or “Reactive Site Loop” or RSL refers to an exposed flexible reactive-site loop found in serpin and which is implicated in the interaction with the putative target protease. From the residue on the amino acid side of the scissile bond, and moving away from the bond, residues are conventionally called P1, P2, P3, etc. Residues that follow the scissile bond are called P1′, P2′, P3′, etc. Usually, the RSL is composed of 6 to 12 amino acid residues.
“Serine protease inhibitors” or serpin according to the invention can be selected from the group comprising the α-1antichymotrypsin (ACT), protein C inhibitor (PCI), α-1antiproteinase (AAT), human α-1antitrypsin-related protein precursor (ATR), α-2-plasmin inhibitor (AAP), human anti-thrombin-III precursor (ATIII), protease inhibitor 10 (PI10), human collagen-binding protein 2 precursor (CBP2), protease inhibitor 7 (PI7), protease inhibitor leuserpin 2 (HLS2), human plasma protease C1 inhibitor (C1 INH), monocyte/neutrophil elastase inhibitor (M/NEI), plasminogen activator inhibitor-3 (PAI3), protease inhibitor 4 (PI4), protease inhibitor 5 (PI5), protease inhibitor 12 (PI12), human plasminogen activator inhibitor-1 precursor endothelial (PAI-1), human plasminogen activator inhibitor-2 placental (PAI2), human pigment epithelium-derived factor precursor (PEDF), protease inhibitor 6 (PI6), protease inhibitor 8 (PI8), protease inhibitor 9 (PI9), human squamous cell carcinoma antigen 1 (SCCA-1), human squamous cell carcinoma antigen 2 (SCCA-2), T4-binding globulin (TBG), Megsin, and protease inhibitor 14 (PI14), fragments thereof, molecular chimeras thereof, combinations thereof and/or variants thereof.
Since most of these serpins have different names, Applicant includes below a table I summarizing their specifications:
Advantageously, the serine protease inhibitor of the invention may be a serine protease trypsin-like enzyme and preferably a Kallikrein inhibitor. Kallikrein inhibitors of the invention are selected amongst hK2, hK3, hK4, hK5, hK6, hK7, hK8, hK9 hK10, hK11, hK12, hK13, hK14 or hK15 inhibitors. Preferably kallikreins inhibitors are selected among hK2, hK5, hK7, and hK14 inhibitors.
In case the kallikrein inhibitor is an inhibitor directed against hK2, said inhibitor can be selected among those disclosed in International Patent Application PCT/IB2004/001040, which content is incorporated herein by reference in its entirety. Preferably, the kallikrein inhibitor of the invention may be selected from the group comprising MD820, MD62, MD61, MD67 and MDCI. Most preferably this inhibitor is MD67. This application discloses a recombinant inhibitor protein of a protease comprising an inhibiting polypeptidic sequence and at least one polypeptidic sequence of a substrate-enzyme interaction site specific for a protease as well as a method for producing the recombinant inhibitor protein of a protease. Preferably the recombinant Serine protease inhibitor is selected from the group comprising the SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14 or a biologically active fragment thereof having a Serine protease inhibitor activity.
As an example of serine protease inhibitor according to the invention, Applicants have surprisingly found 7 new recombinant inhibitor proteins specific for the protease hK2 as resumed below in table II, these inhibitors are:
These inhibitor proteins have been obtained by modifying the RSL of α1-antichymotrypsin (rACT), which is known to inhibit a large panel of human enzymes such as chymotrypsin, mast cell chymase, cathepsin G, prostatic kallikreins hK2 and PSA (hK3), in order to change the specificity of this serpin. Peptide sequences, selected as substrates for the enzyme hK2 by phage display technology as explained in International Patent Application PCT/IB2004/001040, have been used to replace the scissile bond and neighbour amino acid residues of the RSL. Usually, recombinant inhibitors were produced in bacteria and purified by affinity chromatography.
Additionally, Applicants have also found that replacing residues P3-P3′ located in RSL structure of rACTWT by substrate pentapeptide coding for the RSL of Protein C inhibitor (PCI) lead to the production of a recombinant inhibitor (MDCI) which is able to inhibit kallikreins hK2 and hK3.
In case the kallikrein inhibitor is an inhibitor directed against hK14, then said inhibitor can be selected among those disclosed in the International Patent Application PCT/IB2005/000504, which content is incorporated herein by reference in its entirety. Preferably, said recombinant inhibitor may be selected from the group comprising AATG1, AATG1G, AATC11, AATC11G, AATE5, AATE8, AATF11, AATF3, AATG9, ACTG1, AcTG1G, ACTC11, ACTC11G, ACTE5, ACTE8, ACTF11, ACTF3, ACTG9, ACTG1V, ACTWT and ACTC11D. Preferably, said inhibitor protein of an hK14 protease is AATG1 AATG1G, AATC11, AATC11G, AATE5, AATE8, AATF3, AATG9, ACTG1G, ACTC11, ACTC11G, ACTE5, ACTE8, AGTF11, ACTF3, ACTG9, ACTG1V, or ACTC11D.
This application discloses a recombinant inhibitor protein of an hK14 protease having an inhibiting polypeptidic sequence and at least a polypeptidic sequence of a substrate-enzyme interaction site specific for said hK14 protease. Preferably, said recombinant inhibitor protein of an hK14 protease has, under physiological conditions,
In addition, the inhibiting polypeptidic sequence of the protease inhibitor may also be selected from a cysteine protease since there are now a number of well-documented instances of inhibition of cysteine proteases by serpins (Gettins P. G. W., 2002 “Serpin structure, mechanism, and function” in Chem. Rev, 102, 4751-4803). These examples include inhibition of cathepsins K, L and S by the serpin squamous cell carcinoma antigen1, inhibition of prohormone thiol proteinase by the α-1antichymotrypsin, and inhibition of members of the caspase family, including caspase 1 (interleukine 1β converting enzyme), caspase 3, and caspase 8 by the viral serpin crmA and caspases 1, 4 and 8 by the human serpin PI9.
Usually, the serine protease inhibitor is a recombinant inhibitor protein. Thus, when recombinant techniques are employed to prepare a Serine protease inhibitor, nucleic acid molecules or fragments thereof encoding the polypeptides are preferably used.
Therefore the present invention also relates to a purified and isolated DNA sequence encoding the Serine protease inhibitor as described above.
“A purified and isolated DNA sequence” refers to the state in which the nucleic acid molecule encoding the recombinant inhibitor protein of a protease of the invention, or nucleic acid encoding such recombinant inhibitor protein of a protease will be, in accordance with the present invention. Nucleic acid will be free or substantially free of material with which it is naturally associated such as other polypeptides or nucleic acids with which it is found in its natural environment, or the environment in which it is prepared (e. g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in vivo.
DNA which can be used herein is any polydeoxynuclotide sequence, including, e.g. double-stranded DNA, single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently-closed DNA, linear DNA, covalently crosslinked DNA, cDNA, chemically-synthesized DNA, semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radiolabeled DNA and fluorochrome-labeled DNA, DNA containing one or more non-naturally occurring species of nucleic acid.
DNA sequences that encode the Serine protease inhibitor, or a biologically active fragment thereof having a Serine protease inhibitor activity, can be synthesized by standard chemical techniques, for example, the phosphotriester method or via automated synthesis methods and PCR methods.
The purified and isolated DNA sequence encoding the Serine protease inhibitor according to the invention may also be produced by enzymatic techniques. Thus, restriction enzymes, which cleave nucleic acid molecules at predefined recognition sequences can be used to isolate nucleic acid sequences from larger nucleic acid molecules containing the nucleic acid sequence, such as DNA (or RNA) that codes for the recombinant inhibitor protein or for a fragment thereof.
Encompassed by the present invention is also a nucleic acid in the form of a polyribonucleotide (RNA), including, e.g., single-stranded RNA, double-stranded RNA, double-stranded RNA wherein one or both strands are composed of two or more fragments, double-stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double-stranded RNA wherein the RNA strands are fully complementary, double-stranded RNA wherein the RNA strands are only partially complementary, covalently crosslinked RNA, enzyme-digested RNA, sheared RNA, mRNA, chemically-synthesized RNA, semi-synthetic RNA, biosynthetic RNA, naturally-isolated RNA, labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNA containing one or more non-naturally-occurring species of nucleic acid.
The purified and isolated DNA sequence encoding a Serine protease inhibitor is preferably selected from the group comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16 to SEQ ID NO:37.
The present invention also includes variants of the aforementioned sequences, that is nucleotide sequences that vary from the reference sequence by conservative nucleotide substitutions, whereby one or more nucleotides are substituted by another with same characteristics.
Also encompassed in the present invention is the use of a purified and isolated DNA sequence encoding a Serine protease inhibitor in the preparation of a medicament for the treatment of a skin disease.
Alternatively, the Kallikrein inhibitors or the serine protease inhibitors of the invention comprise a detectable label or bind to a detectable label to form a detectable complex.
“Detectable labels” are detectable molecules or detection moiety for diagnostic purposes, such as enzymes or peptides having a particular binding property, e.g. streptavidin or horseradish peroxidase. Detection moiety further includes chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e. g. labelled avidin.
Preferably, detectable labels include fluorescent labels and labels used conventionally in the art for MRI-CT imagine. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
The Kallikrein inhibitors or the serine protease inhibitors of the invention may carry a radioactive label as the detection moiety, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 121I, 124I, 125I, 131I, 111In, 211At, 198Au, 67Cu, 225Ac, 213bu, 99Tc and 186Re. When radioactive labels are used, known currently available counting procedures may be utilized to identify and quantitate the specific binding members.
In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
The radioactive labels are useful in in vitro diagnostics techniques, ex vivo and in in vivo radioimaging techniques. In a further aspect, the radioactive labels are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells.
In the instance of in vivo imaging, the labels of the present invention may be conjugated to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent. Examples of chelating groups include EDTA, porphyrins, polyamines crown ethers and polyoximes.
Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium and erbium.
The present invention is also directed to a pharmaceutical composition comprising the serine protease inhibitor as described herein as an active agent, optionally in combination with one or more pharmaceutically acceptable carriers.
