WO2007046781A1

WO2007046781A1 - Furin inhibitors

Info

Publication number: WO2007046781A1
Application number: PCT/US2005/026086
Authority: WO
Inventors: Robert E. Smith
Original assignee: Smith, Judith
Priority date: 2004-07-23
Filing date: 2005-07-22
Publication date: 2007-04-26
Also published as: EP1799244A4; US20090131328A1; AU2005334458B2; US8263563B2; AU2005334458B9; AU2005334458A8; WO2007046781A8; EP1799244A1; AU2005334458A1; CA2586086A1

Abstract

Inhibitors for the endoprotease furin are provided for the prevention, diagnosis, treatment, and study of human and animal pathologies, which involve furin activity. These pathologies include infections caused by bacteria and virus that exploit host furin activity. These pathologies also include diseases that involve the expression of host proproteins that are processed by furin as a part of growth, development, and maintenance of the host organism including certain cancers of the head and neck.

Description

FURIN ESTHIBITORS

PRIORITY CLAIM

This application claims the benefit US Provisional Patent Application Serial No 60/590,577, filed on July 23, 2004, which is incorporated herein by reference in its entirety.

GOVERNMENT LICENSE RIGHTS

The testing of this invention was supported in part by the United States Government under National Institute of Health Grant No DK37274 Accordingly, the government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds that modulate fuπn activity and methods of using these compounds in the prevention, treatment, diagnosis, and study of diseases that affect humans and animals

BACKGROUND OF THE INVENTION

Most proteins produced by eukaryotic organisms are produced as larger proproteins that are generally either less active, or entirely inactive. Many proproteins are processed in transit through the secretory pathway of the Golgi apparatus where specific proteases cleave peptide bonds at specific amino acid sequences to produce functionally mature proteins Still other propeptides are first transported to specific regions of the cell or the cell membrane where they are cleaved at specific amino acid sequences to produce mature proteins

Proteins first produced as larger propeptides then cleaved into more functional polypeptides include but are not limited to serum albumin, cell surface receptors, adhesion molecules, peptide hormones such as pro-insulin, neuropeptides, growth factors, and components of the clotting cascade

One especially widespread and indispensable protease active in both the secretory pathway and on, or near the cell surface, is the endoprotease fuπn. Fuπn, is itself produced as a proprotein (SEQ ID NO 1), cycles between the Golgi apparatus, endosomes, and cell membrane Fuπn is active in both embryogenesis and m mature cells At steady state, fuπn is localized principally in the trans- Golgi network (TGN)/Endosomal system Depending upon its location in the system, furin catalyses a number of different reactions, all involving proteolytic cleavage of proproteins. For example, in the TGN/biosynthetic pathway fuπn cleaves a propeptide to give active pro-β nerve growth factor (pro-β-NGF). Similarly, fuπn cleaves propeptides thereby activating pro-bone morphogeny protein-4 (pro-BMP-4) and the "single-chain" insulin pro-hormone to form the higher activity latter-form entity

A number of pathogens also exploit host cell fuπn activity to help activate proteins involved in pathology For example host cell furin cleaves the Ebola Zaire pro-glycoprotein (pro-GP) protein as part of the virus's infectious cycle Furin located in the host cell membrane cleaves proproteins produced by bacterial pathogens to create active forms of the bacterial proteins such as anthrax protective antigen (PA), and Clostridium septicum α-toxin Additionally, furin in the early endosome, cleaves propeptides to produce active bacterial proteins such as diptheπa toxins, shigala toxin, shigala -like toxin 1, and Pseudomonas exotoxin A. Fuπn processes the coat protein of Human Immunodeficiency Virus (HIV) and PA toxin produced by Bacillus anthrasis For a more through discussion of fuπns and furin activity, the reader is directed to "Fuπn at the Cutting Edge. From Protein Traffic to Embryogenesis and Disease" Gary Thomas, Nature Reviews Molecular Cell Biology VoI 3, October 2002 pg 753-766, which is hereby incorporated by reference in its entirety

In addition to the propeptides already discussed furin and other subtihsn- like proteases, also cleave proproteins that produce active forms of hormones and growth factors (e g , proactivm A, hepatocyte-growth factor), plasma proteins (albumin, factor VII, factor IX, factor X), receptors (insulin pro-receptor). Additional pathogen derived propeptides processed by furin and other subtihsn- hke proteases include, for example, viral proteins such as HIV-I coat protein gpl60, and influenza virus haemagglutinin as well as bacterial proteins such as diphteπa toxin, and anthrax toxin For further discussion of the role of fuπns in cellular metabolism and pathology the reader is directed to the following references all of which are hereby incorporated by reference in their entirety, (Decroly et al , J Biol Chem 269 12240-12247, 1994, Stieneke-Grober et al , EMBO J 11 2407-2414, 1992, Barr, Cell 66 1-3, 1991, Wasley et al , J. Biol Chem 268-8458-8465, 1993, Klimpel et al., Proc Natl. Acad Sci USA 89-10277- 10281, 1992, Tsuneoka et al , J. Biol Chem 268 26461-26465, 1993, Bresnahan et al , J Cell Biol 111 2851-2859, 1990, Hosaka et al , J Biol Chem 266 12127- 12130, 1991, Vey et al , J Cell Biol 127 1829-1842, 1994.

Because of furin's importance in both cellular development and maintenance and its role in pathology, fuπn has become the focus of considerable study This interest has resulted m the development of some furin inhibitors useful in the study of furin activity and in the treatment of diseases that involve fuπn activity Currently available furin inhibitors include the furin propeptide itself (SEQ ID NO 1), specific alkylating agents, a polypeptide consisting of L- arginines, and polypeptide derivatives of αi -antitrypsin For further discussion of furin inhibitors the reader is directed to U S Patent 6,022,855, "Polyarginines Are Potent Furin Inhibitors" A Cameron, J Appel, R A Houghten, and I Lindberg, The Journal of Biological Chemistry, \o\ 275, No Nov 24, pg 36741- 36749, and U S. patent application publication No. 2003/0087827, all of which are herein incorporated by reference in their entirety

A typical Fuπn propeptide is described in U S Patent No 6,272,365 Bl. The typical sequence representative of a human fuπn propeptide (SEQ ID No. 1 submitted by K Strausberg, et al (gi 15082544) is available from the National Center for Biotechnology Information (NCBI)

Other alkylating agents such as ketomethylene and octapeptidyl chloromethane derivatives are effective inhibitors of furin, unfortunately they are too toxic to be of general therapeutic value For a further discussion of these reagents the reader is directed to see, for example, S Jallenberger, et al , Nature 1992, pp 358-361, vol 360, which is herein incorporated by reference in its entirety.

The αi -antitrypsin derivatives used as furin inhibitors are less toxic to eukaryotic host cells than are the currently used alkylating agents However, α i- antitrypsin derivatives are large polypeptides, not readily taken up by cells The most practical means of delivering α i -antitrypsin derivative fuπn inhibitors is by gene therapy This delivery system includes all of the complications and risks generally associated with gene therapy.

