METHODS FOR INHIBITING CELLULAR UPTAKE OF THE ANTHRAX LETHAL TOXIN (LT) PROTEIN COMPLEX
20170071950 ยท 2017-03-16
Assignee
Inventors
- Xiaofeng Li (Farmington, CT, US)
- Dakai Liu (South Setauket, NY, US)
- Yazhou Zhang (South Setauket, NY, US)
- Richard Jin (Pennington, NJ, US)
- Riddhi Bhattacharyya (West Babylon, NY, US)
- Wei Cheng (Valley Stream, NY, US)
Cpc classification
G01N2333/51
PHYSICS
A61P19/08
HUMAN NECESSITIES
G01N33/6803
PHYSICS
International classification
Abstract
The present invention identifies compounds that disrupt the interaction between anthrax proteins and LRP5/6 receptors, resulting in a reduction in anthrax toxicity. The compounds act to disrupt the intracellular transport of toxin complexes into a target cell. The present invention also provides methods for testing the effect of compounds on Wnt activity, through the use of in vitro experiments involving cells that have in at least one gene mutation involved in the Wnt pathway.
Claims
1. A method for inhibiting cellular uptake of the anthrax lethal toxin (LT) protein complex by cells expressing LRP5 or LRP6, the method comprising: contacting said cells with an effective amount of a compound selected from the group consisting of: ##STR00028## wherein R.sup.13 is a linear or branched alkyl group or substituted or unsubstituted cycloalkyl group; ##STR00029## wherein at least one of up to each except one of R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.8, R.sup.11, R.sup.12 and R.sup.13 is a hydrogen atom and wherein each of the remaining of R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.8, R.sup.11, R.sup.12 and R.sup.13 that is not a hydrogen atom is selected from hydroxy, a halogen, a branched C.sub.1-C.sub.16 alkyl group, a substituted linear or branched C.sub.1-C.sub.16 alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclic group, a substituted heterocyclic group, an aryl alkyl group, a substituted aryl alkyl group, a heteroarylalkyl group, a substituted heteroarylalkyl group, an alkoxy group, a substituted alkoxy group, an alkene group, a substituted alkene group, an acyl group, an amine group, an amide group, a nitrate, a nitrate ester, a carboxyl group, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, a sulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphate ester, a phosphonate, a phosphonate ester, a phosphamide, a phosphoramide, a thiophosphate, a thiophosphate ester, a thiophosphonate, or a thiophosphonate ester, wherein R.sup.1 and R.sup.11, R.sup.11 and R.sup.12, R.sup.12 and R.sup.3, R.sup.3 and R.sup.4, R.sup.13 and R.sup.6 may independently be fused together to form one or more rings, or any combination of the foregoing; ##STR00030## wherein Rt.sup.13 and R.sup.14 are each independently H or a linear or branched alkyl group; or ##STR00031## wherein R.sup.15 is a linear or branched alkyl group, wherein the compound binds to LRP5 or LRP6 expressed by said cells, which binding to said LRP5 or LRP6 by the compound inhibits the binding of anthrax lethal toxin (LT) protein complex to said LRP5 or LRP6.
2. A method for inhibiting cellular uptake of the anthrax lethal toxin (LT) protein complex by cells expressing LRP5 or LRP6, the method comprising: contacting said cells with an effective amount of a compound having the formula: ##STR00032## wherein the compound binds to LRP5 or LRP6 expressed by said cells, which binding to said LRP5 or LRP6 by the compound inhibits the binding of anthrax lethal toxin (LT) protein complex to said LRP5 or LRP6.
3. The method of claim 1, wherein the compound is ##STR00033## wherein at least one of up to each except one of R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.8, R.sup.11, R.sup.12 and R.sup.13 is a hydrogen atom and wherein each of the remaining of R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.8, R.sup.11, R.sup.12 and R.sup.13 that is not a hydrogen atom is selected from hydroxy, a halogen, a branched C.sub.1-C.sub.16 alkyl group, a substituted linear or branched C.sub.1-C.sub.16 alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclic group, a substituted heterocyclic group, an aryl alkyl group, a substituted aryl alkyl group, a heteroarylalkyl group, a substituted heteroarylalkyl group, an alkoxy group, a substituted alkoxy group, an alkene group, a substituted alkene group, an acyl group, an amine group, an amide group, a nitrate, a nitrate ester, a carboxyl group, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, a sulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphate ester, a phosphonate, a phosphonate ester, a phosphamide, a phosphoramide, a thiophosphate, a thiophosphate ester, a thiophosphonate, or a thiophosphonate ester, wherein R.sup.1 and R.sup.11, R.sup.11 and R.sup.12, R.sup.12 and R.sup.3, R.sup.3 and R.sup.4, R.sup.13 and R.sup.6 may independently be fused together to form one or more rings, or any combination of the foregoing.
4. The method of claim 1, wherein the compound is ##STR00034## wherein R.sup.13 is a linear or branched alkyl group or substituted or unsubstituted cycloalkyl group.
5. The method of claim 4, wherein R.sup.13 is a linear or branched C.sub.2-4 group or a cycloalkyl C.sub.3-8 group.
6. The method of claim 1, wherein the compound is ##STR00035## wherein at least one of R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.8, R.sup.11, R.sup.12, R.sup.13 or R.sup.14 is a hydrogen atom and wherein at least one of R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.8, R.sup.11, R.sup.12, R.sup.13 or R.sup.14 comprises an atom other than a hydrogen atom.