Preferably the composition, as a pharmaceutical composition, according to the invention is to be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream or CSF, or directly into the site of the disease, or close to this site. The precise dose will depend upon a number of factors, including whether the composition is for diagnosis, prognosis, prophylaxis of or for treatment, the size and location of, for example, desquamation, the precise nature of the composition, and the nature of the detectable or functional label attached to the Kallikrein inhibitor or the serine protease inhibitor.
The present pharmaceutical composition comprises as an active substance a pharmaceutically effective amount of the composition as described, optionally in combination with pharmaceutically acceptable carriers, diluents and adjuvants.
“A pharmaceutically effective amount” refers to a chemical material or compound which, when administered to a human or animal organism induces a detectable pharmacological and/or physiologic effect.
The pharmaceutically effective amount of a dosage unit of the polypeptide usually is in the range of 0.001 ng to 100 μg per kg of body weight of the patient to be treated.
The pharmaceutical composition may contain one or more pharmaceutically acceptable carriers, diluents and adjuvants.
Acceptable carriers, diluents and adjuvants which facilitates processing of the active compounds into preparation which can be used pharmaceutically are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, hydroxyethylcellulose (NATROSOL® (hydroxyethylcellulose (HEC)) or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN® (polysorbate or polyoxyethylene sorbitol ester), PLURONIC® (polyoxyalkylene ether) or polyethylene glycol (PEG).
The form of administration of the pharmaceutical composition may be systemic or topical. For example, administration of such a composition may be various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, buccal routes or via an implanted device, and may also be delivered by peristaltic means.
The pharmaceutical composition, as described herein, may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support. In addition the matrix may be comprised of a biopolymer such as NATROSOL® (hydroxyethylcellulose (HEC)).
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.
It is understood that the suitable dosage of the present composition will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.
The appropriate dosage form will depend on the disease, the inhibitor, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots.
Since amino acid modifications of the amino acids (of the inhibitor for example) are also encompassed in the present invention, this may be useful for cross-linking the inhibitor to a water-insoluble matrix or the other macromolecular carriers, or to improve the solubility, adsorption, and permeability across the blood brain barrier. Such modifications are well known in the art and may alternatively eliminate or attenuate any possible undesirable side effect of the peptide and the like.
Another subject matter of the present invention is to provide a kit for the diagnosis, prognosis, prophylaxis or treatment of skin disease in a mammal, said kit comprising a recombinant serine protease, optionally with reagents and/or instructions for use.
The kit of the present invention may further comprise a separate pharmaceutical dosage form comprising other pharmaceutical compositions and combinations thereof.
Generally, the Kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
Alternatively, or additionally, the Kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
The present invention also discloses the use of the composition of the invention, as a pharmacological tool in the development and standardization of in vitro and in vivo test systems for the diagnosis, prognosis, prophylaxis or treatment of skin diseases in mammals.
Also encompassed by the present invention is a detection assay for the diagnosis, prognosis, prophylaxis or treatment of skin diseases in a tissue sample comprising contacting the tissue sample with the composition of the invention, determining and measuring the amount of detected label and correlating this amount to the presence or absence of a disease in said tissue sample.
Yet another object of the present invention is to provide a method for killing a skin cell expressing kallikrein molecules, comprising contacting the cell with the composition of the invention so as to kill the cell, destroying or avoiding the survival of cells expressing kallikrein molecules.
It is also an object of the present invention to provide a method for inhibiting skin cell expressing serine protease and in particular kallikrein molecules, comprising contacting said skin cell with the composition of the invention.
Yet another object of the present invention is to provide a cosmetic composition comprising a Serine protease inhibitor, or a biologically active fragment thereof having a Serine protease inhibitor activity as described herein as well as the use of this composition for the improvement of an undesirable skin condition.
Preferably, the Serine protease inhibitor is a recombinant inhibitor protein of the invention.
Usually, the Serine protease is selected from the group comprising kallikrein, plasmin, chymotrypsin (Chtr), urokinase (uPA), tryptase and neutrophile elastase (HNE) enzymes and/or a combination thereof.
Preferably, the kallikrein is selected from the group comprising hK2, hK5, hK7, and hK14 and/or a combination thereof.
The invention also provides the use of a Serine protease inhibitor, or a biologically active fragment thereof having a Serine protease inhibitor activity, in the preparation of cosmetic composition for the improvement of an undesirable skin condition.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.
The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.
Development of Recombinant ACT Inhibitors Specific to Human hK2 Using Phage Display Selected Substrates.
The content of Application PCT/IB2004/001040 (Université de Lausanne) is incorporated herein by reference in its entirety
Material
hK2 and hK3 (PSA) were purified from human semen as previously described (Frenette G, Gervais Y, Tremblay R R, Dube J Y. 1998 “Contamination of purified prostate-specific antigen preparations by kallikrein hK2” J Urol 159, 1375-8), anti-hK2 and anti-PSA monoclonal antibodies were a gift from Professor RR Tremblay, Laval University, Canada. Human chymotrypsin (Chtr), urokinase plasminogen activator (uPA), human kallikrein hK1, human plasma kallikrein (PK), human neutrophil elastase (HNE) and commercial ACT (human plasma α-1-antichymotrypsin) were purchased from Calbiochem. Z-Phe-Arg-AMC, Suc-Ala-Ala-Pro-Phe-AMC (AAPF SEQ ID NO: 135), Z-Gly-Gly-Arg-AMC, MeOSuc-Ala-Ala-Pro-Val-AMC (AAPV SEQ ID NO: 136) were purchased from Calbiochem. CFP-TFRSA-YFP (TFRSA SEQ ID NO: 137) fluorescent substrate was developed as previously described (Mahajan N P et al. 1999 “Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis” Chem Biol 6, 401-9). The cDNA for human al-antichymotrypsin (ACT) was a generous gift from Dr. Harvey Rubin (University of Pennsylvania).
Site-Directed Mutagenesis
Following the subcloning of ACT cDNA into pQE-9 expression vector (Qiagen, Germany) and the introduction of an His6 tag (SEQ ID NO: 164) at the N-terminal of rACTWT, two restriction sites Sac II and MluI, were incorporated 18 bp upstream and 18 bp downstream of P1 codon in RSL domain respectively. These sites were created by silent mutation using oligonucleotides 5′-GTGATTTTGACCGCGGTGGCAGCAG-3′ (SEQ ID NO:111) for Sac II and 5′-GCACAATGGTACGCGTC TCCACTAATG-3′ (SEQ ID NO:112) for Mlu I site and following the quickchange mutagenesis protocol supplied by Stratagene.
Construction of the Substrate Phage Display Library
Substrate phage libraries were generated using a modified pH0508b phagemid (Lowman et al. 1991 “Selecting high-affinity binding proteins by monovalent phage display” Biochemistry 12, 10832-8). The construction consists of a His6 (SEQ ID NO: 164) tag at either end of a Gly-Gly-Gly-Ser-repeat-rich region (GGGS SEQ ID NO: 165) that precedes the carboxyl-terminal domain (codons 249-406) of the M13 gene III. The random pentamers were generated by PCR extension of the template oligonucleotides with appropriate restriction sites positioned on both side of the degenerate codons: 5′TGAGCTAGTCTAGATAGGTGGCGGTNNSNNSNNSNNSNNSGGGTCGACGTCGGTCA TAGCAGTCGCTGCA-3′ (SEQ ID NO:113) (where N is any nucleotide and S is either G or C) using 5′ biotinylated primers corresponding to the flanking regions: 5′TGAGCTAGTCTAGATAGGTG-3′ (SEQ ID NO:83) and 5′-TGCAGCGACTGCTATGA-3′ (SEQ ID NO:84).
PCR templates are digested and purified as described previously (Smith G. P, Scott J. K. 1993 “Libraries of peptides and proteins displayed on filamentous phage” Methods Enzymol. 217, 228-57), inserted into XbaI/SalI digested pH0508b vector, and electroporated into XL1-Blue (F−). The extent of the library was estimated from the transformation efficiency determined by plating a small portion of the transformed cells onto Luria-Bertani plates containing ampicillin and tetracycline (100 and 15 μg·mL−1, respectively). The rest of the transformed cells were used to prepare a phage library by incubating overnight by adding an M13K07 helper phage at a concentration giving a multiplicity of infection of 100 plaque forming units (p.f.u.) per mL. Phages were collected from the supernatant and purified by poly(ethylene glycol) precipitation. Of these, 200 clones were selected arbitrarily for sequencing to verify the randomization of the library.
Phage-Displayed Pentapeptide Library Screening
This new pentapeptide library was subjected to eight rounds of screening with hK2. One hundred microliters of Ni2+-nitrilotriacetic acid coupled to sepharose beads (Ni2+-nitrilotriacetic acid resin) was washed with 10 mL NaCl/Pi containing 1 mg·mL−1 BSA. Phage particles (1011) were added to the equilibrated Ni2+-nitrilotriacetic acid resin and allowed to bind with gentle agitation for 3 h at 4° C. The resin was subsequently washed (NaCl/Pi/BSA 1 mg·mL−1, 5 m
Construction and Expression of Recombinant Wild Type ACT and its Variants.
