Patent Grant
9797882
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 3,791 byte ASCII (text) file named “Seq_List” created on Jul. 9, 2014.
This application relates to screening methods for identifying compounds that inhibit the TWEAK-Fn14 pathway. Also provided are compositions comprising heterocyclic compounds and pharmaceutically acceptable salts thereof that are useful for the treatment of cell proliferative disorders and other disease states.
The TNF-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine involved in many cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis and inflammation (Wiley S R, Winkles J A (2003) Cytokine Growth Factor Rev 14: 241-249). TWEAK is a type II transmembrane protein that consists of an N-terminal cytoplasmic domain followed by a single transmembrane domain that is separated by a stalk region from the C-terminal Tumor Necrosis Factor (TNF) homology domain (THD) (Chicheportiche Y, Bourdon P R, Xu H, Hsu Y M, Scott H, et al. (1997) J Biol Chem 272: 32401-32410; Feng S L, Guo Y, Factor V M, Thorgeirsson S S, Bell D W, et al. (2000) Am J Pathol 156: 1253-1261). Membrane TWEAK is processed by a protease of the furin family resulting in a soluble ligand containing the THD. The THD functions in ligand trimerization and receptor binding causing TWEAK to signal as a trimerized molecule (Kolzsch J (1990) Z Arztl Fortbild (Jena) 84: 1199-1202; Kolfschoten G M, Pradet-Balade B, Hahne M, Medema J P (2003) Biochem Pharmacol 66: 1427-1432). Importantly, both membrane-bound and soluble TWEAK (sTWEAK) proteins are fully functional and can mediate similar cellular signaling effects by binding to cellular receptors (Brown S A, Ghosh A, Winkles J A (2010) J Biol Chem 285: 17432-17441).
TWEAK acts by binding to the fibroblast growth factor-inducible 14 (Fn14) receptor, the smallest member of the tumor necrosis factor receptor (TNFR) superfamily (Wiley S R, Winkles J A (2003) Cytokine Growth Factor Rev 14: 241-249; Meighan-Mantha R L, Hsu D K, Guo Y, Brown S A, Feng S L, et al. (1999) J Biol Chem 274: 33166-33176). TWEAK-mediated Fn14 signaling triggers a wide range of physiological activities in cells and tissues including blood clotting, cell proliferation, cell migration, inflammation, and angiogenesis (Harada N, et al. (2002) Biochem Biophys Res Commun 299: 488-493; Polek T C, et al. (2003) J Biol Chem 278: 32317-32323). The Fn14 receptor contains a single cysteine-rich domain (CRD) in the extracellular ligand-binding region and a short cytoplasmic tail possessing a single TNFR-associated factor (TRAF) binding site (Wiley S R, Winkles J A (2003) Cytokine Growth Factor Rev 14: 241-249; Meighan-Mantha R L, et al. (1999) J Biol Chem 274: 33166-33176). Notably, TWEAK is the only known TNF superfamily member that can bind to Fn14. Site-directed mutagenesis has demonstrated that TWEAK binding to the Fn14 CRD requires evolutionarily conserved amino acid residues (Asp45, Lys48 and Met50) and all three of the predicted disulfide bonds (Brown S A, Hanscom H N, Vu H, Brew S A, Winkles J A (2006) Biochem J 397: 297-304). Optimal TWEAK-mediated activation of Fn14 is important for promoting productive tissue responses after injury, but excessive TWEAK-Fn14 activation can induce pathological tissue responses, leading to progressive damage and degradation (Burkly L C, Michaelson J S, Zheng T S (2011) Immunol Rev 244: 99-114).
Over-expression of Fn14 has been reported in multiple cancers including glioblastoma, breast, pancreatic, esophageal, lung, and liver carcinomas (Feng S L, Guo Y, Factor V M, Thorgeirsson S S, Bell D W, et al. (2000) Am J Pathol 156: 1253-1261; Han H, Bearss D J, Browne L W, Calaluce R, Nagle R B, et al. (2002) Cancer Res 62: 2890-2896; Tran N L, McDonough W S, Donohue P J, Winkles J A, Berens T J, et al. (2003) Am J Pathol 162: 1313-1321; Watts G S, Tran N L, Berens M E, Bhattacharyya A K, Nelson M A, et al. (2007) Int J Cancer 121: 2132-2139; Willis A L, Tran N L, Chatigny J M, Charlton N, Vu H, et al. (2008) Mol Cancer Res 6: 725-734; Whitsett T G, Cheng E, Inge L, Asrani K, Jameson N M, et al. (2012) Am J Pathol 181: 111-120). In glioblastoma, Fn14 mRNA and protein expression is unregulated in migratory cells in vitro and invading cells in vivo (Tran N L, McDonough W S, Savitch B A, Fortin S P, Winkles J A, et al. (2006) Cancer Res 66: 9535-9542). Fn14 expression increases with increasing tumor grade with highest expression observed in glioblastoma multiforme (Grade IV). In contrast, the expression of Fn14 is minimal to absent in normal brain tissue. Moreover, TWEAK binding to Fn14 triggers glioma cell invasion and survival (Tran N L, McDonough W S, Savitch B A, Fortin S P, Winkles J A, et al. (2006) Cancer Res 66: 9535-9542).
TWEAK-Fn14 signaling plays a key role in various disease states and therefore holds significant therapeutic potential as a novel molecular target for developing anti-cancer and anti-autoimmune therapeutic agents in humans. It has been shown that this interaction plays a pivotal role in various immunological conditions like rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, renal injury, ischaemic stroke, as well as cardiac dysfunction and failure (Burkly L C, et al. (2007) Cytokine 40: 1-16; Winkles J A (2008) Nat Rev Drug Discov 7: 411-425; Jain M, Jakubowski A, et al. (2009) Circulation 119: 2058-2068). Several studies have confirmed the therapeutic potential of this pathway in human esophageal and pancreatic cancers, autoimmune disorders (Zheng T S, Burkly L C (2008) J Leukoc Biol 84: 338-347), muscle atrophy and injury (Bhatnagar S, Kumar A (2012) Curr Mol Med 12: 3-13) and chemokine-dependent inflammatory kidney disease (Campbell S, Burkly L C, Gao H X, Berman J W, Su L, et al. (2006) J Immunol 176: 1889-1898). The ever increasing knowledge and data on various downstream reactions stimulated by TWEAK-Fn14 interaction has recently been compiled into a complete repository (Bhattacharjee M, Raju R, Radhakrishnan A, Nanjappa V, Muthusamy B, et al. (2012) J Signal Transduct 2012: 376470). This paves the way for identification of yet unknown components of the signaling pathways.
To date, there are five anti-TNF antibody-based drugs already on the market and 16 out of approximately 22 ligand/receptor pairs under clinical development, constituting one of the most successful classes of biological therapeutics (Tansey M G, Szymkowski D E (2009) Drug Discov Today 14: 1082-1088). These protein-based therapeutics have some notable disadvantages including problems associated with drug delivery, stability, and cost. On the other hand, very few small molecule inhibitors targeting TNFR family members have been identified. Known small molecule inhibitors for the TNFR family act by disrupting trimerization of their respective ligands, as is the case for TNFα (He M M, Smith A S, Oslob J D, Flanagan W M, Braisted A C, et al. (2005) Science 310: 1022-1025) and CD40 (Silvian L F, Friedman J E, Strauch K, Cachero T G, Day E S, et al. (2011) ACS Chem Biol 6: 636-647). Benicchi and coworkers have also focused on the development of a Homogenous Time Resolved Fluorescence (HTRF) assay for identification of small-molecule inhibitors for TWEAK-Fn14 interaction and reported the identification of hits at a rate of 0.007% (Benicchi T, Iozzi S, Svahn A, Axelsson H, Mori E, et al. (2012) J Biomol Screen 17: 933-945). Currently, the potential therapeutic benefit of inhibiting key nodes of the TWEAK-Fn14 signaling pathway remains untapped due to the absence of small molecule tools to interrogate this pathway.
The present disclosure is directed to screening methods to facilitate the discovery of small molecules targeting the TWEAK-Fn14 pathway based on the determination of the molecular basis of the interaction between TWEAK and Fn14 and the elucidation of key structural elements of this interaction. Thus, the present invention addresses the unmet need for effective screening methods to identify compounds that target the TWEAK-Fn14 pathway and provides a number of therapeutic agents identified by these screening methods.
The importance of the Fn14 CRD has been established utilizing an NMR solution structure of this domain and functional mutation studies (Brown S A, Hanscom H N, Vu H, Brew S A, Winkles J A (2006) Biochem J 397: 297-304).
The present invention is based on the characterization of the TWEAK-Fn14 interaction. Six structural models of TWEAK were built based on experimental structures of low homology templates from the TNF superfamily. Protein-protein docking, followed by data-driven prioritization, yielded two promising TWEAK-Fn14 binding hypotheses. Site directed mutagenesis confirmed one hypothesis providing a structural basis for target-based identification of small molecule inhibitors of the TWEAK-FN14 interaction. Validated models served as a basis for in silico library design. A targeted library of molecules was assembled and screened iteratively, leading to enrichment in activity for compounds with similar scaffolds. The identified compounds target the TWEAK-Fn14 interaction and provide therapeutic agents for the treatment of cancer including glioblastoma and other diseases.
In certain implementations, the present invention relates to a method of screening a compound for modulating activity at a TNF-like weak inducer of apoptosis (TWEAK) binding site on a cysteine-rich domain (CRD) of fibroblast growth factor-inducible 14 (Fn14), the method generally comprises the steps of:
In other implementations, the present invention is directed to a method of screening a compound for modulating activity at a TWEAK binding site on a CRD of Fn14, the method comprising:
In other aspects, the present invention provides a method of screening a compound for modulating activity at a TWEAK binding site on a CRD of Fn14, the method comprising:
The present invention also provides a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein
each of R1 and R2 is independently —H, —OH, ═O, —NH2, —N(alkyl)(alkyl), hydrocarbyl, —O-hydrocarbyl, aryl, —O-aryl, aralkyl, —O-aralkyl, heteroaryl, —O-heteroaryl, heterocyclyl, —O-heterocyclyl, halo, —OCF3, —C(O)O(alkyl), —OC(O)(alkyl), —C(O)NH2, —C(O)NH(alkyl), —C(O)N(alkyl)(alkyl), —NHC(O)(alkyl), —N(alkyl)C(O)(alkyl), —OC(O)O(alkyl), —OC(O)NH2, —OC(O)NH(alkyl), —OC(O)N(alkyl)(alkyl), —CHNH, —CHN(alkyl), —SO2NH2,
both R3 and R4 are absent or are ═O;
R5 is H, —OH, hydrocarbyl, aryl, aralkyl, heteroaryl, heterocyclyl, or
each of R6, R8, R9, and R12 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted —O-alkyl, or substituted or unsubstituted aryl, wherein
R6 and R9 together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having 6 members and containing at least one carbon-carbon double bond,
R8 and R9 together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having 5 members and containing at least one carbon-carbon double bond, and
R9 and R12 together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having 6 members and containing at least one carbon-carbon double bond;
R7 is H, hydrocarbyl, aryl, aralkyl, heteroaryl, heterocyclyl, or
each of R10, R11, R13 and R14 independently is absent or is —H, —OH, halo, —C1-6alkyl, or —O—C1-6alkyl; and
each of Z1, Z2, and Z3 is independently CH or N.
In yet other embodiments, the present invention relates to a compound of Formula (II);
or a pharmaceutically acceptable salt thereof, wherein
R1 is —H, —OH, ═O, —NH2, —N(alkyl)(alkyl), hydrocarbyl, —O-hydrocarbyl, aryl, —O-aryl, aralkyl, —O-aralkyl, heteroaryl, —O-heteroaryl, heterocyclyl, —O-heterocyclyl, halo, —OCF3, —C(O)O(alkyl), —OC(O)(alkyl), —C(O)NH2, —C(O)NH(alkyl), —C(O)N(alkyl)(alkyl), —NHC(O)(alkyl), —N(alkyl)C(O)(alkyl), —OC(O)O(alkyl), —OC(O)NH2, —OC(O)NH(alkyl), —OC(O)N(alkyl)(alkyl), —CHNH, —CHN(alkyl), —SO2NH2.