7. The method of claim 6, wherein R.sup.1, R.sup.3, R.sup.4, R.sup.6, R.sup.8, R.sup.11, R.sup.12, R.sup.13 and R.sup.14 independently comprise hydrogen, oxygen, hydroxy, a halogen, a linear or branched C.sub.1-C.sub.16 alkyl group, a substituted linear or branched C.sub.1-C.sub.16 alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclic group, a substituted heterocyclic group, an arylalkyl group, a substituted arylalkyl group, a heteroarylalkyl group, a substituted heteroarylalkyl group, an alkoxy group, a substituted alkoxy group, an alkene group, a substituted alkene group, an acyl group, an amine group, an amide group, a nitrate, a nitrate ester, a carboxyl group, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, a sulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphate ester, a phosphonate, a phosphonate ester, a phosphamide, a phosphoramide, a thiophosphate, a thiophosphate ester, a thiophosphonate, or a thiophosphonate ester, wherein R.sup.1 and R.sup.11, R.sup.11 and R.sup.12, R.sup.12 and R.sup.3, R.sup.3 and R.sup.4, R.sup.13 and R.sup.6 may independently be fused together to form one or more rings, or any combination of the foregoing.
8. The method of claim 1, wherein the compound is ##STR00036## wherein R.sup.13 and R.sup.14 are each independently H or a linear or branched alkyl group.
9. The method of claim 8, wherein R.sup.13 and R.sup.14 are independently H or a linear or branched C.sub.1-5 alkyl group.
10. The method of claim 1, wherein the compound is ##STR00037## wherein R.sup.15 is a linear or branched alkyl group.
11. The method of claim 10, wherein R.sup.15 is a linear or branched C.sub.1-5 alkyl group.
12. The method of claim 1, wherein the cells are exposed to anthrax lethal toxin (LT) protein complex after the contacting step.
13. The method of claim 1, wherein the cells are exposed to anthrax lethal toxin (LT) protein complex before the contacting step.
14. The method of claim 2, wherein the cells are exposed to anthrax lethal toxin (LT) protein complex after the contacting step.
15. The method of claim 2, wherein the cells are exposed to anthrax lethal toxin (LT) protein complex before the contacting step.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0018]
DETAILED DESCRIPTION OF THE INVENTION
[0019] In previous art, mutations in subjects or animal models have been used for the purpose of elucidating pathways for various conditions and diseases. A novel aspect of the present invention is that mutant animals or cells can be used for testing the efficacy of potential pharmacological agents after carrying out a physical or virtual screening of a library. In a preferred embodiment of the present invention, a mutation is located in one or more genes of the Wnt canonical or non-canonical signaling pathway. In the course of carrying out a virtual screening, it is understood that the library itself can be a physical library (as exemplified by screening of the NCI library in U.S. Patent Application Serial No. 2005/0196349, herein incorporated by reference) or it can be a virtual library (as exemplified in Example 6 with compounds Enz-1 to Enz-72 of U.S. patent application Ser. No. 11/598,916, herein incorporated by reference). Any compound that can potentially bind to the protein target of interest and affect the interaction between the target protein and another protein may be selected as a member of the library. Examples of such compounds can include but not be limited to organic molecules, antibodies, peptides, and nucleic acids. The peptides can include, but not be limited to, a library of peptides of random nature, a permutational series of amino acids, fragments of antibodies to the protein of interest and fragments of a protein that interacts with the protein of interest. The nucleic acids can include but not be limited to aptamers and a library of protein binding sequences.
[0020] When the user has access to a physical library, the structure of each member may be used in a virtual screening process and candidates of interest may be subsequently tested. In contrast, there is no absolute necessity to have such molecules immediately in the possession of the user and if some data has already been collected on particular compounds, their structure may be used to design a virtual library with variations in the positions on core structures followed by a virtual screening process. In this variation, a wide variety of related compounds may be analyzed simultaneously and only the particular compounds that score highest after the virtual screening process need to be synthesized and tested in biological assays.
[0021] In the human genome, gene duplication events have led to the existence of a certain degree of redundancy such that multiple copies of similar proteins carry out similar functions. In some of these cases, there are more or less complete copies that are expressed from different genomic sites and in other cases there are families of proteins where there may be differences between otherwise identical copies that reflect evolutionary developments that have led to alterations of some properties, a process that is sometimes referred to as genetic drift. In some cases, this differentiation has led to specialization where particular functions are carried out only by certain members of such families. In other cases, there may be functional overlaps where either of two proteins of a given protein family can carry out a specific step. As a further complication, two unrelated proteins may also be carrying out a particular step in common due to convergent evolution. When two different proteins (related or unrelated) are able to carry out or initiate a common process, there may be difficulties in identifying pharmacological agents that can modulate this process. In such an instance, there may be masking by the presence and activity of a second protein when screening for the activities of a pharmaceutical agent specifically selected for potential inhibition of a target protein, i.e. even when the first protein is effectively blocked by a particular drug candidate, a lack of effective repression of the second protein can lead to little or no effect being seen in the assay system. Thus, a molecule that is highly selective for the first protein may be completely missed by the screening procedure. In the present invention, it is disclosed that animals and cells with mutations in the Wnt signaling system of either natural or artificial origin may provide a more effective means of selecting drug candidates. Whereas in previous art, a partial or complete loss of function of a particular gene was used for delineation of the role of a gene or as a model system for development of therapies that compensate for its loss, the present invention uses the loss of function in one gene to allow identification of pharmacological agents that affect a different gene. In a preferred embodiment of the present invention, these mutations are in proteins involved in the Wnt signaling pathway. Thus, to give a non-limiting example, when a drug is being tested for an ability to inhibit the activity of LRP6, the present invention discloses the utility of carrying out a biological assay procedure in an LRP5 (/) environment.