Six variants, which correspond to a change in the reactive site loop in positions between P3 and P3′ (see Table III below), were generated by PCR extension of the template oligonucleotides:
(where underlined sequences encode new cleavage sites in the reactive site loop), using primers corresponding to the flanking regions: 5′-TACCGCGGTCAAAATC-3′ (SEQ ID NO:67) and 5′-TCACGCGTGTCCAC-3′ (SEQ ID NO:68). PCR products were digested with Sac II and Mlu I restriction enzymes and then subcloned into digested rACTWT construct. Recombinant serpins were produced in TG1 E. coli strain. Cells were grown at 37° C. in 2×TY media (16 g tryptone, 10 g yeast extract, 5 g NaCl per L) containing 100 μg/ml ampicillin to A600=0.5. Isopropylthio-β-galactoside (IPTG) was then added to a final concentration of 0.5 mM allowing the expression of recombinant serpins for 16 h at 16° C. The cells from 100 ml of culture were harvested by centrifugation, resuspended in cold PBS and then passed through a french press to recover the total soluble cytoplasmic proteins. Cell debris were removed by centrifugation and Ni2+-nitilotriacetic affinity agarose beads were added to supernatant for 90 min at 4° C. to bind recombinant serpins. The resin was subsequently washed with 50 mM Tris pH 8.0, 500 mM NaCl, 25 mM Imidazole and the bound proteins were eluted for 10 min with 50 mM Tris pH 8.0, 500 mM NaCl and 150 mM Imidazole. Once purification was completed, rACT were dialysed against 50 mM Tris pH 8.0, 500 mM NaCl, 0.05% Triton X-100 for 16 h at 4° C. The protein concentration was determined for each purification by Bradford assay and normalized by densitometry of Coomassie Blue-stained SDS-PAGE gels (Laemmli U K. 1970 “Cleavage of structural proteins during the assembly of the head of bacteriophage T4” Nature 227, 680-5).
L*
L
R*
S
R
A
R
R*
S
I
D
R
G
R*
S
E
K
L
R*
T
T
M
T
R*
S
N
A
E
R*
V
S
P
aSubstrate peptides selected by kallikrein hK2 using a phage-displayed random pentapeptide library.
Inhibition Assays and Stoichiometry of Inhibition (SI)
The stoichiometry of inhibition (SI) values were determined for the inhibition of rACT WT and its variants with hK2 and different other enzymes. An initial test was made with a molar excess of rACT (100 fold) over hK2, PSA, hK1, chymotrypsin (Chtr), plasma kallikrein (PK), urokinase (uPA) and human neutrophile elastase (HNE) enzymes. The reaction was carried out for 30 min at 25° C. (90 min at 37° C. for PSA) in reaction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0,05% Triton X-100, 0,01% BSA) and residual enzyme activity was measured by adding fluorescent substrates (Z-Phe-Arg-AMC for hK1, hK2 and PK, Suc-Ala-Ala-Pro-Phe-AMC for Chtr (AAPF SEQ ID NO: 135), Z-Gly-Gly-Arg-AMC for uPA, MeOSuc Ala-Ala-Pro-Val-AMC for HNE (AAPV SEQ ID NO: 136), and CFP-TFRSA-YFP (TFRSA SEQ ID NO: 137) for PSA). Activity of enzyme in presence of inhibitors was compared to uninhibited reaction. For reactions where an inhibition was observed, SI was determined by incubating different concentrations of recombinant serpins. Using linear regression analysis of fractional activity velocity of inhibited enzyme reaction/velocity of uninhibited enzyme reaction) versus the molar ratio of the inhibitor to enzyme ([Io]/[Eo]), the stoichiometry of inhibition, corresponding to the abscissa intercept, was obtained.
Kinetics
The association rate constants for interactions of hK2, chymotrypsin, PK and HNE with different rACTs were determined under pseudo-first order conditions using the progress curve 80% (Morrison J F, Walsh C T. 1988 “The behavior and significance of slow-binding enzyme inhibitors” Adv. Enzymol. Relat. Areas Mol. Biol 61, 201-301). Under these conditions, a fixed amount of enzyme (2 nM) was mixed with different concentrations of inhibitor (0-800 nM) and an excess of substrate (10 μM). Each reaction was made in reaction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% Triton X-100, 0.01% BSA) at 25° C. for 45 min and the rate of product formation was measured using a FLx800 fluorescence 96-well microplate reader (Biotek, USA). In this model, inhibition is considered to be irreversible over the course of reaction and the progress of enzyme activity is expressed by product formation (P), beginning at a rate (vz) and is inhibited over time (t) at a first-order rate (kobs), rate constant that is dependent only on inhibitor concentration.
P=(vz/kobs)×[1−e(−k
For each inhibitor, a kobs was calculated, for four different concentrations of inhibitors, by non linear regression of the data using equation 1. By plotting the kobs versus inhibitor concentration [I], a second-order rate constant, k′, equal to the slope of the curve (k′=Δkobs/Δ[I]), was determined. Due to the competition between inhibitor and the substrate, equation 2 below is used to correct the second order rate constant k′ by taking in account the substrate concentration [S] and the Km of the enzyme for its substrate, giving the ka.
ka=(1+[S]/Km)×k′ eq 2
The Km of hK2 for Z-FR-AMC, chymotrypsin for Suc-AAPF-AMC (AAPF (SEQ ID NO:135), PK for Z-FR-AMC and HNE for MeOSuc-AAPV-AMC (AAPV SEQ ID NO:136) were 67 μM, 145 μM, 170 μM and 130 μM respectively.
Western Blot Analysis of Complex Formation and Inhibitor Degradation.
Kallikrein hK2 was incubated 3 hours at 37° C. with different recombinant ACTs at a [I]o:[E]o ratio of 100:1 in 50 mM Tris, 200 mM NaCl, 0,05% Triton X-100. Protein samples were heated at 95° C. for 5 min, separated by SDS-PAGE (12% acrylamid 19:1 T:C ratio) and then electroblotted onto Hybond-ECL (Amersham Pharmacia) nitrocellulose. The free-hK2 and hK2-ACT complexes were detected using a mouse anti-hK2 monoclonal antibody and an alkaline phosphatase-conjugated goat anti-mouse secondary antibody. Western blot was visualized using the ECL detection kit (Amersham Pharmacia Biotech). hK2 was also incubated with ACT8.3 or ACT6.7 30 min at 25° C. (kinetic conditions) at a [I]o: [E]o ratio of 10:1 in 50 mM Tris, 200 mM NaCl, 0,05% Triton X-100. Proteins were detected by western blot, using an anti-His6 monoclonal antibody (“His6” disclosed as SEQ ID NO: 164) followed by detection with the secondary antibody and protocol described above.
Production of Soluble Recombinant Wild Type and Variant ACTs
Wild type serpin α1-antichymotrypsin was used to develop specific inhibitors of the kallikrein hK2. Residues P3-P3′ located in RSL structure of rACTWT were replaced by substrate pentapeptides, previously selected by phage display technology as described above. Six variants of rACT shown in table IV, have been designed and constructed. The scissile bond in substrate peptides was aligned according to Leu-358-Ser-359 into RSL of the serpin. rACTWT and its variants were expressed in E. coli TG1 as fusion proteins containing an His tag in N-terminal position. Each of them was produced at low temperature allowing protein accumulation mainly in active soluble form. Purified under native conditions, the level of production varied between 1.0 to 2.5 mg/L. The purity of purified serpins, such as for example Variant 6.1 and wild type ACT, as estimated by SDS-PAGE analysis is more than 98%.
rACT Variants are Mainly Specific to Kallikrein hK2
A panel of enzymes including human neutrophil elastase, chymotrypsin-like (Chtr, PSA or hK3) and trypsin-like (hK2, hK1, PK, uPA) proteinases have been screened to determine inhibitory specificity of rACT variants (Table IV).
aAmino acid sequence cleaved in RSL (Reactive Serpin Loop) of recombinant ACTs corresponding to selected substrate peptide by hK2.
BProtease and serpins were incubated for 30 min at 25° C. (90 min at 37° for PSA) at a [I]o/[E]o ratio of 100:1. Percent inhibition correspond to 100 × (1 − (velocity in presence of inhibitor/velocity of uninhibited control)).
Incubating with an excess of inhibitors ([I]o/[E]o of 100:1) for 30 minutes, hK2 is completely inhibited by rACT6.2, rACT8.3, rACT6.7 and rACT6.1, whereas rACT8.20 and rACT5.18 inhibited 95% and 73% of enzyme activity, respectively. Under this condition, wild type rACT showed no inhibition activity toward hK2. Among these variants, two (rACT8.3 and rACT5.18) are specific to hK2, inhibiting no other tested enzyme. Two other variants, rACT6.7 and rACT6.2, inhibited as well PK at 36% and 100% respectively. As wild-type ACT, variant rACT8.20 inhibited the two chymotrypsin-like proteases Chtr and PSA but additionally also PK and HNE. None of the recombinant serpins showed inhibitory activity against the kallikrein hK1 and uPA.
Stoichiometries of Inhibitory of Variant ACTs for hK2 are Improved Drastically in Comparison to Wild Type ACT
The determination of the stoichiometry of inhibitory was accomplished under physiological conditions of pH and ionic strength for all enzymes to ensure the most valuable comparison. Recombinant wild type ACT gave a SI value of 2 (table V) with chymotrypsin which is identical to the value obtained with commercial ACT under similar conditions (data not shown).
aSI values reported were determined using linear regression analysis to extrapolate the I/E ratio.
bSecond order rate constants for serpin-proteinase reactions were measured under pseudo-first- or second order conditions as described in “Experimental Procedure”.
cAmino acid sequence of P3-P3′ residues in RSL (Reactive Serpin Loop) of recombinant ACT corresponding to selected substrate peptide by hK2
In order to determine the SI values of all the recombinant variants, Applicants have incubated hK2 (5 nM) with different concentrations (6.25-500 nM) of rACT8.20, rACT6.2, rACT8.3, rACT6.7, rACT6.1, rACT5.18, rACTWT, at 25° C. for 30 min in reaction buffer. Residual activities (velocity) for hK2, were assayed by adding the fluorescent substrate (10 μM) Z-FR-AMC. Fractional velocity corresponds to the ratio of the velocity of inhibited enzyme (vi) to the velocity of the uninhibited control (vo). The SI was determined using linear regression analysis to extrapolate the I/E ratio (i.e. the x intercept).
All newly constructed variants of ACT showed lower SI values with hK2 than wild type ACT. From these variants rACT6.7, rACT6.1 and rACT6.2 had the lowest stoichiometry of inhibition values for hK2 (9, 19 and 25 respectively). Whereas rACT6.2 and rACT6.1 had also the lowest SI values (18 and 16) for PK, the SI for rACT6.7 was much higher (277). The two recombinant ACTs specific for hK2, rACT8.3 and rACT5.18 had a higher SI ratio of 34 and 139, respectively. The SI value of rACT8.20 inhibitor was superior to 100 for all tested proteases including hK2.
