EP1355637A2 - Matriptase hemmstoffe zur behandlung von krebs - Google Patents

Matriptase hemmstoffe zur behandlung von krebs

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Publication number
EP1355637A2
EP1355637A2 EP01944426A EP01944426A EP1355637A2 EP 1355637 A2 EP1355637 A2 EP 1355637A2 EP 01944426 A EP01944426 A EP 01944426A EP 01944426 A EP01944426 A EP 01944426A EP 1355637 A2 EP1355637 A2 EP 1355637A2
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EP
European Patent Office
Prior art keywords
matriptase
subject
compound
hgf
compounds
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English (en)
French (fr)
Inventor
Chen-Yong Lin
Robert B. Dickson
Shaomeng University of Michigan WANG
Istvan Enyedy
Sheau-Ling Lee
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Georgetown University Medical Center
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Georgetown University Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)

Definitions

  • the present invention relates to methods for diagnosing and treating conditions involving matriptase activity, particularly cancer.
  • the invention is based on the elucidation of matriptase activity and involvement in the development of precancerous and cancerous conditions.
  • the invention relates to the design of bioassay testing methods for cancer diagnosis based on the detection of matriptase activity, identification of compounds capable of inhibiting matriptase activity and design of cancer therapy protocols based on the inhibition of matriptase employing small molecule inhibitors.
  • MMPS matrix metalloproteases
  • plasmin/urokinase type plasminogen activator system plasmin/urokinase type plasminogen activator system
  • lysosomal proteases cathepsins D and B reviewed byMignatti et al., Physiol. Rev. 73: 161-95 (1993).
  • the plasmin/urokinase type plasminogen activator system is composed of plasmin, the major ECM-degrading protease; the plasminogen activator, uPA; the plasmin inhibitor ⁇ 2-anti-plasmin, the plasminogen activator inhibitors PAI-1 and PAI-2; and the cell membrane receptor for uPA (uPAR) (Andreasen et al, Int. J. Cancer 72: 1-22 (1997)).
  • the MMPs are a family of zinc-dependent enzymes with characteristic structures and catalytic properties.
  • the plasmin/urokinase type plasminogen activator system and the 72-kDa gelatinase (lVlMP-2)/membrane-type MMP system have received the most attention for their potential roles in the process of progression of breast cancer and other carcinomas.
  • both systems appear to require indirect mechanisms to recruit and activate the major ECM-degrading proteases on the surface of cancer cells.
  • uPA is produced in vivo (Nielson et al, Lab. Invest. 74: 168-77 (1996); Pyke et al, Cancer Res. 53: 1911-15 (1993); Polette et al, Virchows Arch.
  • stromelysin-3 MMP-11
  • MTMMP MMP-14
  • stromal origins of these well-characterized extracellular matrix-degrading proteases may suggest that cancer progression is an event which either depends entirely upon stromal-epithelial cooperation or which is controlled by some other unknown epithelial-derived proteases.
  • Search for these epithelial-derived proteolytic systems that may interact with plasmin/urokinase type plasminogen activator system and/or with MMP family could provide a missing link in the understanding of malignant progression.
  • Matriptase was initially identified from T-47D human breast cancer cells as a major gelatinase with a migration rate between those of Gelatinase A (72-kDa, MMP- 2) and Gelatinase B (92-kDa, MMP-9).
  • matriptase has been proposed to play a role in the metastatic invasiveness of breast cancer. (See U.S. Patent 5,482,848, the contents of which are incorporated herein by reference in their entirety.)
  • the primary cleavage specificity of matriptase was identified to be arginine and lysine residues, similar to the majority of serine proteases, including trypsin and plasmin.
  • matriptase like trypsin, exhibits broad spectrum cleavage activity, and such activity is likely to contribute to its gelatinolytic activity.
  • HGF activator inhibitor- 1 HGF activator inhibitor- 1
  • HAI-1 Hepatocyte growth factor activator inhibitor- 1
  • HGF activator a blood coagulation factor XJ-like serine protease.
  • the mature form of this protease inhibitor has 478 amino acid residues, with a calculated molecular mass of 53,319.
  • a putative transmembrane domain is located at its carboxyl terminus.
  • HAI-1 contains two Kunitz domains (domain I spans residues 246-306; domain II spans residues 371 to 431) separated by a LDL receptor domain (residues 315 to 360).
  • the presumed PI residue of active-site cleft is likely to be arginine-260 in Kunitz domain I and lysine 385 in domain II by alignment with bovine pancreatic trypsin inhibitor (BPTI, aprotinin) and with other Kunitz-type inhibitors.
  • BPTI bovine pancreatic trypsin inhibitor
  • HAI-1 has specificity against trypsin-type proteases.
  • HAI-1 was originally purified from the conditioned media of carcinoma cells as a 40-kDa fragment doublet, rather than the proposed, mature, membrane-bound, 53-kDa form (Shimomura et al, J. Biol. Chem. 272: 6370-76 (1997)).
  • the protein inhibitors of serine proteases can be classified into at least 10 families, according to various schemes. Among them, serpins, such as maspin (Sheng et al, Proc. Natl. Acad. Sci. USA 93: 11669-74 (1996)) and Kunitz-type inhibitors, such as urinary trypsin inhibitor (Kobayashi et al, Cancer Res. 54: 844-49 (1994)) have been previously implicated in suppression of cancer progression. The Kunitz- type inhibitors form very tight, but reversible complexes with their target serine proteases. The reactive sites of these inhibitors are rigid and can simulate optimal protease substrates.
  • the interaction between a serine protease and a Kunitz-type inhibitor depends on complementary, large surface areas of contact between the protease and inhibitor.
  • the inhibitory activity of the recovered Kui ⁇ tz-type inhibitor from protease complexes can always be reconstituted.
  • the Kunitz-type inhibitors may be cleaved by cognate proteases, but such cleavage is not essential for their inhibitory activity.
  • serpin-type inhibitors also form tight, stable complexes with proteases; in most of cases these complexes are even more stable than those containing Kunitz-type inhibitors. Cleavage of serpins by proteases is necessary for their inhibition, and serpins are always recovered in a cleaved, inactive form from protease reactions.