[0022] In a system where either of two proteins is capable of transmitting a signal, this process may also be of a reciprocal or sequential nature. For instance, once an effective drug has been identified in a cell or animal where the presence of a mutation has made signal generation dependent upon only the first protein due to partial or complete elimination of the activity of the second protein, the same procedure can then be carried out in a second stage with cells or strains that are defective in the first protein and screening a library for compounds that have the ability to block the second protein. Thus, in a system where a particular step can be carried out in parallel by more than one protein, a potential benefit of the present invention can be treatment by a combination of therapeutic agents that are optimized for each target. On the other hand, once a compound is identified that is effective with the first target, a series of modifications can be carried out with this compound to identify a pharmacological agent that is effective on the second target as well as the first, thereby taking on a multi-targeting role.
[0023] There are also situations where a pharmaceutical agent can inhibit the actions of a target protein in a cell, but the presence of a second protein, unrelated to the first, may compensate for this effect and nullify any results. In this case, the same strategy outlined above may be used where a mutant lacking the second protein can allow a more fruitful investigation of screening for agents that affect the target protein. As also described above, a second search can then be carried out later for a small molecule that can separately affect the second protein such that a desirable effect can be obtained by a combination of agents that affect the first and second protein targets individually.
Interactions with Anthrax
[0024] It has already been established that the LRP5 and LRP6 receptors are involved in a number of different protein/protein interactions for carrying out signal transduction events. As described above, it has also been found that a binding event to the LRP6 receptor can lead to transportation of a complex through the cellular membrane to produce the toxic effects of anthrax (Wei et al., 2006). By directing antibodies to two different sites on the LRP6 protein, blockage of one site was shown to be effective in reducing the effects of anthrax toxicity while blocking of the second site seemed to offer no protective benefits. The ineffective site was located on a region corresponding to the third repeat of Domain I (amino acids 204-213) while the resistance inducing site was located in the third repeat of Domain II (amino acids 515-534) implying that binding of the anthrax complex to Domain II may be an important factor in the toxicity of anthrax. As such, the same methods that have been previously described for identification of molecules that modulate interactions of LRP5 and LRP6 receptor with other proteins (U.S. Patent Application No. 2005/0196349) may also be used to identify a molecule that can interfere with anthrax induced toxicity.
[0025] The oligopeptides used by Wei et al. for raising the polyclonal antibodies against the YWTD repeat Domain II region were derived from LRP6. It is unknown whether there was activity against the corresponding sequence in LRP5 since the homologous sequence in LRP5 only matched 13 out of the 20 amino acids. However, it is possible that the presence of conserved amino acids in Domain II (as well as the similarity in structure) allowed blockage of the corresponding LRP5 site by the polyclonal antibody. In contrast, there may not have been expression of LRP5 in that particular cell line and as such, it did not have to be blocked. Since structures and functions are so similar, it is probable that when LRP5 is expressed, it may also act as a co-factor for anthrax toxicity. It is therefore an object of the present invention to identify molecules that bind to LRP5 as well as to LRP6. As described above, compounds may be identified that bind to both LRP5 and LRP6, or there may be compounds that bind to LRP5 and LRP6 separately.
[0026] There are two approaches that may be used in the present invention. The first approach is a site-selective method where the binding site on LRP5 and LRP6 that has been identified as both a binding site for a native protein and for the anthrax protein complex is screened using information gained from the native protein. In this approach it is assumed that a molecule which is able to block the action of the normal protein may also offer protection against the anthrax protein complex that binds to the same site. The benefit to this is that it takes advantage of screenings and findings from investigations of small molecules that modulate interactions between the YWTD repeat domain of LRP and the native protein. An example of this method is using the candidates that have been identified as modulating the interaction between LRP5 and Dkk and testing for an additional property of being able to offer protection against anthrax toxicity. Molecules that have been selected on the basis of inhibiting the interaction of Dkk with LRP6 may also be used for this purpose.
[0027] An alternative approach is to carry out a more focused ligand-selective method where the same methodology that has been used for identifying the site on LRP5 for Dkk interaction is used to more specifically identify the site used for anthrax. As described in U.S. Patent Application No. 2005/0196349, key amino acids in LRP5 involved in the binding of Dkk to YWTD Domain II were identified by alanine scanning prior to carrying out a virtual screening that used the amino acid locations as interaction sites. Due to the similarity between Domains II and III, molecules that have been selected for binding to one site may be able to bind to the other site and similarly, compounds selected for their ability to bind to LRP5 may also bind to LRP6. However, a more selective approach for the present invention would consist of carrying out a mutational program to identify the sites of amino acids in Domain II and Domain III of both LRP5 and LRP6 that may be critical for the translocation of anthrax into a cell. It may be of further benefit to combine this aspect of the present invention with other previously disclosed methods involving the use of mutants. In this combined approach, mutants are used to develop a cell line where both LRP5 and LRP6 are eliminated, and anthrax susceptibility is determined by transformation with appropriate LRP5 or LRP6 constructs. In addition, the structure of compounds that show inhibitory activity may be used to carry out further rounds of virtual screening as previously disclosed in U.S. Patent Application No. 2005/0196349.
[0028] Wei et al. used a peptide with amino acids 1314-1613 of LRP6 to generate a third polyclonal antibody that also displayed effectiveness in protection against anthrax toxicity. These amino acids comprised a small portion of the extracellular domain (1314-1370) as well as a portion of the intracellular domain (1394-1613). Presumably, it is the extracellular portion that was recognized by the antibodies in the experiments carried out by Wei et al., and this result represents either an additional binding site of the anthrax complex, an alteration in secondary structure that interferes with binding to a distant binding site, or an interference with the endocytosis process. Regardless of the mechanism, these results imply that this region may also serve as a target for the identification of a small molecule that could interfere with anthrax toxicity.
