Patent Application
20220017572
The invention is in the field biochemistry and pharmaceuticals, more specifically pertaining to a structural chemical platform for providing peptide-like compounds for use as a medicament. Particularly, the invention relates to protease inhibitors which are useful for therapy, especially to therapy in relation to viral infections.
This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Oct. 2, 2019, is named Untitled ST25.txt, and is 15.39 KB bytes in size.
Proteases are involved in many metabolic or catabolic reactions in the cell. Hence, they are also involved or deemed to be involved in pathological processes, especially when they become active at times or places where they are not supposed to become active. Proteases are currently classified into six broad groups:
The threonine and glutamic-acid proteases were not described until 1995 and 2004, respectively. The mechanism used to cleave a peptide bond involves making an amino acid residue in which the serine, cysteine, and threonine (proteases) or a water molecule (aspartic acid, metallo- and glutamic acid proteases) are nucleophilic so that it can attack the peptide carboxyl group. One way to make a nucleophile is by a catalytic triad, where a combination of a histidine with an aspartic acid residue is used to activate serine, cysteine, or threonine as a nucleophile.
Within each of the broad groups the proteases have been classified, by Rawlings and Barrett, into families of related proteases. For example within the serine proteases families are labelled Sx where S denotes the serine catalytic type and the x denotes the number of the family, for example S1 (chymotrypsins). An up to date classification of proteases into families is found in the MEROPS database (merops.sanger.ac.uk).
Protease reactions often occur in cascades where a compound is made active by deleting a part of it, which then acts as a protease for activating a second protein, and so on. The classical example of such a pathway is the blood coagulation cascade that ends with the conversion of fibrinogen into fibrin. It will be clear that any activation of such a protease cascade at a time or place where it is not needed will be dangerous to health.
For this and other reasons, some researchers have addressed the ‘degradome’ which is defined as the complete set of proteases present in an organism (Quesada, V. et al., 2009, Nucl. Acids Res. 37:D239-D243). Results of studies on these degradomes have resulted in a database on a website (degradome.uniovi.es) which website also provides detailed information about generic diseases of proteolysis. An overview of mammalian and more specifically human proteases that are involved in diseases and which thus would serve as a target for pharmaceutical protease inhibitors is given in Table A. This list is not complete, but serves to illustrate the extremely wide scope of the field
It is submitted that finding molecules that safely inhibit proteases would be of benefit for treating diseases such as mentioned in table A.
Further, it is well documented that tumor progression, i.e. proliferation, migration, invasion and metastasis is dependent on regulatory proteases on several levels. This includes intracellular maturation of proteins, such as furin, or turnover such as proteasomes, secreted metalloproteinases (MMP) involved in extracellular matrix turnover, as well as membrane bound proteases (ADAM) involved in regulation of growth factors.
Therefore antagonists of such proteases aimed to inhibit cancer progression are desired pharmaceutical compounds.
As can be derived from Table A proteases of the same categories expressed by epithelial mesenchymal and myeloid cells play a role in the resolution of inflammation and tissue repair. Therefore, protease inhibitors targeting such enzymes as MMP, ADAM can be of use in chronic inflammatory disease such as cystic fibrosis (CF), asthma, COPD, rheumatoid arthritis, Crohn's disease and other chronic inflammatory diseases.
For example, ADAM17 may be seen as a key regulator of several pathways involved in CF lung pathology, which makes it a potential therapeutic target for CF and related chronic lung disease (
In the past decade, significant progress was made towards the development of specific ADAM inhibitors with therapeutic potential, and a wide variety of experimental compounds with efficacy in the nanomolar range and acceptable bioavailability and toxicity in animal studies are available [4, 5]. Several clinical applications are under investigation. However, none of the compounds has been approved for phase III studies yet.
The application of such compounds is associated with several caveats. Since the active sites of the ADAM and MMP metalloproteinases are highly related, the specificity of the inhibitors is not absolute. Side effects may occur through inhibition of other than the intended pathway. Conversely, closely related ADAMS, such as ADAM10 and ADAM17, have different but overlapping target protein target spectra [1]. This suggests that functional redundancy should be expected, and absolute selectivity might actually be a disadvantage in clinical application of inhibitors.
However, not only mammalian proteases are important in this respect. A further particular example of proteases involved in pathogenic processes are viral proteases, which are the enzymes used by viruses to cleave nascent proteins for final assembly of new virions.
In a number of infective diseases, such as those caused by the Flaviviridae family of pathogenic viruses (Dengue, West Nile, Hepatitis C), the viral protein has to be split (
Because of the ever increasing threat of viral infections it is desirable to have good inhibitors for the proteases, but in view of their similar substrate preferences it is difficult to design inhibitors that interact preferentially with viral proteases instead of host proteases.
(viral) Proteases not only play a role in pathological processes. Proteases are also not desired in production processes for proteins. Such processes can take place in mammalian or other cell cultures, but also in bacterial cultures. In the latter case it can happen that such bacterial cultures are infected with bacterial phages that also use proteases in their life cycle. Thus, application of protease inhibitors in the field of protein production processes also is desired.
Therefore there is a need for new small molecule protease inhibitors. Generally, however, application of such inhibitors is limited due to a lack of specificity towards the members of the enzyme families, which have different but overlapping expression patterns and physiological functions. Further, systemic delivery of such molecules is often prohibitive due to toxic and off-target effects, whereas targeted delivery is not feasible.
Also, it would be desired to provide a chemical platform which would provide a structural set-up for making peptide-like compounds that can be delivered to cells, and wherein the peptide structure can be tailored so as to accommodate use in treating a set of different protease-mediated diseases.
Further, it would be desired to provide peptide-like compounds suitable for use in the treatment of Dengue.
Chemically-modified peptides are known for various uses. A background reference is WO 01/98362 which describes antimicrobial peptides having a sequence of two to seven amino acids, wherein both the carboxyl terminus and the amino terminus are suggested to be modified with a great variety of side-chains. The antimicrobial use is described with reference to a wide range of application areas, viz. for inhibition and termination of microbial growth, particularly bacterial growth, for industrial, pharmaceutical, household, and personal care use. The reference does not address protease inhibition.
A background reference related to a protease is GB 1 564 317. Herein dipeptide derivatives are disclosed, wherein the amino terminal side is substituted with an aromatic, aliphatic or cycloaliphatic group up to six carbon atoms (viz. phenyl, substituted phenyl, lower cyclo-alkyl, or n-(C1-C6-alkyl. The carboxyl terminal side is substituted with an amide, sulfonyl amide or sulfinyl amide group. These compounds are said to be suitable for use in the treatment of degenerative diseases associated with the action of elastase-like enzymes.
A further reference related to inhibitors of the enzyme elastase, is U.S. Pat. No. 4,528,133. Disclosed are tripeptide and tetrapeptide alkylamides, which have a short alkyl side chain on the amino terminal side, and an alkylcarbonylamino group with 2 to 12 carbon atoms, an alkenyl with 6 to 12 carbon atoms, or a benzyloxycarbonylamino group on the carboxyl terminal side.
Another background reference is WO 2008/137758. Therein modified amino acids or peptides having 2-20 amino acids are disclosed, wherein either or both of the carboxyl and amino termini have a lipophilic tail. The disclosed compounds serve as enhancing agents for the delivery of various drugs, typically nucleic acid molecules to be delivered to cells. In WO 2008/137758 the aforementioned compounds are not used as drug substances.
Another background reference is WO 2009/046220, which relates to lipopeptides for delivery of nucleic acids. Compounds are referred to that comprise a peptide having 2 to 100 amino acid residues, and having a lipophilic group attached to at least one terminus of the peptide, or at least one amino acid residue of the peptide. The peptides are disclosed for a use as an excipient in the delivery of nucleic acids, whereby the nucleic acid is a therapeutically active substance, and the peptide is a formulation aid.