The compounds of the present invention may be used to treating a cell proliferative disorder including glioblastoma, breast cancer, pancreatic cancer, esophageal cancer, lung cancer, and liver cancer. The compounds may also be used to treat a disease state such as, for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, renal injury, ischaemic stroke, cardiac dysfunction, cardiac failure, an autoimmune disorder, muscle atrophy and injury, and chemokine-dependent inflammatory kidney disease.
Some embodiments of the invention may provide a method of screening a compound for modulating activity at a TWEAK binding site on a CRD of Fn14, with the method generally comprising the steps of:
In some embodiments, the method may comprise screening the compounds validated in step a) to assess non-specific cytotoxicity using a cell viability assay. Moreover, the method may be used such that compounds that are validated in step a) may comprise an IC50 of at least 10 μM. In some embodiments, the method may further comprise performing an ELISA assay with Fn14, a soluble form of TWEAK (sTWEAK), and the compounds validated in step c) to calculate the reduction in sTWEAK binding due to the addition of the compounds validated in step c) with a standard curve.
In certain aspects, the cell-based luciferase reporter assay comprises human embryonic kidney cells. The cell-based luciferase reporter assay may comprise cells that stably co-express full length Fn14 and a firefly luciferase reporter driven by NF-κB response elements.
In other aspects, measuring inhibition of migration of cells comprises culturing cells in medium containing the compounds validated and confirmed in steps a) and b) and monitoring radial migration of the cells. In some embodiments, the cells are glioblastoma or breast cancer cells.
As used herein, the verbs “comprise” and “include” and their conjugations are used in their non-limiting sense to mean that items following the words are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
“Compound” and “compounds” as used herein refers to a compound encompassed by the generic formulae disclosed herein, any subgenus of those generic formulae, and any forms of the compounds specified by the generic and subgeneric formulae, such as a pharmaceutically acceptable salt. Unless specified otherwise, the term further includes the isotopes, racemates, stereoisomers, and tautomers of the compound or compounds.
“Isotopes” refer to pharmaceutically acceptable isotopically-labeled compounds wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Suitable isotopes include isotopes of hydrogen, such as 2H and 3H. Substitution with heavier isotopes such as deuterium, i.e. 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
“Racemates” refers to a mixture of enantiomers.
“Solvate” or “solvates” of a compound refer to those compounds, where compounds are as defined herein, that are bound to a stoichiometric or non-stoichiometric amount of a solvent. Solvates of a compound includes solvates of all forms of the compound such as the oxide, ester, prodrug, or pharmaceutically acceptable salt of the disclosed generic and subgeneric formulae. Preferred solvents are volatile, non-toxic, and/or acceptable for administration to humans. The present invention provides solvates of the compounds disclosed herein.
“Stereoisomer” or “stereoisomers” refer to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds of this invention may exist in stereoisomeric form if they possess one or more asymmetric centers or a double bond with asymmetric substitution and, therefore, can be produced as individual stereoisomers or as mixtures. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of Advanced Organic Chemistry, 4th ed., J. March, John Wiley and Sons, New York, 1992).
“Tautomer” refers to alternate forms of a compound that differ in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a ring atom attached to both a ring —NH— moiety and a ring ═N— moiety such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.
“Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts derived from a variety of organic and inorganic counter ions well known in the art and includes, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium. When the molecule contains a basic functionality, acid addition salts of organic or inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, oxalic acid, 4-toluenesulfonic acid, camphorsulfonic acid, methanesulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Salts can also be formed when an acidic proton present in the parent compound is either replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts are suitable for administration in a patient and possess desirable pharmacological properties. Suitable salts further include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.
The term “alkyl,” as used herein unless otherwise defined, refers to a straight, branched, or cyclic saturated group derived form the removal of a hydrogen atom from an alkane. Representative straight chain alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and n-heptyl. Representative branched alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl and 1,2-dimethylpropyl. Representative cyclic alkyl groups include cyclohexyl, cyclopentyl, and cyclopropyl.
As used herein, and unless otherwise specified, the term “heteroalkyl” refers to a stable straight or branched chain consisting of the stated number of carbon atoms and from one or more, in one embodiment, one to three, heteroatoms selected from the group consisting of O, N, Si, and S, and wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom can optionally be quaternized. In one embodiment, the heteroatom(s) O and N can be placed at any interior position of the heteroalkyl group. In one embodiment, the heteroatom(s) S and Si can be placed at any position of the heteroalkyl group (e.g., interior or terminal position), including the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2—S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, and —CH═CH—N(CH3)—CH3. Up to two heteroatoms can be consecutive, such as, for example, —CH2—NH—O—CH3 and —CH2—O—Si(CH3)3. In certain embodiments, the heteroalkyl is optionally substituted as described herein elsewhere. In some embodiments, the heteroalkyl is optionally substituted with one or more halo.
The term “alkenyl” refers to a straight, branched, or cyclic hydrocarbon group containing at least one double bond. Representative alkenyl groups include, but are not limited to, ethylene, propylene, 1-butylene, 2-butylene, isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene, 2-hexene, 3-hexene and isohexene.
The term “alkynyl” refers to a straight or branched chain hydrocarbon containing at least one triple bond. Representative alkynyl groups include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, isobutyne, sec-butyne, 1-pentyne, 2-pentyne, isopentyne, 1-hexyne, 2-hexyne, 3-hexyne and isohexyne.
The term “hydrocarbyl,” as used herein unless otherwise defined, refers to a substituent derived from the removal of hydrogen atom from a hydrocarbon molecule. Non-limiting examples of hydrocarbyl include alkyl, alkenyl, alkynyl; cyclic groups consisting of hydrogen and carbon such as aryl as described herein, including both aromatic and non-aromatic groups as described herein; and aralkyl described herein.
The term “aryl” as used herein unless otherwise defined, refers to an aromatic group. Non-limiting examples of aryl include phenyl, naphthyl, pyridyl, phenanthryl, anthryl, furanyl, azolyl, imidazolyl, and indolyl. In one embodiment, the aryl group is substituted with one or more of the following groups: -halo, —O—(C1-C6 alkyl), —OH, —CN, —COOR′, —OC(O)R′, —N(R′)2, —NHC(O)R′ or —C(O)NHR′ groups wherein each R′ is independently —H or unsubstituted —C1-C6 alkyl. Unless specified otherwise, the aryl is unsubstituted.
The term “heteroaryl” as used herein unless otherwise defined, refers to an aromatic group, wherein the aromatic group contains at least one ring atom that is not carbon. Non-limiting examples of heteroaryl include pyridyl, furanyl, azolyl, imidazolyl, thiophenyl, and indolyl. In one embodiment, the aryl group is substituted with one or more of the following groups: -halo, —O—(C1-C6alkyl), —CN, —COOR′, —OC(O)R, —N(R′)2, —NHC(O)R′ or —C(O)NHR′ groups wherein each R′ is independently —H or unsubstituted —C1-C6 alkyl. Unless specified otherwise, the heteroaryl is unsubstituted.
The term “aralkyl” as used herein unless otherwise defined, refers to an alkyl group, which is substituted with an aryl group. Non-limiting examples of an aralkyl group include benzyl, picolyl, naphthylmethyl.
The term “heterocyclyl” as used herein unless otherwise defined, refers to a cyclic group, wherein the cyclic group contains at least one ring atom that is not carbon. Representative examples heterocyclyl group include, but are not limited to, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiomorpholinyl, thiophenyl, triazinyl, triazolyl. In one embodiment, the aryl group is substituted with one or more of the following groups: -halo, —O—(C1-C6 alkyl), —OH, —CN, —COOR′, —OC(O)R′, —N(R′)2, —NHC(O)R′ or —C(O)NHR′ groups wherein each R′ is independently —H or unsubstituted —C1-C6 alkyl. Unless specified otherwise, the heterocyclyl is unsubstituted.
The term “alkoxy,” as used herein unless otherwise defined, refers to —O-(alkyl), wherein alkyl is as defined above. Representative examples of a C1-C6 alkoxy include, but are not limited to, —OCH3, —OCH2CH3, —OCH2CH2CH3, —OCH(CH3)CH3, —OCH2CH2CH2CH3, —OCH2CH(CH3)CH3, —OCH(CH3)CH2CH3, —OC(CH3)3, —OCH2CH2CH2CH2CH3, —OCH2CH(CH3)CH2CH3, —OCH2CH2CH2CH2CH2CH3, and —OCH2CH2CH(CH3)CH2CH3.
The terms “halo” and “halogen,” as used herein unless otherwise defined, refers to —F, —Cl, —Br or —I.
Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.
It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups with two other substituted aryl groups are limited to -substituted aryl-(substituted aryl)-substituted aryl.
Similarly, it is understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 fluoro groups). Such impermissible substitution patterns are well known to the skilled artisan.
Turning next to the compositions of the invention, the term “pharmaceutically acceptable carrier or excipient” means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe, and possesses acceptable toxicities. Acceptable carriers or excipients include those that are acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.
With reference to the methods of the present invention, the following terms are used with the noted meanings:
The terms “treating” or “treatment” of a disease includes inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
A preferred embodiment of the invention is treatment of a disease that consists of relieving the disease.
As used herein, the term “subject” or “patient” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In other embodiments, the subject is a human.
The term “an effective amount” or “an amount sufficient to” means the amount of the 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. “An effective amount” or “an amount sufficient to” includes the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
The term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.
In some embodiments, the method of the present invention relates to screening a compound for modulating activity at a TNF-like weak inducer of apoptosis (TWEAK) binding site on a cysteine-rich domain (CRD) of fibroblast growth factor-inducible 14 (Fn14), the method comprising:
SEQ ID NO: 1 shown below is the amino acid sequence of TWEAK in humans and corresponds with GenBank Accession Number AAC51923.1. The anchoring residue Y176 is identified with a box.
SEQ ID NO: 2 shown below is the amino acid sequence for Fn14 in humans and corresponds with GenBank Accession Number AAF69108.1.
In certain embodiments, the protein-protein docking model is Y176 Model 1 and/or Y176 Model 2 shown in Table 2. The screened compound may have modulating activity if the compound matches the pharmacophoric sites corresponding to Y176 and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten additional pharmacophoric sites.
In some implementations, the present invention is directed to a method of screening a compound for modulating activity at a TWEAK binding site on a CRD of Fn14, the method comprising:
In certain aspects, the methods of the present invention utilize the computational tools of Glide. Glide offers the full range of speed vs. accuracy options, from the HTVS (high-throughput virtual screening) mode for efficiently enriching million compound libraries, to the SP (standard precision) mode for reliably docking tens to hundreds of thousands of ligand with high accuracy, to the XP (extra precision) mode where further elimination of false positives is accomplished by more extensive sampling and advanced scoring, resulting in even higher enrichment. Glide also provides a rational workflow for virtual screening from HTVS to SP to XP, enriching the data at every level such that only an order of magnitude fewer compounds need to be studied at the next higher accuracy level. In addition, Glide reliably finds the correct binding modes for a large set of test cases. It outperforms other docking programs in achieving lower RMS deviations from native co-crystallized structures. Glide also exhibits excellent docking accuracy and high enrichment across a diverse range of receptor types.