[0029] In previous art, blocking the lethal effects of anthrax infection/exposure has been the subject of a tremendous amount of research. Although the bacillus itself is susceptible to a number of different antibiotics, the effects of the toxin can create lethality even after the disease organism itself has been eliminated. That is, after a certain stage of infection, antibiotics have no effect when the anthrax toxins are present in sufficient amounts. As such, recent efforts have been more directed towards blocking the effects of the toxin itself rather than destroying the organism that carries it. This has been a brute force screening approach for testing the effects of a library of compounds on cells and animals. Assays were carried out that either looked at particular steps of the anthrax toxin pathway or simply assessed overall lethality.
[0030] An undirected approach has been to take previously described drugs that effect multiple targets to reduce anthrax toxicity and test them for an additional ability to be used in anthrax intervention. For instance, the anti-cancer drug cisplatin is known to affect a wide range of processes during treatment of various disorders and it was shown that cisplatin could block anthrax toxicity when it was used to treat the PA prior to its administration (Moayeri et al., 2006 Antimicrob Agents and Chemotherapy 50; 2658-2665). In vivo experiments also displayed an effect when the drug was co-administered with lethal (anthrax) toxin (LT). However, practical results for the use of this drug are lacking. Co-administration was a critical factor. The administration of cisplatin two hours before or two hours after administration of the lethal toxin eliminated this protection.
[0031] Since one of the steps of anthrax toxicity is the protease action on critical cellular targets by the anthrax LF protein, this activity has been the subject of both random screening and rational drug design efforts where the structure of the LF protein has been used to identify appropriate inhibitors. Examples of the former have included the testing of 10,000 drug-like molecules using a non-selective physical screening approach for the identification of LF inhibitors (Schepetkin et al., 2006 J Med Chem 49; 5232-5244). A more selective variation of this approach has been to take into consideration the presence of anionic rich regions on the LF protein, and physically test for inhibition by a small library of cationic compounds (Goldman et al., 2006 BMC Pharmacology 6:8-15). An example of a rational drug design approach has been the combination of crystallography, molecular docking (virtual screening) and data mining to identify compounds that could bind to LF and thereby inhibit its protease activity (Panchal et al., 2004 Nat Struct Mol Biol 11; 67-72). Other examples of the drug design approach have included the use of the crystallographic predicted structure of LF to select a primary scaffold from a group of three hundred scaffolds that represent various drug families (Forino et al., 2005 Proc Nat Acad Sci (USA) 102; 9499-9504), thereby limiting the amount of searching required. Once a primary structure was selected, a search was made for related compounds that were commercially available. In vitro testing followed to determine the parts of the core compound that needed to be retained for the maintenance of inhibitory activity. Subsequently, structure activity relationship (SAR) analysis was carried out to design novel compounds that could be tested further. (see Johnson et al., 2006 J. Med Chem 12; 27-30). A mixed approach has incorporated the use of a random peptide library to identify the optimal peptide substrate, followed by the design of peptide analogs that could act as inhibitors (Turk et al., 2004 Nat Struct Mol Biol 11; 60-66). This work was continued by carrying out crystallography studies of the inhibitor bound to LF in order to refine designs for more drug candidates. In addition, as previously described, that drugs that have proved to be useful in other contexts (cisplastin) have also been retested for their application to anthrax. Others have examined the ability of some previously developed metalloprotease inhibitors to inhibit anthrax toxicity due to blockage of the activity of LF on cytosolic targets (Kocer et al. 2005 Infection and Immunity 73; 7548-7557).
[0032] The application of compounds directed to intracellular targets is problematic because there must be active or passive transport of the compound into the cell. As such, there may be a number compounds that may affect anthrax toxic activity that are ineffective in cellular assays because of an inability to enter the cell. Since anthrax toxin action is initiated by events taking place on the cell surface, compounds that affect events taking place in this extracellular environment can avoid such problems and provide a greater realm of potential pharmacological agents. As such, rather than aiming directly at LF enzymatic activity, a search has also been carried out for compounds that would bind to the PA protein such that the entry of LF into the cell would be blocked (Karginov et al., 2005 Proc Nat Acad Sci USA 102; 15,075-15,080). As previously mentioned, cisplatin was used for the inhibition of anthrax toxicity. Although it was partially chosen for working in the intracellular environment as a known protease inhibitor, it seems that its effectiveness in blocking anthrax in vivo may be taking place by blocking translocation of LF into the cytosol (Moayeri et al., 2006).
[0033] Various events occur prior to the translocation of the anthrax toxin complex into the cell. Consequently, these pre-translocation events are also potential targets for pharmacological intervention. A prerequisite for translocation is a protease cleavage of the PA protein by the endogenous protease furin. In one report on the use of furin as a target for a drug such as endogenous protease inhibitor (inter-alpha-inhibitor protein) was found to increase the survival of treated animals (Opal et al., 2005 Infect Immun73; 5101-5105). Other furin inhibitors have also been isolated that are either modified proteins or functionalized small peptides (Komiyama et al., 2005 Antimicrob Agents Chemotherapy 49; 3875-3882). The reagents described in this study showed protection against toxin lethality for at least 5 hours, but after 8 hours the course of lethality resumed, i.e. these agents did not seem to prevent toxin lethality per se but only delayed it. This resurgence of lethality could partially be prevented by the co-administration of a second reagent, chloroquine, at the same time as the furin inhibitor. In addition to incomplete protection, there could also be an immune reaction to these peptides. The use of peptides and/or proteins may also have problems with stability where specific storage requirements are needed. This could be problematic when application of these reagents may be needed immediately in bio-warfare conditions where a pill or desiccated powder may be more useful.