Yet another teaching of a similar use as in the foregoing references, is provided by Damen, M. et al. (J. Contr. Release, 145:33-39, 2010). Therein gemini-like amphiphilic peptides are disclosed for use as vectors for transport of polynucleotides into cells. Here too, the peptides are a formulation aid, with the polynucleotides serving as therapeutically active substances.
A further background reference is Tomohiro Hikima et al., International Journal of Pharmaceutics, Vol. 443, 2013, 288-292, which relates to the gemini surfactant sodium dilauramidoglutamine lysine. This surfactant is investigated as a chemical enhancer for the skin penetration of L-ascorbic acid 2-glucoside. It is not itself used as a drug substance. The disclosed compound does not have a conventional peptide bond. Lysine is connected to one glutamate by its alpha-amino group, and to another glutamate by its epsilon-amino group.
Ten Brink et al., J. Pept.Sci, 2006, 12, 686-692 presents a protocol to label the C-terminus of a peptide with a moiety that is functionalized with a primary amine. Several of such modified peptides are exemplified. The reference foresees a use of the C-modified peptides in click chemistry, biological assays, in making noncovalent stabilized peptides and giant amphiphiles, or as functional building blocks.
The variety of teachings in the art does not allow meeting the present desires. Some of these teachings are too general in nature to provide guidance specifically towards antiviral compounds, let alone to protease inhibition. Others are too specific in nature to provide the desired versatility to create a to provide a structural chemical platform, and other teachings turn into a direction of a non-therapeutic use of modified peptides, viz. as an excipient or formulation aid.
In order to better address one or more of the foregoing desires, the invention, in one aspect, provides geminoid peptide-like compounds according to Formula I:
R1—C(═O)—Zn—NR3-R2 (I)
in which R1 and R2 are each independently saturated, partly saturated or unsaturated, straight, branched or cyclic alkyl chains, wherein R1 has a number of C atoms of 11 or more, preferably 11 to 19, and R2 has a number of C atoms of 12 or more, preferably 12 to 20; R3 is hydrogen or C1-C6 alkyl; n is an integer from 1-15;
each Z independently is an amino acid residue, wherein Zn comprises an N-terminus attached to C(═O) and a C-terminus that is attached to NR3, for use as a medicament.
In another aspect, the invention presents a pharmaceutical composition comprising, as the sole drug substance, a geminoid peptide-like compound according to Formula I, for use as a medicine.
In another aspect, the invention presents novel geminoid peptide-like compounds according to Formula I as defined above, wherein R1 comprises 11 to 13 carbon atoms, R2 comprises 12 to 14 carbon atoms, and n is 4.
In a still further aspect, the invention presents novel geminoid peptide-like compounds according to Formula I as defined above, wherein R1 comprises 13 carbon atoms and R2 comprises 14 carbon atoms.
In yet another aspect, the invention concerns novel geminoid peptide-like compounds according to Formula I as defined above, with the proviso that said compound is not any of the compounds:
General Remark
In the following description, the compounds of formula (I) are sometimes defined with reference to a shorthand notation, whereby the H atoms in R1 and R2, the C(═O) group attached to R1 and the NR3 group attached to R2, are not shown. This refers to a notation Cp—Zn—Cq, whereby Zn has the aforementioned meaning, and p and q are integers such that Cp indicates the number of carbon atoms in R1—C(═O), and Cq indicates the number of carbon atoms in R2. Hereby the left-hand side of the molecule as described is R1 and the right-hand side is R2. E.g., the notation C16-KAAAK-C16 (SEQ ID NO:52) implies R1=n-C15H31, R2=n-C16H33; the C═O group linking R1 to the left-hand K is not shown; R3 is H and the NH group linking the right-hand K to R2 is not shown.
1×106 Vero E6 cells were plated per well into a 6-wells plate and incubated overnight at 37° C. Next day, the DENV-2/NGC virus stock was diluted to 104 TICD50/ml, 103 TICD50/ml, 102 TICD50/ml and 1 ml of the respective virus dilutions was added to each well. Wells contained approximately 80% confluent monolayers of Vero cells. After an incubation period of 1 hour at 37° C., cells were washed twice with medium (DMEM) and medium containing 2% methyl cellulose was added to the wells. To this medium C16-KAK-C16 10 uM was added (W) in DMSO, or an equivalent amount of DMSO (0.05%) was added (W/O). Plate was incubated at 37° C. for two days. Methyl cellulose overlays were removed and cells were fixed with absolute ethanol. Cells were subsequently incubated with specific DENY monoclonal antibody for 1 hour at 37° C., followed by incubation with HRPO-labeled rabbit-anti mouse conjugate. Positive plaques were counted after incubation with AEC substrate chromogen.
The experiment was performed as described in the legend of
In a broad sense, the invention is based on the judicious insight to provide geminoid peptides, having hydrocarbon side chains at both the carbonyl and the amino terminus of the peptide, with a number of carbon atoms of at least 12. It has been found that this allows providing useful antiviral geminoid peptides, particularly being protease inhibitors.
It is thereby emphasized that the invention relates to a medical use of geminoid peptides that hitherto have not been known for such use. Rather, several background references on geminoid peptides relate to a use as a formulation aid. The invention particularly presents a composition for use as a medicine (i.e., a pharmaceutical composition) comprising a geminoid peptide-like compound according to Formula (I) as defined above, and in all of its embodiments described hereinbefore and hereinafter, as the sole drug substance.
In this application the term ‘geminoids’ or ‘gemini-like peptides’ or ‘bi(s)-alkylated peptide’ or ‘BAPS’ is used for those compounds that have a number of amino acids connected through a peptide binding, wherein the C-terminal and the N-terminal peptide both are provided with an alkyl chain. The general synthesis and properties of BAPs has been described in ten Brink et al. (2006) and Damen et al. (2010).
These compounds have the general formula (I):
R1—C(═O)—Zn—NR3-R2 (I)
in which R1 and R2 are each independently saturated, partly saturated or unsaturated, straight, branched or cyclic alkyl chains with a number of C atoms of 12 or more, preferably 12 to 20; R3 is hydrogen or C1-C6 alkyl; n is an integer from 1-15;
each Z independently is an amino acid residue, wherein Zn comprises an N-terminus attached to C(═O) and a C-terminus that is attached to NR3.
Preferably Z is —NR3—C(R4R5)—C(═O)—, in which R4 is selected from side chains occurring in natural amino acids and R5 is selected from the group consisting of hydrogen, C1-C6 straight or branched, saturated, partly saturated or unsaturated alkyl, and alkoxy.
Each Z independently preferably is an amino acid selected from the group consisting of natural amino acids, beta-alanine (bAla), 4-aminomethyl phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl phenylalanine (Iaf), 3-pyridyl alanine (Pya), 4-piperidyl alanine (Ppa), 4-aminomethyl cyclohexyl alanine (Ama), 4-aminocyclohexyl alanine (Aca), ornithine (Orn), citrulline, hydroxylysine (Hyl), allo-hydroxylysine (aHyl), 6-N-methyllysine (MeLys), desmosine (Des), isodesmosine (Ide), 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), 2-aminobutyric acid (Abu), 4-aminobutyric acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), 2,2′-diaminopimelic acid (Dpm), 2-3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), allo-isoleucine (AIle), sarcosine (MeGly), N-methylisoleucine (MeIle), N-methylvaline (MeVal), norvaline (Nva), and norleucine (Nle). Preferably each Z is independently a natural amino acid.
Preferably n is an integer from 1-10, and more preferably from 3-8, more preferably from 3-7, more preferably from 3-6, and more preferably from 3-5.
As an example the structure of such a compound is given below, the compound R1—C(═O)—KZnK—NH—R2, wherein a left (N-terminal) and a right (C-terminal) Z amino acid is provided by a lysine residue, which can be connected via a further number of amino acids.