The Glide algorithms and methods (Suite 2011: Glide, version 5.7, Schrödinger, LLC, New York, N.Y., 2011; Suite 2012: Glide, version 5.8, Schrödinger, LLC, New York, N.Y., 2012) are described in:
In certain implementations, the disclosed methods comprise calculating a Glide score using a high-throughput virtual screening (HTVS) algorithm of Glide, a standard precision (SP) algorithm of Glide, and/or an extra precision (XP) algorithm of Glide. In some embodiments, a high HTVS, SP, or XP Glide score is less than −1.0, −1.1, −1.2, −1.3, −1.4, −1.5, −1.6, −1.7, −1.8, −1.9, −2.0, −2.1, −2.2, −2.3, −2.4, −2.5, −2.6, −2.7, −2.8, −2.9, −3.0, −3.1, −3.2, −3.3, −3.4, −3.5, −3.6, −3.7, −3.8, −3.9, −4.0, −4.1, −4.2, −4.3, −4.4, −4.5, −4.6, −4.7, −4.8, −4.9, −5.0, −5.1, −5.2, −5.3, −5.4, −5.5, −5.6, −5.7, −5.8, −5.9, −6.0, −6.1, −6.2, −6.3, −6.4, −6.5, −6.6, −6.7, −6.8, −6.9, −7.0, −7.1, −7.2, −7.3, −7.4, −7.5, −7.6, −7.7, −7.8, −7.9, −8.0, −8.1, −8.2, −8.3, −8.4, −8.5, −8.6, −8.7, −8.8, −8.9, −9.0, −9.1, −9.2, −9.3, −9.4, −9.5, −9.6, −9.7, −9.8, −9.9, or −10.0.
In certain embodiments, an HTVS score, an SP score, and an XP score are calculated for compounds within a library. In some embodiments, an SP score is calculated for the about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, highest scoring compounds from the HTVS algorithm. In some embodiments, an XP score is calculated for the about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, highest scoring compounds from the SP algorithm.
In some embodiments, energy-optimized pharmacophores (e-pharmacophores) are obtained by mapping the energetic terms from the Glide XP scoring function onto atom centers. The ligand is docked with Glide XP and the pose is refined. The Glide XP scoring terms are computed, and the energies are mapped onto atoms. Next, pharmacophore sites are generated, and the Glide XP energies from the atoms that comprise each pharmacophore site are summed. The sites are then ranked based on these energies, and the most favorable sites are selected for the pharmacophore hypothesis. These pharmacophores are then used as queries for virtual screening.
Pharmacophore sites may be automatically generated from the protein-ligand docked complex with Phase (Phase, v.3.0, Schrodinger, LLC) using the default set of six chemical features: hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobe (H), negative ionizable (N), positive ionizable (P), and aromatic ring (R). Phase treats most cationic groups as being exclusively positive ionizable. Hydrogen-bond acceptor sites may be represented as vectors along the hydrogen bond axis in accordance with the hybridization of the acceptor atom. Hydrogen-bond donors may be represented as projected points, located at the corresponding hydrogen-bond acceptor positions in the binding site. Projected points allow the possibility of structurally dissimilar active compounds forming hydrogen bonds to the same location, regardless of their point of origin and directionality.
In certain embodiments, each pharmacophore feature site is first assigned an energetic value equal to the sum of the Glide XP contributions of the atoms comprising the site. This allows sites to be quantified and ranked on the basis of these energetic terms. Glide XP descriptors include terms for hydrophobic enclosure, hydrophobically packed correlated hydrogen bonds, electrostatic rewards, π-π stacking, π . . . cation, and other interactions. In some embodiments, sites where less than half of the heavy atoms contribute to the pharmacophore feature are excluded from the final hypothesis. Thus, if only two heavy atoms in a six-membered ring exhibit energetic interactions, the ring may not be considered a pharmacophore feature. The Glide algorithms and their application in virtual screening of compounds are explained in greater detail in Shen et al. (2013) Mol Biosyst 9:1511-1521; Singh et al. (2012) J Mol Model 18:39-51; and Tamilvanan et al. (2013) Bioinformation 9(6):286-292.
Referring to
In some embodiments, the first process may comprise a cell-based nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) luciferase reporter assay, wherein inhibition of luciferase signal following addition of the one or more compounds and purified TWEAK to NF-κB luciferase cells validates the modulating activity of the compound at the TWEAK binding site on the CRD of Fn14. In some aspects, compounds that exhibit modulating activity of the TWEAK-Fn14 interaction can be further tested using the other processes.
In some embodiments, the second process may comprise a cell-based NF-κB luciferase reporter assay. For example, the second process may include validation of the modulating activity at the TWEAK binding site on the CRD of Fn14 of the one or more compounds. In some embodiments, compounds that may exhibit an IC50 of about less than or equal to 10 μM can be selected for further testing. In other embodiments, the IC50 values can be any other values, such as 1 μM, 5 μM, 15 μM, 25 μM, etc.
Moreover, in some embodiments, the second process may also include a counterscreen to confirm the modulating activity at the TWEAK binding site on the CRD of Fn14 of the one or more compounds that have been validated using the prior methods or processes. For example, the lack of inhibition of luciferase signal following addition of the one or more compounds and purified tumor necrosis factor alpha (TNFα) to NF-κB luciferase cells can confirm specificity of the modulating activity at the TWEAK binding site on the CRD of Fn14. In some embodiments, compounds that exhibit an IC50 in these counterscreening assays of about greater than or equal to 10 times the IC50 from the validation assays described above can be selected for further testing.
In addition, the second process may further comprise a counterscreen to assess whether the modulating activity at the TWEAK binding site on the CRD of Fn14 of the one or more compounds is not correlated with non-specific global cytotoxicity. For example, some or all of the compounds that exhibit a CC50 of about 10 times less than the IC50 on reporter cells can be removed from additional testing.
In some embodiments, the third process may comprise one or more assays to elucidate the structure-activity relationship of the one or more compounds that are substantially or completely validated and/or confirmed by the first and second processes. For example, the third process may comprise an analysis of the scaffold class of the compounds to provide an assessment of chemical tractability and probability of success in biological systems of modulating activity of TWEAK binding site on the CRD of Fn14. In addition, the third process may also include using the scaffold class of the compounds to potentially identify more potent and selective compounds than the compounds that were initially identified using some embodiments of the invention.
In some embodiments, the fourth process may comprise assays that can be used to further assess the activity of the compounds identified by the disclosed methods and processes by measuring inhibition of migration of glioma cells resulting from addition of the one or more compounds and purified TWEAK to the glioma cells, wherein inhibition of migration of the glioma cells validates the modulating activity of the compounds at the TWEAK binding site on the CRD of Fn14. The fourth process may also comprise additional assays (e.g., enzyme-linked immunosorbent assays) to further confirm modulating activity and/or mechanism of action of the compounds.
In certain implementations, the present invention relates to a method of modulating the TWEAK-Fn14 pathway in a subject, the method comprising administering to the subject an Fn14 antagonist comprising a compound of Formula (I):
each of R6, R8, R9, and R12 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted —O-alkyl, or substituted or unsubstituted aryl, wherein
R6 and R9 together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having 6 members and containing at least one carbon-carbon double bond,
R8 and R9 together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having 5 members and containing at least one carbon-carbon double bond, and
R9 and R12 together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having 6 members and containing at least one carbon-carbon double bond;
R7 is H, hydrocarbyl, aryl, aralkyl, heteroaryl, heterocyclyl, or;
In yet other embodiments, the present invention relates to a method of modulating the TWEAK-Fn14 pathway in a subject, the method comprising administering to the subject an Fn14 antagonist comprising a compound of Formula (II):
In certain embodiments, the disorder that can be treated, prevented, or ameliorated is cancer or a proliferative disorder, including but not limited to, breast cancer (e.g., triple negative breast cancer, ER+ breast cancer, or ER− breast cancer), basal cell carcinoma, skin cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, medulloblastoma, glioma (including glioblastoma, oligodendroglioma, astrocytoma, ependymoma), neuroblastoma, colorectal cancer, ovarian cancer, liver cancer, pancreatic cancer (e.g., carcinoma, angiosarcoma, adenosarcoma), gastric cancer, gastroesophageal junction cancer, prostate cancer, cervical cancer, bladder cancer, head and neck cancer, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), solid tumors that cannot be removed by surgery, locally advanced solid tumors, metastatic solid tumors, leukemia (e.g., acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or chronic myeloid leukemia (CML)), or recurrent or refractory tumors. In one embodiment, the disorder that can be treated, prevented, or ameliorated includes, but is not limited to, basal cell nevus syndrome (Gorlin syndrome). In one embodiment, the disorder that can be treated, prevented, or ameliorated includes, but is not limited to, basal cell carcinoma associated with Gorlin syndrome.
In one embodiment, the disorders, diseases, or conditions treatable with a compound provided herein, include, but are not limited to, (1) inflammatory or allergic diseases, including systemic anaphylaxis and hypersensitivity disorders, atopic dermatitis, urticaria, drug allergies, insect sting allergies, food allergies (including celiac disease and the like), and mastocytosis; (2) inflammatory bowel diseases, including Crohn's disease, ulcerative colitis, ileitis, and enteritis; (3) vasculitis, and Behcet's syndrome; (4) psoriasis and inflammatory dermatoses, including dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, viral cutaneous pathologies including those derived from human papillomavirus, HIV or RLV infection, bacterial, flugal, and other parasital cutaneous pathologies, and cutaneous lupus erythematosus; (5) asthma and respiratory allergic diseases, including allergic asthma, exercise induced asthma, allergic rhinitis, otitis media, allergic conjunctivitis, hypersensitivity lung diseases, and chronic obstructive pulmonary disease; (6) autoimmune diseases, including arthritis (including rheumatoid and psoriatic), systemic lupus erythematosus, type I diabetes, myasthenia gravis, multiple sclerosis, Graves' disease, and glomerulonephritis; (7) graft rejection (including allograft rejection and graft-v-host disease), e.g., skin graft rejection, solid organ transplant rejection, bone marrow transplant rejection; (8) fever; (9) cardiovascular disorders, including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, cardiomyopathy, congestive heart failure, atherosclerosis, coronary artery disease, restenosis, and vascular stenosis; (10) cerebrovascular disorders, including traumatic brain injury, stroke, ischemic reperfusion injury and aneurysm; (11) cancers of the breast, skin, prostate, cervix, uterus, ovary, testes, bladder, lung, liver, larynx, oral cavity, colon and gastrointestinal tract (e.g., esophagus, stomach, pancreas), brain, thyroid, blood, and lymphatic system; (12) fibrosis, connective tissue disease, and sarcoidosis, (13) genital and reproductive conditions, including erectile dysfunction; (14) gastrointestinal disorders, including gastritis, ulcers, nausea, pancreatitis, and vomiting; (15) neurologic disorders, including Alzheimer's disease; (16) sleep disorders, including insomnia, narcolepsy, sleep apnea syndrome, and Pickwick Syndrome; (17) pain; (18) renal disorders; (19) ocular disorders, including glaucoma; and (20) infectious diseases, including HIV.