Variant ACTs Form Stable Complexes with hK2 without Degradation of Inhibitors
hK2 was incubated 3 h at 37° C. with rACT8.20, rACT6.2, rACT8.3, rACT6.7, rACT6.1, rACT5.18 and wild type rACT, at a I:E ratio of 100:1. Western Blot analysis of the reaction products of rACTs with hK2 (rACT8.20), rACT6.2, rACT8.3, rACT6.7, rACT6.1, rACT5.18 and wild type rACT has been done under reducing conditions using a mouse anti-hK2 antibody to determine the fate of inhibitors after the interaction with the enzyme. When hK2 is incubated with ACT variants, free hK2 (E) disappeared completely to form a covalent complex (EI). This covalent complex demonstrated a high stability as it did not break down over a 16 h incubation period (data not shown). Wild type ACT inhibited more slowly hK2, which was mainly uncomplexed after 3 hours of incubation. Elevated SI values measured with hK2 were not due to non-complex forming degradation of ACT variant inhibitors.
Further on ACT8.3 or ACT6.7 were incubated with hK2 under kinetic conditions (30 min at 25° C.) at a I:E ratio of 10:1. The complex formation was analysed by western blot under reducing conditions using a mouse monoclonal anti-his tag. All inhibitor proteins were either complexed with hK2 or present as uncleaved form, indicating that the possible substrate pathway for the serpin-enzyme interaction is marginal.
Variant ACTs Showed Highest Association Constants with hK2
The rate of inhibitory reaction with variant ACTs was determined for each protease showing reactivity with these inhibitors. To that end, interaction of hK2 and recombinant serpins was measured under pseudo-first order conditions using progress curve method. hK2 (2 nM) and substrate Z-FR-AMC (10 μM) were added to varying amounts (20n-800 nM) of inhibitors rACT8.20, rACT5.18 and inhibitors rACT6.2, rACT8.3, rACT6.7, rACT6.1 (data not shown). Representative progress curves were subjected to non linear regression analysis using eq 1 and the rate (kobs) was plotted against the serpin concentrations. After determination of kobs, association constants (ka) were calculated using Km of the proteases for their corresponding substrates (table VI). The ka value of wild type ACT with chymotrypsin was identical as to published data (Cooley et al. 2001 “The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites” Biochemistry 40, 15762-70). The recombinant rACT6.7 showed a highest ka (8991 M−1 s−1) with hK2 whereas that obtained with PK was 45 fold inferior. In contrast, recombinant rACT6.2 gave equivalent ka with hK2 and PK demonstrating a lack of discrimination between the two proteases. ka values of hK2 specific recombinant inhibitors rACT8.3 and rACT5.18 were lower, 2439 and 595 M−1 s−1 respectively, whereas non specific ACT8.20 exhibited a ka of 1779 M−1 s−1, for hK2, superior compared to Chtr, PK and HNE. One of the recombinant serpins, rACT6.1, was reacting at higher velocity with PK than with hK2.
Residues P3-P3′ located in RSL structure of rACTWT were replaced by substrate pentapeptide coding for the RSL of Protein C Inhibitor (PCI) (Table VI) as described in example 1.
L
R
F
R
S
A
L
AGA
Briefly, to produce the recombinant protein ACTPCI (MDCI), TG1 cells were transformed with the corresponding constructions followed by growth in appropriate culture media. Cells were then induced to an optimal density to express recombinant inhibitors for 16 h at 16° C. Recombinant inhibitor ACTPCI was extracted from cytoplasm bacteria and separated by affinity chromatography using Ni-NTA column as described for the previous example.
Analysis of Recombinant ACT Expression by SDS-PAGE.
The purity of the different inhibitors developed in example 1 and 2 was tested by SDS-PAGE analysis under reducing conditions.
Evaluation of the Inhibitors.
These inhibitors were further analysed to assess their specificity and affinity to inhibit the human kallikreins hK2 and hK3 and plasma kallikrein, trypsin, urokinase, elastase, thrombin, hK14 and human kallikrein 8 (Table VII). These two enzymes possess different enzymatic specificities (hK2: trypsin-like, hK3: chymotrypsin-like) but are naturally inhibited by ACT. While ACT is considered to be the natural hK3 inhibitor in blood circulation, its inhibition of hK2 is weaker.
Analysis of the inhibitory reaction between rACTs and the human kallikreins were analysed by Western Blot (data not shown). For each variants of ACT, 1 μg of inhibitor was incubated with 100 ng of either hK2 or hK3 during 1 hour at 37° C. under physiological conditions.
The amino acid changes within the reactive loop using substrate sequences selected for hK2 specificity transformed ACT into an inhibitor highly specific for hK2 (MD820, MD61, MD62) without inhibiting hK3. These results confirm those previously shown in Table IV. Only MDCI, based on the reactive loop of the inhibitor of the Protein C (PCI) is able to inhibit both kallikreins tested (hK2 and hK3).
MD61 and MD62 are inhibitors with very high affinity for hK2 inhibiting all hK2 protein in less than 3 minutes (under the same conditions) compared to wild type or commercial α1-antichymotrypsin, which requires more than 12 hours of incubation to inhibit the same amount of hK2 (data not shown).
Development of substrate active sites specific to human hK14.
The content of Application N+ PCT/IB2006/000574 (Université de Lausanne) is incorporated herein by reference in its entirety.
Materials
The following materials were obtained from commercial sources: elastase, trypsin, chymotrypsin, and plasma kallikrein (Calbiochem), human laminin 10 & 11 (Chemicon), human collagen IV (Life Technologies), T4 DNA ligase (Invitrogen), T4 polynucleotide kinase (Qbiogene), Ni2+-nitrilotriacetic acid agarose beads (Qiagen), restrictions enzymes (Roche, Amersham Pharmacia, Promega), anti-His antibody (Sigma). Oligonucleotide synthesis was carried out by Invitrogen and DNA sequencing by Synergene Biotech GmbH. Human kallikrein 2 and prostate specific antigen were purified from human seminal plasma as previously described (Frenette et al., 1997; Frenette et al., 1998). Matrilin-4 is a gift from R. Wagener (Cologne, Germany).
Cloning of KLK14 into P. pastoris Expression Vector pPICZαA
First-strand cDNA synthesis was performed by reverse transcriptase using the SUPERSCRIPT® (reverse transcriptase) preamplification system (Gibco BRL, Gaithersburg, Md.) with 2 μg of total human cerebellum RNA (Clontech, Palo Alto, Calif.) as a template. The final reaction volume was 20 μL. To confirm the efficiency of RT-PCR, 1 μL of cDNA was subsequently amplified by PCR with primers specific for actin, a housekeeping gene (ActinS: 5′ ACAATGAGCTGCGTGTGGCT (SEQ ID NO:114), ActinAS: 5′ TCTCCTTAATGTCACGCACGA (SEQ ID NO:115)). Actin PCR products with an expected length of 372 base pairs (bp) were visualized on a 2% agarose gel stained with ethidium bromide. PCR amplification of KLK14 cDNA encoding the 227 amino acids of the mature hK14 protein (corresponding to amino acids 25-251 of Genbank accession no. AAK48524) was carried out in a 50 μL reaction mixture containing 1 pt of cerebellum cDNA as a template, 100 ng primers (FPL6: 5′ AGG ATG AGG AAT TCA TAA TTG GTG GCC AT (SEQ ID NO:69) and RPL6: 5′ CCC ACC GTC TAG ACC ATC ATT TGT CCC GC (SEQ ID NO:70)), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM deoxynucleoside triphosphates (dNTPs) and 0.75 μL (2.6 U) of Expand Long Template PCR polymerase mix (Roche Diagnostics, Mannheim, Germany), using an Eppendorf master cycler. The PCR conditions were 94° C. for 2 min, followed by 94° C. for 10 s, 52° C. for 30 s, 68° C. for 1 min for 40 cycles, and a final extension at 68° C. for 7 min. Following PCR, amplified KLK14 was visualized with ethidium bromide on 2% agarose gels, extracted, digested with EcoRI/XbaI and ligated into expression vector pPICZαA of the EASYSELECT™ Pichia pastoris expression system (Invitrogen, Carlsbad, Calif.) at corresponding restriction enzyme sites using standard techniques (Sambrook et al., 1989). The KLK14 sequence within the construct was confirmed with an automated DNA sequencer using vector-specific primers in both directions.
Protein Production
PmeI-linearized pPICZαA-KLK14, as well as empty pPICZαA (negative control), were transformed into chemically competent P. pastoris yeast strain X-33 after which they integrated into the yeast genome by homologous recombination. Transformed X-33 cells were then plated on YPDS (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 2% agar) plates containing Zeocin™, a selective reagent. A stable yeast transformant was selected as per the manufacturer's recommendations, inoculated in buffered minimal glycerol-complex (BMGY) medium [1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen base, 40 mg/litre biotin, and 1% glycerol] overnight at 30° C. on a plate agitator at 250 rpm, diluted to OD600=1.0 in BMMY (same as BMGY except that 1% glycerol is replaced with 0.5% methanol) and incubated under the same conditions as above for 6 days with a daily supplement of 1% methanol. The supernatant was collected by centrifugation at 4000×g for 20 min.