Furthermore, in this application the notation C16-KAAAK-C16 (SEQ ID NO:52) implies R1=n-C15H31, R2=n-C16H33; the C═O group linking R1 to K is not shown; R3 is H and the NH group thus linking K to R2 is not shown); Zn is represented by the sequence KAAAK (Lys-Ala-Ala-Ala-Lys; (SEQ ID NO:52)). Note that due to the presence of the linker C═O, the number of C atoms at the right and the left of the amino acid sequence is 16. Thus, the short hand notation reads C16-KAAAK-C16 (SEQ ID NO:52).
In one aspect, the invention is directed to the compounds of formula (I) for use as a medicament. Particularly, this use is as a medicament in the treatment of viral infection. Accordingly, the invention also pertains to a method of treatment of a viral infection, by the administration, to a subject in need thereof, an effective amount of a compound according to the above-identified formula (I).
In part, the invention relates to novel compounds. In one aspect, these compounds are characterized by satisfying the above formula I, wherein the number of carbon atoms in R1—C(═O) and R2 is 14. In another aspect, these compounds are characterized by satisfying the above formula I, wherein the number of carbon atoms in R1—C(═O) and R2,each independently, is 12 to 14, and n is 4. The foregoing compounds are believed to provide an optimum in terms of combined properties such as a viral inhibitory effect and ease of formulation.
In an alternative embodiment, the novel compounds are those satisfying the above formula I, with the proviso that said compound is not any of the following compounds:
The compounds herein excluded are those which have been incidentally disclosed by Damen et al. or ten Brink et al. The skilled person will understand that the capital letters refer to an internationally accepted way of indicating amino acids. A list of natural amino acids, with their general abbreviations is given in Table 1.
Z is an amino acid residue. The term “residue” is hereby used to indicate that both the carboxyl and the amino groups of each Z are bound, either to another Z, or to the C(═O) or —NR3 groups shown in Formula I. More specifically, in the above-mentioned geminoid peptide-like compounds Z is based on an amino acid chosen from the group of natural amino acids, beta-alanine (bAla), 4-aminomethyl phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl phenylalanine (Iaf), 3-pyridyl alanine (Pya), 4-piperidyl alanine (Ppa), 4-aminomethyl cyclohexyl alanine (Ama), 4-aminocyclohexyl alanine (Aca), ornithine (Orn), citrulline, hydroxylysine (Hyl), allo-hydroxylysine (aHyl), 6-N-methyllysine (MeLys), desmosine (Des), isodesmosine (Ide), 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), 2-aminobutyric acid (Abu), 4-aminobutyric acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), 2,2′-diaminopimelic acid (Dpm), 2-3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), allo-isoleucine (AIle), sarcosine (Me Gly), N-methylisoleucine (MeIle), N-methylvaline (MeVal), norvaline (Nva), and norleucine (Nle). Also preferred are geminoid peptide like compounds wherein n is an integer from 1-10 and more preferably from 3-8, more preferably from 3-7, more preferably from 3-6 more preferably from 3-5. Further preferred are geminoid peptide-like compounds wherein NR3 is NH. Further preference is expressed for geminoid peptide-like compounds wherein Z is a natural amino acid. It is also preferred to use geminoid peptide-like compounds wherein the alkyl chains are partly saturated.
Further preferred are geminoid peptide-like compounds wherein Zn is a part of the molecule that is capable of binding to a protease recognition site on a substrate, preferably wherein said protease recognition site is chosen from the group of recognition sites specified in Tables B and C, AKRRSQ (SEQ ID NO:1), RmXR, in which m is an integer of 2 or higher and X is any amino acid, SPLAQAVKSSSRK (SEQ ID NO:2), GSDMELPLPRNITEGEARGSVILTVKPIFEEF (SEQ ID NO:3) and GSKTEEISEVNLDAEFRHDS (SEQ ID NO:4).
In an interesting embodiment of the various applicable aspects of the invention as broadly described above, R1—C(═O) and R2 in the compounds of formula (I), each independently, have a number of carbon atoms of at least 14, preferably at least 16. Preferably, the number of carbon atoms for the groups R1—C(═O) and R2, each independently, is 24 or lower, such as 22, 20, 18, 16, 14, or 12. Preferably, the number of carbon atoms for the groups R1—C(═O) and R2, each independently, is 12 to 19, more preferably 13 to 18, more preferably 15 to 17.
In another interesting embodiment, either or both of R1 and R2 are straight chain hydrocarbons, preferably mono-unsaturated. In yet another embodiment, either or both of R1 and R2 are branched chain hydrocarbons, preferably saturated.
Preferably, the integer n in the compounds of formula (I) is 4 or 8, most preferably 4.
In an interesting embodiment, Zn in the compounds of Formula (I) is devoid of proline in the second position. In another interesting embodiment, proline is absent.
It is noted that, in accordance with conventional peptide nomenclature, the peptide sequence is numbered from the N-terminal side to the C-terminal side of the peptide.
In a further interesting embodiment, serine is not present in a position at the N-terminal side of an arginine or a lysine (i.e., in conventional numbering, a serine is not present before an arginine or a lysine). In a still further interesting embodiment, a serine is present and at least one argine or lysine, wherein the serine is positioned at the C-terminal side of the argine or lysine.
In a further interesting embodiment, Zn in the compounds of Formula (I) has a hydrophobic amino acid in the first position. Natural hydrophobic amino acids are glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. Preferred hydrophobic amino acids are leucine and phenylalanine.
Further preferred are geminoid peptide-like compounds having the general formula:
C15C(═O)—KAqK—NH—C16 (II)
with q being an integer of from 1 to 15, preferably 1 to 7, more preferably from 1 to 5, more preferably from 1 to 4, more preferably from 1 to 3, and more preferably 1 or 2, and wherein C15 is a saturated, partly saturated or unsaturated straight, branched or cyclic alkyl chain of 15 carbon atoms and C16 is a saturated, partly saturated or unsaturated straight, branched or cyclic alkyl chain of 16 carbon atoms.
The therapeutic use of said geminoid peptide-like compounds is, for instance, in treating protease mediated disease. The therapeutic use is preferably in antiviral therapy, in inflammation and in ADAM17 mediated diseases, such as ulcerative colitis, rheumatoid arthritis, cystic fibrosis, COPD, IPF, Crohn's disease, multiple sclerosis and atherosclerosis. If the use is in antiviral therapy, preferably said antiviral therapy is therapy against Flaviviridae, more preferably therapy against dengue.
Further part of the invention are non-therapeutic uses of a geminoid peptide-like compound, having a general formula according to Formula I as defined above as protease inhibitors, as anti-septics, particularly for the disinfection of surfaces, and as anti-microbial agents in cell-culturing.
Salts and solvates. It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of a geminoid, as described herein, for example, a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19. Examples of suitable salts include: those derived from the following inorganic acids (such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous acid); those derived from organic acids (such as 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camp horsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric acid); those derived from polymeric acids (such as tannic acid and carboxymethyl cellulose). Unless otherwise specified, a reference to a geminoid or geminoids also includes salt forms thereof.
It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of a geminoid. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g., geminoid, salt of geminoid) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc. Unless otherwise specified, a reference to geminoid also includes solvate forms thereof.
One aspect of the invention pertains to a pharmaceutical composition comprising a geminoid according to Formula I or a salt or solvate thereof.
A further aspect of the invention pertains to a pharmaceutical composition comprising a geminoid according to Formula I or a salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. Examples of suitable pharmaceutically acceptable carriers, diluents, and excipients are described below.
Unique characteristics of Geminoids We submit, based on evidence presented below that the compounds according to the above described general formula can be used as specific protease inhibitors in various clinical and non-clinical contexts.