In certain aspects, the cancer treatable with the methods provided herein includes, but is not limited to, (1) leukemias, including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome or a symptom thereof (such as anemia, thrombocytopenia, neutropenia, bicytopenia or pancytopenia), refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts (RAEB), RAEB in transformation (RAEB-T), preleukemia, and chronic myelomonocytic leukemia (CMML), (2) chronic leukemias, including, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; (3) polycythemia vera; (4) lymphomas, including, but not limited to, Hodgkin's disease and non-Hodgkin's disease; (5) multiple myelomas, including, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; (6) Waldenström's macroglobulinemia; (7) monoclonal gammopathy of undetermined significance; (8) benign monoclonal gammopathy; (9) heavy chain disease; (10) bone and connective tissue sarcomas, including, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancers, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; (11) brain tumors, including, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; (12) breast cancer, including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary cancers, Paget's disease, and inflammatory breast cancer; (13) adrenal cancer, including, but not limited to, pheochromocytom and adrenocortical carcinoma; (14) thyroid cancer, including, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; (15) pancreatic cancer, including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; (16) pituitary cancer, including, but limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; (17) eye cancer, including, but not limited, to ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; (18) vaginal cancer, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; (19) vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; (20) cervical cancers, including, but not limited to, squamous cell carcinoma, and adenocarcinoma; (21) uterine cancer, including, but not limited to, endometrial carcinoma and uterine sarcoma; (22) ovarian cancer, including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; (23) esophageal cancer, including, but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; (24) stomach cancer, including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; (25) colon cancer; (26) rectal cancer; (27) liver cancer, including, but not limited to, hepatocellular carcinoma and hepatoblastoma; (28) gallbladder cancer, including, but not limited to, adenocarcinoma; (29) cholangiocarcinomas, including, but not limited to, pappillary, nodular, and diffuse; (30) lung cancer, including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma, and small-cell lung cancer; (31) testicular cancer, including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, and choriocarcinoma (yolk-sac tumor); (32) prostate cancer, including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; (33) penal cancer; (34) oral cancer, including, but not limited to, squamous cell carcinoma; (35) basal cancer; (36) salivary gland cancer, including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; (37) pharynx cancer, including, but not limited to, squamous cell cancer and verrucous; (38) skin cancer, including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, and acral lentiginous melanoma; (39) kidney cancer, including, but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, and transitional cell cancer (renal pelvis and/or uterer); (40) Wilms' tumor; (41) bladder cancer, including, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, and carcinosarcoma; and other cancer, including, not limited to, head and neck cancer, myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangio-endotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinomas (See Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).
Particular embodiments provide treating a subject having cancer using one or more of the methods provided herein, together with surgery. Particular embodiments provide treating a subject having cancer using one or more of the methods provided herein, together with chemotherapy. Particular embodiments provide treating a subject having cancer using one or more of the methods provided herein, together with immunotherapy. Particular embodiments provide treating a subject having cancer using one or more of the methods provided herein, together with targeted therapy. Particular embodiments provide treating a subject having cancer using one or more of the methods provided herein, together with radiation therapy. Particular embodiments provide treating a subject having cancer using one or more of the methods provided herein, together with two or more of the treatments selected from surgery, chemotherapy, immunotherapy, targeted therapy, and radiation therapy.
In certain embodiments, the subject to be treated with one of the methods provided herein has not been treated with anticancer therapy prior to the administration of a compound provided herein. In certain embodiments, the subject to be treated with one of the methods provided herein has been treated with one or more anticancer therapies prior to the administration of a compound provided herein. In certain embodiments, the subject to be treated with one of the methods provided herein has been treated with a cancer therapeutic agent, as described herein. In certain embodiments, the subject to be treated with one of the methods provided herein has developed drug resistance to anticancer therapy. In certain embodiments, the subject to be treated with the methods provided herein has a relapsed cancer. In certain embodiments, the subject to be treated with the methods provided herein has a refractory cancer. In certain embodiments, the subject to be treated with the methods provided herein has a metastatic cancer.
In one embodiment, provided herein is a pharmaceutical composition comprising a compound of Formula (I) or Formula (II) as defined herein elsewhere, or an enantiomer, a mixture of enantiomers or a mixture of diastereomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, and at least one pharmaceutically acceptable excipient, adjuvant, carrier, buffer, or stabiliser.
In one embodiment, the pharmaceutically acceptable excipient, adjuvant, carrier, buffer, or stabiliser is non-toxic and does not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, such as cutaneous, subcutaneous, or intravenous injection.
In one embodiment, the pharmaceutical compositions are provided in a dosage form for oral administration, which comprise a compound provided herein, e.g., a compound of Formula (I) or Formula (II), or an enantiomer, a mixture of enantiomers or a mixture of diastereomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, and one or more pharmaceutically acceptable excipients or carriers. The pharmaceutical compositions provided herein that are formulated for oral administration may be in tablet, capsule, powder, or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, or mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. A capsule may comprise a solid carrier such as gelatin.
In another embodiment, the pharmaceutical compositions are provided in a dosage form for parenteral administration, and one or more pharmaceutically acceptable excipients or carriers. Where pharmaceutical compositions may be formulated for intravenous, cutaneous or subcutaneous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has a suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride injection, Ringer's injection, or Lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants, and/or other additives may be included as required.
In yet another embodiment, the pharmaceutical compositions are provided in a dosage form for topical administration, which comprise a compound provided herein, and one or more pharmaceutically acceptable excipients or carriers.
In one embodiment, the pharmaceutical compositions can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, e.g., Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology, 2nd Ed., Rathbone et al., eds., Marcel Dekker, Inc.: New York, N.Y., 2008).
In one embodiment, the pharmaceutical compositions provided herein can be provided in a unit-dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to physically discrete a unit suitable for administration to a human and animal subject, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of an active ingredient(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of a unit-dosage form include an ampoule, syringe, and individually packaged tablet and capsule. A unit-dosage form may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of a multiple-dosage form include a vial, bottle of tablets or capsules, or bottle of pints or gallons.
In one embodiment, the pharmaceutical compositions provided herein can be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.
In another embodiment, the pharmaceutical compositions provided herein further comprise one or more chemotherapeutic agents as defined herein.
In other aspects, the compounds of the present invention may be used to ameliorate or treat a cancer selected from the group consisting of a brain tumor, glioblastoma, breast cancer, prostate cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, melanoma, pancreatic cancer, liver cancer, and renal cancer.
The present invention is also directed to a pharmaceutical composition comprising an Fn14 antagonist and a pharmaceutically acceptable vehicle, excipient, and/or carrier. In certain embodiments, the pharmaceutical composition further comprises a therapeutic agent for the treatment of cancer or an autoimmune disease wherein the combination of the Fn14 antagonist and the therapeutic agent results in a synergistic effect. The therapeutic agent may be any one of of temozolomide, camptothecin, cisplatin, an Akt inhibitor, an NFκB inhibitor, an Mc1-1 inhibitor, a BCL-XL inhibitor, a BCL-2 inhibitor, a BCL-W inhibitor, an Src inhibitor, or a Lyn inhibitor. Such inhibitors are described in Druv et al., Carcinogenesis 35(1): 218-226, 2014; Tran et al., J Biol Chem 280(5): 3483-3492, 2005; Fortin et al., Mol Cancer Res 7: 1871-1881, 2009; and Whitsett et al., Mol Cancer Res 12: 550-559, 2014; the contents of which are hereby incorporated by reference.
In some aspects, the compounds of the present invention may be used to ameliorate or treat an autoimmune disease such as autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmunocytopenia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis, Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendo-crinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism, systemic lupus erythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Graves' Disease, Myasthenia Gravis, and insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, adrenergic drug resistance with asthma or cystic fibrosis, chronic active hepatitis, primary biliary cirrhosis, endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, an inflammatory disorder, a granulomatous disorder, an atrophic disorder, or an alloimmune disease.
Chemotherapeutic agents in the general sense thereof, are compounds, which can be used for the treatment of a disease or disorder that arises from bacterial, viral or parasitic infection or that is due to transformation of normal cells (cancer). One particular indication of chemotherapy is cancer. Chemotherapeutic agents can act for example by killing cells that divide more rapidly than other cells, and thus target cancer cells which commonly divide more rapidly than non-cancerous cells. Most chemotherapeutic agents work by impairing cell division at one of several stages of the cell cycle. Thus, they are able to target those cells that divide more rapidly. Chemotherapeutic agents can be either cytostatic, i.e., they slow down or abrogate the growth or division of cells; other chemotherapeutic agents can cause damage to cells and kill them; in that case they are termed cytotoxic. Most cytotoxic drugs inflict a damage that per se does not suffice to kill a cell but that generates a stimulus to initiate programmed cell death (apoptosis).
In general, major classes of chemotherapeutic drugs are alkylating agents, anti-metabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors and other anti-tumour agents. Most commonly, as mentioned above, these drugs affect one or several stages of the cell cycle; they can also affect DNA synthesis or DNA integrity. Other chemotherapeutics do not directly interfere with DNA. These are newer classes of chemotherapeutics and can include monoclonal antibodies and tyrosine kinase inhibitors lite imatinib mesylate. Other examples are chemotherapeutic hormones and hormone antagonists, e.g. glucocorticosteroids.
Examples for alkylating agents, which alkylate nucleophilic functional groups are mechtorethamine, cyclophosphamide, chlorambucil, melphalane, trofosfamide, ifosfamide, carmustine, lomustine, dacarbazine, temozolomide, mitomycine C and many others. Cisplatin, carboplatin, oxaliplatin and other platinum containing compounds form stable complexes with DNA.
Cytotoxic anti-metabolites are folic acid analogues (e.g., methotrexat/aminopterin, raltitrexed, pemetrexed), purine analogs (e.g., 6-mercaptopurine, azathioprine, thioguanine, fludarabine, cladribine) or pyrimidine analogs (cytarabine, gemcitabine, 5-fuk uracil and its prodrugs, deazacytkiine). Antimetabolites either inhibit DNA-synthesis by interfering with crucial steps in the cfe novo synthesis of purine and pyrimidine nucleotides or they become incorporated into DNA during the S-phase of the cell cycle, where they interfere with DNA-folding, DNA-repair or methylation. Alternatively, some compounds also become incorporated into RNA. Examples for alkaloids and terpenoids which are derived from plants and block cell division by preventing microtubule function are vinca-aialoids and taxanes. Particularly well known vinca-alkaloids are vincristine, vinblastine, vinorelbine and vindesine. Podophyllotoxin is an additional example of a plant-derived compound. An example for a taxane is docetaxel or paclitaxel. Another example is abraxane, an albumin bound paclitaxel. Estramustin is an example of a synthetic compound that targets tubulin.
Examples of topoisomerase inhibitors, which are inhibitors of enzymes that maintain the topology of DNA, include camphtotecines like irinotecan and topotecan (type 1 topoisomerase inhibitors) or amsacrin, etoposide, etoposide phosphate and teniposide (topoisomerase-type 2 inhibitors).
Finally, examples of antineoplastic intercalating agents include dactinomyctn, doxorubicin, epirubicin, bleomycin and others.
A comprehensive overview is comprised in Goodman and Gilman, The Pharmacological Basis of Therapeutics, 121h Edition, “General Principles of Cancer Chemotherapy”.
The following are examples for alkylating agents: Mechtorethamine, Cyclophosphamide Ifosfamide, Melphalan, Chlorambucil, Procarbazine (N-methylhydrazine, MIH), Busulfan, Camustine (BCNU), Streptozocin (streptozotocin), Bendamustine, Dacarbazine (DTIC; dimethyttriazenol midazole carboxamide), Temozolomide, Cisplatin, carboplatin, oxalplatin.
Antimetabolites are exemplary represented by Methotrexate (Amethopterin), Pemetrexed, Fluorouracil (5-fluorouracil; 5-FU), capecitabine, Cytarabine (cytosine arabinoside), Gemcitabine, 5-aza-cytidine, Deoxy-5-aza-cytidine, Mercaptoptirine (6-mercaptopurine; 6-MP), Pentostatin (2′-deoxycoformycin), Rudarabine, Clofarabine, and Nelarabine.
Natural Products can be selected from: Vinblastine, Vinorelbine, Vincristine, Paclitaxel, docetaxel, Etoposide, Teniposide, Topotecan, Irinotecan, Dactinomycin, (actinomycin D), Daunorubicin (daunomycin, rubidomycin), Doxorubicin, Yondelis, Mitoxantrone, Bleomycin, Mitomycin C, and L-Asparaginase.
Examples for Hormones and Antagonists are: Mitotane, Prednisone, Hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, Dietyhlstilbestrol, ethinyl estradiol, Tamoxifen, toremifene, Anastrozole, letrozole, exemestane, Testosterone propionate, fluoxymesterone, Flutamide, casodex, Leuprolide, while examples for further agents are: Hydroxyurea, Tretinoin, arsenic trioxide, Histone deacetylase inhibitor (vorinostat), Imatinib, Dasatinib, nilotinib, Gefrtinib, ertoinib, Sorafenib, Sunitinib, Lapatinib, Bortezomib, interferon-alpha, lnterteukin-2, Thalidomide, Lenaiidomide, Temsiroiimus, and Everolimus.