Although the general association between specific disease states and fuπn activity is known, it is unlikely that all disease states associated with furin activity have been discovered Because of the essential role fuπn plays in metabolism, development, and a wide variety of pathologies there is an urgent need for compounds and methods for regulating furin activity It is one object of the invention to provide such compounds and methods

SUMMARY OF THE INVENTION

Briefly describing the present invention, there is provided compounds and methods for diagnosing, preventing, and treating medical conditions involving fuπn activity In one embodiment compounds and methods are used to diagnose, prevent, and treat diseases involving fuπn activity.

One embodiment comprises an n-mer or a pharmaceutically acceptable salt thereof, of a compound with the formula

[Compound 1]

wherein, the n-mer is a polypeptide comprised of the D-enantiomers of the compounds illustrated in Fig 1 joined by peptide bonds, n is greater than or equal to 1 and less than or equal to 6, and

X is either hydrogen (H) or fluorine (F)

One embodiment comprises a hexamer of the D-enantiomers of the compound illustrated in Fig 1 , having the following formula or a pharmaceutically acceptable salt thereof

[Compound 2]

wherein,

X is either hydrogen, fluorine or a combination thereof, and

Z is Hydrogen or an N-terminal blocking group selected from the group comprising α -lipoic acid, pyroglutamic acid, 4-morpholinylcaronyl, CBZ, or a derivative of propionic acid

Still another embodiment comprises a compound with the general formula.

Z-O wherein O, for example, comprises an n-mer of the D-enantiomer of the amino acid arginine, or pharmaceutically acceptable salt thereof, having for example the formula:

[Compound 3] and

Z is hydrogen or an N-terminal blocking group, or the directing group α - hpoic acid, pyroglutamic acid, 4-morpholinylcaronyl, CBZ, or a propionic acid derivative, and z is bonded to the N-terminus of the terminal arginme

Still another embodiment is a compound or pharmaceutically acceptable salt thereof, with the formula.

Z-U-C

[Compound 4] wherein,

C is a group selected from the group comprising

X is hydrogen (H) or fluorine (F),

U is a peptide with the general formula (J)_n- Arg in which (J)_n is an n mer of amino acids joined together via peptide bonds, Q is at least one amino acid selected from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe,

Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, Z is either hydrogen or an N-terminal blocking group selected from the groups comprising α -hpoic acid, pyroglutamic acid, 4-morpholinylcaronyl, CBZ, or a propionic acid derivative, Z is bonded to the N-terminus of the peptide

U; and R' is selected from the group comprising hydrogen, fluorine and either

or

N

wherein n' is equal to or less than 4, and R" is selected from the group comprising hydrogen, fluorine or either

or

C — AFC The N-termmal blocking group may be attached to either an amino acid or a peptide chain. In one embodiment the N-terminal blocking group is a group that has intrinsic anti-inflammatory activity, for example, 2-Acetoxybenzenecarboxylic acid For a more complete listing of N-terminal blocking groups that can be used in various embodiments the reader is directed to Gilman, Goodman, Gilman, "The Pharmacological Basis of Therapeutics", Sixth Ed. MacMilhan, Chapter 29, which is herein incorporated by reference in its entirety.

Yet another embodiment is the compound or a pharmaceutically acceptable salt thereof, with the following formula.

[Compound 5] wherein,

U is a polypeptide with the general formula (J)_n-Arg in which (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, and

Z is selected from the group consisting of D-lipoic acid, pyroglutamic acid, biotin, hydrogen, or fluorine, z is bonded to the N-terminus of the peptide U Another embodiment is the compound or pharmaceutically acceptable salt thereof, with the formula

[Compound 6] wherein,

U is a peptide with the general formula (J)_n-Arg in which (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, and

Z is selected from the group consisting of α-hpoic acid, pyroglutamic acid, biotin, hydrogen and fluorine and Z is bonded to the N-terminus of the peptide U.

Still another embodiment is the compound or pharmaceutically acceptable salt thereof, with the following formula

[Compound 7]

wherein,

U is a polypeptide with the general formula (J)_n-Arg in which (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, and Z is selected from the group consisting of α-hpoic acid, pyroglutamic acid, biotin, hydrogen and fluorine Still another embodiment is the compound or pharmaceutically acceptable salt thereof, with the following formula

[Compound 8] wherein,

U is a polypeptide with the general formula (J)_n-Arg in which (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one ammo acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, and

Z is selected from the group consisting of α-hpoic acid, pyroglutamic acid, biotin, hydrogen and fluorine, is bonded to the N-terminus of the peptide U Yet another embodiment is the compound or pharmaceutically acceptable salt thereof, with the following formula"

[Compound 9] wherein,

U is a polypeptide with the general formula (J)_n-Arg in which (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, and

Z is selected from the group consisting of α-hpoic acid, pyroglutamic acid, biotin, hydrogen and fluorine, Z is bonded to the N-terminus of the peptide U,

R' is hydrogen, or fluorine, or H₂)"¹

C

NH "" NH₂ or

N

, and A is selected from the group of amino-fluoro-acetic acid, OPh

Another embodiment is the compound or pharmaceutically acceptable salt thereof, with the following formula.

[Compound 10]

wherein,

U is a peptide with the general formula (J)_n-Arg in which (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid selected from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, and,

Z is selected from the group consisting of α-lipoic acid, pyroglutamic acid, biotm, hydrogen and fluorine Z is selected from the group comprising α-hpoic acid, pyroglutamic acid, morpholino, CB₂, biotin, α lipoic acid, quinoly, groups and the like, Z is bonded to the N-terminus of peptide U; and

R' is selected from the group comprising hydrogen, fluorine,

NH

and

n' is greater than or equal to 0 and less than or equal to 4; and X is hydrogen, fluorine or a combination thereof

In still another embodiment, therapeutically effective amounts of an appropriate fuπn inhibitor are administered in a therapeutic manner to a medical patient having a medical condition involving fuπn activity Medical conditions involving furin activity include, but are not limited to, neuro-degenerative diseases, malignancies, and the like.

Medical conditions due to bacteria or viruses include, but are not limited to, infection with specific bacteria or viruses or exposure to various bacterial or viral toxins produced by any means Means for producing diseases due to bacteria or viruses include exposure to bacterial or viral toxins produced through genetic manipulation or chemical synthesis Additional medical conditions that may be treated with fuπn inhibitors include exposure to virus or bacteria or viral or bacterial derived toxins before a human or animal patient becomes symptomatic for exposure to the pathogen

In still another embodiment, appropriate fuπn inhibitors may be used to diagnose disease involving furin activity.

In yet other embodiments, appropriate fuπn inhibitors may be used to study furin activity as well as pathologies and components of pathology that involve fuπn activity

BRIEF DESCRIPTION OF THE FIGURES

Fig 1 is a schematic representation of the major steps in the synthesis of one compound according to one embodiment, which includes SEQ ID NO 2

Fig 2 is a schematic representation of the major steps in the synthesis of one of the compounds according to an embodiment of the present invention

Fig 3 is a schematic representation of the major steps in the synthesis of one of the compound according to one embodiment, which includes SEQ ID NO. 3

Fig. 4 is a schematic representation of the major steps in the synthesis of one compound according to one embodiment, which includes SEQ ID NO. 3.