One of the main advantages of the present compounds of the invention is the specificity that is offered by the peptide sequence Zn, which can be optimized to be identical to or a derivative of a sequence that is capable of being targeted to an actual protease cleavage site on a substrate. Thus, preferably, the compounds of the invention comprise a moiety Z that provides for a chemical structure, preferably an amino acid structure, that is targeted to a specific protease active domain. A further major advantage of the present compounds is formed by the nanoparticle aggregation of the compounds and their adaptable interaction with cellular membranes. These two properties together offer unique opportunities for functional targeting tissue specificity and sub-cellular delivery. It is submitted that the interaction with cellular membranes and nanoparticle formation has already been described by Damen et al. (J. Controlled Release 145: 33-39, 2010), which information, especially the synthesis of the compounds as described in paragraphs 2.2 and 2.3 of the scientific document and the results depicted in
One aspect of the present invention pertains to the use of a geminoid according to Formula I or a salt or solvate thereof as an anti-protease agent, also indicated as protease inhibitor. In general the term ‘protease inhibitor’ relates to a compound that inhibits a protease. Many proteases are highly specific, acting on single or small families of substrates, but many single substrates can also be cleaved by several proteases. For many proteases the actual amino acid sequence that acts as the substrate is known. These substrate sequences often are short sequences (maximizing 4-8 amino acids). As is shown in the experimental part, the substrate sequence, or a derivative thereof can be designed to form the main core of the geminoid compound (the part Zn of the general formula). In such a way a compound can be constructed that is ideally suited to bind with a single protease.
In a number of infective diseases, such as those caused by the Flaviviridae family of pathogenic viruses (Dengue, West Nile, Hepatitis C), the viral protein has to be split (
The active site of the dengue protease is in the N-terminal part of NS3 which is also a serine protease with catalytic triad Asp79-His51-Ser135, but requires NS2A (CF40) for activity; the inhibition reported here was studied on a NS3-NS2A construct (CF40-GGGGSGGGG-NS3; (SEQ ID NO:54)) which has also been structurally characterized [Erbel et al., 2006; Luo et al. 2008].
The substrate specificity of proteases can be studied with FRET substrates of the Abz-EEDnp type, where the fluorescence of the N-terminal Abz (aminobenzoyl) group is quenched by the C-terminal EDDnp (ethylenediamine-dinitrofluorophenyl) group until the peptide is split (
For ADAM17 a highly susceptible recognition site is formed by SPLAQA∧VKSSSRK (SEQ ID NO:2), the aggrecanase recognition sequence from aggrecan is GSDMELPLPRNITEGE∧ARGSVILTVKPIFEEF (SEQ ID NO:3), and the BACE recognition sequence from β-amyloid precursor protein is GSKTEEISEVNL∧DAEFRHDS (SEQ ID NO:4) (the ∧ indicates the protease cleavage site).
Further specific recognition sites and cleavage sites for some serine proteases are given in the below table B.
Ile-Val-Glu-Gly
Ile-Ile-Gly-Gly
Ile-Val-Gly-Gly
Val-Val-Gly-Glu
Ser-Asn-Gly-Glu
Ser-Phe-Arg-Phe
Ile-Val-Gly-Gly
Val-Val-Gly-Gly
Ile-Val-Gly-Gly
Val-Val-Gly-Gly
Ile-Val-Gly-Gly
Ile-Val-Gly-Gly
Val-Val-Gly-Gly
Ile-Ile-Gly-Gly
Substrate cleavage sites for various caspases are given in the below table C.
There is thus a large variation in specific sites that can be used for constructing the protease inhibitors according to the invention. The sequences as given above can be used, but also sequences that are derived from these sequences, i.e. by adding, deleting or substituting one or more of the amino acids. Substitutions can take the form of natural amino acids, but also the non-natural amino acids as listed above may be used. An example is the 1,2,3-triazole moiety that can be obtained by the Cu-catalysed so-called ‘click’ reaction between an amphiphilic peptide fragment appended with an alkyne and another one with an azide. Other less reactive analogues of the amide bond are the compounds in which the carboxylic acid part of the amide has an alpha-keto group (so another, but more reactive, carbonyl next to the carbonyl involved in the covalent bond with N), or in which the amine part of the amine bond is replaced by a hydrazine (so 2 N atoms between the carbonyl and the C of the next amino acids instead of 1 as in the amide). It is also possible that amino acid like moieties according to the —NCOR4R5 schedule as defined above are inserted.
Accordingly, the invention comprises methods to inhibit proteases by using a geminoid compound according to Formula I. Such methods may be performed, for example, in vitro, as part of an assay. Such methods may also be performed, for example, in vivo, by administration of a geminoid according to Formula I or a salt or solvate thereof to a patient. Another aspect of the present invention pertains to a geminoid compound according to Formula I or a salt or solvate thereof, for use in a method of treatment of the human or animal body by therapy.
Another aspect of the present invention pertains to a geminoid compound according to Formula I or a salt or solvate thereof, for use in a method of treatment, for example, in a method of treatment or prophylaxis of (including, e.g., reducing the risk of) a disease condition as described herein. Another aspect of the present invention pertains to a geminoid compound according to Formula I or a salt or solvate thereof, for use in a method of treatment of a disease condition as described herein. Another aspect of the present invention pertains to a geminoid compound according to Formula I or a salt or solvate thereof, for use in a method of prophylaxis of (including, e.g., reducing the risk of) a disease condition as described herein.
Another aspect of the present invention pertains to use of a geminoid compound according to Formula I or a salt or solvate thereof in the manufacture of a medicament for use in a method of treatment or prophylaxis, for example, in a method of treatment or prophylaxis of (including, e.g., reducing the risk of) a disease condition as described herein.
Another aspect of the present invention pertains to use of a geminoid compound according to Formula I or a salt or solvate thereof in the manufacture of a medicament for use in a method of treatment, for example, in a method of treatment of a disease condition as described herein.
Another aspect of the present invention pertains to use of a geminoid compound according to Formula I or a salt or solvate thereof in the manufacture of a medicament for use in a method of prophylaxis, for example, in a method of prophylaxis of (including, e.g., reducing the risk of) a disease condition as described herein.
Another aspect of the present invention pertains to a method of treatment or prophylaxis, for example, a method of treatment or prophylaxis of (including, e.g., reducing the risk of) a disease condition as described herein, comprising administering to a patient in need of said treatment or prophylaxis a therapeutically- or prophylactically-effective amount of a geminoid compound according to Formula I or a salt or solvate thereof, preferably in the form of a pharmaceutical composition.
Another aspect of the present invention pertains to a method of treatment, for example, a method of treatment of a disease condition as described herein, comprising administering to a patient in need of said treatment a therapeutically-effective amount of a geminoid compound according to Formula I or a salt or solvate thereof, preferably in the form of a pharmaceutical composition.
Another aspect of the present invention pertains to a method of prophylaxis, for example, a method of prophylaxis of (including, e.g., reducing the risk of) a disease condition as described herein, comprising administering to a patient in need of said prophylaxis a prophylactically-effective amount of a geminoid compound according to Formula I or a salt or solvate thereof, preferably in the form of a pharmaceutical composition. In one embodiment, the disease condition is a disease condition that is mediated by a protease, such as a viral protease, intracellular proteases such as furin or proteasomes, extracellular metalloproteases, such as MMP, Neutrophil elastase (NE), and membrane-bound metalloproteinases including ADAMS (e.g. ADAM17, ADAM10, ADAM33), and Meprins. The term “treatment” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition.
Unless otherwise specified, treatment as a prophylactic measure (i.e., prophylaxis) is encompassed by the term “treatment”. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term “treatment” but is more specifically described by the term “prophylaxis”. Both absolute prophylaxis and probabilistic prophylaxis are encompassed by the term “prophylaxis”. Thus, “prophylaxis” of a disease condition encompasses “reducing the risk of’ that disease condition.