It will be appreciated that therapeutic agents and pharmaceutical compositions in accordance with the present invention can be employed in combination therapies. In some embodiments, the present invention encompasses “therapeutic cocktails” comprising compositions in accordance with the invention. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same purpose. In some embodiments, the therapies employed may achieve different effects (e.g., control of any adverse side effects) or they may achieve a synergistic effect when administered together.
Pharmaceutical compositions in accordance with the present invention may be administered either alone or in combination with one or more other therapeutic agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the invention. It will further be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. Additionally, the invention encompasses delivery of pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
The particular combination of therapies (therapeutics and/or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and/or the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an agent in accordance with the invention may be administered concurrently with another therapeutic agent used to treat the same disorder), and/or they may achieve different effects (e.g., control of any adverse side effects). In some embodiments, compositions in accordance with the invention are administered with a second therapeutic agent that is approved by the U.S. Food and Drug Administration.
In some embodiments, compositions in accordance with the invention may be administered in combination with any therapeutic agent or therapeutic regimen that is useful to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of an autoimmune disorder. For example, various agents which inhibit inflammation (e.g., steroids) can be used to treat autoimmune disorders in general. In some embodiments, compositions in accordance with the invention may be administered in combination with agents which inhibit inflammation (e.g., steroids) in order to treat autoimmune disorders.
In specific embodiments, compositions in accordance with the invention may be administered in combination with any therapeutic agent or therapeutic regimen that is useful to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of diabetes. For example, compositions in accordance with the invention may be administered in combination with traditional diabetes therapies including, but not limited to, insulin administration.
In specific embodiments, compositions in accordance with the invention may be administered in combination with any therapeutic agent or therapeutic regimen that is useful to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of rheumatoid arthritis. For example, compositions in accordance with the invention may be administered in combination with soluble TNF receptor, anti-TNFα receptor, analgesics, non-steroidal anti-inflammatory agents (NSAIDs), and/or other agents useful for treatment of rheumatoid arthritis.
In specific embodiments, compositions in accordance with the invention may be administered in combination with any therapeutic agent or therapeutic regimen that is useful to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of Crohn's disease. For example, compositions in accordance with the invention may be administered in combination with anti-TNFα receptor and/or other agents useful for treatment of Crohn's disease.
In specific embodiments, compositions in accordance with the invention may be administered in combination with any therapeutic agent or therapeutic regimen that is useful to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of multiple sclerosis. For example, compositions in accordance with the invention may be administered in combination with interferon β-1b, interferon β-1a, and/or other agents useful for treatment of multiple sclerosis.
In specific embodiments, compositions in accordance with the invention may be administered in combination with any therapeutic agent or therapeutic regimen that is useful to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of granulomatous disease and/or osteoporosis. For example, compositions in accordance with the invention may be administered in combination with interferon β-1b and/or other agents useful for treatment of granulomatous disease and/or osteoporosis.
One of ordinary skill in the art will understand that the examples presented above are not meant to be limiting. The principles presented in the examples above can be generally applied to any combination therapies for treatment of autoimmune disease.
Pharmaceutical compositions in accordance with the present invention may be administered either alone or in combination with one or more other therapeutic agents.
In some embodiments, compositions in accordance with the invention may be administered in combination with any therapeutic agent or therapeutic regimen that is useful to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of an autoimmune disorder. For example, compositions in accordance with the invention may be administered in combination with traditional diabetes therapies including, but not limited to, insulin administration. To give another example, compositions in accordance with the invention may be administered in combination with soluble TNF receptor, anti-TNFα receptor, analgesics, non-steroidal anti-inflammatory agents (NSAIDs), and/or other agents may be useful for treatment of rheumatoid arthritis.
In yet another embodiment, provided herein is the use of compound of Formula (I) or Formula (II) or an enantiomer, a mixture of enantiomers or a mixture of diastereomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, in the manufacture of a medicament for the treatment of one or more disorders disclosed herein. In certain embodiments, the medicament is in tablet, capsule, powder, or liquid form. In certain embodiments, the medicament is formulated as described herein.
The amount of a compound of the invention that is effective in the treatment of a particular condition disclosed herein can depend on the nature of the condition, and can be determined by standard clinical techniques. In vitro or in vivo assays can be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions can also depend on the route of administration or the severity of the condition, and can be decided according to the judgment of the practitioner and each subject's circumstances. However, suitable dosage ranges for oral administration are generally about 0.001 mg to 2000 mg of a compound of the invention per kg body mass. In some embodiments, the oral dose is 0.01 mg to 100 mg per kg body mass, 0.1 mg to 50 mg per kg body mass, 0.5 mg to 20 mg per kg body mass, or 1 mg to 10 mg per kg body mass. In some embodiments, the oral dose is 5 mg of a compound of the invention per kg body mass. The dosage amounts described herein refer to total amounts administered; that is, if more than one compound of the invention is administered, the dosages can correspond to the total amount of the compounds of the invention administered. Oral compositions can comprise 10% to 95% active ingredient by mass.
In one embodiment, the pharmaceutical compositions provided herein for oral administration can be provided in solid, semisolid, or liquid dosage forms for oral administration. As used herein, oral administration also includes buccal, lingual, and sublingual administration. Suitable oral dosage forms include, but are not limited to, tablets, fastmelts, chewable tablets, capsules, pills, strips, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, bulk powders, effervescent or non-effervescent powders or granules, oral mists, solutions, emulsions, suspensions, wafers, sprinkles, elixirs, and syrups. In addition to the active ingredient(s), the pharmaceutical compositions can contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, flavoring agents, emulsifying agents, suspending and dispersing agents, preservatives, solvents, non-aqueous liquids, organic acids, and sources of carbon dioxide.
In one embodiment, binders or granulators impart cohesiveness to a tablet to ensure the tablet remaining intact after compression. Suitable binders or granulators include, but are not limited to, starches, such as corn starch, potato starch, and pre-gelatinized starch (e.g., STARCH 1500); gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as acacia, alginic acid, alginates, extract of Irish moss, panwar gum, ghatti gum, mucilage of isabgol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone (PVP), Veegum, larch arabogalactan, powdered tragacanth, and guar gum; celluloses, such as ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC); microcrystalline celluloses, such as AVICEL-PH-101, AVICEL-PH-103, AVICEL RC-581, AVICEL-PH-105 (FMC Corp., Marcus Hook, Pa.); and mixtures thereof. Suitable fillers include, but are not limited to, talc, calcium carbonate, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The amount of a binder or filler in the pharmaceutical compositions provided herein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art. The binder or filler may be present from about 50 to about 99% by weight in the pharmaceutical compositions provided herein.
In one embodiment, suitable diluents include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar. Certain diluents, such as mannitol, lactose, sorbitol, sucrose, and inositol, when present in sufficient quantity, can impart properties to some compressed tablets that permit disintegration in the mouth by chewing. Such compressed tablets can be used as chewable tablets. The amount of a diluent in the pharmaceutical compositions provided herein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art.
In one embodiment, suitable disintegrants include, but are not limited to, agar; bentonite; celluloses, such as methylcellulose and carboxymethylcellulose; wood products; natural sponge; cation-exchange resins; alginic acid; gums, such as guar gum and Veegum HV; citrus pulp; cross-linked celluloses, such as croscarmellose; cross-linked polymers, such as crospovidone; cross-linked starches; calcium carbonate; microcrystalline cellulose, such as sodium starch glycolate; polacrilin potassium; starches, such as corn starch, potato starch, tapioca starch, and pre-gelatinized starch; clays; aligns; and mixtures thereof. The amount of a disintegrant in the pharmaceutical compositions provided herein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art. The amount of a disintegrant in the pharmaceutical compositions provided herein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art. The pharmaceutical compositions provided herein may contain from about 0.5 to about 15% or from about 1 to about 5% by weight of a disintegrant.
In one embodiment, suitable lubricants include, but are not limited to, calcium stearate; magnesium stearate; mineral oil; light mineral oil; glycerin; sorbitol; mannitol; glycols, such as glycerol behenate and polyethylene glycol (PEG); stearic acid; sodium lauryl sulfate; talc; hydrogenated vegetable oil, including peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; zinc stearate; ethyl oleate; ethyl laureate; agar; starch; lycopodium; silica or silica gels, such as AEROSIL® 200 (W.R. Grace Co., Baltimore, Md.) and CAB-O-SIL® (Cabot Co. of Boston, Mass.); and mixtures thereof. The pharmaceutical compositions provided herein may contain about 0.1 to about 5% by weight of a lubricant.
In one embodiment, suitable glidants include, but are not limited to, colloidal silicon dioxide, CAB-O-SIL® (Cabot Co. of Boston, Mass.), and asbestos-free talc. Suitable coloring agents include, but are not limited to, any of the approved, certified, water soluble FD&C dyes, and water insoluble FD&C dyes suspended on alumina hydrate, and color lakes and mixtures thereof. A color lake is the combination by adsorption of a water-soluble dye to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye. Suitable flavoring agents include, but are not limited to, natural flavors extracted from plants, such as fruits, and synthetic blends of compounds which produce a pleasant taste sensation, such as peppermint and methyl salicylate. Suitable sweetening agents include, but are not limited to, sucrose, lactose, mannitol, syrups, glycerin, and artificial sweeteners, such as saccharin and aspartame. Suitable emulsifying agents include, but are not limited to, gelatin, acacia, tragacanth, bentonite, and surfactants, such as polyoxyethylene sorbitan monooleate (TWEEN® 20), polyoxyethylene sorbitan monooleate 80 (TWEEN® 80), and triethanolamine oleate. Suitable suspending and dispersing agents include, but are not limited to, sodium carboxymethylcellulose, pectin, tragacanth, Veegum, acacia, sodium carbomethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable preservatives include, but are not limited to, glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol. Suitable wetting agents include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Suitable solvents include, but are not limited to, glycerin, sorbitol, ethyl alcohol, and syrup. Suitable non-aqueous liquids utilized in emulsions include, but are not limited to, mineral oil and cottonseed oil. Suitable organic acids include, but are not limited to, citric and tartaric acid. Suitable sources of carbon dioxide include, but are not limited to, sodium bicarbonate and sodium carbonate.
It should be understood that many carriers and excipients may serve several functions, even within the same formulation.
In one embodiment, the pharmaceutical compositions provided herein for oral administration can be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets. Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach. Enteric-coatings include, but are not limited to, fatty acids, fats, phenyl salicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation. Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Film coating imparts the same general characteristics as sugar coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press-coated or dry-coated tablets.
In one embodiment, the tablet dosage forms can be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled-release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.
In one embodiment, the pharmaceutical compositions provided herein for oral administration can be provided as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, or calcium alginate. The hard gelatin capsule, also known as the dry-filled capsule (DFC), consists of two sections, one slipping over the other, thus completely enclosing the active ingredient. The soft elastic capsule (SEC) is a soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol. The soft gelatin shells may contain a preservative to prevent the growth of microorganisms. Suitable preservatives are those as described herein, including methyl- and propyl-parabens, and sorbic acid. The liquid, semisolid, and solid dosage forms provided herein may be encapsulated in a capsule. Suitable liquid and semisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. The capsules may also be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
In one embodiment, the pharmaceutical compositions provided herein for oral administration can be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in-water or water-in-oil. Emulsions may include a pharmaceutically acceptable non-aqueous liquid or solvent, emulsifying agent, and preservative. Suspensions may include a pharmaceutically acceptable suspending agent and preservative. Aqueous alcoholic solutions may include a pharmaceutically acceptable acetal, such as a di(lower alkyl)acetal of a lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxyl groups, such as propylene glycol and ethanol. Elixirs are clear, sweetened, and hydroalcoholic solutions. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative. For a liquid dosage form, for example, a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.