Fig 5 is a schematic representation of the major steps in the synthesis of one compound according to one embodiment, which includes, SEQ ID NO 3

Fig 6(a) is a photomicrograph (100 X magnification) of wild type A-7 cells grown in vitro in the absence of either fuπn inhibitor or vaccinia virus

Fig 6(b) is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro

Fig 6(c) is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 5 μM of one of the compounds, made in accordance with one embodiment.

Fig. 6(d) is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 25 μM of one of the compounds made in accordance with one embodiment

Fig 6(e)is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 50 μM of one of the compounds made in accordance with one of the embodiments

Fig 7(a) is a photomicrograph (100 X magnification) of wild type A-7 cells grown in vitro in the absence of either a fuπn inhibitor or vaccinia virus

Fig 7(b) is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro Fig. 7(c) is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 5 μM of one of the compounds made in accordance with one of the embodiments

Fig. 7(d) is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 25 μM of one of the compounds made in accordance with one of the embodiments

Fig. 7(e) is a photomicrograph (100 X magnification) of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 50 μM of one of the compounds made in accordance with one of the embodiments

Fig 8(a) is side by side photomicrographs (100 X magnification) illustrating the results of two separate assays of wild type A-7 cells grown in vitro in the absence of either fuπn inhibitor or vaccinia virus

Fig 8(b) is side by side photomicrographs (100 X magnification) illustrating the results of two separate assays of type A-7 cells, inoculated with vaccinia virus and grown in vitro

Fig 8(c) is side by side photomicrographs (100 X magnification) illustrating the results of two separate assays of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 5 μM of one of the compounds made in accordance with one of the embodiments

Fig 8(d) is side by side photomicrographs (100 X magnification) illustrating the results of two separate assays of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 25 μM of one of the compounds made in accordance with one of the embodiments.

Fig 8(e) is side by side photomicrographs (100 X magnification) illustrating the results of two separate assays of type A-7 cells, inoculated with vaccinia virus and grown in vitro in the presence of 50 μM of one of the compounds made in accordance with one of the embodiments DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to various embodiments and specific language will be used to describe the same It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications of the invention, and such further applications of the principles of the invention as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the invention relates

Furins are found in all vertebrates and are typically of about 794 amino- acid and characterized as a Type-1 transmembrane protein. Classified as a member of the protein convertase family it is also a member of the subtilisin superfamily of serine endoproteases The active site of the enzyme rigorously conserves the Aspartic acid, Histidine, Serine (Asp, His, Ser) catalytic triad characteristic of proteases in the serine proteases superfamily Typically, fuπn activity is calcium dependent and peaks over the pH range of 5-8 Standard Units (SA) By way of example and not of limitation, a sequence of one human form of a propepetide encoding a fuπn is presented in SEQ ID NO 1 Again by way of illustration and not limitation, additional fuπns and molecules that include fuπn activity and can be used in conjunction with the present invention include those disclosed in U S Patent Number 6,210,929 to Schlokat et al , issued on 3 April 2001, 6,274,365 Bl to van de Ven et al , issued on August 14, 2001, and International Publication WO/91/06314, published 16 May 1991, both of which are incorporated herein by reference in their entirety.

For further discussions of this polypeptide including a comparison of the furin and fuπn like proteins from various organisms, please see U S. Patent No 6,271,365 Bl, which is incorporated herein by referenced in its entirety

The consensus proteolytic cleavage site for furin proteases is Arg-Xan- Xan-Arg, wherein Xan is preferentially a basic amino acids. For a more thorough discussion of fuπns the reader is directed to, "Proteolytic Processing of the Hepatitus B Virus e Antigen Precursor," Messagoet F , Salhi S , Eon, P , and Rossignol J , Journal of Biological Chemistry Vol. 278, Issue 2, 891-895 Jan 10, 2003, incorporated herein by reference in its entirety

Various embodiments of the invention relate to methods of treating medical conditions, characterized by the involvement of proteins processed by the protease fuπn One embodiment provides for the treatment of pathologies caused by the over expression (or expression at inappropriate times) of a host encoded propeptides processed by fuπn

The term peptide, as used herein, refers to a single amino acid joined to other groups via peptide or peptide like bonds The term peptide also refers to short polypeptides, such as for example, amino acid residues that may or may not include both D and L enantiomers and are bonded to one another or to other groups by peptide or peptide like bonds

Another embodiment provides for the treatment of medical conditions involving, or exacerbated by, fuπn processing of propeptides encoded by infectious agents These infectious agents include microorganisms such as specific pathogenic protozoa and bacteria, as well as specific viruses

A number of pathogenic organisms also produce proteins in proprotein form, which are processed into their mature, more active form by eukaryotic endoproteases Because of its role in activating pathogen encoded propeptide, fuπn plays an essential role in the pathology of a number of infectious agents.

Examples of pathogenic bacteria that exploit fuπn activity to process their proproteins include Psuedomonas areugenosa, Bacillus anthrasis, and members of the genus Clostridium including C botuhnum and C tetani For a more exhaustive list see Table I

Viruses, which exploit host fuπn activity for processing of viral encoded propeptides include, for example, Borna virus, Human Immunodeficiency Virus (HIV), Infectious Bronchitis virus, vaccinia virus, Hepatitus B, Ebola Zaire, Japan B Encephalitis virus, arboviruses (including the virus responsible for yellow fever), and Coronavirus (including the Coronavirus responsible for Severe Acute Respiratory Syndrome, SARS) By way of illustration and not limitation, fuπn cleavage sites for some propeptides produced by some viral and bacterial pathogens are summarized in Table 1.

As used herein, amino acid residues may be designated as Pi, P₂, etc , wherein Pi and Pi ' refer to groups nearest to and on opposite sides of the scissile bond, P₂ refers to the amino acid residue next to Pi and nearer the blocking group, etc. For example, in a dipeptide inhibitor, Pj is the amino acid nearest to the scissile bond cleared by the protease, if the N-terminal of the dipeptide is blocked the Pi residue may be the group nearest to the blocking group.

As used herein, the one letter and three letter amino acid designations as are used in Table 1 and throughout the examples are those well-known in the art. For a specific reference, the reader is directed to see for example, Biochemistry, 2^nd ed , Lubert Stryer, W H Freeman & Co , 1975, pg 16, herein incorporated by reference in its entirety

Table 1

While many of the inhibitors are discussed in terms of molecules including between one and four amino acids in positions Pi through P₄. The inclusion of additional groups can, in some instances, be used to further refine the binding affinity of the molecules For example, in some instances, other amino acids or amino acid analogues in positions beyond Pi through P₆ may be added to the molecule to modulate the binding affinity of the molecule in a given therapeutic or experimental setting.