The term “therapeutically-effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. Similarly, the term “prophylactically-effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
The term “treatment” includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, a geminoid compound according to Formula I or a salt or solvate thereof may also be used in combination therapies, e.g., in conjunction with other agents, for example, other anti-viral agents, antibiotic agents, anti-cancer agents, etc. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets. For example, it may be beneficial to combine treatment with a geminoid compound according to Formula I or a salt or solvate thereof with one or more other (e.g., 1, 2, 3, 4) agents or therapies, for example, treatment with one or more of: AZT, Tamaflu®, Tofacitinib (JAR inhibitor), Velkade or related (Proteasome inhibitor).
In one embodiment, a geminoid compound according to Formula I or a salt or solvate thereof is combined with one or more (e.g., 1, 2, 3, 4) additional therapeutic agents. One aspect of the present invention pertains to a geminoid compound according to Formula I or a salt or solvate thereof, in combination with one or more additional therapeutic agents. The particular combination would be at the discretion of the physician who would select dosages using his or her common general knowledge and dosing regimens known to a skilled practitioner.
The agents (i.e., a geminoid compound according to Formula I or a salt or solvate thereof, plus one or more other agents, including one or more other geminoid compounds) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s). The agents (i.e., a geminoid compound according to Formula I or a salt or solvate thereof, plus one or more other agents, including one or more other geminoid compounds) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use, as described below.
A geminoid compound according to Formula I or a salt or solvate thereof may also be used as part of an assay, for example, an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound.
A geminoid compound according to Formula I or a salt or solvate thereof may also be used as a standard or comparator, for example, in an assay, in order to identify other active compounds.
Another aspect of the present invention pertains to a kit comprising (a) a geminoid compound according to Formula I or a salt or solvate thereof, preferably provided in the form of a pharmaceutical composition and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the active compound.
The written instructions may also include a list of indications for which a geminoid compound according to Formula I or a salt or solvate thereof is a suitable treatment.
The geminoid compound according to Formula I or salt or solvate thereof, or the pharmaceutical composition comprising a geminoid compound according to Formula I or a salt or a solvate thereof may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action). Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual, transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol or powder, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.
The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilled platypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a Iagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g, a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a fetus. In a preferred embodiment, the subject/patient is a human. Further, the subject can be a plant, chosen form a monocotyledonous plant, such as a grain plant or a bulbous plant, a dicotyledonous plant, a fern, a moss, or even a micro-organism, if said micro-organism suffers from viral pathogens. Accordingly also bacteria, suffering from bacteriophages, can be considered as subject for the present invention.
While it is possible for the active compound (i.e, a geminoid compound according to Formula I or a salt or solvate thereof) to be administered alone, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one active compound, as defined above, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound. The term “pharmaceutically acceptable” as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
Suitable carriers, diluents, excipients, etc can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
The formulations may be prepared by any methods well known to the skilled person in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary. The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof. Formulations may suitably be in the form of tablets (including, e.g., coated tablets), granules, powders, lozenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, pessaries, suppositories, liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more active compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.
The active compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients.
One preferred pharmaceutical formulation is when the active compound is presented in a liposome or other microparticulate which is designed to target the active compound, for example, to blood components or one or more organs. The geminoid compound according to Formula I is especially suitable for such a formulation, since it is well attached to the liposome particle due to the fatty alkyl chains.
Formulations suitable for oral administration (e g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, etectuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses. Due to the amphiphilic character the geminoid compounds are soluble both in aqueous and non-aqueous solvents and typically suitable for emulsions.
Formulations suitable for buccal administration include mouthwashes, lozenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Lozenges typically comprise the active compound in a flavored basis, usually sucrose, mint and acacia or tragacanth. Pastilles typically comprise the active compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the active compound in a suitable liquid carrier.
Formulations suitable for sublingual administration include tablets, lozenges, pastilles, capsules, and pills. Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), mouthwashes, lozenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
One particularly preferred oral delivery route is transmucosal for the upper respiratory pathways by using an aerosol.
Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavours, flavour enhancing agents, and sweeteners. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.
Ointments are typically prepared from the active compound and a paraffinic or a water-miscible ointment base. Creams are typically prepared from the active compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
Emulsions are typically prepared from the active compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. A hydrophilic emulsifier may be included together with a lipophilic emulsifier which acts as a stabilizer, but in view of the amphiphilic character of the geminoids according to the invention, such additions do not seem necessary. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound. Formulations suitable for intranasal administration, where the carrier is a solid, include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluorornethane, dichloro-tetrafluoroethane, carbon dioxide, or other suitable gases.
Formulations suitable for ocular administration include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound.
Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active compound, such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e g., solutions, suspensions), in which the active compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection Typically, the concentration of the active compound in the liquid is from about 1 ng/mL to about 10 mg/mL, for example from about 10 ng/mL to about 1 mg/mL The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
It will be appreciated by one of skill in the art that appropriate dosages of the active compound (i.e., a geminoid compound according to Formula I or a salt or solvate thereof), and compositions comprising the active compound, can vary from patient to patient and from targeted protease to targeted protease. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
In general, a suitable dose of the active compound is in the range of about 50 pg to about 1 gram (more typically about 100 pg to about 25 mg) per kilogram body weight of the subject per day. Where the active compound is a salt or solvate, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
Next to systemic delivery also local delivery is contemplated and a further alternative may be targeted administration. Targeted administration can be achieved by binding a homing moiety that is specific for a particular site or molecule in the subject to the geminoid compound of the invention, thus providing a targeted geminoid compound. Homing moieties that target various possible targets (such as tumor tissue, nucleolar localization, etc.) are known to the skilled person and can be coupled to the geminoid compound using conventional chemical binding techniques.
The geminoid compounds according to Formula I can be used as substrate for proteases and as such can function as protease inhibitors. As such they can be tailored to different specific targets with high affinity by modeling the peptide part. The compounds are not only versatile with respect to the peptide part, but also with respect to the alkyl chains. The lipid part of the molecule ensures high delivery and target binding properties (as has been demonstrated by Damen et al., 2010).
Proteases that can be targeted are viral proteases, such as dengue protease, other flavivirus proteases, serine proteases, such as furin and kallikrein, extracellular metalloproteases, such as MMP, Neutrophil elastase (NE), and membrane-bound metalloproteinases including ADAM (e.g. ADAM17, ADAM10, ADAM33), blood proteases such as thrombin and plasmin, fecal proteases that cause pruritis and other proteases as mentioned in Table A or elsewhere in the present specification,
In principle any protease can be targeted, because proteases are characterized by their reactivity towards a very specific substrate, the specific target epitope/sequence of the protein that is cleaved by the protease. It is believed (and shown in the experimental section of the present application) that the geminoid compounds of the present invention are capable of being recognized by the protease for which they have been designed, i.e. bind to the protease, and thus act as a (competitive) inhibitor for the protease. Accordingly, the compounds of the present invention are suitable as pharmaceutically active compounds against a variety of diseases, which are caused or aggravated by proteases.
One particular important area is the interaction of the compounds of the present invention with MMP metalloproteinases and ADAM compounds.
Metalloproteinases involved in chronic inflammatory disease are endogenous secreted or membrane bound enzymes, which are involved in the resolution of inflammation, tissue injury and repair. These proteases regulate pro-inflammatory cytokines, growth factors, extracellular matrix remodeling enzymes, and dynamic cell-cell interactions required for repair and differentiation. These enzymes have been identified previously as important therapeutic targets. Specifically, chronic airway inflammation and recurrent exacerbations are hallmarks of cystic fibrosis (CF) and other chronic lung disease (COPD, IPF), of which the mechanisms are explained below. The frequent cycles of damage and repair under inflammatory stress result in a progressive and apparently irreversible tissue remodeling and loss of function.