  • serpins are considered to be suicide substrate inhibitors, and their inhibitory activity will be lost after encounters with proteases.
  • the suicide nature of serpin inhibitors may result in regulation of proteolytic activity in vivo by direct removal of unwanted proteases via other membrane-bound endocytic receptors (in the case of uPA inhibitors).
  • the Kunitz type inhibitors may simply compete with physiological substrates (such as ECM components), and in turn, reduce their availability for proteolysis. These differences may result in different mechanisms whereby these proteases perform their roles in ECM-degradation and cancer progression.
  • BBI Bowman-Birk inhibitor
  • small molecule inhibitors provide valuable tools on functional studies in various systems. It is highly desirable to provide small molecules that can inhibit matriptase activity. Based on the potential effects thereof on carcinoma progression, cell migration, proliferation and apoptosis, it would be beneficial if compounds could be obtained which selectively block the proteolytic activity of matriptase. More specifically, it would be beneficial if compounds could be obtained which antagonize cascade protease activators produced by cleavage of matriptase substrates. Such compounds have significant therapeutic potential, in particular for treatment of cancer and other conditions involving carcinoma progression and migration and abnormal cell differentiation and proliferation. Compounds having improved selectivity, solubility and stability are particularly desirable.
  • the present invention provides a method of inhibiting carcinoma progression wherein matriptase plays a role in a subject in need of such inhibition comprising administering to a subject an effective amount of a compound comprising two groups capable of being positively charges at physiological pH, which are the same or different.
  • the groups are linked by a chemical group having a length of between 5 and 30 A, and preferably between 15 and 24 A.
  • the groups are preferably selected from the following groups:
  • Preferred compounds include the compounds of Examples I through VI which have the structures below, wherein X and Y can be any substituents.
  • the present invention provides a method of inhibiting carcinoma progression wherein matriptase plays a role in a subject in need of such inhibition comprising administering to a subject an effective amount of a compound selected from the group consisting of compounds A, B, C, D, E, F, G and analogs thereof.
  • Compounds A, B, C, D, E, F and G have the following respective structures:
  • the invention provides a method of treating malignancies, pre-malignant conditions, and pathologic conditions in a subject which are characterized by the expression of single-chain (zymogen) and/or two-chain (activated) form of matriptase comprising administering a therapeutically effective amount of a compound selected from the group consisting of compounds A, B, C, D, E, F, G and analogs thereof.
  • the invention provides a method of therapy which results in the inhibition of matriptase in a subject in need of such inhibition which comprises administering a therapeutically effective amount of at least one compound selected from the group consisting of compounds A, B, C, D, E, F, G and analogs thereof.
  • the invention provides a method of treating cancer comprising administering a therapeutically effective amount of at least one compound selected from the group consisting of compounds A, B, C, D, E, F, G and analogs thereof.
  • the invention provides a method of diagnosing cancer comprising exposing a tissue sample to an antibody or immunogenic fragment thereof which recognizes and binds to a product of matriptase mediated proteolysis of a matriptase substrate.
  • the invention provides an in vivo method of diagnosing the presence of a pre-malignant lesion, a malignancy or other pathologic condition in a subject comprising the steps of:
  • the invention provides an in vitro method of diagnosing the presence of a pre-malignant lesion, a malignancy, or other pathologic condition, in a subject, which is characterized by the presence of a product of matriptase mediated proteolysis of a substrate of matriptase comprising the steps of:
  • Figs. 1 A and IB show how matriptase converts single-chain HGF into smaller fragments, which can be recognized by anti- _-chain HGF and _-chain HGF antibodies;
  • Fig. 1A shows the silver-stained protein patterns of HGF incubated overnight without (0) or with increasing amount of matriptase ( );
  • Fig. IB shows Western immunoblot. HGF incubated overnight without (-) or with (+) matriptase was immunoblotted with anti-JHGF (_ chain) or anti-JHGF (_ chain);
  • Figs. 2 A and 2B show how matriptase-cleaved HGF stimulates scattering on MDCK cells, plates show wells in the 96-well plates with about 3000 MDCK cells after 20 h in the absence (CRL) or presence of matriptase-cleaved HGF (HGF/MTP) or untreated HGF (HGF) at the dilutions shown, with leupeptin being included in every culture;
  • Fig. 3 shows how matriptase-cleaved HGF stimulates c-Met tyrosine phosphorylation
  • A549 cells were treated in the absence of HGF (NO HGF), matriptase-cleaved HGF (HGF/MTP), or HGF alone (HGF) for 5 min at 37 _C; equal amounts of lysed cell protein were immunoprecipitated with anti-c-Met antibody followed by immunodetection using anti-phosphotyrosin antibody (Tyr-p) as described below; in controlling for the amounts of c-Met in each sample, immunoblots were then stripped and detected with anti-c-Met antibody (c-Met); wherein the shown arrow points to the p 145 kDa _ chain of c-Met under reducing conditions;
  • Fig. 4 shows how plasminogen is not a substrate for matriptase, this figure shows the silver-stained protein patterns of plasminogen incubated overnight without (0) or with increasing amount of matriptase ( ); with the highest amount of matritpase used is 8-fold of the lowest amount of matriptase that cleaves HGF;
  • Figs. 5A and 5B show how Pro-uPA is activated by matriptase cleavage
  • Fig. 5 A shows how single-chain pro-uPA is converted into two-chain form uPA by matriptase
  • pro-uPA was incubated overnight active matriptase in the absence (0) or presence of increasing amount of matriptase ( ); the cleaved products were analyzed by electrophoresis followed by silver-staining
  • Fig. 5B shows how matriptase cleavage of pro-uPA generates an active protease
  • pro-uPA was either incubated for 30 min with matriptase (closed circles) or incubated 1 min with plasmin r
  • Fig. 6 shows a ribbon model 3-D structure of matriptase; the structure is obtained through homology modeling and refinement by molecular dynamics simulation;
  • Fig. 7 shows a model 3-D structure of matriptase with compound A; the structure is obtained through molecular dynamics docking using the generalized effective potential;
  • Fig. 8 shows a model 3-D structure of matriptase with compound B; the structure is obtained through molecular dynamics docking using the generalized effective potential
  • matriptase is meant a trypsin-like protein, with a molecular weight of between 72-kDa and 92-kDa and is related to SEQ ID NO: 4 or is a fragment thereof. It can include both single-chain and double-chain forms of the protein.