Protein Purification
Recombinant hK14 was purified from yeast culture supernatant by cation exchange using a 5 mL HiTrap™ carboxymethyl (CM) Sepharose Fast Flow column on the AKTAFPLC chromatography system (Amersham Biosciences, Piscataway, N.J.). First, the supernatant was filtered with a 0.22 μm disposable filter and concentrated 50-fold by ultrafiltration with an AMICON™ (protein concentrating and desalting, regenerated cellulose, ultrafiltration centrifugal filter) YM10 membrane (Millipore Corporation, Bedford, Mass.). The filtered, concentrated supernatant was then introduced into the injector of the AKTAFPLC system and loaded onto the CM sepharose column, previously equilibrated with 5 mL of 10 mM MES buffer (pH 5.3) at a flow rate of 0.8 ml/min. The column was washed with the aforementioned equilibration buffer and the adsorbed hK14 was eluted with a 150 mL continuous linear KCl gradient from 0 to 1 M in 10 mM MES (pH 5.3) at a flow rate of 3 ml/min. Elution fractions of 5 ml were collected and analyzed. Fractions containing hK14 were pooled and further concentrated 10 times using Biomax-10 Ultrafree®-15 Centrifugal Filter Device (Millipore Corporation, Bedford, Mass.). The protein concentration of the purified hK14 was determined by the bicinchoninic acid method (Smith et al., 1985), which uses bovine serum albumin as calibrator (Pierce Chemical Co., Rockford, Ill.). The purity of the recombinant hK14 protein was analyzed by SDS-PAGE (Laemmli, 1970) followed by Coomassie blue staining and/or Western blot analysis using a previously produced polyclonal rabbit antibody raised against hK14 (Borgono et al., 2003) and its identity was confirmed by tandem mass spectrometry, as described in detail for recombinant hK10 (Luo et al., 2001).
Phage-Displayed Pentapeptide Library Screening.
A monovalent type phagemid supplied by Dr Lowman (Genentech company, San Fransisco, Calif.) was previously modified in order to generate a substrate phage library containing six His residues (SEQ ID NO: 164) N terminal to the random pentapeptide fused to the g3p (Cloutier et al, 2002). The six His residues (SEQ ID NO: 164) allow the phage fixation to the Ni-NTA column.
Preparation of the duplex that is inserted into the phagemid was performed by PCR reaction of a degenerated oligonucleotide, in which the 5 random amino acids are coded by NSS (N=A, T, G, C and S=G, C). The resulting library was composed of 1.8×108 transformants, which is largely enough to get all random sequences represented.
This phage display substrate library was subjected to six rounds of screening with hK14. Briefly, substrate phages (1011) were incubated with sixty microliters of Ni2+-nitrilotriacetic acid resin in PBS 1× containing BSA at 1 mg/mL, washed four times (PBS 1×, BSA 1 mg/mL, 5 mM imidazole, 0.1% TWEEN® 20 (polysorbate 20 or polyoxyethylene sorbitol ester)) to remove unbound phages and then exposed to 65 nM (final concentration) of hK14 for 45 minutes at 37° C. in 50 mM Tris, 100 mM NaCl, 0.05% Triton, pH 7.5. The released phages were subsequently amplified using XL1-Blue Escherichia coli and then used after purification for subsequent rounds of selection. 32 individual clones from the last round of selection were sequenced for determination of their corresponding amino acid sequences.
Expression of CFP-YFP Fluorescent Substrate
Recombinant fluorescent substrates, using cyan fluorescent protein as donor and yellow fluorescent protein as acceptor, were constructed as described recently (Felber et al., 2004). CFP-XXXXX-YFP-6×His (“6×His” disclosed as SEQ ID NO: 164) recombinant proteins were constructed with varying pentapeptides (in bold) between CFP and YFP proteins using synthetic genes possessing the appropriate restriction sites (BssHII; SalI). The constructs contain the following amino acid sequences between CFP and YFP proteins: Gly-Ala-Leu-Gly-Gly-XXXXX-Gly-Ser-Thr (GALGGXXXXXGST (SEQ ID NO:116)). To produce recombinant proteins, TG1 cells were transformed with the corresponding constructs and purified by affinity chromatography using Ni2+-NTA agarose beads. The purity and quantity of the purified CFP-YFP recombinant substrates were evaluated by SDS gel electrophoresis according to Laemmli followed by Coomassie Blue staining and Western blot analysis using a specific anti-His primary antibody (1/3000 dilution), a mouse anti-Fab secondary antibody (1/50000 dilution) and the ECL system (Amersham) for detection. All clones were sequenced prior to evaluation.
Direct Determination of the Kcat/Km and Specificity Studies Using CFP-YFP Fluorescent Substrates
Substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and Kcat/Km values calculated as previously described (Felber et al., 2004). Briefly, fluorescence of CFP-X5-YFP proteins was measured in black 96-well plates using a microplate fluorescence reader (Bio-Tek Instruments, Inc.) with excitation at 440 nm (±15) and emissions at 485 nm (±10) and 528 nm (±10). Each recombinant substrate, at a concentration of 150 nM, was incubated with hK14, chymotrypsin, trypsin, PSA, hK2, plasma kallikrein or elastase at a final concentration of 8 nM, 0.1 nM, 0.3 nM, 2 μM, 10 nM, 10 nM and 0.5 nM respectively. The reaction was performed for 60 min at 37° C. in reaction buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.05% Triton-X100). The enzyme concentration for initial-rate determinations was chosen at a level intended to hydrolyze specifically the substrate linker and not a GGGGG (SEQ ID NO:117) substrate, which was used as negative control. The appearance of fluorescence, corresponding to product formation, was measured spectrometrically with excitation at 440 nm (±15) and emission at 485 nm (±10). The slope was converted into units of nmol of product generated per sec, based on a calibration curve obtained from the complete hydrolysis of each peptide, evaluated on SDS-PAGE. The kinetic parameter kcat/Km was determined under pseudo-first order conditions using a substrate concentration far below the estimated Km (Felber et al., 2004).
The cleavage products were separated by SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon polyvinylidene difluoride membrane (Bio-Rad), and subjected to automated Edman degradation with an Applied Biosystems (model ABI493A) sequenator to determine the cleavage site.
Selection of Phage Substrate for hK14
The substrate phage library was panned against hK14 to select substrates cleaved by its hydrolytic activity. Cleaved phages were amplified in E. coli TG1 cells and then subjected to five more rounds of enzyme digestion and screening. The amount of released phages increased with each round, indicating the presence of a higher number of hK14-susceptible phages after each round of selection. The amino acid sequences of 32 phage peptides from the last round of selection were determined by sequencing. The sequences corresponding to the substrate regions are listed in Table 1. From all selected and cleaved peptides, 69% possess a basic residue in P1 position, as expected for a putative trypsin-like activity of hK14, whereas 31% of peptides have a tyrosine residue specific for a chymotrypsin-like enzyme in P1.
Kinetic Characterization of Substrate Hydrolysis by hK14
To verify that the sequences from the phage display analysis were indeed substrates for hK14, and to identify the cleavage site, all selected peptides were constructed in fluorescent substrate form. Applicants substrate system is based on the transfer of energy from CFP to YFP which are linked by the substrate. Cleavage of the linker by a protease separates the two fluorophores and results in a loss of the energy transfer. Thus, hydrolysis of the substrate can be evaluated by the measurement of increasing fluorescence intensity of the donor at 485 nm, corresponding to the wavelength of CFP emission (Mitra et al., 1996; Felber et al., 2004).
All substrates were hydrolyzed by hK14 with variable level of efficacy and Kcat/Km values ranged from 2 000 to 481 000 M−1 s−1. The specificity of cleavage was demonstrated with CFP-GGGGG-YFP (SEQ ID NO:117) which is not hydrolyzed by hK14 (not shown).
Results clearly indicate that the preferred P1 amino acid for hK14 susceptibility is Arg (Table 1) since the best hK14 substrates with Kcat/Km greater than 200,000 M−1 s−1 possess an Arg in P1 position. Interestingly, from the four peptides cleaved most efficiently by hK14, two contained a Gln at the P2 position. In contrast, a broad variety of amino acids were found in P1′ position, demonstrating no significant preference at this position. However, two substrates possess an aspartic acid in P1′ position and are cleaved relatively efficiently.
On the other hand, all substrates with a Lys at the P1 position were cleaved at low rate with a Kcat/Km equal to or below 34 000 M−1 s−1. Similarly, the cleavage rate for substrates with a P1 tyrosine was very low except for one substrate, peptide G9, which had a Kcat/Km of 134 000 M−1 s−1. With the exception of P1′ position, where glycine residue is found in about 50% of P1 Lysine or Tyrosine substrates, no amino acid was recovered more frequently at the other positions. Nevertheless, it has to be stated that the majority of glycine residues found in position P1′ were originating from the phage linker region flanking the selected pentapeptide substrates, where Lys or Tyr residues are found in position 5 of the selected peptide.
Specificity of Preferred Selected Substrates
Since many of the selected substrates contained motifs potentially susceptible to cleavage by other proteases, Applicants measured the degree to which hK2, plasma kallikrein, PSA, chymotrypsin, trypsin and elastase could cleave these hK14 substrates (Table VIII). Each substrate was tested at enzyme concentration leading to specific cleavage in the substrate linker and not hydrolyzing the GGGGG (SEQ ID NO:117) control substrate.
Not surprisingly, most of trypsin-like substrates are cleaved by trypsin with a variable efficacy which was not strictly in correlation with hK14 preferences. For instance, the two pentapeptides VGSLR (SEQ ID NO:118) and RQTND (SEQ ID NO:119) were the best substrates for hK14 but were not very efficiently cleaved by trypsin in comparison to other peptides like LSGGR (SEQ ID NO:124) exhibiting a Kcat/Km of almost 5,000,0000 M−1·s−1. In contrast, peptides possessing a Gln in P2 position were excellent substrates for hK14 as well as for trypsin. Only two hK14 substrates with low trypsin-like hK14 activity, RVTST (SEQ ID NO:128) and VVMKD (SEQ ID NO:129), but four out of five substrates with chymotrypsin like hK14 activity were not cleaved by trypsin.
All chymotrypsin-like substrates were cleaved by chymotrypsin more efficiently than with hK14, except for the substrate TVDYA (SEQ ID NO:130) which gave almost the same kcat/Km with hK14, chymotrypsin and elastase. Elastase also proteolyzed the two selected peptides TSYLN (SEQ ID NO:134) and YQSLN (SEQ ID NO:133), which is also cleaved weakly by PSA. Preferred substrates displayed a high selectivity for hK14 in comparison to other human kallikreins such as hK1, hK2, PSA and PK. Only hK2 proteolyzed most of the trypsin-like substrates with Kcat/Km values always at least 5 fold lower than for hK14. For example, NQRSS (SEQ ID NO:120) peptide is 27 and 78 fold more selective for hK14 than for hK2 and PK, respectively and F3 peptide demonstrates high hK14 specificity and no cleavage with another kallikrein could be detected.