Current studies aimed at the elucidation of the signaling pathways that control tissue repair in chronic lung disease, in particular CF, have suggested that in particular ADAM17-TACE and the related ADAM10 are involved in the regulation of pro-inflammatory signaling through their substrate TNFa, and of tissue remodeling through the regulation of the EGFR and IL6RA receptors. Supported by preliminary data in a mouse model of CF and cultured bronchial epithelial cells (Scholte et al in preparation), it is proposed that a mechanism schematically depicted in
Other targets in this category are involved in extracellular matrix turnover and deposition, including a family of collagenases and elastases (MMP8, MMP9, neutrophil elastase), and natural inhibitors of these (TIMP, al-antitrypsin).
These enzymes have been identified as important therapeutical targets [1, 2] and many inhibitors have been developed, none of which has reached phase II clinical trial [5].
A further part of the invention is formed by the non-therapeutical use of the protease inhibitors according to the present invention. Non-therapeutic uses according to the present invention are the use of the protease inhibitors according to the present invention as research tools, e.g. for screening the presence of proteases. For such a use the geminoid protease inhibitors may be labeled, e.g. by binding to a labeling moiety or by using a radioactive moiety in the synthesis of the geminoid compound. Further, the compounds can be used in assays for specific detection of the substrate against which the geminoid compound is targeted. Also here, labeling of the compound may be applied.
A further use may be cosmetic, e.g. the use against acne or against pruritis.
Further, the protease inhibitors of the present invention may be used in food processing, as e.g. described by Garcia-Carreno, F. L. (1991, Biotechnol. Educat. 2:150-153) or be added to food or feed to increase digestibility.
Another non-therapeutic use is based on the versatility of the compounds of the invention to serve as a structural chemical platform for further screening of derivative compounds. Particularly, the compounds according to Formula I as defined above, in all its embodiments, can suitably be used in a screening method for finding further biologically active geminoid peptide-like compounds, wherein one or more compounds according to Formula I are subjected to screening in an assay for the desired activity, preferably in comparison with chemical derivatives of said one or more compounds.
According to a still further non-therapeutic use, the geminoid compounds of formula I, and the various above-described embodiments thereof, is as anti-microbial agents in cell-culturing. In yet another aspect of the invention, the geminoid compounds of formula I, and the various above-described embodiments thereof, are uses as anti-sceptics, particularly for the disinfection of surfaces, such as table surfaces, or surfaces in kitchens or bathroom, such as in households, or in public environments such as restaurants, and the like. For such a use, the compounds will generally be comprised in a carrier, typically dissolved or dispersed in an aqueous carrier, particularly in water, and can then be applied in a conventional manner, such as by spraying or via application by a cloth or other suitable article for applying a liquid disinfectant onto a surface.
As an example of a valid application of specific and targetable geminoids in human disease we present an analysis of the role of membrane-bound proteases in the development of chronic lung disease. In chronic lung disease, activation of inflammatory responses is associated with mucus hyper secretion, epithelial metaplasia and irreversible remodelling (thickening) of the airways. Genetic factors predispose patients to excessive inflammation, tissue injury and inadequate repair. Regulatory proteases (MMP, ADAM, Meprin) play a major role in these responses. An extreme example of this is cystic fibrosis (CF), where a mutation in the CFTR chloride transport channel causes a devastating and untreatable form of chronic lung disease in more than seventy thousand patients worldwide.
Data from earlier studies in our labs show that progressive airway remodelling is observed in CT scans of CF infants, even before chronic bacterial colonisation, which is generally considered a hallmark of CF lung disease [6, 7]. While upper airway disease has traditionally received most attention in CF, it has become clear with the advent of improved imaging and lung function measurement techniques that the distal airways, in particular the membranous bronchioles are involved at a very early stage in CF [8].
The relationship between the complex pathology and the primary defect, a mutation in the CFTR chloride channel protein, is still the subject of intense investigation. The molecular mechanisms involved in progressive airway remodelling in the CF lung as discussed below yield new therapeutic approaches which can be fulfilled by the compounds of the present application.
In this context it is important to note that CF is not only a disease of the secretory epithelia. CFTR deficiency affects the behavior of alveolar macrophages [9-11] and neutrophils [12, as well as airway smooth muscle cells {Michoud et al., Am. J. Resp. Cell Molec. Biol. 40 (2009) 217, 2009]. This may independently contribute to the ‘exaggerated’ inflammatory and remodeling responses observed in patients and animal models of CF. The ‘trigger happy’ state of the immune system may contribute to the intensity of exacerbations in CF lung disease in human patients. Abnormal responses of fibroblasts and smooth muscle cells, either cell autonomous or by interaction with CF epithelial cells may determine the CF airway connective tissue pathology.
The large variation in CF lung disease progression even among patients with the same CFTR mutations, and the identification of genetic factors that influence pathology (modifier genes) further illustrates the fact that CFTR is not the only relevant therapeutic target [13].
Current efforts in the CF field are aimed at activation of the most common mutant form of CFTR, F508del (70% of all CF alleles). Although promising results were reported, none of the available compounds have shown significant long term remission in patients. Therefore, alternative approaches, including novel anti-inflammatory treatments are actively pursued [14].
The molecular mechanisms involved in the development of CF lung disease are poorly understood, despite intensive research in the past two decades. Experiments in cellular and animal models, including work in our laboratory (Scholte et al in preparation, Buijs-Offerman Thesis Erasmus MC 2011), has suggested that CFTR dysfunction affects a network of interrelated regulatory signaling molecules and their receptors, which regulates cell fate decisions. Based on our current investigations we propose that the CF deficient airways are not only challenged by recurrent infections but also respond differently to epithelial injury and inflammation.
Together, our data suggest that CF mutant mice suffer from delayed resolution of injury and inflammation, associated with enhanced activity of the EGFR and IL6 pathways. We propose that a similar mechanism is at work in the human CF lung contributing to chronic inflammation, epithelial metaplasia and connective tissue remodeling.
IL6 signalling requires the IL6R receptor IL-6RA and the co-receptor IL6 signal transducer (Gp130/IL6st). In fibroblasts and smooth muscle cells the EGFR and IL6R signals converge in activation by phosphorylation of the acute response factor STAT3, a transcription factor involved in fibrotic responses and inflammatory lung disease [15]. Recently STAT3 was recognized as an important element in progressive CF lung disease by a meta-analysis of transcriptional responses of CF compared to normal tissues [16] (
ADAMs (A Disintegrin And Metalloproteinase) form a family of ubiquitous membrane associated proteases, involved in many aspects of human development and pathology. The ADAM isoforms each interact with a different range of target proteins, many of which are involved in cell signaling, including cell adhesion proteins and receptors, cytokines and and growth factors. The canonical ADAM17/TACE, activates the pro-inflammatory factor TNFa, and is investigated as a target for inflammatory disease [2]. An ADAM17/TACE conditional (loxed) mutation in myeloid cells successfully prevented endotoxin shock in a mouse model [19]. Since CF mutant mice display a hyper inflammatory phenotype, at least in part due to abnormal behavior of alveolar macrophages [9, 20], it seems likely that inhibition of the ADAM17-TNFa pathway could attenuate this aspect of the CF phenotype.
ADAM17 is also required to activate the precursors of EGFR agonists like amphiregulin (AREG), epiregulin (EREG), heparin binding EGF (HB-EGF) and TNFα by shedding their active domain from the cell membrane, allowing autocrine and paracrine EGFR signaling [3]. EGFR activation is related to airway repair and goblet cell hyperplasia [21]. In the progression liver fibrosis amphiregulin activation of EGFR is shown to be important [22]. In experimental lung fibrosis another ADAM17 substrate and EGFR agonist TNFα plays a major role [23].
The IL6-RA receptor, which is produced by epithelial cells, is an ADAM17 substrate as well [24, 25]. This allows transactivation of IL6st (Gp130) on airway smooth muscle cells, which do not express IL6-RA, causing local VEGF release [26] (
In sum, targeting ADAM17 by an inhibitor appears an attractive approach.