Linking a fuπn inhibitor to the N-termini of the proteolytic cleavage site, for the example, the N-terminus of an amino acid in the P₄ position can affect the biophysical and biochemical properties of the molecule Such N-terminal groups can, for example, help direct the fuπn inhibitors to specific regions of the cell, for example, to the Golgi apparatus or the membrane

These types of directing groups are useful when the fuπn inhibitors are used in vivo and they are especially useful when it is desirable to differentially inhibit fuπn activity either on the cell surface or in the Golgi apparatus Useful directing groups for the practice of the instant invention include, but are not limited to, α lipoic acid, pyroglutamic acid and certain D-amino acids

One aspect of the invention provides fuπn inhibitors that are particularly effective in both in vivo and in vitro applications

Leaving groups that may be used in various embodiments include, for example, OPH

In various embodiments, a number of different leaving groups and a number of different amino acid recognition sites have been named Fuπn activity is an essential component of virtually all healthy host cells Accordingly, broadly inhibiting fuπn activity is not necessarily desirous in all therapeutic settings To that end, a number of different leading groups and a number of different recognition sites have been provided One strategy for the use of such substrate recognition sites and leading groups is to tailor a specific therapeutic compound to treat a specific pathogenic state. For example, in order to treat an infection caused by bacteria, which relies upon host fuπn processing of a bacterial encoded proprotein, it may not be necessary or desirous in all situations to provide a fuπn inhibitor that effectively competes with the fuπn's native substrate. It may be more effective to provide a fuπn inhibitor that has a higher affinity for a fuπn- active site than does the bacteria proprotein And at the same time, a lower affinity for the fuπn active site than does propeptides encoded by the host The appropriate choice of substrate recognition site can be used to limit collateral damage otherwise done to the host cell by blanket inhibition of host fuπn activity

Similarly, it is possible to select an appropriate leading (directing group) to cause an accumulation of furin inhibitor where it will have the greatest therapeutic impact Conversely, it may be possible to use the directing group to steer the bulk of the inhibitor away from areas of the cell where the inhibitor is most likely to cause adverse side effects and little therapeutic gam

For example, judicious use of the proper substrate recognition sites and directing groups in conjunction with the inhibitors and the other compounds of various embodiments of the invention can be used to selectively prevent or treat various infections and other pathogenic states with a minimum of adverse side effects.

One embodiment provides compounds and methods for the prevention, diagnosis, and treatment of diseases caused by bacteria that exploit host fuπn activity Bacteria that exploit host fuπn activity include, but are not limited to, Psuedomonas areugenosa, Corynebacterium dφtheriae, Bacillus anthrasis, and members of the genus Clostridiu, including, for example, C botuhnum and C tetani

One embodiment provides compounds and methods for the prevention, diagnosis, and treatment of diseases caused by viruses that exploit host fuπn activity Viruses that exploit host fuπn activity include, but are not limited to, Borna virus, Human Immunodeficiency Virus (HIV), Infectious Bronchitis virus, Ebola Zaire, Japan B Encephalitis virus, arboviruses including, for example, the virus responsible for Yellow Fever, and Coronaviruses including the Coronavirus responsible for Severe Acute Respiratory Syndrome (SARS)

Still another embodiment provides compounds and methods for treating, preventing, and diagnosing human and animal diseases that involve pathogenic expression of native proproteins processed by fuπn. These conditions include, but are not limited to, degenerative joint diseases (such as rheumatoid arthritis), various forms of dementia including, but not limited to, Alzheimer's disease, familial British dementia (FBD) and familial Danish dementia (FDD), and tumor metastasis Other pathologies that may be treated using these fuπn inhibitors include, for example, non-small-cell lung carcinomas, squamous-cell carcinomas of the head and neck, and Glioblastomas

By way of explanation and not limitation, it is also to be appreciated that during the course of the reaction of the enzyme with some of the inhibitors, the carbonyl of the inhibitor is thought to rehybπdize to sp³ and to form a ketal intermediate with the thiol functionabiltiy of the enzyme When the reaction between some of the inhibitors and the enzyme occurs under acidic conditions, other ketals can exchange with this intermediate either by going through the ketone or by ketal-ketal exchange Accordingly, ketals can be substituted for carbonyls in the peptidyl inhibitors of the present invention. Similarly, other compounds such as hydrazones, hemiketals, oximes, lmines, cyanohydπns, enolethers, enamines, hemithioketals, and the like are to be considered carbonyl equivalents and may be substituted for a carbonyl (or to give a carbonyl) under the acidic conditions of these inhibition reactions Utilization of such derivatives can also be vehicles to either improve the bioavailability of the inhibitor drug or keep it from crossing a cellular membrane depending upon the hydrophobic nature of the masking function.

It is also to be appreciated that the development and synthesis of compounds having isosteric replacements of amide bonds is now a standard practice m the development of biologically active peptides once the optimum peptide sequence has been identified Accordingly, one aspect of the present invention includes compounds having one or more modified amide bonds in the peptide sequence so long as conformation and binding are maintained while secondary enzymatic hydrolysis is prevented For a list of such modifications, see for example, Kaltenbronn, 33, J Med Chem , 838 In addition, inhibitors having a hydrazine replacement for the P] nitrogen as reported by Giordano for other halogen methyl ketones are also intended to be claimed

Some embodiments relate to inhibiting fuπn processing of proproteins (propeptides) in humans, animals, and in organs, tissues, or cells maintained in culture by administering effective amounts of an inhibitor of the endoprotease

Still other embodiments involve methods of treating diseases involving fuπn endoprotease activity by administering to a human, or an animal, patient therapeutically effective amounts of a fuπn inhibitor in a suitable drug delivery vehicle

Yet another embodiment provides methods of treating diseases involving fuπn endoprotease activity, and/or serine protease activity by administering to a human patient or animal a therapeutically effective dose of a furin inhibitor in a suitable drug delivery vehicle

One embodiment is a method of diagnosing disease in humans or animals using an assay comprising a fuπn inhibitor as, for example, a reagent

The various compounds disclosed herein in various embodiments may be prepared for administration to both human and animal patients as appropriate for each compound and therapeutic or prophylactic situation by methods well known in the pharmaceutical art. If administered as a solid, the compounds may be mixed with a carrier or excipient. The specific carrier or excipeint used may be a solid, semi-solid, or liquid material that can serve as a vehicle or medium for the active ingredient Suitable carriers or excipients are well known in the art The pharmaceutical composition may be adapted for intravenous, oral, inhalation, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, aerosols, inhalants, suppositories, solutions, suspensions, or the like

The compounds of the present invention may be administered intravenously, for example, as aqueous solutions including suitable carriers. Suitable carriers are well known to the art and include ethanol, Tween-80, solutol, Cremophor and the like The compounds of the present invention may also be administered orally, for example, with an inert diluent or capsules or compressed into tablets For the purpose of oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like The actual amount of the compounds in such preparations but may be varied depending upon the particular form and therapeutic application and dosing strategy The amount of the compound present in compositions is such that a suitable dosage will be obtained Preferred compositions and preparations of the present invention may be determined by methods well known in the art

Tablets, pills, capsules, troches, and the like may also contain one or more of the following adjuvants^* binders such as povidone, hydroxypropyl cellulose, microcrystalline cellulose, gelatin and the like, excipients or diluents such as- starch, lactose, microcrystalline cellulose, dicalcium phosphate and the like, disintegrating agents such as. croscarmellose, crospovidone, sodium starch glycolate, corn starch and the like, lubricants such as magnesium stearate, steric acid, talc or hydrogenated vegetable oil, ghdants such as colloidal silicon dioxide, wetting agents such as sodium lauryl sulfate and polysorbate 80, and sweetening agents such as^* sucrose, aspartame or saccharin may be added or a flavoring agent such as. peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, oils or the like. Other dosage unit forms may contain other various materials that modify the physical form of the dosage unit, for example, as coatings.