Materials
The following materials were obtained from commercial sources: elastase, trypsin, chymotrypsin, thrombin and plasma kallikrein (Calbiochem), T4 DNA ligase (Invitrogen), T4 polynucleotide kinase (Qbiogene), Ni2+-nitrilotriacetic acid agarose beads (Qiagen), restrictions enzymes (Roche, Amersham Pharmacia, Promega), anti-His antibody and an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Sigma). Fluorescent substrates Z-Phe-Arg-AMC, Suc-Ala-Ala-Pro-Phe-AMC (AAPF (SEQ ID NO:135), Z-Gly-Gly-Arg-AMC and MeOSuc-Ala-Ala-Pro-Val-AMC (AAPV SEQ ID NO:136) were purchased from Calbiochem, Boc-Val-Pro-Arg-AMC from Bachem, Abz-Thr-Phe-Arg-Ser-Ala-Dap(Dnp)-NH2 (SEQ ID NO: 203) from Neosystem. Oligonucleotide synthesis was carried out by Invitrogen and DNA sequencing by Synergene Biotech GmbH. Human kallikrein 2, 5, 13 and 14 were produced in a yeast system (Yousef et al., 03c; Kapadia et al., 03; Borgono et al., 03). Human kallikrein 6 was produced in a 293 human embryonic kidney cell system and human kallikrein 8 with a baculovirus vector and HighFive insect cells (Little et al., 97; Kishi et al., 03). HK6 and hK8 were activated with Lys-C(Shimizu et al., 98).
Construction of Expression Vectors for Recombinant Wild-Type AAT, ACT and their Variants.
Human AAT cDNA (Invitrogen, UK) was amplified by PCR using the oligonucleotides 5′-TATGGATCCGATGATCCCCAGGGAGA-3′ (SEQ ID NO:71) and 5′-CGCGAAGCTTTTATTTTTGGGTGGGA-3′ (SEQ ID NO:72). The BamHI-HindIII fragment of the amplified AAT gene was cloned into the vector pQE9 (Qiagen, Germany) resulting in plasmid pAAT, which contains an open reading frame of the mature AAT with an N-terminal His6-tag (SEQ ID NO: 164). Silent mutations producing KasI and Bsu36I restriction sites were introduced in pAAT 24 bp upstream and 11 bp downstream of the P1 codon of the RSL domain, respectively. The restriction sites were created using the oligonucleotides 5′-ACTGAAGCTGCTGGCGCCGAGCTCTTAGAGGCCATA-3′ (SEQ ID NO:73) for the KasI and 5′-GTCTATCCCCCCTGAGGTCAAGTTC-3′ (SEQ ID NO:74) for the Bsu36I site following the QuikChange mutagenesis protocol supplied by Stratagene. Construction of the plasmid expressing wild-type ACT was described previously (Cloutier et al., 2004). rAAT and rACT variants were produced by replacement of the RSL region with corresponding DNA fragments amplified from appropriate template oligonucleotides:
Templates were amplified using primers corresponding to their respective flanking regions, 5′-GCTGGCGCCATGTTTCTAGAG-3′ (SEQ ID NO:79; AAT variants1) and 5′-TTGTTGAACTTGACCTCAGG-3′(SEQ ID NO:80; AAT variants 2) for AAT variants and 5′-GTACCGCGGTCAAA-3′(SEQ ID NO:81; ACT variants 1) and 5′-TCACGCGTGTCCAC-3′(SEQ ID NO:82; ACT variants 2) for ACT variants. Resulting PCR fragments were cloned as KasI/Bsu36I fragments into pAAT and as MluI/SacII fragments into rACTWT constructs and confirmed by DNA sequencing. Changes in the reactive site loop between positions P4 and P2′ are shown in Table IX.
Expression and Purification of Recombinant Serpins
Recombinant serpins were produced in Escherichia coli strain TG1. Cells were grown at 37° C. in 2× TY media (16 g tryptone, 10 g yeast extract, 5 g NaCl per L) containing 100 μg/ml ampicillin to O.D.600=0.5-0.7. Isopropyl thio-β-D-galactoside (IPTG) was added to a final concentration of 0.5 mM for production of rACT proteins and 0.1 mM for rAAT proteins and recombinant serpins were expressed for 16 h at 18° C. Cells were harvested by centrifugation and resuspended in 0.1 volume of cold PBS 2×. After 45 min of incubation with lysozyme (0.5 mg/ml) on ice, total soluble cytoplasmic proteins were extracted by four cycles of freeze/thaw and total DNA was degraded with DNase I. Cell debris was removed by centrifugation (25 min., 17′500 g) and Ni2+-nitrilotriacetic affinity agarose beads were added to the supernatant for 90 min at 4° C. to bind recombinant serpins. The resin was washed three times with 50 mM Tris, pH 7.5, 150 mM NaCl, 20 mM imidazole and bound proteins were eluted with 50 mM Tris, pH 7.5, 150 mM NaCl, 150 mM imidazole. Eluted proteins were dialyzed against 50 mM Tris, pH 7.5, 150 mM NaCl, 0.01% Triton X-100 for 16 h at 4° C. and protein purity was assessed by Coomassie Blue-stained SDS-PAGE. Protein concentrations were determined by the bicinchoninic acid method (Smith et al., 1985), using bovine serum albumin as standard (Pierce Chemical Co., Rockford, Ill.). AATE8, ACTE8 and AATG9, ACTG9 were titrated with trypsin and chymotrypsin, respectively.
Stoichiometry of Inhibition (SI)
SI values of rAAT, rACT, and their variants were determined with hK14 incubating the protease with varying concentrations of inhibitor. After an incubation of 4 hours at 37° C. in reaction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Triton X-100, 0.01% BSA), the residual activity was detected by the addition of fluorescent substrate (Boc-Val-Pro-Arg-AMC). Fluorescence was measured with excitation at 340 nm (±15) and emission at 485 nm (±10) in black 96 well plates using a microplate fluorescence reader FLx800 (Bio-Tek Instruments, Inc.). The SI value corresponds to the abscissa intercept of the linear regression analysis of fractional velocity (velocity of inhibited enzyme reaction (vi)/velocity of uninhibited enzyme reaction (v0)) vs. the molar ratio of the inhibitor to enzyme ([I0]/[E0]).
Kinetic Analysis
The association rate constants for interactions of hK14, with different inhibitors were determined under pseudo-first order conditions using the progress curve method (Morrison and Walsh, 1988). Under these conditions, a fixed amount of enzyme (2 nM) was mixed with different concentrations of inhibitor (0-80 nM) and an excess of substrate (20 μM). Reactions were performed in reaction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% Triton X-100, 0.01% BSA) at 37° C. for 45 min and the rate of product formation was measured using a FLx800 fluorescence 96-well microplate reader (Biotek, USA). Inhibition is considered to be irreversible over the course of reaction and the progression of enzyme activity is expressed as product formation (P), beginning at a rate (vz) and is inhibited over time (t) at a first-order rate (kobs), where the rate constant is only dependent on the inhibitor concentration.
P=(vz/kobs)×[1−e(−k
For each inhibitor, a kobs was calculated for four different concentrations of inhibitor, by non linear regression of the data using equation 1. By plotting the kobs versus inhibitor concentration [I], a second-order rate constant, k′, equal to the slope of the curve (k′=Δkobs/Δ[I]), was determined. Due to the competition between the inhibitor and the substrate, equation 2 below is used to correct the second order rate constant k′ by taking into account the substrate concentration [S] and the Km of the enzyme for its substrate, giving the ka.
ka=(1+[S]/Km)×k′ eq 2
The Km of hK14 for MeOSuc-VPR-AMC was 8 μM. However, it will be understood that, depending on the purity grade and specific activity of the hK14 protease, the Km may vary.
SDS-PAGE Analysis of Enzyme-Inhibitor Complexes
A constant amount of the different inhibitors (ranging from 1 to 2 ug) was incubated for 4 h in reaction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% Triton X-100) with different amounts of hK14 corresponding to 0.5, 1 and 2 times the SI value. Samples were heated at 90° C. for 10 minutes, resolved on a 10% SDS gel under reducing conditions and visualized by Coomassie Blue staining.
Inhibitory Specificity of Recombinant rAAT and rACT Variants (Table IX)
2 nM of trypsin, chymotrypsin, plasma kallikrein, human neutrophil elastase and thrombin and 10 nM of hK2, hK3, hK5, hK6, hK8, hK13 and hK14 were incubated for 30 minutes at 37° C. with 100 nM and 500 nM of recombinant inhibitors, respectively. Residual activities were detected by the addition of fluorescent substrates (Z-Phe-Arg-AMC for trypsin and plasma kallikrein, Suc-Ala-Ala-Pro-Phe-AMC (SEQ ID NO: 135) for chymotrypsin, Z-Gly-Gly-Arg-AMC for thrombin and MeOSuc-Ala-Ala-Pro-Val-AMC (SEQ ID NO: 136) for human neutrophil elastase and Abz-Thr-Phe-Arg-Ser-Ala-Dap(Dnp)-NH2 (SEQ ID NO: 203) for human kallikreins).
Stability of the Complex
HK14 (2 nM) was incubated with different amounts of inhibitors, corresponding to 0, 1 and 2 times the SI. After incubations for 4, 8 and 24 h at 37° C. in reaction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Triton X-100, 0.01% BSA), the residual activity was detected by addition of 20 μM of the fluorescent substrates Boc-Val-Pro-Arg-AMC. The slope (velocity) of each inhibitory reaction was divided by the slope of the corresponding reaction without inhibitor.