In view of the many biological functions of ADAM17 in different physiological compartments [1], systemic delivery of an inhibitor is likely to have multiple and possibly adverse and contradictory effects. Further it is difficult to find small molecules that show sufficient specificity for their targets. Indeed, although many small molecule ADAM inhibitors have been developed none of these have reached Phase III. Therefore, an approach that allows targeted delivery of highly selective geminoids as is possible and contemplated in the present application would be preferable. In the case of lung diseases like CF luminal delivery by aerosol is preferred to intravenous or oral delivery
In CF and related lung disease targets are not limited to ADAM17. ADAM10 is closely related to ADAM17 and has an overlapping target spectrum. It is involved in epithelial fate decisions during tissue repair and inflammation. Also ADAM33 was recently identified as genetic determinant of Asthma and COPD [28, 29].
Meprin, another extracellular protease is also involved in CF pathology at the level of EGFR [30] and sodium channel regulation [31]; thus offering an alternative target for specific inhibition. Neutrophil elastase, which is secreted by degranulation of activated Neutrophils and causes excessive tissue damage during chronic lung inflammation is also considered to be a target of intervention.
Methods and Materials
General. Aldehyde functionalized resin (4-(4-Formyl-3-methoxyphenoxy) butyryl AM resin, loading 0.98 mmol/g) was obtained from Novabiochem and amino acids were purchased from Bachem and Novabiochem. All other chemicals were acquired from Fluka, Aldrich and Baker. The chemicals were used as received, unless stated otherwise. The polyethylene syringe barrels containing a 20 micron porous polyethylene frit were acquired from Supelco.
Mass spectra were recorded on a Thermofinnigan LCQ-ESI-ion trap. The samples were dissolved in methanol. 1H-NMR spectra were recorded on a Bruker DMX-300 MHz at room temperature. The samples were dissolved in DMSO-d6. In 1H-NMR spectra, the assigned protons are in italics; s=singlet; t=triplet; qu=quintet; m=multiplet; b=a broad peak. Spectra are written in the following format: chemical shift (peak type, number of protons, subjective assignment).
Synthesis. Compounds B as depicted in
A reductive amination of 1.2 g aldehyde resin (1.2 mmol) as described using 2.0873 ml palmitylamine (9 mmol), 0.56 g NaCNBH3 (9 mmol) and 550 μl AcOH in 50 ml of a 1:1 mixture of DMF/MeOH. The resin was transferred to a syringe marked (A) and one sixth of the resin was removed for experiment 8.5.4. Then, the first amino acid was coupled twice using 1.9881 g and 1.53 g Fmoc-Arg(Pmc)-OH (3.0 mmol and 2.3 mmol) 3.60 ml 1M HOBt/DMF (3.60 mmol) and 3.30 ml 1M DIPCDI/DMF (3.30 mmol). A chloranil test was negative. The resin was capped. From syringe (A), one fifth of the resin was put in a new syringe (B). To syringe (A) Fmoc-Ala-OH (592 mg, 1.5 mmol) was coupled and to syringe (B) Fmoc-Arg(Pmc))-OH (397 mg, 0.6 mmol) was coupled. Then, from syringe (A) one fourth of the resin was put in a new syringe (C). Then, Fmoc-Ala-OH (592 mg, 1.5 mmol) was coupled to it to syringe (A), Fmoc-Arg(Pmc)-OH (398 mg, 0.7 mmol) was coupled to syringe (C) and palmitic acid (154 mg, 0.6 mmol) was coupled to syringe (B). From syringe (A) one third of the resin was put in a new syringes (D). Then, Fmoc-Ala-OH (395 mg, 1.0 mmol) was coupled to it to syringe (A), Fmoc-Arg(Pmc)-OH (398 mg. 0.6 mmol) was coupled to syringe (D) and palmitic acid (154 mg, 0.6 mmol) was coupled to syringe (C). Then, from syringe (A) half of the resin was put in a new syringe (E). Then, Fmoc-Ala-OH (197 mg, 0.5 mmol) was coupled to it to syringe (A), Fmoc-Arg(Pmc)-OH (398 mg, 0.6 mmol) was coupled to syringe (E) and palmitic acid (154 mg, 0.6 mmol) was coupled to syringe (D). Next, Fmoc-Arg(Pmc)-OH (398 mg. 0.6 mmol) was coupled to syringe (A) and palmitic acid (154 mg, 0.6 mmol) was coupled to syringe (E) and finally palmitic acid (154 mg 0.6 mmol) was coupled to syringe (A). After ether washing and drying the rein in all syringes, the products were cleaved from the resin.
Precipitated from ether and lyophilized from water. Yield: 49.2 mg.
LCQ-ESI Calculated (C44H89N9O3): 792.24. Found: 792.9 (19%, M+H+), 636.7 (6%, M-Arg+H+), 397.1 (100%, M+2H+)
Precipitated from ether and triturated with ether (2×). Yield: 30.2 mg.
LCQ-ESI Calculated (C47H94N10O4): 863.31. Found: 863.9 (25%, M+H+), 707.7 (8%, M-Arg+H+), 432.5 (100%, M+2H+)
Precipitated from ether and triturated with ether (2×). Yield: 36.6 mg.
LCQ-ESI Calculated (C50H99N11O5): 934.39. Found: 934.8 (25%, M+H+), 778.7 (8%, M-Arg+H+), 468.1 (100%, M+2H+)
Precipitated from ether and triturated with ether (2×). Yield: 104.0 mg.
LCQ-ESI Calculated (C53H104N12O6): 1005.47. Found: 1103.6 (10%), 1005.7 (9%, M+H+), 920.7 (8%), 849.7 (100%, M-Arg+H+), 778.6 (12%), 539.5 (13%), 503.5 (100%, M+2H+), 468.1 (14%)
Precipitated from ether and triturated with ether (2×). Yield: 192.9 mg.
LCQ-ESI Calculated (C56H109N13O7): 1076.55. Found: 1174.76%) 1076.9 (26%, M+H+), 920.8 (46%, M-Arg+H+), 539.1 (100%, M+2H+)
A reductive amination of 1.2 g aldehyde resin (1.2 mmol) as described using 1.585 ml dodecyl amine (13 mmol), 754.8 mg NaCNBH3 (12 mmol) and 680 μl AcOH in 50 ml of a 1:1 mixture of DMF/MeOH. The resin was transferred to a syringe marked (A) and the first amino acid was coupled twice using 1.7570 g and 1.7153 g Fmoc-Lys(Boc)-OH (3.7 mmol) 3.60 ml 1M HOBt/DMF (3.60 mmol) and 3.30 ml 1M DIPCDI/DMF (3.30 mmol). A chloranil test was negative. The resin was capped. From syringe (A), subsequently one sixth was removed for experiment 8.5.3 and one fifth of the resin was put in a new syringe (B). To syringe (A) Fmoc-Ala-OH (790 mg, 2.0 mmol) was coupled and to syringe (B) Fmoc-Lys(Boc)-OH (281.1 mg, 0.6 mmol) was coupled. Then, from syringe (A) one fourth of the resin was put in a new syringe (C). Then, Fmoc-Ala-OH (592 mg, 1.5 mmol) was coupled to it to syringe (A), Fmoc-Lys(Boc)-OH (281.1 mg, 0.6 mmol) was coupled to syringe (C) and lauric acid (120 mg, 0.6 mmol) was coupled to syringe (B). From syringe (A) one third of the resin was put in a new syringes (D). Then, Fmoc-Ala-OH (395 mg, 1.0 mmol) was coupled to it to syringe (A), Fmoc-Lys(Boc)-OH (281.1 mg. 0.6 mmol) was coupled to syringe (D) and lauric acid (120 mg, 0.6 mmol) was coupled to syringe (C). Then, from syringe (A) half of the resin was put in a new syringe (E). Then, Fmoc-Ala-OH (200 mg, 1.1 mmol) was coupled to it to syringe (A), Fmoc-Lys(Boc)-OH (281.1 mg. 0.6 mmol) was coupled to syringe (E) and lauric acid (120 mg, 0.6 mmol) was coupled to syringe (D). Next, Fmoc-Lys(Boc)-OH (281.1 mg. 0.6 mmol) was coupled to syringe (A) and lauric acid (120 mg, 0.6 mmol) was coupled to syringe (E) and finally lauric acid (120 mg, 0.6 mmol) was coupled to syringe (A). After ether washing and drying the rein in all syringes, the products were cleaved from the resin.