[0034] In another example on the selection of extracellular targets, advantage has been taken of the knowledge that protein/protein interactions are an important element in the lethality of anthrax toxin. For instance, a random peptide library was used in a phage display system to screen 7 or 12 amino acid peptides that would bind to Domain I of ANTRX1 and ANTRX 2 (Basha et al., 2006 Proc Nat Acad Sci (USA) 103; 13,509-13,513). At least one peptide selected on the basis of binding to ANTXR1 was later shown to be able to inhibit anthrax toxicity in a cell line (RAW 264.7) that expresses ANTRX2. These studies showed that the simultaneous administration of the peptide as well as the lethal toxin blocked the lethality of the anthrax toxin in vivo. However, the use of peptides also entails problems cited previously that might abrogate their utility.
[0035] As described above, the discovery that LRP6 was also a co-receptor for the translocation of anthrax toxin into a cell was partially based upon the use of antibody to specific regions of LRP6 (Wei et al., 2006). This observation has been used as the basis of a therapeutic mode, where Cohen and Wei have disclosed the use of monoclonal antibodies against LRP6 as reagents that could inhibit anthrax toxicity in vivo (U.S. Patent Application No. 20060257892 filed Feb. 16, 2006). This is similar to certain previously described methods where a specific target is chosen and then a screening of a random library of potential inhibitors is carried out where candidates are evaluated on the basis of their ability to bind to LRP6. Although the main concern of this application is the use of antibodies and variations thereof as reagents, there is also the potential use of small molecules. However, this method is carried out in the same way previously described for searching for antibodies and the discussion on screening is concerned solely with biological assays. Although screening of a random library by a biological assay is the only way to carry out a search for antibodies, this does not hold true for small molecules where more sophisticated ways are available. There is no suggestion or appreciation in the Cohen and Wei application that a much more efficient system is the method described in the present invention that uses the structure of LRP6 (and possibly LRP5) to carry out a virtual screening of a library of compounds prior to carrying out a series of biological assays.
[0036] Effective molecules that are discovered by using the materials and methods of the present invention may be used in conjunction with pharmacological agents that have been selected for intervention in other steps in the process leading to anthrax toxicity. It has previously been shown that reagent combinations have provided more effective protection against anthrax toxicity compared to being used alone (Komiyama et al., 2005). It would be expected that the use of a new target by means of the present invention should allow these compounds to enjoy cumulative or even synergistic effects when used with other anti-anthrax reagents.
[0037] The compounds of the present invention and the compounds identified by the methods described in U.S. Patent Application 2005/0196349 and related applications may be used in conjunction with one or more other drugs in the treatment, prevention, control, amelioration, or reduction of risk of diseases or conditions for which the compounds of the present invention have utility, where the combination of the drugs together are safer or more effective than either drug alone. Examples of combinations of these compounds with other drugs in either unit dose or kit form include combinations with: a) antiresorptive reagents, such as Bisphosphonates (for example, Alendronate sodium, sold under the brand name Fossamax by Merck); b) anabolic reagents, such as Parathyroid hormones (e.g., Teriparatide, a recombinant form of parathyroid hormones sold under the brand name Forteo by Eli Lilly); c) bone regeneration material, such as beta-tricalcium phosphate (i.e. beta-TCP, sold under the brand name Cerasorb by Curasan AG); and 4) other drugs that affect receptors or enzymes that either increase the efficacy, safety, convenience, or reduce unwanted side effects or toxicity of the compounds of the present invention or the compounds in related applications. The foregoing list is illustrative only and not intended to be limiting in any way. An advantage of this approach includes the possibility of synergistic effects where the products of two different modalities may be more beneficial than a single medicine. Also where the same level of relief is achieved by different medicines, this treatment may be carried out by using lower dosages of two or more medicines resulting in a diminishment in potential side effects that would be seen with a higher dose of any single medicine.
[0038] When carrying out the methods of the present invention, treatments may be chosen from a variety of administration methods comprising but not limited to oral, nasal, inhalation, intravenous, intraperitoneal, intramuscular, parenteral, transdermal, sublingual, topical, rectal or subcutaneous means. When carrying out a combination procedure, the treatments may share the same administration or treatment method or they may utilize different methods. The pharmacological agents identified by the present invention may also be administered with other agents as well that can include but not be limited to excipients, drug release-polymers, carriers, and enhancers.
[0039] The molecules or compounds identified by the methods of the present invention may be used to create compositions and/or pharmaceutical compositions that may be administered to subjects or patients (in therapeutically effective amounts) to treat disorders, diseases or conditions that are affected by modulating the activity of any member of the Wnt signaling pathway.
[0040] The compounds or molecules of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E. L. Eliel and S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention.
[0041] The compounds or molecules of the invention, and derivatives, fragments, analogs, homologs pharmaceutically acceptable salts or hydrate thereof, can be incorporated into pharmaceutical compositions suitable for administration, together with a pharmaceutically acceptable carrier or excipient. Such compositions typically comprise a therapeutically effective amount of any of the compounds above, and a pharmaceutically acceptable carrier.
[0042] The compounds or molecules of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The compounds or molecules can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
[0043] The subject or patient to whom the compounds of the present invention is administered is generally a human being, male or female, but may also encompass other mammals, such as dogs, cats, mice, rats, cattle, horses, sheep, rabbits, monkeys, chimpanzees or other apes or primates.