In one embodiment, other useful liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient(s) provided herein, and a dialkylated mono- or poly-alkylene glycol, including, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol. These formulations can further comprise one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfate, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.
In one embodiment, the pharmaceutical compositions provided herein for oral administration can be also provided in the forms of liposomes, micelles, microspheres, or nanosystems. Micellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.
In one embodiment, the pharmaceutical compositions provided herein for oral administration can be provided as non-effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form. Pharmaceutically acceptable carriers and excipients used in the non-effervescent granules or powders may include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.
In one embodiment, coloring and flavoring agents can be used in all of the above dosage forms.
In one embodiment, the pharmaceutical compositions provided herein for oral administration can be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.
In one embodiment, the pharmaceutical compositions provided herein can be administered parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, intravesical, and subcutaneous administration.
In one embodiment, the pharmaceutical compositions provided herein for parenteral administration can be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, e.g., Remington: The Science and Practice of Pharmacy, supra).
In one embodiment, the pharmaceutical compositions intended for parenteral administration can include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.
In one embodiment, suitable aqueous vehicles include, but are not limited to, water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection. Suitable non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, and palm seed oil. Suitable water-miscible vehicles include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.
In one embodiment, suitable antimicrobial agents or preservatives include, but are not limited to, phenols, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffering agents include, but are not limited to, phosphate and citrate. Suitable antioxidants are those as described herein, including bisulfate and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents are those as described herein, including sodium carboxymethylcelluose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents are those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, including α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, and sulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, Kans.).
In one embodiment, when the pharmaceutical compositions provided herein are formulated for multiple dosage administration, the multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.
In one embodiment, the pharmaceutical compositions for parenteral administration are provided as ready-to-use sterile solutions. In another embodiment, the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle prior to use. In yet another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile suspensions. In yet another embodiment, the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use. In still another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile emulsions.
In one embodiment, the pharmaceutical compositions provided herein for parenteral administration can be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.
In one embodiment, the pharmaceutical compositions provided herein for parenteral administration can be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot. In one embodiment, the pharmaceutical compositions provided herein are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.
In one embodiment, suitable inner matrixes include, but are not limited to, polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.
In one embodiment, suitable outer polymeric membranes include but are not limited to, polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.
In one embodiment, the pharmaceutical compositions provided herein can be administered topically to the skin, orifices, or mucosa. The topical administration, as used herein, includes (intra)dermal, conjunctival, intracorneal, intraocular, ophthalmic, auricular, transdermal, nasal, vaginal, urethral, respiratory, and rectal administration.
In one embodiment, the pharmaceutical compositions provided herein can be formulated in any dosage forms that are suitable for topical administration for local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, irrigations, sprays, suppositories, bandages, and dermal patches. The topical formulation of the pharmaceutical compositions provided herein can also comprise liposomes, micelles, microspheres, nanosystems, and mixtures thereof.
In one embodiment, pharmaceutically acceptable carriers and excipients suitable for use in the topical formulations provided herein include, but are not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.
In one embodiment, the pharmaceutical compositions can also be administered topically by electroporation, iontophoresis, phonophoresis, sonophoresis, or microneedle or needle-free injection, such as POWDERJECT™ (Chiron Corp., Emeryville, Calif.), and BIOJECT™ (Bioject Medical Technologies Inc., Tualatin, Oreg.).
In one embodiment, the pharmaceutical compositions provided herein can be provided in the forms of ointments, creams, and gels. Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including lard, benzoinated lard, olive oil, cottonseed oil, and other oils, white petrolatum; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water-soluble ointment vehicles, including polyethylene glycols of varying molecular weight; emulsion vehicles, either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid (see, e.g., Remington: The Science and Practice of Pharmacy, supra). These vehicles are emollient but generally require addition of antioxidants and preservatives.
In one embodiment, suitable cream base can be oil-in-water or water-in-oil. Suitable cream vehicles may be water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase is also called the “internal” phase, which is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.
In one embodiment, gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the liquid carrier. Suitable gelling agents include, but are not limited to, crosslinked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, and CARBOPOL®; hydrophilic polymers, such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.
In one embodiment, the pharmaceutical compositions provided herein can be administered rectally, urethrally, vaginally, or perivaginally in the forms of suppositories, pessaries, bougies, poultices or cataplasm, pastes, powders, dressings, creams, plasters, contraceptives, ointments, solutions, emulsions, suspensions, tampons, gels, foams, sprays, or enemas. These dosage forms can be manufactured using conventional processes as described in Remington: The Science and Practice of Pharmacy, supra.
In one embodiment, rectal, urethral, and vaginal suppositories are solid bodies for insertion into body orifices, which are solid at ordinary temperatures but melt or soften at body temperature to release the active ingredient(s) inside the orifices. Pharmaceutically acceptable carriers utilized in rectal and vaginal suppositories include bases or vehicles, such as stiffening agents, which produce a melting point in the proximity of body temperature, when formulated with the pharmaceutical compositions provided herein; and antioxidants as described herein, including bisulfate and sodium metabisulfite. Suitable vehicles include, but are not limited to, cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol), spermaceti, paraffin, white and yellow wax, and appropriate mixtures of mono-, di- and triglycerides of fatty acids, and hydrogels, such as polyvinyl alcohol, hydroxyethyl methacrylate, and polyacrylic acid. Combinations of the various vehicles can also be used. Rectal and vaginal suppositories may be prepared by compressing or molding. The typical weight of a rectal and vaginal suppository is about 2 to about 3 g.
In one embodiment, the pharmaceutical compositions provided herein can be administered ophthalmically in the forms of solutions, suspensions, ointments, emulsions, gel-forming solutions, powders for solutions, gels, ocular inserts, and implants.
In one embodiment, the pharmaceutical compositions provided herein can be administered intranasally or by inhalation to the respiratory tract. The pharmaceutical compositions can be provided in the form of an aerosol or solution for delivery using a pressurized container, pump, spray, atomizer, such as an atomizer using electrohydrodynamics to produce a fine mist, or nebulizer, alone or in combination with a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. The pharmaceutical compositions can also be provided as a dry powder for insufflation, alone or in combination with an inert carrier such as lactose or phospholipids; and nasal drops. For intranasal use, the powder can comprise a bioadhesive agent, including chitosan or cyclodextrin.
In one embodiment, solutions or suspensions for use in a pressurized container, pump, spray, atomizer, or nebulizer can be formulated to contain ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active ingredient provided herein; a propellant as solvent; and/or a surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
In one embodiment, the pharmaceutical compositions provided herein can be micronized to a size suitable for delivery by inhalation, such as about 50 micrometers or less, or about 10 micrometers or less. Particles of such sizes can be prepared using a comminuting method known to those skilled in the art, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.
In one embodiment, capsules, blisters, and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mix of the pharmaceutical compositions provided herein; a suitable powder base, such as lactose or starch; and a performance modifier, such as 1-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate. Other suitable excipients or carriers include, but are not limited to, dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. The pharmaceutical compositions provided herein for inhaled/intranasal administration can further comprise a suitable flavor, such as menthol and levomenthol; and/or sweeteners, such as saccharin and saccharin sodium.
The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.
Experimental Procedures
Consensus alignment and model building for TWEAK. The templates for homology modeling were selected from RCSB PDB database (Berman H M, Westbrook J, Feng Z, Gilliland G, Bhat T N, et al. (2000) Nucleic Acids Res 28: 235-242). Consensus alignment based on three-dimensional structure was performed in MOE to obtain a structure-derived sequence alignment (v2010.10, Chemical Computing Group Inc. 2012) ((2010) Molecular Operating Environment (MOE). Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7). All template structures were superimposed in 3D, with an initial main-chain atom root mean square deviation (RMSD) of 2.49 Å. In the corresponding sequence alignment, a consensus set of residues was defined based on two criteria: (1) residue identity, retaining those residues with at least 50% of sequence identity per alignment column; and (2) RMSD of main-chain atoms. The former parameter was kept fixed while the latter parameter was decremented, starting at the initial value of 2.49 Å and decreasing by increments of 0.5 Å until reaching 1 Å. At each step, the structural superimposition of the proteins was refined using only the consensus residues, and the sequence alignment was subsequently refined to reflect the structural alignment changes. The resulting sequence alignment was utilized as a fixed template to align the sequence of TWEAK. Homology modeling was performed using MOE with the options of disabling C-terminal and N-terminal outgap modeling and enabling automatic disulphide bond detection. A maximum of 10 intermediate models were created. Model refinement was performed at a medium setting for both intermediate and final model. AMBER99 forcefield was used for all energy minimizations and GB/VI scoring method was used for model scoring. One final refined model was created per template.
Protein-protein docking simulations. Protein-protein docking simulations were performed using well-vetted methodologies implemented in ICM Pro v 3.7-2b, MolSoft LLC 2012 (Abagyan R, Totrov M, Kuznetsov D (1994) J Comp Chem 15: 488-506). The epitopes were selected on the basis of available biological knowledge of the interacting interfaces from April-TACI (PDB ID: 1XU1) and April-BCMA (PDB ID: 1XU2) complexes, and knowledge from Fn14 residues required for binding, as established by Winkles and coworkers (Brown S A, Hanscom H N, Vu H, Brew S A, Winkles J A (2006) Biochem J 397: 297-304). For the receptor protein, pre-calculated grid maps were generated involving van der Waals, electrostatic and desolvation terms using ECEPP/3 molecular mechanics force field (Nemethy G, Gibson K D, Palmer K A, Yoon C N, Paterlini G, et al. (1992) J Phys Chem 96: 6472-6484). For the ligand protein, several starting conformations were sampled and optimized using a pseudo-Brownian Monte-Carlo based method, followed by local energy minimizations (Abagyan R, Totrov M, Kuznetsov D (1994) J Comp Chem 15: 488-506). All the conformations accumulated were merged into a single conformational set compressed by comparison of the atomic coordinates and removal of geometrically similar conformations. The resulting conformations were further optimized by allowing flexibility of the ligand side-chains. The interaction energy function uses the internal energy of the ligand and intermolecular energy based on the optimized potential maps. The multiple levels of optimization performed in this approach reduce the possibility of being trapped in local minima. A single docking run was performed for each receptor-ligand complex and 30-40 poses were obtained for each individual docking run.
Virtual Library Preparation. The peptidomimetic set of ChemDiv (version of May 2011; 13,137 compounds) was prepared using LigPrep version 2.5 in Schrodinger v2011 (Schrodinger LLC, New York) by adding hydrogen atoms and calculating protonation states corresponding to pH of 7.4. This resulted in generation of 21,682 structures. A conformational search was performed using ConfGen Standard search (default parameters), with ConfGen version 2.3 in Schrodinger (Schrodinger LLC, New York) (Watts K S, Dalal P, Murphy R B, Sherman W, Friesner R A, et al. (2010) J Chem Inf Model 50: 534-546), generating a database of 145,995 conformations.
Pharmacophore-based Virtual Screening. Receptor-based pharmacophore generation was performed using Phase version 3.4 implemented in Maestro 9.3 of Schrodinger suite v2011 (Schrodinger LLC, New York) (Dixon S L, Smondyrev A M, Rao S N (2006) Chem Biol Drug Des 67: 370-372). The ligand was defined as the ensemble of TWEAK residues involved in inter-molecular interactions with Fn14. The pharmacophore features were then identified exhaustively for these contacting residues. The features that were not located at the direct protein-protein interaction interface were manually removed. Excluded volumes were included to capture the Fn14 receptor geometry when preparing the pharmacophore model; these were calculated using a scaling factor of 0.9. The conformational ligand database was interrogated for hits matching the generated pharmacophore hypothesis. Pharmacophoric points involving the TWEAK anchoring residue were required and matching of other pharmacophoric points was set as optional.