  • the zymogen form (inactive form) of matriptase is a single-chain protein.
  • the two-chain form of matriptase is the active form of matriptase, which possesses catalytic activity. Both forms of matriptase are found to some extent in milk and cancer cells because extracellular matrix (ECM) remodeling is necessary to both normal and pathologic remodeling processes. Both cancer cells and milk contain the different forms of matriptase. However, in milk the dominant form is the activated form of matriptase which then binds to HAI- 1.
  • matriptase modulating compound or “matriptase modulating agent” is meant a reagent which regulates, preferably inhibits the activation of matriptase (e.g., cleavage of the matriptase single-chain zymogen to the active two-chain moiety) or the activity of the two-chain form of matriptase. This inhibition can be at the transcriptional, translation, and/or post-translational levels. Additionally, modulation of matriptase activity can be via the binding of a compound to the zymogen or activated forms of matriptase.
  • matriptase e.g., cleavage of the matriptase single-chain zymogen to the active two-chain moiety
  • matriptase expressing tissue any tissue which expresses any form of matriptase, either malignant, pre-malignant, normal tissue, or tissue which is subject to another pathologic condition.
  • malignant is meant to refer to a tissue, cell or organ which contains a neoplasm or tumor that is cancerous as opposed to benign.
  • Malignant cells typically involve growth that infiltrates tissue (e.g., metastases).
  • benign is meant an abnormal growth which does not spread by metastasis or infiltration of the tissue.
  • the malignant cell of the instant invention can be of any tissue; preferred tissues are epithelial cells.
  • tumor progression or “tumor metastasis” is meant the ability of a tumor to develop secondary tumors at a site remote from the primary tumor. Tumor metastasis typically requires local progression, passive transport, lodgement and proliferation at a remote site. This process also requires the development of tumor vascularization, a process termed angiogenesis. Therefore, by “tumor progression” and “metastasis,” we also include the process of tumor angiogenesis.
  • pre-malignant conditions or "pre-malignant lesion” is meant a cell or tissue which has the potential to turn malignant or metastatic, and preferably epithelial cells with said potential.
  • Pre-malignant lesions include, but are not limited to: atypical ductal hyperplasia of the breast, actinic keratosis (AK), leukoplakia, Barrett's epithelium (columnar metaplasia) of the esophagus, ulcerative colitis, adenomatous colorectal polyps, erythroplasia of Queyrat, Bowen's disease, bowenoid papulosis, vulvar intraepithelial neoplasia (NIN), and displastic changes to the cervix.
  • AK actinic keratosis
  • NIN vulvar intraepithelial neoplasia
  • tumor formation-inhibiting effective amount is meant an amount of a compound, which is characterized as inhibiting activation of matriptase or matriptase activity, and which when administered to a subject, such as a human subject, prevents the formation of a tumor, or causes a preexisting tumor, or pre-malignant condition, to enter remission. This can be assessed by screening a high-risk patient for a prolonged period of time to determine that the cancer does not arise and/or the pre-malignant condition is reversed.
  • a tumor formation- inhibiting effective amount also includes an amounts which provides ameliorative to relief to the subject.
  • the tumor formation-inhibiting effective amount can also be assessed based on its effect on blood circulation of inhibitors, such as BBIC.
  • Preferred tumor formation-inhibiting effective amounts of agents such as BBIC are in the range of 100 ⁇ g/kg to 20 mg/kg body weight of the subject. More preferred ranges include 1 ⁇ g/kg to 10 mg/kg body weight of the subject.
  • labeling agent is meant to include fluorescent labels, enzyme labels, and radioactive labels.
  • radioisotope for use in humans for purposes of diagnostic imaging.
  • Preferred radioisotopes for such use include: 67 Cu, 67 Ga, 99 Te, 131 L 123 L 125 I, m h , 188 Re, 186 Re and 90 Y.
  • fluorescent label is meant any compound used for screening samples (e.g., tissue samples and biopsies) which emits fluorescent energy.
  • Preferred fluorescent labels include fluorescein, rhodamine and phycoerythrin.
  • biological sample is meant a specimen comprising body fluids, cells or tissue from a subject, preferably a human subject.
  • the biological sample contains cells, which can be obtained via a biopsy or a nipple aspirate, or are epithelial cells.
  • the sample can also be body fluid that has come into contact, either naturally or by artificial methods (e.g. surgical means), a malignant cell or cells of a pre-malignant lesion.
  • matriptase expressing tissue is meant any biological sample comprising one or more cells which expresses a form or forms of matriptase.
  • subject is meant an -tnimal, preferably mammalian, and most preferably human.
  • antibody is meant to refer to complete, intact antibodies, and Fab fragments and F(ab) 2 fragments thereof. Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies (niAb), chimeric antibodies and humanized antibodies.
  • antibody fragments e.g., Fab fragments and F(ab) 2 fragments
  • immunogenic fragment a portion of a matriptase protein which induces humoral and/or cell-mediated immunity but not immunological tolerance.
  • epipe is meant a region on an antigen molecule (e.g., matriptase) to which an antibody or an immunogenic fragment thereof binds specifically.
  • the epitope can be a three dimensional epitope formed from residues on different regions of a protein antigen molecule, which, in a native state, are closely apposed due to protein folding.
  • "Epitope” as used herein can also mean an epitope created by a peptide or hapten portion of matriptase and not a three dimensional epitope.
  • the present invention is based on recent discoveries relating to a novel serine protease system, matriptase and its cognate inhibitor (the hepatocyte growth factor activator inhibitor 1). Considering its unique characteristics: the epithelial and cancer cells origin, the membrane integral property, and the multiple putative regulatory domains, the invention is generally based on the hypothesis that matriptase functions as an upstream activator in matrix progression. In particular, the present invention is based on the discovery that matriptase can convert single chain hepatocyte growth factor/scatter factor precursor to the active factor that can induce scatter of Madin- Darby canine kidney epithelial cells and can activate c-Met tyrosine phosphorylation in A549 human lung carcinoma cells. In addition, it has been discovered that matriptase can activate urokinase plasminogen activator but not plasminogen.