[0044] The terms administration of or administering a compound should be understood to mean providing a compound of the invention to the individual in need of treatment in a form that can be introduced into that individual's body in a therapeutically useful form and therapeutically useful amount, including, but not limited to: oral dosage forms, such as tablets, capsules, syrups, suspensions, and the like; injectable dosage forms, such as IV, IM, or IP, and the like; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and the like; and rectal suppositories.
[0045] The terms therapeutically effective amount means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. As used herein, the term treatment refers to both to the treatment and to the prevention or prophylactic therapy of the mentioned conditions, particularly in a patient who is predisposed to such disease or disorder.
[0046] The term treating in its various grammatical forms in relation to the present invention refers to preventing, (i.e., chemoprevention), curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition. For example, treatment may involve alleviating a symptom (i.e., not necessary all symptoms) of a disease or attenuating the progression of a disease. Because some of the inventive methods involve the physical removal of the etiological agent, the artisan will recognize that they are equally effective in situations where the inventive compound is administered prior to, or simultaneous with, exposure to the etiological agent (prophylactic treatment) and situations where the inventive compounds are administered after (even well after) exposure to the etiological agent.
EXAMPLES
[0047] Examples provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limited of the reasonable scope thereof.
Example 1
Protection Against Anthrax Toxicity
A. Test Compounds
[0048] In US Patent Application Serial No. 2005/0196349, a virtual screening was disclosed that identified compounds that could interact with Domain III of LRP5; potentially interesting compounds were then tested in a biological assay for an ability to modulate Wnt activity. Two of the compounds that were a result of this process were IC15 and IIIC3. As described in U.S. patent application Ser. No. 11/598,916, compound IIIC3 was used to design a series of related compounds by varying functional groups on a core structure. One of the products that gave positive results by both virtual screening scores and biological assays was the compound Enzo MO1. As described above, compounds that have been selected to bind to Domain III of LRP5 might be able to give protection against anthrax toxicity.
B. Preparation of Test Compound Stocks
[0049] Compounds IC15, IIIC3 and EnzoM01 were prepared as 312.5 M concentrated stock solutions and diluted into culture media as 4 stocks such that a final concentration of 10 and 40 M would be present during the assay.
C. Preparation of Anthrax Toxin Stocks
[0050] PA and LF proteins (0.1 mg/vial, List Biological Labs Inc., Campbell, Calif.) were reconstituted with 100 l of H.sub.2O to give a final concentration of 1 mg/ml. These were aliquoted into separate 10 l samples that were maintained at 20-80 C. until required. Working solutions of PA and LF were made by diluting 1 mg/ml of each toxin stock into media to give a final concentration of 10 g/ml. PA and LF were diluted and mixed together just prior to use to give a 4 toxin mix that resulted in either a 0.1 g/ml or 0.2 g/ml final concentration during the assay.
D. Biological Assay
[0051] Two murine macrophage-like cell lines, J774A.1 (ATCC TIB-67) and RAW264.7 (ATCC TIB-71), were used to examine the effects of the selected compounds on anthrax toxicity. Cells were grown in DMEM medium supplemented with 4 mM GlutaMax-1, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 10% FBS, 1% Penicillin/Streptomycin and buffered with HEPES followed by seeding 510.sup.3 cells/well into a 96 well plate. After overnight growth, 50 l of a test compound diluted into media was added to the 100 l of medium already present in each well and incubated for 30 minutes. At this point, 50 l of 4 toxin was added to give a final volume of 200 l. At various time points (3 hours or overnight), medium was collected and cells washed once with 180 l of 1PBS, followed by addition of 20 l of MTS reagent (Promega, Madison Wis.) mixed with 100 l of RPMI 1640 medium (phenol red free) supplemented with 1% FBS to measure vitality. Incubation was then carried out for 2-4 hours followed by absorbance readings at 490 nm and 630 nm. The results of the 490 nm results are tabulated in
E. Discussion of Results
[0052] It can be seen that under the conditions used, the J774 cell line is more sensitive to anthrax toxicity than the RAW264 cells. For the J774 cells, some effects of protection were provided by M01 and IIIC3 during the three hour incubation period which was essentially lost by extending the incubation to overnight exposure or increasing the toxin from 0.1 g to 0.2 g. For the RAW cells, little or no resistance was seen with any of these compounds after the three hour incubation, whereas in the overnight exposure, IC15 seemed to offer limited protection in the presence of either 0.1 g or 0.2 g of toxin. Such differential effects may be due to the nature of the sensitivity of J774 compared to RAW264 or it may be related to different expression patterns of anthrax receptors for these cell lines.
[0053] The following compounds disclosed in U.S. application Ser. No. 11/598,916 may be used in the methods of the invention.
[0054] NCI 8642 (also referred to as IIIC3), has the structure:
##STR00001##
[0055] Retaining the core structure, and indicating where various substitutions can take place, a generalized formula for a family of analogues of this compound can be as follows (I):
##STR00002##
wherein at least one of R1, R3, R4, R6, R8, R11, R12 or R13 is a hydrogen atom and wherein at least one of R1, R3, R4, R6, R8, R11, R12 or R13 comprises an atom other than a hydrogen atom. In a particular embodiment, R1, R3, R4, R6, R8, R11, R12 and R13 independently comprise hydrogen, oxygen, hydroxy, a halogen, a linear or branched (C1-C16) alkyl group, a substituted linear or branched (C1-C16) alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclic group, a substituted heterocyclic group, an aryl alkyl group, a substituted aryl alkyl group, a heteroarylalkyl group, a substituted heteroararylalkyl group, an alkoxy group, a substituted alkoxy group, an alkene group, a substituted alkene group, an acyl group, an amine group, an amide group, a nitrate, a nitrate ester, a carboxyl group, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, a sulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphate ester, a phosphonate, a phosponate ester, a phosphamide, a phosphoramide, a thiophosphate, a thiophosphate ester, a thiophosphonate, or a thiophosponate ester, wherein R1 and R11, R11 and R12, R12 and R3, R3 and R4, R13 and R6 may independently be fused together to form one or more rings, or any combination of the foregoing. When the nitrogen of the amine group comprising R11 and R12 is charged and further comprises R15, wherein R15 is as described previously for R1, R3, R4, R6, R8, R11, R12 and R13. In a particular embodiment, the compound has the structure (VIII):
##STR00003##
wherein R13 is a linear or branched alkyl group or substituted or unsubstituted cycloalkyl group. In a most particular embodiment R13 is a linear or branched C2-4 group. In another particular embodiment R13 is a cycloalkyl C3-8 group.