Structure-Based Virtual Screening. The protein preparation workflow of Maestro 9.3 (Schrodinger LLC v2011, New York) was employed to prepare the Fn14 receptor by adding missing H atoms and refining the structure using default parameters. A grid-enclosing box was centered at the centroid of the three binding site residues involved in TWEAK binding as indicated by mutation data from Winkles, i.e., Asp45, Lys48, Asp62 (Brown S A, Hanscom H N, Vu H, Brew S A, Winkles J A (2006) Biochem J 397: 297-304). Structure-based virtual screening was performed with the conformational ligand database, and the ligands were kept flexible during the docking stage. A three-step docking process was executed: (1) a first parsing was performed by docking of compounds with the fastest HTVS algorithm of Glide (Schrodinger LLC, New York, v2011) (Friesner R A, Banks J L, Murphy R B, Halgren T A, Klicic J J, et al. (2004) J Med Chem 47: 1739-1749), and scoring of compounds. (2) The top 50% of the virtual hits from step 1 were docked using the standard precision algorithm of Glide and were subsequently scored. (3) Lastly, 10% of the top scoring compounds of step 2 were re-docked using XP algorithm, scored, and considered as hits.
Cell culture. The human astrocytoma cell line T98G and human HEK293 cells (American Type Culture Collection) were maintained in DMEM (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA) at 37° C. with 5% CO2. For assays involving TWEAK treatment, cells were cultured in reduced serum (0.5% FBS) for 16 hr prior to TWEAK stimulation.
Expression constructs. The coding sequence for the soluble form of TWEAK, designated sTWEAK, encoding amino acids K97-H249 was amplified by polymerase chain reaction and ligated in-frame either downstream of a 3× FLAG epitope in p3XFLAG-CMV (Sigma, St. Louis, Mo.) or upstream of a 3×HA epitope in pcDNA3 (Invitrogen, Carlsbad, Calif.). The sTWEAK Y176D, Y176A, Y176F, and W231G variants were generated using the Quickchange II site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). All substitutions were verified by DNA sequence analysis. Expression constructs for in vitro transcription were generated using Gateway Technology (Invitrogen). First, coding sequences for sTWEAK and sTWEAK variants were amplified by polymerase chain reaction with oligonucleotide primers containing the appropriate aatB recombination sequences and the aatB flanked PCR products transferred to the entry vector pDONR 221. Resulting pDONR clones were transferred to the T7 based in vitro transcription expression vector pANT7 (Ramachandran N, Hainsworth E, Bhullar B, Eisenstein S, Rosen B, et al. (2004) Science 305: 86-90). All proteins were expressed as epitope tagged proteins.
Synthesis of sTWEAK and Mutant sTWEAK proteins. sTWEAK and sTWEAK variant proteins were synthesized using a 1-step coupled human in vitro transcription/translation (IVTT) kit (Pierce Biochemicals) or a 1-step coupled rabbit in vitro transcription/translation (IVTT) kit (Promega) according to the manufacturer's instructions.
Double Sandwich ELISA. sTWEAK and sTWEAK variant proteins were synthesized as described above. The expression level of each IVTT synthesized sTWEAK protein was analyzed by western blot analysis and used in approximately equal amounts in the ELISA. To assay the binding of wild type sTWEAK and sTWEAK variants to Fn14, the human Fc fragment-tagged Fn14 extracellular domain (R&D Systems) was captured in an Immuno 96-microwell white plate by adding 100 μL of 0.025 μg/mL Fn14-Fc to wells coated with goat anti-human Fcγ fragment specific monoclonal antibody. After capture, the wells were washed 3× with Dulbecco's Phosphate Buffered Saline (DPBS) containing 0.05% Tween-20. Unbound sites were blocked by addition of 100 μL of blocking solution containing 0.05% Tween-20, 1% BSA, and 3% normal goat serum (NGS) in DPBS for 1 hr at room temperature. 0.01 μL of sTWEAK or sTWEAK variant made with the human IVTT kit or 2 μL of sTWEAK or sTWEAK variant made with the rabbit IVTT kit was diluted in 100 μL of sample diluent (DPBS+1% BSA+0.005% Tween-20) and then added to the wells for 2 hr followed by addition of 100 μL of 50 ng/mL biotinylated TWEAK detection antibody (R&D Systems) in sample diluent. Following incubation for additional 2 hr at room temperature, wells were washed 3× with DPBS containing 0.05% Tween-20 and bound biotinylated TWEAK antibody was detected by incubation with an HRP-conjugated streptavidin. The total luminescent signal was obtained using Femto ELISA kit (Pierce Biochemical) and compared to the standard curve signal obtained from the binding of 0-4000 pg/mL recombinant TWEAK (Peprotech inc) to Fn14-Fc. Using 5-parameter logistic curve fitting for standard curve analysis (Sigmaplot 11.0, SyStat Software Inc.), binding of sTWEAK or TWEAK variant to Fn14-Fc was determined. The data represents that observed for at least four replicate assays.
The small molecule screening was performed using the ELISA assay described above with minor modifications. Briefly, after capturing Fn14-Fc in the microwell plate, 80 n1 of drug solution in sample diluent was added to desired wells and incubated for 2 hrs at room temperature. Subsequently, 20 μL of 2500 pg/mL (5×) TWEAK was added to each well to achieve final TWEAK concentration of 500 pg/mL and incubated for additional 2 hr at room temperature. Bound TWEAK was detected as described in the protocol above. All small molecule inhibitors were screened at 25 nM final concentration (final DMSO concentration of 0.125%) in duplicate. Cyclohexamide, a non-specific small molecule, at 25 nM concentration was used as negative control. The anti-Fn14 antibody ITEM-4, added at 2.5 ng/mL, was used as a positive control for complete blockade of of TWEAK binding. Reduction in TWEAK binding due to compound addition or controls was calculated by using standard curve (Separate standard curve was obtained for every screening plate).
Non-Denaturing/Native Gel Electrophoresis. Native gel electrophoresis kit and reagents were purchased from Invitrogen and electrophoresis and western blotting was performed according to the manufacturer's protocol. Briefly, IVTT protein lysates (1 μL) were mixed with 1 μL of 10% n-dodecyl-β-D-maltoside (DDM), 0.5 n1 of 5% NativePAGE™ G-250 additive, 2.5 μL of 4× NativePAGE™ Sample Buffer (4×), and deionized water to make the total volume to 10 μL. Electrophoresis was performed for 2 hr at 16 mA at room temperature using NativePAGE™ Novex® 4-16% Bis-Tris Gels. Calibration was achieved by separation of NativeMark™ protein standards of known molecular masses. After gel electrophoresis, proteins were transferred to PVDF membrane for immunoblotting with an anti-FLAG antibody (Sigma, St. Louis, Mo.).
NF-κB Luciferase reporter Assay. The capacity of sTWEAK or sTWEAK variants to activate Fn14 signaling was evaluated using engineered reporter cell lines that express luciferase upon NF-κB activation. Two reporter cell lines were utilized for these experiments. HEK293 NF-κB luciferase (courtesy of Dr. Jeff Winkles) were generated by transfecting HEK293 cells with a reporter plasmid containing 5 copies of a consensus NF-κB binding site upstream of a minimal CMV promoter driving expression of firefly luciferase. The second cell line, designated HEK293 NF-κB luciferase Fn14 FL, was generated by stably transfecting the HEK293 NF-κB luciferase cell line with full length Fn14. To assay the binding of sTWEAK or sTWEAK variants to Fn14, reporter cells were seeded in tissue culture treated white 96-well plates at 1×104 cells/well in 80 μL Opti-MEM media (Invitrogen) and incubated for 48 hr at 37° C. After 48 hr incubation, 20 μL of 5× purified recombinant TWEAK (Peprotech Inc.; 150 ng/mL) in 1 mg/mL BSA in PBS was added to each well and incubated for 5 hr at 37° C. as positive control. Similarly, equivalent amounts of sTWEAK or sTWEAK variant prepared via IVTT as determined using ELISA assay described above was added in 20 μl. A IVTT solution lacking a cDNA template in 1 mg/mL BSA in PBS and 1 mg/mL BSA in PBS alone was used as an additional control. At the end of 5 hr incubation, the luminescent signal was determined using Bright-Glo assay kit (Promega, Madison, Wis.) according to the manufacturer's instructions.
Small molecules that demonstrated ≧15% inhibition of TWEAK binding to Fn14 in the ELISA screen were validated using the cell based NF-κB luciferase reporter assay with minor modifications. Briefly, Fn14-NF-κB-Luc reporter cells, (Fn14 overexpressing HEK293 NF-κB luciferase cells), were seeded in tissue culture treated white 96-well plates at 1×104 cells/well in 80 μL Opti-MEM media (Invitrogen) and incubated for 48 hr at 37° C. After 48 hr incubation, 10 μL of the drug solution (200 μM) in DMSO was added to the designated wells at a final concentration of 20 μM. After 1 hr of drug incubation at 37° C., 10 μL of 10× purified recombinant TWEAK (Peprotech Inc.; 300 ng/mL) in 1 mg/mL BSA in PBS was added to each well and incubated for 5 hr at 37° C. DMSO alone was used as a negative control whereas anti-TWEAK antibody suspended in DMSO was used as a positive control for the assay. Luminescent signal was determined using Bright-Glo assay kit (Promega, Madison, Wis.) according to the manufacturer's instructions and normalized to negative control. A counter-screen assay was carried out using TNFα to stimulate NF-κB activity in NF-κB-Luc reporter cells (HEK293 NF-κB luciferase cells). The counter-screen assay was performed similar to the drug screening assay described above, except 10 μL of 10× purified recombinant TNFα (R&D Scientific; 300 ng/mL) in 1 mg/mL BSA in PBS was added to each well instead of TWEAK for NF-κB activation. Small molecules, that showed inhibition of the luciferase signal following TWEAK stimulation but not after TNFα stimulation, were further validated by performing a dose response analysis. The selected small molecule inhibitor was tested at concentrations ranging from 0.75 μM to 250 μM for its ability to suppress TWEAK induced NF-κB activity in Fn14 overexpressing HEK293 NF-κB luciferase cells compared to its ability to suppress TNFα induced NF-κB activity in HEK293 NF-κB luciferase cells. IC50 values for dose response curve were determined using the curve fitting functionality of GraphPad Prism software.
Cell Migration Assay. The effect of pharmacological inhibition of TWEAK/Fn14 signaling on glioma cell migration was analyzed using a modified Boyden chamber (Neuroprobe, Cabin John, Md.) as described previously (Lamszus, K., et al. (1998) Journal international du cancer 75, 19-28; Brockmann, M. A., et al. (2003) Neurosurgery 52, 1391-1399). Each well contains a 8-μm pore size Nucleopore filter that had been coated with 50 μg/mL PureCol® (Bovine Collagen) (Advanced Biomatrix, Poway, Calif.). T98G glioma cells were treated with selected drug compound for 1 hr and then seeded at 4.8×104 cells in 100 μL of DMEM with 0.1% bovine serum assay medium to the top well of the chamber. TWEAK was added to the lower wells of the chamber using DMEM with 0.1% bovine serum albumin as assay medium. After incubation for 5 hours at 37° C., nonmigrated cells were scraped off the upper side of the filter, and filters were stained with 4′,6-diamidino-2-phenylindole (DAPI). Nuclei of migrated cells were counted in 5 high-power fields (HPF) with a 20× objective. Values were assessed in triplicate.