  • the subject invention is directed to all conditions wherein matriptase plays a role.
  • the invention provides novel cancer diagnosis and therapy methods based in part on a novel mechanism in the epithelial-mensenchymal interaction that an epithelial membrane activator regulates the activation of the stromal-origin factors in the extracellular matix degradation and in the cell migration.
  • the present inventors have conducted extensive research the initial results of which allowed for the discovery of matriptase and the elucidation of some aspects of its activity.
  • the discoveries associated with matriptase have been incorporated in a research program aimed at providing diagnosis and therapy protocols for the treatment of various cancers, particularly breast cancer. These protocols are based in part on the elucidation of the mechanistic aspects associated with matriptase activity and the utilization of computer modeling tools in combination with biological testing in the identification of antibody diagnostic agents and small molecule matriptase inhibitors as therapeutic agents.
  • Matriptase is a trypsin-like serine protease with two regulatory modules: two tandem repeats of the complement subcomponent Clr/s domain and four tandem repeats of LDL receptor domain (Lin et al, J. Biol. Chem. 274: 18231-6 (1999)).
  • two tandem repeats of the complement subcomponent Clr/s domain and four tandem repeats of LDL receptor domain (Lin et al, J. Biol. Chem. 274: 18231-6 (1999)).
  • human milk was studied. It was found that milk-derived matriptase strongly interacts with fragments of HAI-1 to form SDS-stable complexes.
  • the mosaic protease is characterized by trypsin-like activity and two regulatory modules (e.g., LDL receptor and complement subcomponent Clr/s domains).
  • Matriptase was initially purified from T-47D human breast cancer cells. h breast cancer cells, matriptase was detected mainly as an uncomplexed form; however, low levels of matriptase were detected in SDS-stable, 110- and 95-kDa complexes that could be dissociated by boiling. In striking contrast, only the complexed matriptase was detected in human milk.
  • the complexed matriptase has now been purified by a combination of ionic exchange chromatography and immunoaffinity chromatography.
  • Amino acid sequences obtained from the matriptase-associated proteins reveal that they are fragments of an integral membrane, Kunitz-type serine protease hibitor that was previously reported to be an inhibitor (termed HAI-1) of hepatocyte growth factor activator.
  • HAI-1 Kunitz-type serine protease hibitor
  • matriptase and its complexes were also detected in four milk- derived, SN-40 T-antigen-immortalized mammary luminal epithelial cell lines, but not in two human foreskin fibroblasts nor in HT1080 fibrosacroma cell line.
  • the milk- derived matriptase complexes are likely to be produced by the epithelial components of lactating mammary gland in vivo, and the activity and function of matriptase may be differentially regulated by its cognate inhibitor, comparing breast cancer with the lactating mammary gland.
  • matriptase and its cognate Kunitz-type serine protease inhibitor can be characterized as an extracellular, trypsin-like protease , system, hi vivo expression of matriptase and its inhibitor was observed in breast cancer cells, breast epithelial cells, and in other human carcinomas including ovarian, endometrial, and colon, h no case were matriptase or the inhibitor found in fibroblasts or fibrosarcomas.
  • Matriptase is a type 2 integral membrane serine protease that contains tandem repeats of putative regulatory domains: the LDL receptor domains and the CUB (Clr/s, Uegf and Bone morphogenetic protein-1) domains (2;3) (also see updated sequence in the GenBank/EBI Data Bank with accession number AF118224).
  • the cognate inhibitor of matriptase is a type 1 integral membrane protein containing two Kunitz domains separated by a LDL receptor domain.
  • the inhibitor has been characterized as an inhibitor of hepatocyte growth factor activator (HGF).
  • HGF hepatocyte growth factor activator
  • matriptase is likely to be resulted from the shedding of the surface-bound matriptase.
  • matriptase is involved in cell migration and progression by acting as a direct upstream regulator. Without wishing to be bound by any particular theory, it is believed that matriptse is presented on the tumor or epithelial cell surface and interacts with a variety of factors and digests a broad range of substrates. These actions, together, may induce dramatic changes in the extracellular environment and in the surrounding cells contributing to cell migration, progression, and metastasis.
  • HGF also known as scatter factor (SF)
  • SF scatter factor
  • HGF/SF can stimulate cell mptility, promote matrix progression, and induce morphogenesis.
  • HGF activator inhibitor 1 HGF activator inhibitor 1
  • Human lung carcinoma cell line A549 was from ATCC.
  • Madin-Darby canine kidney (MDCK H) epithelial cell lines and the single-chain form HGF protein were the generous gifts from Dr. George Vande Woude (NCI, NIH, Fredrick, MD).
  • Single-chain form of human urokinase plasminogen activator (pro-uPA) was purchased from American Diagnostic h e (Greenwich. CT).
  • Plasminnogen, plasmin, and fluorescent substrate peptides N-tert- Butoxy-Carbonyl (N-t-Boc)-Gm-Ala-Arg-7-Amido-4-Methylcoumarin (AMC), N-t- Boc-Leu-Gly-Arg-AMC, N-t-Boc-_-Benzyl (Bz)-Glu-Gly-Arg-AMC, N-t-Boc-_-Bz- Glu-Ala-Arg-AMC, N-Succinyl (Suc)-Ala-Phe-Lys-AMC, Suc-Leu-Leu-Val-Tyr- AMC, Suc-Ala-Ala-Pro-Phe-AMC, and Suc-Ala-Ala-Ala-AMC were from Sigma chemical company (St.
  • the enzyme activity of matriptase was measured at room temperature in a reaction buffer containing 100 mM Tris-HCl (pH 8.5) and 100 _g/ml of BSA using fluorescent peptide as substrate, hi brief, 10 _1 of enzyme solution and 10 _1 of peptide substrate were added to a cuvette containing 180 _1 of the reaction buffer. The mixture was mixed well, placed back to a fluorescent spectrophotometer (HITACH F4500), and the release of fluorescence resulting from hydrolysis of the peptide substrate was recorded with excitation at 360 nm and emission at 480 nm.