Accordingly, tablets or pills may be coated with sugar, hydroxypropyl methylcellulose, polymethacrylates, or other coating agents Syrups may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used

The compounds of the present invention may also be administered in the form or an aerosol, mist, vapor or the like When administered, as for example an aerosol, the compounds present in a therapeutically significant amount may be combined with any non-toxic non-cross reacting carrier as is known in the art

EXAMPLES

The following examples are included by way of illustration and not limitation All of the reagents use in the following examples are preferable reagent grade or better

Where used, primarily in the synthesis examples, the amines, amino cepham acid, cycloserine and cyclic aspartic imide were all purchased from Acme Biosciences, Belmont, California

Where used the reagent dihydrochloride was prepared in accordance with the method of Jozef Oleksyszyn as published in the J Med Chem, 1994, 37, 226, which is incorporated herein by reference in its entirety

EXAMPLE 1

Referring now to FIG 1 , the synthesis of PGluArgValArgArgOPH (SEQ ID NO 27) 100 mg ( 1.0 equivalents) of PgluArg(Pbf)ValARg(Pbf)Arg(PBf)OH was dissolved in THf and cooled to -15⁰C 0 01 ml (1 3 equivalents) of NMM was added to the cooled mixture followed by 0 01 ml (1 1 equivalents) of IBCF The mixture was stirred for 20 minutes Diazomethane, freshly made from diazald was added to the mixture and the reaction was stirred for one hour at -1O⁰C and then for 20 hours at room temperature. Next the solvent was removed and the residue purified by a single preparative TLC plate The product eluted from the TLC plate in a solution of 9% MeOH in CH₂Cl₂ The reaction yielded 60 mg of diazomethyl ketone.

The diazomethyl ketone (60 mg, 1 0 equivalent) was dissolved in THF Ether. CH₂Cl₂ (2 2 2) and cooled to O⁰C 0 01 ml (1 2 equivalents) of HBr/HOAc as added to the mixture The mixture was stirred for 30 minutes After 30 minutes the solvent was removed and the residue was dried using a vacuum pump. The dried residue was dissolved in 3 ml of DMF comprising 1 0 equivalents of bromide, 6 mg (2 5 equivalents) of potassium fluoride and 5 mg (1 0 equivalents) of 2,6-difluorophenol and the mixture was stirred overnight Next, 50 ml of Ethyl acetate was added. The organic layer was washed with a saturated solution of NaCl and dried over MgSO₄ The solvent was removed and the residue was purified using a single preparative TLC plate. The intermediate eluted from the plate in a mixture of 9% MeOh m CH₂Cl₂ to give the protected difluorophenyl methyl ketone.

To remove the protecting groups, 95% TFA was added to the ketone and the mixture was stirred for 45 minutes The solvent was removed and the residue was tπtulated with ether to give the final product PGIu Arg VaI ArgArgOPH. The product had a molecular weight of 821 Daltons as determined my mass spectrometry The reaction yielded was 30 mg of the named product

EXAMPLE 2

Synthesis of ZArgValArg(4-AmPhGly)(OPh)₂ Referring now to Figure 2, 1.05 equivalents of DCI was added to 3 ml of DMF comprising 1 0 equivalents of ZArg(Pbf)ValArg)Pbf)OH at room temperature The mixture was stirred for one hour then 1.0 equivalent of dihydochloride was added followed by 1 0 equivalents of tπethylamine The mixture was stirred for two days

After two days of stirring, 5 ml of a saturated solution of NaHCO₃ was added and the solution was extracted with two, 20 ml washes of ethyl acetate The organic layer was washed with a saturated solution of NaCl and dried over MgSO₄ The solvent was removed and the residue was purified using preparative TLC The reaction yielded protected phosponate

Next, 95% TFA was added to the protected phosphonate and the mixture was stirred for 45 minutes The solvent was removed and the residue was tritulated with ether to give the titled product The molecular weight of the product was 927 Dalton as determined my Matrix Assisted Laser Desportion Ion Spectroscopy (MALDI) mass spectrometry

EXAMPLE 3

The synthesis of X-ArgValArgArgArgCH₂Y, X is either alpha lipoic acid or proparglycine and Y is a specific amine Referring now to FIG 3, 100 mg (1 equivalent) of α-hpoicAcid-Arg(Pbf)ValArg(Pbf)Arg(Pbf)Arg(Pbf)OH was dissolved in THF and cooled to -15⁰C 0.01 ml (1 3 equivalents) of NMM was added to the cooled mixture followed by 0 01 ml (1 1 equivalents) of IBCF The mixture was stirred for 20 minutes Diazomethane, freshly made from diazald, was added to the mixture and the reaction was stirred for one hour at -1O⁰C and then for 20 hours at room temperature. Next, the solvent was removed and the residue purified by a single preparative TLC plate The product eluted from the TLC plate in a solution of 9% MeOH in CH₂Cl₂. The reaction yielded 60 mg of diazomethyl ketone.

The diazomethyl ketone (60 mg, 1 0 equivalent) was dissolved in THF. Ether CH₂Cl₂ (2 2:2) and cooled to O⁰C then 0 01 ml (1.2 equivalents) of HBr/HOAc as added to the mixture and the mixture was stirred for 30 minutes. After 30 minutes, the solvent was removed and the residue was dried under a vacuum The dried residue was dissolved in 3 ml of DMF, that included 1 0 equivalents of bromide, 6 mg (2 5 equivalents) of potassium fluoride and 5 mg (1 0 equivalents) of 2,6-difluorophenol, and the mixture was stirred overnight

Next, 50 ml of Ethyl acetate was added, and the organic layer was washed with a saturated solution of NaCl and dried over MgSO₄ The solvent was removed and the residue was purified using a single preparative TLC plate. The intermediate eluted from the plate in mixture of 9% MeOh in CH₂Cl₂ to give the protected difluorophenyl methyl ketone

To remove the protecting groups, 95% TFA was added to the ketone and the mixture was stirred for 45 minutes The solvent was removed and the residue was tπtulated with ether to give the compound illustrated in FIG 3.