No precipitation from ether. Yield: 16.0 mg.
LCQ-ESI Calculated (C36H73N5O3): 624.00. Found: 1269.7 (2M+Na+), 1247.6 (2M+H+), 624.5 (M+H+), 312.8 (M+2H+).
No precipitation from ether. Yield: 50.0 mg.
LCQ-ESI Calculated (C39H78N6O4): 695.07. Found: 1411.5 (2M+Na+), 1389.6 (2M+H+), 718.7, 695.7 (M+H+), 348.4 (M+2H+)
Precipitated from ether and triturated with ether (2×). Yield: 116.8 mg.
LCQ-ESI Calculated (C42H83N7O5): 766.15. Found: 1553.5 (2M+Na+), 1531.7 (2M+H+), 788.9 (M+Na+), 766.7 (M+H+), 383.9 (M+2H+)
Precipitated from ether and triturated with ether (2×). Yield: 45.6 mg.
LCQ-ESI Calculated (C45H88N8O6): 837.23. Found: 1695.6 (11%), 995.2 (11%), 859.7 (100%, M+Na+), 837.7 (89%, M+H+), 527.5 (9%), 419.3 (91%, M+2H+)
Precipitated from ether and triturated with ether (2×). Yield: 134.9 mg.
LCQ-ESI Calculated (C48H93N9O7) 908.31. Found: 1066.3 (7%, ?) 930.9 (100%, M+Na+), 908.8 (58%, M+H+), 598.5 (9%), 454.9 (52%, M+2H+).
Enzyme Assays
The enzyme was dissolved in 1 mL MES buffer (10 mM), 1 mM CaCl2, pH 7.0 at 36.5° C. Substrate was added in a concentration 10 times the Km, and the inhibitor compounds of type C (C16-KGnK-C16, C16-KAnK-C16, and C16-RAnR-C16) were added in increasing concentrations (1.0, 5.0, 10.0, 20.0, 40.0 μL) from a stock solution of 2 mg in 1 mL DMSO. The residual activity was measured in a Hitachi F2500 spectrofluorimeter, and plots were fitted using the Grafit® software (Erithracus Software, Horley, Surrey, UK). All the assays were calculated by the Morrison equation for competitive inhibition (eq. 1, [Morrison, 1969]). The amount of substrate used for the tight binding titration experiments follow all the requirements, where [S]<<Km for all enzymes, so the Kiapp=Ki.
However, for furin we used a correction of Ki app, where [S]˜Km (eq. 2).
Ki=Kiapp/1+[S]/Km Eq. 2
Inhibition of Proteases with Alkylated Peptides (‘Geminoids’)
Range of Proteases Inhibited by Geminoids, Determination of Ki with Z-RR-MCA (Dengue 2 Protease) and Ac-RVRR-MCA (Furin) (SEQ ID NO:60).
The first group of gemini-like peptide amphiphiles to be screened was that of C16-K(G or A)nK-C16 (compound type C, with R1=n-C15H31, R2=n-C16H33). Experiments on trypsin, thrombin, and plasmin are given in Table 4. The residual activity left upon inhibition with these geminoids decreased in the order trypsin≈thrombin>plasmin, but the inhibition of dengue 2 protease and human furin was even stronger; in addition to the results shown here, the geminoids also inhibited recombinant human thimet oligopeptidase (TOP), human cathepsin D, recombinant human cathepsin L, subtilisin A, and angiotensin converting enzyme with residual activities comparable to those of trypsin, and human kallikrein (substrate Abz-KLFSSKQ-EDDnp [Fogaça et al., 2001] SEQ ID NO:59) with Ki values in the micromolar range as for dengue 2 protease and human furin. The results of the Ki determination for trypsin, dengue 2 protease, and human furin are given in Table 1A; some general data for the inhibition by this type of compounds are given in Table 1B. For most compounds in Table 1 the Ki could be derived from competitive inhibition experiments. Judging from the Ki values with the relatively simple substrate Z-RR-MCA, the best inhibitors for dengue 2 protease contain alanine (A) rather than glycine (G).
a)In 50 mM Tris.HCl, pH 9.0, with 20 μM Z-RR-MCA, 37° C.
b)In 10 mM Mes. NaOH, pH 7.0, with 2.35 μM Ac-RVRR-MCA (SEQ ID NO: 60), 37° C.
Inhibition of Dengue 2 Protease Studied with Optimum Substrate.
The best substrate for dengue protease is Abz-AKRR↓SQ-EDDnp (SEQ ID NO:1) [Gouvea et al., 2007]. The results of inhibition studies with this substrate are shown in
Contrary to the experiments with furin and Ac-RVRR-MCA (SEQ ID NO:60) shown in
The effect of preincubation of the enzyme with inhibitor was studied in the experiments shown in
a)Experimental procedure: 1) buffer + enzyme were pre-equilibrated for 2 min in cuvette at 37° C., 2) substrate was added and the hydrolysis measured for 200 s, 3) inhibitor was then added in different amounts.
b) Procedure: the enzyme activity was determined in absence and in presence of inhibitor,
c) Procedure derived from the Nature Protocol [Feng & Shoichet, 2006]: 1) pre-incubation of buffer, enzyme, 0.01% Triton X-100, and inhibitor at 37° C., 2) substrate added after 5 min., 3) same procedure repeated without inhibitor.
2. Inhibition with Gemini Surfactants
In the gemini surfactants of type B (Kirby et al., 2003;
a) Extra Lys connected by amide bonds to the —NH2 in the head groups.
Inhibition of Dengue Virus Replication in VERO Cells by C16-KAK-C16
Based on the apparent Ki data obtained in vitro (Table 1A), we selected C16-K(A)nK-C16 for a test of dengue virus replication in cell in culture. DENV-2/NGC and VERO cells were used for the experiment. First, a toxicity study was performed in VERO cells with different Geminoids (
C16-KAK-C16 was subsequently tested in a standard Dengue replication (plaque) assay in VERO cells (
The results show an differential effect of different geminoids on cell phenotype (
The best inhibitor for dengue 2 protease of the lysine-based gemini surfactants (B) type (see
The amphiphilic nature of the inhibitor could have various advantages for their application as drugs, such as formation of nanoparticles in the blood, and the possible translocation into the cell. Cationic peptides are also studied for their antimicrobial properties, and their ability to penetrate the cell as nanoparticles with the cationic membrane translocation ‘TAT’ peptide sequence on the outside [Liu et al., 2009] is an important factor in their efficiency. It is not unlikely that the amphiphilic cationic peptides can be taken up by the cell by endocytosis, analogous to what has been proposed for lipoplexes with cationic gemini surfactants in transfection [Kirby et al., 2003; Bell et al., 2003]. Indeed, the effect of the geminoids on cell morphology at high concentration (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016987 | Jun 2016 | NL | national |
This application claims the benefit of U.S. application Ser. No. 16/310,343, filed Dec. 14, 2018, which is a national stage application of International Application Number PCT/NL2017/050403, which was filed on Jun. 16, 2017, which claims priority to Netherlands Application Number 2016987 filed on Jun. 17, 2016, each of the disclosures of which are herein incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16310343 | Dec 2018 | US |
| Child | 17374437 | US |