  • HITACH F4500 fluorescent spectrophotometer
  • kinetic parameter _ Substrate concentration versus initial reaction velocity were analyzed by the Michaelis-Menten equation and plotted using SigmaPlot software. Double reciprocal (Lineweaver-Burk) plots thus derived were used to determine Nmax and Km values.
  • DME Dulbecco's Modified Eagle
  • FCS fetal calf serum
  • Met phosphorylation detection _ A549 cells were grown confluent in RPMI medium supplemented with 10% FCS. After 3 -hour serum-starvation, cells were incubated 5 min incubation at 37 _C with 450 ng/ml HGF or matriptase-cleaved HGF in RPMI medium supplemented with 5% FCS. Where it is needed, leupeptin was included in the medium at 100 _g/ml. Medium were removed, cells were rinsed with lx PBS and collected by centrifugation following trypsinization. After washed one more time with lx PBS, cell pellets were frozen in dry-ice.
  • the frozen cell pellets were either stored at -80 _C for later extraction or immediately extracted as described below.
  • Cells were thawed on ice, extracted by suspension in buffer containing 50 mM Tris-HCl ( ⁇ H7.5), 5 mM EDTA, 150 mM sodium floride, 10 mM sodium pyrophosphate, 10 mM sodium ortovanadate, 100 _g/ml PMSF, 10 _g/ml leupeptine, 10 _g/ml apropeptenin, and 1 % Triton X- 100.
  • Extracts were clarified by centrifugation for 15 min at 12,000 xg in a microfuge, and the protein concentration was determined by BCATM protein assay kit (PIERCE, Rochford, IL) using bovine serum albumin (BSA) as standard. About 2 mg protein of extracts were immuoprecipitated using anti-c-Met antibody and Pansorbin (Calbiochem, La Jolla, CA). The protein-antibody-Pansorbin immunocomplex was collected by centrifugation, washed twice with extraction buffer then with lx PBS, and then dissociated by boiling in SDS-sample buffer.
  • BCATM protein assay kit PIERCE, Rochford, IL
  • BSA bovine serum albumin
  • the Pansorbin was removed by centrifugation, the supernatant fractions were subjected to 8% SDS electrophoresis, and the proteins were detected by Western immunoblot with anti-phosphotyrosin antibody. The same immunoblot were then stripped inlOO mM 2-mercaptoethonal, 2% SDS, and 62.5 mM Tris-HCl ( ⁇ H7.6) for 30 min at 50 _C and re-probed with antic-Met antibody.
  • Gin- Ala- Arg is a standard substrate for trypsin
  • Leu-Gly-Arg is a substrate for uPA
  • Leu-Leu-Nal-Tyr and Ala-Ala-Pro-Phe are the substrates for chymotrypsin
  • Ala- Ala-Ala is the substrate for elase. No cleavage activity was detected with these substrates at concentration of 200 _M.
  • Table 1 shows that, as expected from the sequence analysis, matriptase, like trypsin, prefers to cleave peptides at Arg or Lys.
  • the best substrate peptide for matriptase is Boc-Gln-Ala-Arg-Amc, the best substrate for trypsin. No released fluorescence was detected from the substrate for chymotrypsin or elastase (table 1, peptide substrates 7, 8, 9).
  • Matriptase appears to prefer to bind to peptides containing small side chain amino acids, such as Ala and Gly, at P2 site (table 1, peptide substrates 1-5).
  • Peptides containing P2 Ala are better substrates for matriptase than peptides containing P2 Gly (compare peptides 1, 2 with peptides 3-5).
  • matriptase The binding affinity of matriptase to the formers is about 30-fold higher that to the latter.
  • a change from Gin to Glu at the P3 site significant reduces the Nmax (compare peptide 1 with 2) without causing significant change on Km.
  • Information from matriptase peptide substrate specificity led us to look for macromolcular substrates that require cleavage at Arg for activation.
  • HGF/SF is secreted as an inactive, single chain precursor by stromal cells, and it is activated by proteolytic conversion to the two-chain form factor by cleavage at Arg-495 (19) in the extracellular environment.
  • HAH is also the inhibitor of matriptase.
  • HGF/SF appears to be an ideal substrate candidate for matriptase.
  • HGF/SF can be cleaved by matriptase
  • inactive HGF/SF purified from the condition medium of fibroblast cells cultured in the absence of serum was taken in the following experiments.
  • Fig. 1 A shows that this HGF/SF preparation is primarily composed of the single-chain form protein about 97 kDa on SDS-PAGE (lane a).
  • 64 kDa and 33 kDa apparent molecular weight corresponding to the expected sizes of activation cleaved _- and _- chains of HGF/SF, respectively.
  • Figs. 2A and 2B show the images of plates of MDCK II cells after incubation with various dilutions of untreated (HGF) or matriptase-treated (HGF/MTP) HGF/SF.
  • HGF untreated
  • HGF/MTP matriptase-treated
  • HGF operates its function through binding to its cell surface receptor, Met.
  • HGF/MTP matriptase-cleaved HGF
  • HGF untreated HGF
  • No HGF untreated HGF
  • Phosphotyrosine of c-Met in cells incubated with untreated HGF appears to caused by the residual active HGF contamination in the preparation.
  • Leupeptin did not affect the total c-Met expression, the c-Met phosphorylation, or the total pattern of tyrosine phosphorylation (data not shown).
  • Plasmin has long been regarded as the enzyme that converts pro-uPA to active uPA.
  • the level of active uPA is not reduced in the urine of mice bearing a targeted disruption of the plasminogen gene , suggesting the existence of plasmin independent pro-uPA activation.
  • Plasma kallikrein, trypsin-like proteases from human ovarian tumors, a T cell-associated serine protease, cathepsins B and L, nerve growth factor _, human mast cell trypase, and prostate specific antigen have also been reported to activate pro-uPA.
  • the roles of these enzymes in vivo, relevant to activation of pro-uPA in vivo are not clear.
  • Fig. 5 A showed that after incubation with the matriptase, the 55 kDa single-chain pro-uPA was converted into smaller fragments.