EXAMPLE 4

Referring now to FIG 4, the compound illustrated in FIG 4 was synthesized by substantially the same method presented in Example 3 The only major difference was in the choice of amine EXAMPLE 5

Referring now to FIG. 5, the compound illustrated in FIG. 5 was synthesized by substantially the same method presented in Example 3 The only major difference was in the choice of amine

EXAMPLE 6

Determining the prophylactic effect of some fuπn inhibitors, therapeutically effective amounts of compounds according to the formula of Compound 4 are administered to a group of genetically identical mice. As a control, a group of identical mice are maintained under identical conditions, and are not administered a therapeutically effective amount of Compound 4 Both sets of mice are challenged with an infectious amount of Bacillus anthrasis

The two groups of mice, maintained under identical conditions, are followed for a period often days, and the mortality of the two groups is assessed The mice receiving a therapeutically effective dose of Compound 4 suffer fewer fatalities than the mice m the control group

EXAMPLE 7

Determining the effect of Compound 4 using the in vitro assay method described by Cameron (Cameron, A.; Apel, J., Houghten, R A , and Lmdberg, I., "Polyarginines are Potent Furin Inhibitors", J Biol Chem., VoI 275, Issue 47, 36741-36749, November 24, 2000 incorporated herein by reference in its entirety), human furin is over expressed and purified

Truncated versions of the furin enzyme are produced using the dyhydropholate reductase amplification method to over express truncated furin This cell line secretes approximately 8 micrograms of furin into the culture medium Purity is assessed using SDS Page Gel Electrophoresis, and staining with Coomassie Blue dye

The actual activity assay is carried out in vivo as follows A series of furin inhibitors as given for example in Table 2 are synthesized and tested for inhibition against furin The assay for furin activity is performed in pH 5 buffer using the P Ertkr-MCA. An assay for furin activity is performed at pH 7, in 100 millimolar HEPES at 5 millimolar CaCl₂ in 0.5% brig 35. All assays are performed at 37 degrees C in 96 well plates Flurorescne is measured using a fluorometer with an excitation wavelength of 380 nanometers and an emission wavelength of 460 nanometers The total assay volume is on the order of 50 microliters

The final substrate concentration for all assays is about 200 micromoler In some of the samples, furin is pre-incubated with ftiπn inhibitor for 30 minutes at room temperature prior to the addition of the substrate molecule Assays are performed in duplicate or triplicate A number of compounds are tested as inhibitors of furin activity against a model substrate. Some of the compounds tested demonstrate varying degrees of concentration dependent inhibition of furin

EXAMPLE 8

The toxicity of various furin inhibitors disclosed in the embodiments of the invention is tested as follows. Various concentrations of inhibitor over a therapeutically effective range are administered to a series of mice over a period of time The overall health of the mice is observed and assessed Over a period of 10 days, various compounds used in the assay are not toxic at therapeutically effective concentrations used m the assay

EXAMPLE 9

One procedure for studying the efficacy of anti-tumor agents is to measure the inhibition of tumor growth and development in mice See, for example, Corbett, et al., In vivo Methods for Screening and Preclinical Testing, Use of rodent solid tumors for drug discovery , In- Anticancer Drug Development Guide Preclinical Screening, Clinical Trials, and Approval, B Teicher (ed), Humana Press Inc , Totowa, N.J , Chapter 5, pages 75-99 (1997), Corbett, et al., Int J Pharmacog , 33, Supplement, 102-122 (1995), incorporated herein by reference in its entirety.

Various compounds disclosed in various embodiments of the invention are tested for their ability to inhibit cancerous tumor growth in mammals such as mice For example, murine tumors or human xenografts are implanted in mice essentially as described by Corbett in "In vivo Methods for Screening and Preclinical Testing, Use of rodent solid tumors for drug discovery " 2^nd Ed., Humana Press, 2004

Briefly, the murine tumor or human xenograft is implanted subcutaneously using either 12-gauge trocar implants or counted number of cells The location for the trocar insertion is midway between the auxiliary and inguinal region along the side of the mouse The trocar is slipped approximately 3/4 of an inch subcutaneously up toward the axilla before discharging the tumor fragment, and pinching the skin as the trocar is removed Alternatively, human tumor cells can be prepared from a brie of donor tumors (5x10⁶ cells) and implanted subcutaneously in a hind-leg of a male or female nude mouse (Charles River) Either a test compound in vehicle or vehicle alone is administered by intravenous bolus injection (IV), intraperitoneal injection (ip), or oral gavage (po) Each treatment group, as well as a group of untreated control animals, consists of about five animals per group. Subcutaneous tumor response is monitored by tumor volume measurement performed twice weekly over the course of the experiment (60-120 days) Body weights are taken as a general measure of toxicity The subcutaneous tumor data is analyzed by determining the median tumor weight for each treatment group over the course of the experiment and calculating the tumor growth delay as the difference in days for the treatment versus the control tumors to reach a volume of either 500 or 1000 mm³.

Animals treated with compounds of the invention in the appropriate dosing range show a statistically significant reduction is healthier as assessed by body weight Tumors in the treated animals are smaller than tumors in animals in the control group which receive only doses of same inert carrier used to prepare the therapeutic compounds of the invention

EXAMPLE 10

Tests were conducted to determine if a fuπn inhibitor with the same general formula as compound 8, in which (R)_n is Arg-Arg-Arg was able to inhibit infection of A-7 cells with vaccinia virus in vitro

A stock solution of the inhibitor with a concentration of 100 mM was made A control, no inhibitor or viral inoculum (w t ) and three test solutions were made Next, additional inhibitor was added to cell media and incubated along with the cells for one hour at 37⁰C at a final inhibitor concentration of 5, 25, or 50 μM, respectfully

All cells except these in the wild type (w t ) group were inoculated with lOpfu/cell of Vaccinia virus (plaque forming material (pfm)). All cells, including those inoculated with vaccina, were incubated in PBS+ calcium buffer for 60 minutes at room temperature Then the inoculum was removed from inoculated cells and all cells were fed with 2 ml of MEM supplemented with serum, and either with or without 5, 25, or 50 μM of the inhibitor

The cells were then incubated for four hours at 37⁰C Next, the cells were washed with serum free MEM and 1 ml of fresh PBS-M, pH 5 1 at 37⁰C was added to the cells. The cells were allowed to sit for two minutes at 37⁰C The cells were then quickly washed with IX MEM supplemented with 10% serum Next, the first wash was removed and 2 ml of fresh MEM supplemented with 10% serum either with or without the inhibitor was added All cells were held at 37⁰C.

No Syncytia formed after 1 hour A second pH 5 1 shock was performed on all of the cells and they were returned to the 37⁰C environment After another hour Syncytia formed The samples were fixed in parafilm for 15 minutes and washed three times with PBS The samples were stained with 1 10 methyl/crystal violet, and washed to remove excess stain The samples were examined for evidence of viral infection under a Willovent tissue culture scope using a 1 Ox-lens piece, giving a final magnifier of 10Ox The experiment was run in duplicate The samples were photographed and these photomicrographs are presented in Figs 6, 7 and 8

Figures 6 and 7 are illustrative of separate experiments run under similar conditions. Figure 8 is composed of images gathered from separate experiments displayed side by side As illustrated in Figs 6, 7 and 8 a through e, separate distinct cells are visible m the wild type sample (not exposed to vaccina virus) The cell exposed to the flu virus in the absence of compound shows evidence of lysis. Cell membranes are compromised and multi-nucleated areas are clearly present Referring now to FIG 6, c and d, cells provided with either 5 or 25 μM of the compound show multiple distinct cells The cells are somewhat rounded and touching, but unlike the unprotected cells shown in Fig 6b they are not generally lysed. Cell incubated with 50 μM also exhibited little evidence of cell lysis and merging, although vacuoles are clearly forming in some of the cells

These results are consistent with low concentrations of the furin inhibitor protecting the cells from vaccina virus induced lysis While higher concentrations of inhibitor protect from lysis by vaccina although the inhibitor may be somewhat toxic to the cells at high concentrations. As illustrated in FIG. 7, the tests were repeated and similar results observed at similar concentration of the inhibitor.