Design and Production of Soluble Recombinant Serpins
To develop specific inhibitors for hK14, Applicants substituted five residues surrounding the scissile bond of rAATwt and rACTwt by two substrate pentapeptides, previously selected with hK 14 using phage-display technology (Felber et al., 05). Profiling of hK14 enzymatic activity demonstrated that hK14 has a dual trypsin and chymotrypsin-like activity. Applicants therefore decided to develop inhibitors with two substrate peptides, E8 and G9, specific for trypsin and chymotrypsin-like activity, respectively. The scissile bond of these substrates was aligned according to the P1-P′1 of the rAATwt and rACTwt. The RSL regions of the serpin variants are shown in Table IX (SEQ ID NOS. 177-182, respectively).
L
Q
R*
A
T
V
D
Y*
A
L
Q
R*
A
I
T
V
D
Y*
A
aSubstrate peptides selected by kallikrein hK14 using a phage-displayed random pentapeptide library (Felber et al., 2004).
The recombinant serpins were produced as soluble, active form and were purified under native conditions from cytoplasmic proteins in a one-step procedure over a nickel affinity column. Analysis on SDS-PAGE under reducing conditions revealed a single band for each inhibitor, rAAT and rACT variants, migrating at apparent sizes of 45 to 50 kDa, corresponding with their molecular weight, except for the protein AATE8, which is migrating slightly faster (data not shown). All inhibitors were estimated to be more than 95% pure by densitometric analysis, with a range of production yield of 1 to 5 mg/L.
Stoichiometry of Inhibition, Association Constants and Complex Stability
Determination of stoichiometry of inhibition (SI) was performed under physiological conditions of pH and ionic strength. The SI indicates the number of inhibitor molecule required to inhibit one molecule of hK14. Applicants observed that titration curves were linear, even for SI values >>1, indicating that the reaction is completely finished. The calculated SI values of the serpin variants range from ˜1 to 1.5, except for rAATE8 which resulted in a SI of 7.4 (Table X).
Whereas wild type ACT did not react with hK14 under the tested conditions, AATWT was found to be a good inhibitor for hK14 with a SI of 1. Substitution of ACT RSL region with hK14 substrate peptides not only allowed generating reactivity toward the enzyme but creating inhibitors with high affinity. On the other hand, using AATWT as scaffold, modification of the RSL was less favorable since all inhibitors are less efficient than wild type version (Table X).
aSubstrate peptide selected by phage display technology with hK14 (Felber et al., 2005) and used to modify the rAATwt and rACTwt.
Calculated SI values were consistent with the ratio between cleaved and complexed forms of the serpins after reaction with hK14 as demonstrated by SDS-PAGE analysis (data not shown). Each variant was incubated with different concentrations of hK14 corresponding to a ratio of inhibitor to protease below, equal and above the SI value. The analysis of SDS-PAGE showed the formation of covalent complexes (C) for each serpin variant hK14 pair, with apparent molecular masses consistent with expected values. When hK14 concentration was 0.5 time the SI value, degraded forms of the complex was observed which is certainly generated by the uncomplexed and free hK14.
Besides the formation of an inhibitor complex, reaction with hK14 also produced a fraction of hydrolyzed inhibitor, with a molecular size consistent with the serpin being cleaved at or near the reactive site of the RSL. The amount of this fraction was largely lowered when the SI value is close to 1 (AAT-G9, ACT-E8 and ACT-G9). In contrast, the only variant with a SI values >>1 (rAATE8) exhibited a substrate behavior with hK14, resulting mainly in accumulation of the cleaved form of the inhibitor rather than formation of the irreversible complex. As expected, the presence of intact inhibitor was observed when the ratio [I]o/[E]o was above the SI with a weak band of complex.
Surprisingly, most of complexes were found to be SDS stable (data not shown) even if a relatively slow breakdown of the complex was observed with AATG9 resulting in the reappearance of hK14 activity after 8 hours of incubation.
Kinetic analysis of the inhibition of human hK14 by recombinant serpins were performed under pseudo-first-order conditions using an excess of inhibitor at various molar ratios of hK14. The time-dependent inactivation of the enzyme through reaction with serpin was monitored continuously, following the decrease in the rate of substrate turnover. Progress curves for reactions with different serpin concentrations were fitted to equation 1 to calculate values describing the rate constant (kobs). The association rate constants (ka) were determined from the slope of kobs values versus the concentration of the hK14 inhibitors. Independently of the inhibitor scaffold (AAT or ACT), the recombinant serpins modified with the substrate E8 showed superior ka values than the equivalent G9 inhibitor.
Serpins modified with the chymotrypsin-like substrate, rAATG9 and rACTG9, demonstrated only a moderate affinity for hK14, with association constants of respectively 217,000 and 74,000 M−1 s−1 while rACTE8 possessed association constants of 575,000 M−1 s−1.
Inhibitory Specificity of Recombinant rAAT and rACT Variants
In order to define the inhibitory specificity of hK14 inhibitors, Applicants investigated the reaction of purified variants with a broad panel of proteinases. First at all, proteinases with broad specificities were examined, including trypsin, chymotrypsin, plasma kallikrein, human neutrophil elastase and thrombin. Additionally, Applicants assessed the specificity of hK14 inhibitors towards enzymes belonging to the same protease family, i.e. hK2, hK3, hK5, hK6, hK8 and hK13 (Table XI). Following 30 minutes of incubation of hK14 with an excess of inhibitors ([I]o/[E]o of 50:1), no residual activity was detected with all modified serpins and rAATwt.
Under these conditions, only rACTwt showed weak inhibitory activity against hK14, with only 17% of inhibition. Serpins modified with the E8 substrate showed a moderate specificity since several other enzymes were inhibited by these inhibitors. A very high specificity was observed with AATG9 and ACTG9 and none of the tested enzymes was inhibited, except for chymotrypsin and to a lower extent for hK5.
Production and Purification of Active hK14
Human Kallikrein 14 was produced and purified as previously described.
Selection of substrate peptides for hk14 using phage display technology
The substrate phage library was panned against hK14 to select substrates hydrolyzed by its hydrolytic activity. Cleaved phages were amplified in E. coli TG1 cells and then subjected to five more rounds of enzyme digestion and screening. The amount of released phages increased with each round, thus verifying a higher number of hK14-susceptible phages after each round of selection. The amino acid sequences of 32 phage peptides from the last round of selection were determined by sequencing and the obtained sequences corresponding to the substrate regions were listed in Table 8. From all selected and cleaved peptides, 69% possess at least one basic residue in P1 position as expected with the putative trypsin-like activity of hK14 whereas 31% of peptides have a tyrosine residue specific to chymotrypsin-like enzyme in P1.
Kinetic Characterization of Substrate Hydrolysis by hK14
To verify that the sequences from the phage display analysis were indeed substrates for hK14 and to identify the cleavage site, all selected peptides were constructed in fluorescent substrate form. All substrates were hydrolyzed by hK14 with variable level of efficacy and kcat/Km ranged from 2,000 to 481,000 M−1 s−1. The specificity of cleavage was demonstrated by CFP-GGGGG-YFP (SEQ ID NO:117) which is not hydrolyzed by hK14.
Results clearly indicate that the preferred P1 amino acid for hK14 susceptibility is Arg (Table XIII) since all of the best hK14 substrates with kcat/Km superior to 200 000 M−1 s−1 possess an Arg in P1 position. Interestingly, from the four peptides cleaved most efficiently by hK14, two contained Glu at the P2 position. In contrast, a broad variety of amino acids occurred in P′1 position demonstrating no significant preference at this position. However, two substrates possess an aspartic acid in P′1 position and are cleaved relatively efficiently.
On the other hand, all substrates with a Lys at the P1 position were cleaved at very low rate with a Kcat/Km below 34 000 M−1 s−1. Similarly, the cleavage rate for the substrate with a P1 Tyrosine was very low excepted one substrate, peptide G9, which has a Kcat/Km of 134 000 M−1 s−1. With the exception of P′1 position, where glycine residue is recovered in almost 50% of P1 Lysine or Tyrosine substrates, none amino acid in particular was recovered more frequently at the other positions.
Since many of the selected substrates contained some motifs susceptible to be cleaved by other proteases, Applicants measured the degree to which hK2, plasma kallikrein, PSA, chymotrypsin, trypsin and elastase could cleave these hK14 substrates (Table XII). Each substrate was tested at enzyme concentration giving a specific cleavage in the substrate linker.
Not surprisingly, most of trypsin-like substrates are cleaved by trypsin with a variable efficacy which was not strictly in correlation with hK14 preferences. For instance, the two pentapeptides VGSLR (SEQ ID NO:118) and RQTND (SEQ ID NO:119) were best substrates for hK14 but were not very efficiently cleaved by trypsin in comparison to other peptides like LSGGR (SEQ ID NO:124) peptide giving a Kcat/Km of almost 5,000,0000 M−1·s−1 with trypsin. In contrast, peptides possessing a Gln in P2 position were best substrates as well as for hK14 than for trypsin. Only two hK14 substrates, RVTST (SEQ ID NO:128) and VVMKD (SEQ ID NO:129), in exception to chymotrypsin-like substrates were not cleaved by trypsin.
Chymotrypsin-like substrates were cleaved by chymotrypsin more efficiently than with hK14 excepted TVDYA (SEQ ID NO:130) substrate which gave almost the same Kcat/Km with hK14, chymotrypsin and elastase. This last enzyme also proteolyzed the two selected peptides YQSLN (SEQ ID NO:133), which is also cleaved weakly by PSA, and TSYLN (SEQ ID NO:134). Selected substrates displayed a high selectivity for hK14 in comparison to other human kallikreins such as hK1, hK2, PSA and PK. Only hK2 proteolyzed most of the trypsin-like substrates with Kcat/Km values always at least 5 fold less than for hK14. For example, NQRSS (SEQ ID NO:120) peptide is 27 and 78 fold more selective for hK14 than for hK2 and PK, respectively.