  • One of these cleavage products clearly appeared on the protein gel as the 33 kDa molecule, which resembles the size of the active uPA protease (Fig. 5A).
  • the cleaved product exhibited enzymatic activity toward the fluorescent peptide substrate, Boc- Leu-Gly-Arg-AMC, for uPA (Fig. 5B, compare the closed circles with closed triangles).
  • matriptase By using the active matriptase isolated from human milk, we report in this study that matriptase cleaves and converts HGF into a biologically functional factor that can induce c-Met activation of and stimulate epithelial cells scattering, hi addition, we also noted that matriptase can activate pro-uPA but not plasminogen. These results further support matriptase to be an upstream regulator in matrix progression. Most significantly, these results reveal a novel mechanism in the control of tissue remodeling that involve an upstream epithelial membrane activator and downstream stromal effectors. Tissue remodeling is a process observed both in physiological processes such as embryonic development, morphogenesis, and wound healing, and in pathological situations such as cancer progression and metastasis.
  • epithelial-mensenchymal transition transforms rigid epithelial cells to the mobile menchymal cell that can migrate distances; 2) extracellular matrix degradation that open path for the migrating cells.
  • HGF is a potent inducer of epithelial-menchymal transition; engagement of HGF to its epithelial compartment c-Met triggers various signaling subsequently induces various cellular responses.
  • HGF is secreted as inactive precursor by stromal cells and is proteolytically activated in the extracellylar environment. Therefore, activation of HGF/SF needs to occur in the close vicinity of the epithelial cells.
  • the network of the serine proteases uPA/uPA-receptor/plasminogen and the zinc-dependent metalloproteinases have been proposed to be responsible for the majority of proteolysis of pericellular proteins.
  • both systems are largely synthesized by the stromal cells and require indirect mechanisms for their recruitment and activation on the surfaces of epithelial cells.
  • an epithelial-derived protease like matriptase would preferably provide the missing link in this process.
  • the process of extracellular matrix degradation requires proteases cascades; proteolytic activation of one protease depends on fhe proteolytic activation of its activator. A central question in this process is that of whether there is a primary initiating enzyme.
  • matriptase can activate both pro-uPA and HGF/SF suggesting that it should be considered as a central regulator in epithelial cell remodeling and/or progression of extracellular matix.
  • Matriptase contains a serine protease activation motif; its activation is likely to require the proteolytic cleavage at Arg-Nal.
  • Autoactivation has been reported with the recombinant protease domain of matriptase.
  • These domains may assist in accumulating other factors at the contact sites between cancer and stromal cells; these domains may also be involved in the activation of matriptase.
  • Clues may lie in its ⁇ -terminal non-catalytic domains, particularly the CUB domains. It has been shown that CUB-EGF module in the Clr and Cls subunits play essential role in the assembly and auto-activation of the Cl complex.
  • the same domains in matriptase may also be critical for its function. Specifically, they may contribute to an intermolecular interaction between matriptase molecules. Thus these domains may be involved in the auto-activation of matriptase, like they do in Cl complex.
  • Matriptase appears to have selectivity for the macromolecular substrates. In our experiments, matriptase did not cleave plasminogen, despite that the high sequence homology between plasminogen and HGF. Another group recently has also reported this substrate selectivity. In the same report, it was also showed that matriptase/MT- SP1 has selectivity for a basic residue at P3 or P4 site.
  • the sequence at the activation cleavage site of HGF and plasminogen is P4-(Lys)-P3-(Gln)-P2-(Gly)-Pl-(Arg) and P4-(Cys)-P3-(Pro)-P2-(Leu)-Pl-(Arg), respectively.
  • plasminogen contains a Lys-binding site that serves to mediate its localization to fibrin and to cellular surfaces. Plasminogen circulates in the blood in a globular and closed conformation; upon binding to the surface, it shifts to an extended and opened conformation. This conformation change promotes its recognition by its activator and its rapid convert to plasmin.
  • the kringle domains on HGF/SF also contribute to its binding to its receptor c-Met.
  • HGF/SF cleavage-activation of HGF/SF does not depend on its binding to c-Met. It is possible that the single chain form HGF/SF exhibit a more open conformation than does plasminogen; and that matriptase can distinguish this subtle structural difference.
  • the focus of the subject invention is to provide therapies based on compounds capable of interfering with matriptase protease activity
  • the invention provides therapies based on existing compounds which are identified through computational modeling as inhibitors of matriptase activity.
  • the compounds are identified through structure-based three-dimensional (3D) database searching.
  • the compounds identified through database searching are processed through biological tests to identify one or more lead compounds for clinical testing and/or rational drug design refinement.
  • Computationally predicting a compound's binding affinity to a host protein involves utilizing the three dimensional (3-D) structures of the host protein and the compound.
  • the 3-D structure of the compound is obtained from a database of chemical compounds.
  • the 3-D structure of the host protein can also be obtained from a protein database.
  • the invention provides potent and specific matriptase inhibitors through a structure-based drug discovery approach.
  • the methodology employed in the discovery of matriptase inhibitors is disclosed in U.S. Patent Application No. 09/301,339, filed on April 29, 1999, the contents of which are hereby incorporated by reference in their entirety.
  • a model structure can be constructed based on the known tertiary structure of a protein which is homologous to the target protein.
  • the structure of the homologous protein is used to construct a template structure of all or part of the target protein.
  • the structure obtained through homology modeling provides a working structure for further refinement.
  • the working structure for the protein not having a known structure is obtained by refining the template structure.
  • each atom of the backbone of the target protein is assigned a position corresponding to the position of the equivalent backbone atom in the homologous protein.
  • each atom of a side chain of the target protein having an equivalent side chain in the homologous protein is assigned the position corresponding to the position of the atom in the equivalent side chain of the homologous protein.
  • the atom positions of a side chain not having an equivalent in the homologous protein are determined by constructing the side chain according to preferred internal coordinates and attaching the side chain to the backbone of the host protein.
  • the template structure thus obtained is refined by minimizing the internal energy of the template protein.
  • the positions of the atoms of the side chains having no equivalents in the homologous protein are adjusted while keeping the rest of the atoms of the template protein in a fixed position. This allows the atoms of the constructed side chains to adapt their positions to the part of the template structure determined by homology.