EXAMPLE 11

Determination of the ability of the furin inhibitor with the general formula ERVRR-OPH to prevent components of the pathogenic pathway of bacillus anthracis from inhibiting protein synthesis in lung epethahal and macrophage cells Briefly, anthrax toxin consists of three proteins lethal factor (LF) protective antigen (PA) and edema factor (EF) All three of these proteins working in concert are necessary to cause the death of an infected cell In the course of infection PA is thought to bind to an AT receptor on the host cell surface, where PA is subsequently cleaved by furin (or a fuπn-like proteases) to produce two fragments These two fragments are thought to consist of a 20 k-Da N-terminal fragment designated as PA₂₀ and a 63-K-Da C-terminal fragment designated as PA₆₃. For a more detailed discussion of this process, see Panchal, R G. et al Nature Structural and Molecular Biology, VoI 11 No 1, page 67, January 2004, which is incorporated herein by reference in its entirety

In order to test the efficacy of a furin inhibitor to interfere with the mechanism of anthrax toxicity, the following study was carried out Cells in 12- well plates were incubated at 37⁰C for two ours under one of the following three conditions a) no putative prophylactic therapeutic, b) 100 μM Glu-Arg-Val-Arg- Arg-OPH or c) 100 μM decanoyl RVKR-CMK Next all cells, except the cells in a control group, were exposed to 100 ng/ml of PA and 50 ng/ml of a fusion protein consisting of anthrax lethal factor and pseudomonas exotoxin (FP59)

After six hours all cells were rinsed with physiological buffer and pulsed with radioactive ³⁵S for 40 minutes followed by precipitation with Tπ-chloro- acetic acid (TCA) Activity was accessed by scintillation counting All assays were conducted in triplicate including the controls (cells not exposed to anthrax toxin). The amounts of protein biosynthesized were estimated by determining the amount of ³⁵S in the precipitates Activity was accessed by scintillation counting All values reported are normalized to the amount of radiolabeled protein in the control cells The results are summarized in table II

As indicated by the results summarized in Table II, both putative fuπn inhibitors used in the assay appeared to inhibit the ability of the anthrax toxin to affect protein biosynthesis

Table II

Results of pulse chase assay for cell exposed to anthrax toxin in the presence and absence of utative fuπn rotease inhibitors

All references, patents, patent application and the like cited herein and not otherwise specifically incorporated by references in their entirety, are hereby incorporated by references in their entirety as if each were separately incorporated by reference m their entirety

An abstract is included m to aid in searching the contents of the abstract are not intended to be read as explaining, summarizing or otherwise characterizing or limiting the invention in any way.

While the invention has been illustrated and described in detail, this is to be considered as illustrative, and not restrictive of the patent rights The reader should understand that only the preferred embodiments have been presented and all changes and modifications that come within the spirit of the invention are included if the following claims or the legal equivalent of these claims The present invention contemplates modifications as would occur to those skilled in the art It is also contemplated that processes embodied in the present invention can be altered, duplicated, combined, or added to other processes as would occur to those skilled in the art without departing from the spirit of the present invention. Unless specifically identified to the contrary, all terms used herein are used to include their normal and customary terminology in the art

Further, any theory of operation, proof, or finding stated herein is meant to further enhance understanding of the present invention and is not intended to make the scope of the present invention dependent upon such theory, proof, or finding.

While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is considered to be illustrative and not restrictive in character, it is understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.

It is understood that a number of variations may be made to adapt the present invention to a particular medical conditions, diagnostic test, experimental protocol, or the like without changing the basic compositions and methods disclosed herein Therefore, while the invention has been illustrated and described in detail in the foregoing examples and discussion, the same are to be considered illustrative and not restrictive in character. The invention is in no way bound by any theory or explanation of how or why a particular feature of the invention functions

Claims

1 A compound having the following formula.

Z-O or a pharmaceutically acceptable salt thereof, wherein

O is independently selected from the group consisting of the D or L enantiomers of

; and Z is independently selected from the group consisting of H or α-hpoic acid, proparglyglycine, CBZ, and biotin wherein Z is attached to the N-terminal group of O

2. A method of treating a medical condition involving fuπn activity comprising therapeutically administering to a medical patient a therapeutic amount of the fuπn inhibitor according to claim 1

3 The method of claim 2, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier

4 A method according to claim 2, wherein said medical condition is a neuro-degenerative disease

5. A method according to claim 2, wherein said medical condition is a malignancy.

6 The method according to claim 2, wherein said medical condition is due to bacteria

7. The method according to claim 6, wherein said bacteria is independently selected from the group of bacteria consisting of Bacillius anthracis, Coryne bacterium dipthenae, Pseudomonas areugenosa, Clostridium tetani, Clostridium botuhnum

8. The method according to claim 2, wherein said medical condition is due to a virus

9 The method according to claim 8, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus

10 The method according to claim 8, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS)

11 A compound having the following formula

Z-O or a pharmaceutically acceptable salt thereof, wherein

O is comprised of at least one D-arginine amino acid and at least one, but no more than six total arginine amino acid groups, and

Z is independently selected from the group consisting of α-lipoic acid, proparglyglycine, CBZ, biotin, and the D enantiomer of an amino acid, wherein Z is attached to the N-terminal of at least one said ammo acid

12. A method of treating a medical condition involving furin activity comprising therapeutically administering to a medical patient a therapeutic amount of the compound according to claim 11

13 The method according to claim 12, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier

14. The method according to claim 12, wherein said medical condition is a neuro-degenerative disease

15 The method according to claim 12, wherein said medical condition is a malignancy.

16 The method according to claim 12, wherein said medical condition is due to bacteria

17 The method according to claim 16, wherein said bacteria is independently selected from the group of bacteria consisting of Bacilhus anthracis, Corynebacterium dipthenae, Pseudomonas areugenosa, Clostridium tetani, Clostridium botulinum

18 The method according to claim 12, wherein said medical condition is due to a virus.

19 The method according to claim 18, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus.

20. The method according to claim 19, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS).

21 A compound having the formula

Z-U-C wherein,

C is independently selected from the group of compounds consisting of

O

I NH

O

R' is independently selected from the group consisting of,

n' is greater than or equal to 0 and less than or equal to 4,

X is selected from the list of atoms comprising H or F,

U is a peptide with the general formula (J)_n- Arg such that (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, and

Z is independently selected from the group consisting of α -lipoic acid, pyroglutamic acid, 4-morpholinylcaronyl, CBZ, or a derivative of propionic acid, wherein Z is bonded to the N-terminus of said peptide U