[0056] This core compound can be generalized further by retaining the ring structure and allowing substitutions for the carboxyl or ester group shown in the structure above, giving a formula (II) for a series of other analogues as follows:
##STR00004##
wherein at least one of R1, R3, R4, R6, R8, R11, R12, R13 or R14 is a hydrogen atom and wherein at least one of R1, R3, R4, R6, R8, R11, R12, R13 or R14 comprises an atom other than a hydrogen atom
[0057] In a particular embodiment, R1, R3, R4, R6, R8, R11, R12, R13 and R14 independently comprise hydrogen, oxygen, hydroxy, a halogen, a linear or branched (C1-C16) alkyl group, a substituted linear or branched (C1-C16) alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclic group, a substituted heterocyclic group, an aralalkyl group, a substituted arylalkyl group, a heteroarylalkyl group, a substituted heteroarylalkyl group, an alkoxy group, a substituted alkoxy group, an alkene group, a substituted alkene group, an acyl group, an amine group, an amide group, a nitrate, a nitrate ester, a carboxyl group, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, a sulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphate ester, a phosphonate, a phosponate ester, a phosphamide, a phosphoramide, a thiophosphate, a thiophosphate ester, a thiophosphonate, or a thiophosponate ester, wherein R1 and R11, R11 and R12, R12 and R3, R3 and R4, R13 and R6 may independently be fused together to form one or more rings, or any combination of the foregoing.
[0058] In a particular embodiment the compound has the structure (VII):
##STR00005##
wherein R13 and R14 are each independently H or a linear or branched alkyl group. In a more particular embodiment, R13 and R14 are independently H or a linear or branched C1-5 linear or branched alkyl group. In most specific embodiments, R13 is H and R14 is CH3 groups. (Enz M14); R13 and R14 are CH3 groups (Enz M15); R13 are CH3 groups and wherein R14 is C(CH3)3 (Enz M25); R13 is H and R14 is (CH2)2CH(CH3)2. (Enz M35); R13 is H, wherein R11 and R12 are CH3 groups and wherein R14 is CH2CH(CH3)(CH2CH3). (Enz M39).
[0059] The compound encompassed by (VII) may be obtained by
[0060] (a) reacting gallocyanine with an agent to replace the COOH group on gallocyanine with a leaving group; and
[0061] (b) reacting the compound obtained in step (a) with an alkyl amine to obtain said compound (VII).
[0062] The invention is further directed to a novel compound having the structure (VI):
##STR00006##
wherein R15 is a linear or branched alkyl group. In a particular embodiment, R15 is a linear or branched C1-5 alkyl group. In most specific embodiments, R15 is a methyl group (Enz M01); R15 is an ethyl group (EnzM02); R15 is a propyl group (EnzM03); R15 is CH2C(CH3)3 (EnzM12).
[0063] This compound may be obtained by reacting gallocyanine with an alkyl halide under conditions promoting formation of said compound.
[0064] In a similar fashion, a series of compounds that may be of interest may be designed using IC15 and IC5 as starting points:
##STR00007##
[0065] The common anthra-9, 0-quinone structure in these two compounds was used in a secondary screening with UNITY followed by docking with FlexX and biological assays. This led to the identification of IIC8, IIC10, IIC18 and IIC19 (all sharing the anthra-9,10-quinone) as demonstrating effects upon Wnt activity. Thus, in this instance a family of analogues could have the generalized structure (III):
##STR00008##
wherein at least one of R1, R2, R3, R4, R5, R6, R7 or R8 is a hydrogen atom and wherein at least one of R1, R2, R3, R4, R5, R6, R7 or R8 comprises an atom other than a hydrogen atom. In a preferred embodiment, R1, R2, R3, R4, R6, R6, R7, R8 independently comprise hydrogen, oxygen, hydroxy, a halogen, a linear or branched (C1-C16) alkyl group, a substituted linear or branched (C1-C16) alkyl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclic group, a substituted heterocyclic group, an aralalkyl group, a substituted aralalkyl group, a heteroarylalkyl group, a substituted heteroaryllalkyl group, an alkoxy group, a substituted alkoxy group, an alkene group, a substituted alkene group, an acyl group, an amine group, an amide group, a nitrate, a nitrate ester, a carboxyl group, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, a sulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphate ester, a phosphonate, a phosponate ester, a phosphamide, a phosphoramide, a thiophosphate, a thiophosphate ester, a thiophosphonate, or a thiophosponate ester, wherein R1 and R2, R2 and R3, R3 and R4, R5 and R6, R6 and R7, R7 and R8 may independently be fused together to form one or more rings, or any combination of the foregoing.