  • the full template structure is then minimized (relaxed) by allowing all the atoms to move. Relaxing the template 3-D structure of the protein eliminates unfavorable contacts between the atoms of the protein and reduces the strain in the template 3-D structure.
  • a host-guest complex is formed by disposing a compound from a compound database in a receptor site of the protein.
  • the structure of the host-guest complex is defined by the position occupied by each atom in the complex in a three dimensional referential.
  • a geometry-fit group is formed by selecting the compounds which can be disposed in the target binding site without significant unfavorable overlap with the atoms of the protein. For each compound in the geometry fit group, a predicted binding affinity to the receptor site of the host protein is determined by minimizing an energy function describing the interactions between the atoms of the compound and those of the protein. The minimization of the energy function is conducted by changing the position of the compound such that a guest-host complex structure corresponding to a minimum of the energy function is obtained. The compounds having the most favorable energy interaction with the atoms of the binding site are identified for optional further processing, for example through display and visual inspection of compound protein complexes to identify the most promising compound candidates.
  • the displayed complexes are visually examined to form a group of candidate compounds for in vitro testing.
  • the complexes are inspected for visual determination of the quality of docking of the compound into the receptor site of the protein. Visual inspection provides an effective basis for identifying compounds for in vitro testing.
  • Potent and selective inhibitors are tested further in their ability to inhibit colony-formation in soft-agarose.
  • Compounds having good in vitro activity are tested in vivo.
  • Tumor bearing mice are treated with therapy, based on the compounds and the effect on the tumor size is observed.
  • Compounds showing effective tumor reduction are then used in clinical trial protocols.
  • Computational identification of compounds having potential matriptase inhibitory activity To date, the experimental 3D structure matriptase has not been determined.
  • the X-ray structure of human thrombin, entry lhxe from the protein databank was used as template for building the 3D structure of matriptase through homology modeling. Since the sequence identity and similarity between template and model are 34% and 53%, respectively, the 3D structure of matriptase can be modeled accurately.
  • the serine protease domain (b-chain) of matriptase has a catalytic triad formed by Serl91, His42 and Asp97, that is positioned on the surface of the protease.
  • a negatively charged residue, Aspl85 is located at the bottom of the SI binding site, Fig. 6, which is consistent with the experimental data showing the preference of matriptase for substrates with positively charged residues, Arg/Lys at the PI position.
  • the disulfide bond between Cys216 and Cysl87 stabilize the position of As ⁇ l85. Gly215, Cys216, Ala217 and Gln218 are at the entrance of the SI binding pocket. An anionic site formed by Asp46, Asp47 and Asp91 may also be important for inhibitor binding to matriptase. A putative hydrophobic SI' binding site is marked by Leul8, Ala20, Leu21, Ile26 and Trp58. The disulfide bond between Cys27 and Cys43 may stabilize the position of Ile26, which may be important for the geometry of this binding site.
  • the sequence for MTP was obtained from sequencing data. Templates for homology modeling were obtained by searching the Protein Databan, using the program BLAST. As discussed above, the structure of thrombin, entry 1HXE with 34% identities, 53% similarity and 6% gaps, was used as a template for modeling MTP using the program MODELLER.
  • the structure obtained from homology modeling was refined using the molecular dynamics program CHARMM. Hydrogen atoms were assigned to the modeled structure using the program HBUILD from CHARMM. Then it was solvated by inserting it in a 30 A sphere of water and deleting solvent molecules with heavy atoms closer than 2.5 A to any protein heavy atom. This was followed by minimization to relieve bad contacts and 100 ps molecular dynamics simulation. The refined structure of MTP was used for structure-based database search.
  • the refined structure of MTP was used as the target for structure-based 3D database search.
  • DOCK was used for computer aided database screening to identify potential inhibitors.
  • Shape and binding energy scoring functions were used to screen and rank ligands. Filters were used to eliminate molecules that have more than 10 flexible bonds to avoid considering overly flexible molecules, also molecules with fewer than 10 or more than 50 heavy atoms were discarded.
  • the screening of the large NCI database was done in two stages with the ligand flexibility being considered in both. In the first one, two minimization cycles with 50 iterations maximum were considered for each compounds from the database. The best scoring 10,000 molecules were considered in the second stage when 100 minimization cycles and 100 maximum iterations per cycle were done to refine the position of the ligand and its score. The top 2,000 compounds were considered for selecting potential inhibitors for matriptase by inspecting if they contain ionizable groups that will bind to the SI site and the anionic site. After the third screening 69 compounds were selected for biological testing.
  • the refined structure of matriptase obtained from molecular dynamics simulation was used for screening the NCI database.
  • the SI site, the anionic site and the hydrophobic SI' site were used to prepare the template for docking with DOCK, h order to save time, the docking was done in two rounds. In the first one a crude minimization was used and the best scoring 10,000 compounds were considered for the second round with longer minimization. The best scoring 2,000 compounds were considered and 69 were selected for testing.
  • Active matriptase was purified from human milk as will be described later.
  • Active urokinase-type plasminogen activator (uPA) was purified by aminobenzamidine- Sepharose 6B (Amersham Pharmacia, Piscataway, NJ) from a partially purified uPA from human urine (a gift from a local company in Taiwan).
  • Bovin _-trypsin, bovine thrombin, and fluorescent peptide substrates N-tert-Butoxy-Carbonyl (N-t-Boc)-Gln- Ala-Arg-7-Amido-4-Methylcoumarin (AMC), N-t-Boc-Leu-Gly-Arg-AMC, and N-t- Boc-Leu-Arg-Arg-AMC were purchased from Sigma (Sigma Chemical Company, St. Louis).
  • Activated matriptase in the complex form with its endogenous inhibitor HAI-1, was purified from human milk as described previously. To separate the activated matriptase from the bound HAI-1, the complex was dissociated by acid, and resolved in 10% SDS polyacryamide gel electrophoresis. The proteins were stained by Zinc stain Kit (Bio-Rad, Hercules, CA). Gels containing the 70-kDa active matriptase was sliced out, and eluted from gel using Electro-Eluter (Bio-Rad, Hercules, CA) under non-denatured condition (Tris-Glysine buffer pH 8.3). Purified active matriptase was then stored at -80 _C in acid solution.