RQTND
RGKTN
RVTST
RVDTG
YQNSS
All
This study identified two classes of pentapeptide substrates for hK14: trypsin-like and chymotrypsin-like substrates. However, Applicants showed that hK14 has trypsin-rather than chymotrypsin-like cleavage specificity despite the selection of several aromatic residue-containing substrates. The substrates with the highest Kcat/Km have an arginine in P1 position indicating a preference for this amino acid (Table XIV). Lysine, on the other hand, seems to be less suitable than tyrosine in P1 position. If the two amino acids were present in the same peptide, hK14 cleaved after the tyrosine residue. In addition, one of the chymotrypsin-like substrates, TVDYA (SEQ ID NO:130), gave a significantly higher kinetic value, 134,000 M−1·s−1, than all the lysine-P1 substrates, with Kcat/km values not higher than 34,000 M−1·s−1. No selectivity of hK14 was observed for the P1′ position, where different types of amino acids such as small and uncharged, hydrophobic, positively charged or negatively charged residues have been recovered in the best substrates Analysis of other surrounding positions demonstrated that hK14 can be accommodated by a large variety of amino acids. This observation does not mean that hK14 has a large spectrum of activities like trypsin or chymotrypsin but demonstrates an ability to cleave different sequences depending to the context.
The chymotrypsin-like activity of hK14, even if it is inferior to its trypsin-like activity, is interesting. To Applicants knowledge, except for the Phe-Phe link cleaved by hK1 in kallistatin and some derived peptides, this is the first human kallikrein described with a dual activity. The conformation of the specificity pocket in hK14 should therefore accommodate both aromatic and basic amino acid side chains at the substrate P1 position to explain the dual chymotrypsin and trypsin-like activity of hK14.
Development of hK14 Specific Inhibitors
Modifications of the RSL of α1-antichymotrypsin (ACT) and cd-antitrypsin (AT or AAT) have been performed to change the specificity of this inhibitor. Selected substrates (G1, C11, E5, E8, F3, F11, G9) were then transplanted into the reactive site loop of serpins to generate new variants, able to inhibit the human kallikrein hK14. More than one inhibitor variants have been constructed using sequences from peptides G1 and C11.
G
S
R
*
G
S
R
*
G
V
G
S
R
*
RQTNd
R
*
Q
T
N
G
R
*
Q
T
N
R
*
Q
T
N
D
N
Q
R
*
S
L
Q
R
*
A
I
Q
R
R
*
D
P
D
R
*
H
M
T
V
D
Y
*
A
aSubstrate peptides selected by kallikrein hK14 using a phage-displayed random pentapeptide library. Plain type residues are common to rACTWT, underlined residues correspond to substrate peptides relocated in RSL of ACT variants. The scissile bond by hK14 in substrate peptides is designated by bold and putative cleavage site in serpins is marked by asterisks between the P1-P1′ residues.
G
S
L
R
*
G
S
L
R
*
G
V
G
S
L
R
*
RQTND
R
*
Q
T
N
G
R
*
Q
T
N
N
Q
R
*
S
L
Q
R
*
A
Q
R
L
R
*
D
P
D
R
*
M
T
V
D
Y
*
A
aSubstrate peptides selected by kallikrein hK14 using a phage-displayed random pentapeptide library. Plain type residues are common to rAATWT, underlined residues correspond to substrate peptides relocated in RSL of AT variants. The scissile bond by hK14 in substrate peptides is designated by bold and putative cleavage site in serpins is marked by asterisks between the P1-P1′ residues.
The determination of the stoichiometry of inhibitory (SI) and the rate of inhibitory reaction (ka) were performed under physiological conditions of pH and ionic strength to ensure a more relevant comparison. Almost all the newly constructed variants of ACT showed lower SI values with hK14 than wild type ACT. From these variants rACTC11, rACTC11D, rACTG9 and rACTE8 had the lowest stoichiometry of inhibition values for hK14 (4.8, 2.8, 1.5 and 1.2 respectively) and the highest association constants (65000, 74000, 75000 and 575000 M−1 s−1 respectively). Contrary to ACT, the serpin AATwt is a good inhibitor for hK14 with an association constant of 263 000 M−1 s−1. All the AAT variants had a lower association constant than AATwt, but several of them still react at high velocity with hK14, as AATG1, AATG9, AATE8, AATG1g and AATC11 exhibiting a ka of 168 000, 217 000, 242 000, 257 000 and 63 000 M−1 s−1 respectively. Only two AT variants did not inhibit hK14.
A panel of enzymes including trypsin, human neutrophil elastase, chymotrypsin, plasma kallikrein (PK), urokinase (uPA), and thrombin were screened to determine inhibitory specificity of ACT and AAT variants with a SI for hK14 lower than 10 (Table XVIII). When incubated for 30 min with an excess of inhibitor ([I]o/[E]o) of 50:1), hK14 is completely inhibited (100%). Under this condition, wild type ACT showed only 17% inhibition activity toward hK14 contrary to AATwt (100% of inhibition). Among the ACT variants, two (rACTC11 and rACTC11D) show specificity to hK14, inhibiting no other tested enzymes apart from trypsin and chymotrypsin. For AAT variants, these new inhibitors clearly exhibited a higher specificity toward hK14 than AATwt. AATG9 demonstrated to be highly specific to hK14, showing no reactivity with any trypsin-like proteases.
aSerpins and proteases were incubated for 30 min at 37° C. at a [I]o/[E]o ratio of 50:1. Percent inhibition correspond to 100 × (1 − (velocity in presence of inhibitor/velocity of uninhibited control).
Additional hK14 inhibiting ACT variants were screened against a larger panel of tissue kallikreins related to hK14. Partial inhibition was observed against different subsets of tested kallikreins.
aProtease and serpins were incubated for 30 min at 37° C. (90 min at 37° for PSA) at a [I]o/[E]o ratio of 50:1. Percent inhibition correspond to 100 × (1 − (velocity in presence of inhibitor/velocity of uninhibited control)).
The permutation of RSL cleavage site for hK14 phage display selected substrates has changed wild type serpins (ACT and AAT) into highly sensitive inhibitors for hK14, especially AATG9 showing a unique reactivity. To Applicants knowledge, this is the first report describing the development of a specific inhibitor for hK14. The fact that some recombinant inhibitors also inhibited other enzymes than hK14 is not surprising because of the homology of substrate between trypsin-like proteases. Moreover, the velocity of reaction should be determined for recombinant inhibitors toward other enzymes.
Arcanobacterium Haemolyticum,
Mycobacterium Avium-
Mycobacterium Marinum
A potential therapeutic effect of MDPK67b (rACT6.7) on skin diseases has been tested on a Netherton syndrome mouse model as a topical application.
Trial Design
The molecule has been formulated at 2 mg/ml in NATROSOL® (hydroxyethylcellulose (HEC)) 2% (w/v). The formulation has been chosen following in vitro diffusion criteria retaining MDPK67b inhibition property over trypsin (surrogate in vitro substrate). MDPK67b 2 mg/ml, prepared as a solution, is formulated in 2% NATROSOL® (hydroxyethylcellulose (HEC)) (w/v), PBS1× pH7.4 at 4° C. under slow agitation to prevent molecule shearing. The preparation is carefully homogenized under stirring at 4° C. to ensure proper inhibitor repartition within the hydrogel. NATROSOL® (hydroxyethylcellulose (HEC)) has to be added as a powder to MDPK67b solution to avoid clumps and to allow a homogenous formulation without shearing.
To maintain sterility the solutions are autoclaved or filtered through a 0.22 u filter.
MDPK67b potential therapeutic effect has been assessed on a group of 12 transgenic KLK5 mice with different lesion grade severity, starting from a low severity grade (grade 1) to a more severe grade (grade 4) (
Mice have been monitored for changes in lesion grade and lesion size phenotypes. Lesion size has been measured every 3 days and lesion grade was monitored daily
Results (
Comparison of the development of skin lesions on KLK5 transgenic mice of MDPK67b treated versus non-treated mice showed a decrease of lesion sizes within the MDPK67b group (group 2) compared to vehicle group (group 1). Whereas lesion sizes increased in a majority of the vehicle control group, the majority of the MDPK67b treated group showed a decrease in lesion size. A clear size increase was observed in 3 test animals of group1 and 1 within group 2. A slight lesion size increase was observed 1 animal of group 2. No change was reported in 1 animal of group 1. A decrease in lesion size was observed in 1 test animals of group1 and 3 within group 2. The protective effect seems larger in mice with low grade symptoms.
Lesion grade development was also positively affected by topical application of MDPK67b. One MDPK67b treated test animal showed a complete reversion of the phenotype. A partial reversion was seen on a second MDPK67b treated animal. The protective effect seems larger in mice with low grade symptoms.
This application is a continuation of U.S. patent application Ser. No. 14/447,317, filed Jul. 30, 2014, which is a continuation of U.S. patent application Ser. No. 12/863,651, filed Jan. 11, 2009, which is a national stage application under 35 U.S.C. § 371 of PCT Application No. PCT/IB2009/000089, filed Jan. 21, 2009, which claims priority to and the benefit of U.S. provisional patent applications Ser. No. 61/022,386, filed Jan. 21, 2008 and Ser. No. 61/006,576, filed Jan. 22, 2008, each of which is incorporated herein by reference in its entirety. The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 15, 2020, is named 121089-0102_SL.txt and is 184,930 bytes in size.
| Number | Name | Date | Kind |
|---|---|---|---|
| 7105172 | Bolla | Sep 2006 | B1 |
| 8309596 | Flohr | Nov 2012 | B2 |
| 20030054445 | Chen et al. | Mar 2003 | A1 |
| 20060269536 | Deperthes | Nov 2006 | A1 |
| 20140341881 | Deperthes et al. | Nov 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| WO-0007620 | Feb 2000 | WO |
| WO-2004045634 | Jun 2004 | WO |
| WO 2004087912 | Oct 2004 | WO |
| WO-2005117955 | Dec 2005 | WO |
| WO-2006037606 | Apr 2006 | WO |
| WO-2006090282 | Aug 2006 | WO |
| WO-2007072012 | Jun 2007 | WO |
| WO-2009024528 | Feb 2009 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20190183989 A1 | Jun 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61006576 | Jan 2008 | US | |
| 61022386 | Jan 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14447317 | Jul 2014 | US |
| Child | 16217957 | US | |
| Parent | 12863651 | US | |
| Child | 14447317 | US |