Use of IIIC3 as a Core Compound to Design New Variants
[0066] The ability of IIIC3 (NCI 8642) to act upon Wnt activity allows it to be used to design a core model where varying the R groups on this template allows identification of other molecules that may also have effects upon Wnt activity. Thus, if like IIIC3, R1, R3, R4 and R6 were hydrogens and R8 was a Hydroxyl group, a more limited series of compounds could be made with the remaining positions of the following model compound (VIII):
##STR00009##
[0067] To initiate this series, a panel of compounds have been designed where R11 and R12 are methyl groups and R13 is a hydroxyl group as in IIIC3 and the amine group was quarternized, giving the structure (VI):
##STR00010##
[0068] A series of substitutions have been designed for variations of R15 in this compound using both linear and branched alkanes. The structures of the resultant compounds (EnzoM01-EnzoM12) are scanned as described previously to obtain a score for their likelihood of binding. A list of the particular substitutions used in EnzoM01-EnzoM12 as well as the resultant Cscore ratings are given in Table I below:
TABLE-US-00001 TABLE I Compound R15 Cscore IIIC3 4 EnzoM01 CH3 5 EnzoM02 CH2CH3 5 EnzoM03 (CH2)2CH3 5 EnzoM04 CH(CH3)2 4 EnzoM05 (CH2)3CH3 3 EnzoM06 CH2CH(CH3)2 5 EnzoM07 CH(CH3)(CH2CH3) 4 EnzoM08 C(CH3)3 4 EnzoM09 (CH2)4CH3 4 EnzoM10 (CH2)2CH(CH3)2 4 EnzoM11 CH2CH(CH3)(CH2CH3) 5 EnzoM12 CH2C(CH3)3 5
[0069] In another approach, the carboxyl group of NCI 8642 is replaced by a carboxamide group to generate a series of compounds with the general structure (VII):
##STR00011##
[0070] A list of the particular substitutions used in this series (EnzoM13-EnzoM41) as well as the resultant scores are given in Table II below:
TABLE-US-00002 TABLE II Compound R.sup.13 R.sup.14 Cscore EnzoM13 H H 5 EnzoM14 H CH.sub.3 5 EnzoM15 CH.sub.3 CH.sub.3 5 EnzoM16 H CH.sub.2CH.sub.3 5 EnzoM17 H (CH.sub.2).sub.2CH.sub.3 5 EnzoM18 CH.sub.3 CH.sub.2CH.sub.3 4 EnzoM19 CH.sub.3 (CH.sub.2).sub.2CH.sub.3 5 EnzoM20 H C(CH.sub.3).sub.3 5 EnzoM21 H (CH.sub.2).sub.3CH.sub.3 5 EnzoM22 CH.sub.3 (CH.sub.2).sub.3CH.sub.3 5 EnzoM23 H CH.sub.2CH(CH.sub.3) 5 EnzoM24 CH.sub.3 CH.sub.2CH(CH.sub.3).sub.2 5 EnzoM25 CH.sub.3 C(CH.sub.3).sub.3 5 EnzoM26 CH.sub.2CH.sub.3 (CH.sub.2).sub.2CH.sub.3 5 EnzoM27 CH.sub.2CH.sub.3 CH(CH.sub.3).sub.2 5 EnzoM28 CH.sub.2CH.sub.3 (CH.sub.2).sub.3CH.sub.3 5 EnzoM29 CH.sub.2CH.sub.3 CH.sub.2CH(CH.sub.3).sub.2 3 EnzoM30 CH.sub.2CH.sub.3 (CH.sub.2).sub.3CH.sub.3 5 EnzoM31 CH.sub.2CH.sub.3 CH.sub.2CH(CH.sub.3).sub.2 3 EnzoM32 CH.sub.3 (CH.sub.2).sub.4CH.sub.3 5 EnzoM33 CH.sub.3 (CH.sub.2).sub.2CH(CH.sub.3).sub.2 5 EnzoM34 H (CH.sub.2).sub.4CH.sub.3 5 EnzoM35 H (CH.sub.2).sub.2CH(CH.sub.3).sub.2 5 EnzoM36 CH.sub.2CH.sub.3 (CH.sub.2).sub.4CH.sub.3 5 EnzoM37 CH.sub.2CH.sub.3 (CH.sub.2).sub.2CH(CH.sub.3).sub.2 5 EnzoM38 CH.sub.2CH.sub.3 CH.sub.2CH(CH.sub.3)(CH.sub.2CH.sub.3) 2 EnzoM39 H CH.sub.2CH(CH.sub.3)(CH.sub.2CH.sub.3) 5 EnzoM40 H CH.sub.2C(CH.sub.3).sub.3 2 EnzoM41 CH.sub.2CH.sub.3 CH.sub.2C(CH.sub.3).sub.3 4
[0071] In another series of compounds, the carboxyl group is esterified to give the structure (VIII):
##STR00012##
A panel of compounds (EnzoM42-EnzoM70) were designed with various groups; these substitutions and cScores are given in Table III below:
TABLE-US-00003 TABLE III Compound R13 Cscore EnzoM42 CH3 3 EnzoM43 CH2CH3 5 EnzoM44 (CH2)2CH3 5 EnzoM45 CH(CH3)2 5 EnzoM46 (CH2)3CH3 5 EnzoM47 CH2CH(CH3)2 5 EnzoM48 CH(CH3)(CH2CH3) 5 EnzoM49 C(CH3)3 5 EnzoM50
[0072] It can be seen that the variety of substitutions that have been made in just three sites on the core molecule were able to generate a large number of candidates that can be tested by virtual screening without synthesizing a single molecule. Furthermore, when this series of compounds was tested in the same virtual screening program described previously, 44 out of the 70 compounds gave cScore values of 5. This demonstrates the power of the virtual substitution technique in designing new compounds since the compound IIIC3 used to design these molecules only had a relative cScore rating of 4.