  • Inhibitory activity of the compound inhibitors against each protease was measured at room temperature using fluorescent substrate peptides in 100 mM Tris- HCl (pH 8.5) containing 100 _g/ml of bovine serum albumin.
  • To a cuvette containing 170 _1 buffer was added 10 1 of enzyme solution and 10 1 of inhibitors. After pre- incubation, 10 _1 of substrate was added and the solution was mixed well by shaking the cuvette. The residue enzyme activity was then determined by following the change of fluorescence released by hydrolysis of the fluorescent substrates in a fluorescent spectrophotometer (HITACH F4500) with excitation at 360 nm and emission at 480 nm.
  • HITACH F4500 fluorescent spectrophotometer
  • Peptide N-t-Boc-Gln-Ala-Arg-AMC was used as substrate for matriptase and trypsin, peptide N-t-Boc-Leu-Gly-Arg-AMC was used as substrate for uPA, and peptide N-t-Boc-Leu-Arg-Arg-AMC was used as substrate for thrombin.
  • the inhibitory activity of each compound was first investigated under the same concentration to a fixed amount of matriptase. Compound that exhibited inhibition was then further analysis for its Ki value using Dixon plotting. We recorded the rate of hydrolysis in the presence of 6-7 different concentrations of each inhibitor. A straight line of the concentration of inhibitor versus the reciprocal values of the rate of hydrolysis was plotted with SigmaPlot software. Two lines were obtained from two unsaturated substrate concentrations; the X value of the intersection of these lines gives the value of-Ki.
  • these compounds will comprise significant therapeutic application, hi particular, these compounds should inhibit matriptase proteolytic activity and the biological effects associated therewith. More specifically, these compounds should selectively inhibit matriptase carcinoma progression, cell proliferation and/or differentiation and/or promote apoptosis especially of cancer and other neoplastic cells.
  • Table 2 Initial screening of compound inhibitors for matriptase.
  • Table 2 shows the results from an initial screening. Among 69 compounds examined, 44 could inhibit at least 60% of the matriptase activity; 15 of them actually inhibited over 95% of the activity. There were 6 compounds not useful in this study: 3 compounds had high absorbency which made them incompatible with our activity assay; other three compounds, instead of reducing, increased the rate of releasing fluorescence by matriptase hydrolysis. From the 15 compounds that inhibited more than 95% of matriptase activity 5 were analyzed further for their Ki value as described in Methods. TABLE 3
  • Table 3 shows the Ki of five most potent compounds.
  • Compound A that is the most potent is also the most selective when tested against matriptase, trypsin, uPA and thrombin.
  • Compound C that is the unsubstituted analog of compound A is about 5 times less potent against matriptase but is about 3 times more potent inhibitor of thrombin.
  • an iodine at position 2 can improve the potency and selectivity of the inhibitor for matriptase.
  • the length of A, 19 A is comparable to the distance between Aspl85 and the anionic site, that is about 20 A. This data also supports our model.
  • the compounds produced according to the invention will be used to treat conditions wherein inhibition of matriptase is therapeutically beneficial.
  • This will include conditions that involve abnormal cell growth and/or differentiation such, as cancers and other neoplastic conditions.
  • cancers which may be treated according to the invention include colon, pancreatic, prostate, head and neck, gastric, renal, brain and CML.
  • the subject therapies will comprise administration of at least one compound according to the invention in an amount sufficient to elicit a therapeutic response, e.g., inhibition of carcinoma progression.
  • the compound may be administered by any pharmaceutically acceptable means, by either systemic or local administration. Suitable modes of administration include oral, dermal , e.g., via transdermal patch, inhalation, via infusion, intranasal, rectal, vaginal, topical parenteral (e.g., via intraperitoneal, intravenous, intramuscular, subcutaneous, injection).
  • oral administration or administration via injection is preferred.
  • the subject compounds maybe administered in a single dosage or chronically dependent upon the particular disease, condition of patient, toxicity of compound, and whether this compound is being utilized alone or in combination with other therapies. Chronic or repeated administration will likely be preferred based on other chemotherapies.
  • the subject compounds will be administered in a pharmaceutically acceptable formulation or composition.
  • formulations include injectable solutions, tablets, milk, or suspensions, creams, oil-in-water and water-in-oil emulsions, microcapsules and micro vesicles.
  • compositions will comprise conventional pharmaceutical excipients and carriers typically used in drug formulations, e.g., water, saline solutions, such as phosphate buffered saline, buffers, surfactants.
  • the subject compounds may be free or entrapped in microcapsules, in colloidal drug delivery systems such as liposomes, microemulsions, and macroemulsions. Suitable materials and methods for preparing pharmaceutical formulations are disclosed in Remington's Pharmaceutical Chemistry, 16 th Edition, (1980). Also, solid formulations containing the subject compounds, such as tablets, and capsule formulations, may be prepared.
  • Suitable examples thereof include semipermeable materials of solid hydrophobic polymers containing the subject compound which may be in the form of shaped articles, e.g., films or microcapsules, as well as various other polymers and copolymers known in the art.
  • the dosage effective amount of compounds according to the invention will vary depending upon factors including the particular compound, toxicity, and inhibitory activity, the condition treated, and whether the compound is administered alone or with other therapies. Typically a dosage effective amount will range from about
  • 0.0001 mg/kg to 1500 mg/kg more preferably 1 to 1000 mg/kg, more preferably from about 1 to 150 mg/kg of body weight, and most preferably about 50 to 100 mg/kg of body weight.
  • the subjects treated will typically comprise mammals and most preferably will be human subjects, e.g., human cancer subjects.
  • the compounds of the invention maybe used alone or in combination. Additionally, the treated compounds maybe utilized with other types of treatments, e.g., cancer treatments.
  • the subject compounds may be used with other chemotherapies, e.g., tamoxifen, taxol, methothrexate, biologicals, such as antibodies, growth factors, lymphokines, or radiation, etc. Combination therapies may result in synergistic results.
  • the preferred indication is cancer, especially the cancers identified previously.

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