22 A method of treating a medical condition involving fuπn activity comprising therapeutically administering to a medical patient a therapeutic amount of the compound according to claim 21

23. The method of claim 22, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier

24. The method according to claim 22, wherein said medical condition is a neuro-degenerative disease

25 A method according to claim 22, wherein said medical condition is a malignancy

26 The method according to claim 22, wherein said medical condition is due to bacteria

27 The method according to claim 27, wherein said bacteria is independently selected from the group of bacteria consisting of Bacillius anthracis, Corynebacterium diptheriae, Pseudomonas areugenosa, Clostridium tetani, Clostridium botulinum

28. The method according to claim 22, wherein said medical condition is due to a virus

29 The method according to claim 28, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus.

30 The method according to claim 8, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS)

31. A furin endoprotease inhibitor comprising a compound of the formula

wherein,

X is either H, or F, and

U is a peptide with the general formula (J)_n-Arg such that (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, and

Z is independently selected from the group consisting of D -lipoic acid, pyroglutamic acid, 4-morpholinylcaronyl, CBZ, or a derivative of propionic acid, wherein Z is bonded to the N-terminus of said peptide U

32 A method of treating a medical condition involving fuπn activity comprising therapeutically administering to a medical patient a therapeutic amount of the compound according to claim 31.

33 The method of claim 32, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier

34 The method according to claim 32, wherein said medical condition is a neuro-degenerative disease

35. A method according to claim 32, wherein said medical condition is a malignancy.

36. The method according to claim 32, wherein said medical condition is due to bacteria.

37 The method according to claim 36, wherein said bacteria is independently selected from the group of bacteria consisting of Bacillius anthraas, Corynebacterium dψtheriae, Pseudomonas areugenosa, Clostridium tetani, Clostridium botulinum

38 The method according to claim 32, wherein said medical condition is due to a virus

39 The method according to claim 38, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus

40. The method according to claim 39, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS)

41 A fuπn endoprotease inhibitor comprising a compound of the formula

wherein, X is at least one of the following H, or F; and

Z is independently selected from the group consisting of D -hpoic acid, pyroglutamic acid, 4-morphohnylcaronyl, CBZ, or a derivative of propionic acid, wherein Z is bonded to the N-terminus of said peptide U.

42 A method of treating a medical condition involving fuπn activity comprising therapeutically administering to a medical patient a therapeutic amount of the fuπn inhibitor according to claim 41

43. The method of claim 42, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier.

44 The method according to claim 42, wherein said medical condition is a neuro-degenerative disease.

45 A method according to claim 42, wherein said medical condition is a malignancy.

46. The method according to claim 42, wherein said medical condition is due to bacteria

47 The method according to claim 46, wherein said bacteria is independently selected from the group of bacteria consisting of Bacillius anthracis, Corynebacterium diptheriae, Pseudomonas areugenosa, Clostridium tetani, Clostridium botulinum.

48. The method according to claim 42, wherein said medical condition is due to a virus

49 The method according to claim 48, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus

50. The method according to claim 49, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS)

51 A fuπn endoprotease inhibitor comprising a compound of the formula

wherein,

X is at least one of the following H, or F,

Z is independently selected from the group consisting of α -hpoic acid, pyroglutamic acid, 4-morpholinylcaronyl, CBZ, or a derivative of propionic acid, wherein Z is bonded to the N-terminus of said peptide U

52 A method of treating a medical condition involving furin activity comprising therapeutically administering to a medical patient a therapeutic amount of the furin inhibitor according to claim 51

53 The method of claim 52, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier

54 The method according to claim 52, wherein said medical condition is a neuro-degenerative disease

55. A method according to claim 52, wherein said medical condition is a malignancy.

56 The method according to claim 52, wherein said medical condition is due to bacteria

57 The method according to claim 56, wherein said bacteria is independently selected from the group of bacteria consisting of Bacillius anthracis, Corynebacterium dipthenae, Pseudomonas areugenosa, Clostridium tetani, Clostridium botulinum

58. The method according to claim 52, wherein said medical condition is due to a virus

59. The method according to claim 58, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus

60. The method according to claim 59, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS)

61 A fuπn endoprotease inhibitor comprising a compound of the formula

wherein,

X is at least one of the following H, or F,

U is a peptide with the general formula (J)_n-Arg such that (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, and n is greater than or equal to 1 and less than or equal to 6.

62 A method of treating a medical condition promoted by fuπn activity comprising therapeutically administering to a medical patient a therapeutic amount of the fuπn inhibitor according to claim 61

63 The method of claim 62, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier

64 The method according to claim 62, wherein said medical condition is a neuro-degenerative disease

65. A method according to claim 62, wherein said medical condition is a malignancy

66 The method according to claim 62, wherein said medical condition is due to bacteria

67 The method according to claim 66, wherein said bacteria is independently selected from the group of bacteria consisting oϊBacilhus anthracis, Corynebacterium dipthenae, Pseudomonas areugenosa, Clostridium tetam, Clostridium botuhnum

68. The method according to claim 62, wherein said medical condition is due to a virus

69. The method according to claim 68, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus

70 The method according to claim 69, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS)

71 A fuπn endoprotease inhibitor comprising a compound of the formula^*

wherein,

X is at least one of the following H, or F,

U is a peptide with the general formula (J)_n-Arg such that (J)_n is an n mer of amino acids joined together via peptide bonds, J is at least one amino acid from the group consisting of Arg, Lys, GIn, GIu, VaI, Ala, Phe, Thr, His, Ser, and GIy, n is greater than or equal to 1 and less than or equal to 6, Z is independently selected from the group consisting of D -lipoic acid, pyroglutamic acid, 4-morpholinylcaronyl, CBZ, or a derivative of propionic acid, wherein Z is bonded to the N-terminus of said peptide U,

R' is independently selected from the group consisting of,

; and

A is the trap consisting of OPH

72 A method of treating a medical condition involving fuπn activity comprising therapeutically administering to a medical patient a therapeutic amount of the fliπn inhibitor according to claim 6171

73 The method of claim 72, wherein said compound is delivered to a medical patient in a pharmaceutically acceptable carrier

74. The method according to claim 72, wherein said medical condition is a neuro-degenerative disease

75 A method according to claim 72, wherein said medical condition is a malignancy.

76. The method according to claim 72, wherein said medical condition is due to bacteria 77 The method according to claim 76, wherein said bacteria is independently selected from the group of bacteria consisting oϊBacilhus anthracis, Corynebacterium dipthenae, Pseudomonas areugenosa, Clostridium tetani, Clostridium botuhnum

78 The method according to claim 72, wherein said medical condition is due to a virus

79 The method according to claim 78, wherein said virus is independently selected from the group of virus consisting of Borna virus, Human Immunodeficiency Virus, Infectious Bronchitis virus, Ebola Zaire, Ebola, Japanese B Encephalitis, arbovirus, and Coronavirus

80 The method according to claim 79, wherein said Coronavirus causes Severe Acute Respiratory Syndrome (SARS)