Cytotoxicity Assay. Cytotoxic effects of drugs on glioma cells were analyzed by quantifying the ATP, an indicator of metabolically active cells. Briefly, glioma cells were seeded in tissue culture treated white 96-well plates at 3×103 cells/well in 80 μL Opti-MEM media (Invitrogen) and incubated for 24 hr at 37° C. After 24 hr incubation, 20 μL of 5× drug solution at required concentration in Opti-MEM was added to each well and incubated for 72 hr at 37° C. Opti-MEM/DMSO mixture without drug was used as negative control and 20 μM staurosporine was used as positive control. At the end of 72 hr incubation, the number of viable cells were quantified by using CellTiter-Glo assay kit (Promega, Madison, Wis.) according to the manufacturer's instructions. Luminescence signal measured was normalized to negative control to determine % cell viability.
Surface Plasmon Resonance (SPR) Assay. The binding affinity of at least one of the compounds (e.g., L524-0366) to TWEAK and Fn14-Fx was determined using a BIAcore T100 optical biosensor (GE Healthcare) at 25 degrees Celsius at the Arizona Proteomics Consortium. Recombinant human TWEAK (20 μg/mL in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) or recombinant human Fn14-Fc (20 μg/mL in 10 mM NaOAc pH 4.0) were covalently coupled to separate flow cells on a BIAcore CMS sensor chip using standard amine coupling chemistry as per manufacturer's protocol. Final immobilization levels were 6000 Response Units (RU) for TWEAK and 13,000 RU for FN14-Fc. The first flow path of the chip was treated with the same coupling and blocking reagents without protein and was used as a reference for each binding cycle. Functionality of the TWEAK and the Fn14-Fc sensor surfaces were verified by injecting Fn14-Fc and TWEAK over them respectively. Serial dilutions of L524-0366 or cycloheximide (control) from 0 to 50 μM were made in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 1% DMSO). The compounds were injected over the TWEAK and Fn14-Fc sensor surfaces for 60 seconds at a flow rate of 45 μL/minute. Running buffer was injected for 5 minutes at a flow rate of 45 μL/minute to dissociate bound compound molecules from the sensor surface. The fluidics were washed with 50% DMSO after each sample injection and to minimize sample carryover, a buffer wash step was included after each binding cycle. A DMSO calibration curve was used to correct for any bulk responses due to mismatches between sample and running buffer (Frostell-Karlsson, A. et al. (2000) Journal of Medicinal Chemistry 75, 19-28). Equilibrium dissociation constants for the small molecules were calculated by fitting the double reference substracted data to Req=((CRmax)/(KD+C))+RI, where RI is the bulk refractive index contribution.
Fn14 Receptor Selection
An NMR structure of the Fn14 CRD is available in PDB (PDB ID: 2RPJ) (He F, Dang W, Saito K, Watanabe S, Kobayashi N, et al. (2009) Protein Sci 18: 650-656). All 20 models captured in the structure were visually inspected to assess areas of structural flexibility in the putative receptor binding site. This revealed a highly conserved core region (A34-A69) with very few flexible side-chains. This rigid core includes the residues D45, K48, and D62 previously identified as required for TWEAK binding by Winkles et al. and delineating the protein-protein binding interface (Brown S A, Hanscom H N, Vu H, Brew S A, Winkles J A (2006) Biochem J 397: 297-304). Importantly the side chain of R58, located in close proximity of the putative protein-protein interface, presents a high degree of flexibility. We therefore hypothesized that R58 could potentially act as a switch that opens the binding groove. Models 1 and 17 respectively capture a closed and an open geometry as illustrated in
Homology Modeling of TWEAK
Homology modeling of TWEAK was undertaken in the absence of an experimental crystal structure. The C-terminal extracellular domain of TWEAK was predicted to have a (3-pleated sheet structure based on structures of other members of TNF superfamily (Banner D W, D'Arcy A, Janes W, Gentz R, Schoenfeld H J, et al. (1993) Cell 73: 431-445). Several other members of TNF superfamily are characterized by experimental structures in PDB but the low sequence identity with TWEAK limits their selection as a direct template for homology modeling. We overcame this limitation by using consensus-based structural overlay of the template structures available to derive a quality multiple sequence alignment, and by employing this alignment for building homology models (
TWEAK-Fn14 Binding Mode Prediction via Protein-protein Docking
Prior to docking of the TWEAK protein to the Fn14 CRD, benchmarking was performed to parameterize the algorithms and verify the predictive ability of the ICM-Pro algorithms for TNFR and their ligands. For that purpose, we used the crystal structure of the April-BCMA complex (PDB ID: 1XU2) (Hymowitz S G, Patel D R, Wallweber H J, Runyon S, Yan M, et al. (2005) J Biol Chem 280: 7218-7227) as a model system. In a first step, the ligand (BCMA) was translated away from the complex and rotated, but the conformation was unchanged. The ICM-Pro protein-protein docking algorithm identified multiple poses, with the lowest energy solution corresponding to the geometry of the crystal structure. In a second step, the ligand was moved away from the binding site and the side-chains of the interfacing residues were randomized to evaluate the ability to identify a complex in the ensemble of solutions that approaches the experimental complex. We found that the 7th best solution rank-ordered by interaction energy was consistent with the actual binding mode. In light of these outcomes, we concluded that the ICM-Pro algorithm, as parameterized, was suitable to identify plausible poses of TNF ligands bound to their receptors in an ensemble of low energy solutions.
On that basis, protein-protein docking calculations were performed for two Fn14 structures (vide supra) and six TWEAK homology models in order to predict TWEAK-Fn14 binding modes. A total of twelve protein-protein docking simulations were performed, producing hundreds of poses. Initial binding epitopes required to start the protein-protein docking calculation were defined by similarity to the known interacting interfaces from April-TACI and April-BCMA complexes. Each simulation generated between 30 and 40 ligand-receptor poses. TWEAK-Fn14 candidate solutions were analyzed and prioritized following a data-driven decision-making process as summarized in
Experimental Confirmation of Binding by TWEAK Mutagenesis
Binding mode prediction via protein-protein docking indicated TWEAK Y176 or W231 as plausible anchoring amino acid residues mediating the TWEAK-Fn14 interaction. To experimentally validate models generated from protein-protein docking calculation and to determine which predicted residue is critical for TWEAK binding to the Fn14, we performed a double sandwich ELISA to analyze the binding of sTWEAK and sTWEAK variants to Fn14. A schematic of the ELISA is shown in
Non-Denaturing/Native Gel Electrophoresis
The results of the ELISA indicated that substitution of TWEAK Y176 disrupted TWEAK binding to Fn14. To examine whether substitution of Y176 caused significant changes in the secondary, tertiary, or quaternary structure of TWEAK, we performed non-denaturing gel electrophoresis comparing sTWEAK and sTWEAK Y176 variants. As shown in
Luciferase Induction Assay
The ELISA indicated that substitution of Y176 significantly reduced sTWEAK binding to Fn14. Furthermore, native gel electrophoresis further indicated that conservative substitutions of Y176 did not cause obvious alterations in sTWEAK structure and surface charge. However, neither of these assays can accurately predict the effect of the sTWEAK variants on Fn14 cellular signaling. TWEAK binding to the Fn14 receptor results in NF-κB phosphorylation (Tran N L, McDonough W S, Savitch B A, Sawyer T F, Winkles J A, et al. (2005) J Biol Chem 280: 3483-3492). Therefore, we examined the ability of sTWEAK and the sTWEAK Y176 variants to initiate Fn14 signaling using cells expressing a NF-κB luciferase reporter. Stimulation of HEK293 cells expressing the NF-κB luciferase reporter with sTWEAK Y176A or sTWEAK Y176F resulted in luciferase expression that was 87% and 100% less respectively than cells stimulated with sTWEAK (
In Silico Identification of Small Molecule Inhibitors of TWEAK-Fn14 Interaction
The work presented above predicted plausible binding modes of the TWEAK-Fn14 association involving TWEAK residue Y176, and was validated experimentally. These data provided a structural basis to enable further examination of the chemical tractability of the system with the goal to determine whether the characterized TWEAK-Fn14 interaction would be of utility as a therapeutic target for small molecule discovery. To pursue that goal, the structural TWEAK-Fn14 complexes predicted by protein-protein docking involving the Y176 anchoring residue of TWEAK to Fn14 were used as starting point for virtual screening of a library of commercially available small molecules. The peptidomimetic set of ChemDiv (13,137 compounds, v05.2011) was selected for this study and pre-processed as described in methods. A two-pronged virtual screening workflow intersecting the results of a ligand-biased pharmacophore-based and an unbiased structure-based approach was followed, as illustrated in
ELISA Assay and Expansion on Actives
The 60 compounds selected by virtual screen were supplied from the internal compound collection and assayed in the ELISA screen as described in methods. These compounds demonstrated variable inhibitory activity of the Fn14-TWEAK interaction, with individual data points ranging from 0 to 26%. The data set capturing the reduction in Fn14-TWEAK binding demonstrated by each compound was rank-ordered by decreasing average inhibition, and summarized in Table 3. As shown in
Cell Based Luciferase Induction Assay and Validation of Actives
Compounds that demonstrated ≧15% inhibition of TWEAK binding to Fn14 in the ELISA assay were selected for further validation using a cell based NF-κB luciferase induction assay. A total of 19 compounds were selected and screened for their capacity to inhibit cellular NF-κB stimulated luciferase activity in NF-κB-Luc and Fn14-NF-κB-Luc cells stimulated with either TWEAK or TNFα. One compound, L524-0366, demonstrated significant inhibition of TWEAK-induced NF-κB driven luciferase activity in Fn14-NF-κB-Luc cells but only minor inhibition of TNFα-induced NF-κB driven luciferase activity in NF-κB-Luc cells (
Validation of TWEAK-Fn14 Interaction Inhibitor in Phenotypic Assay
The capacity of L524-0366 to inhibit the binding of TWEAK to Fn14 and inhibit TWEAK-Fn14 signaling suggests that it could functionally inhibit endogenous Fn14. Therefore, we examined the capacity of L524-0366 to inhibit TWEAK induced glioma cell migration. While TWEAK significantly stimulated T98G glioma cell migration, L524-0366 (10 μM) completely suppressed TWEAK-induced T98G cell migration (
SPR Assay Validates the Direct Interaction of L524-0366 with Fn14
To define the molecular basis of how L524-0366 inhibits the TWEAK-Fn14 signaling cascade, the interaction of L524-0366 with TWEAK and FN14 was analyzed using an SPR assay. The binding of cycloheximide to Fn14 or TWEAK was used as a control. While cycloheximide did not show significant binding to Fn14 or TWEAK (
The current work elucidated key structural elements of the TWEAK-Fn14 binding interaction using in silico protein modeling and protein-protein docking, followed by experimental validation in vitro. The results identified TWEAK Y176 as an anchor residue in the interaction of TWEAK with the Fn14 CRD. Leveraging this functionally validated information, we demonstrated that the predicted TWEAK-Fn14 interaction interface structural models could guide the virtual selection of small molecule inhibitors that disrupt the TWEAK-Fn14 interaction. By doing so, 60 compounds were identified, of which 4 were confirmed to inhibit TWEAK binding to Fn14 by ≧15% relative to control. These inhibitory activities were confirmed and increased in a second iteration of screening of 69 compounds selected by expanding the chemical spaces of active scaffolds identified in the initial screening iteration. A higher rate of actives was observed in this second screening as well as an overall increase in the average inhibitory activities, with a particular enrichment of actives around structures with similarity to one of the initial hits. Compounds that demonstrated ≧15% inhibition in TWEAK binding to Fn14 were further validated using a cell based functional screen that evaluates the ability of compounds to inhibit TWEAK-Fn14 signaling. One compound (L524-0366) was confirmed to be a specific dose-dependent inhibitor of TWEAK-Fn14 interaction and found to confer its activity by binding specifically to Fn14. Finally, L524-0366 demonstrated functional activity and completely suppressed TWEAK induced glioma cell migration without any potential cytotoxic effects. These results represent a significant step towards proving that the TWEAK-Fn14 interaction is chemically tractable and can serve as a foundation for further exploration utilizing chemical biology approaches focusing on functional validation of this interaction as a therapeutic target of interest in invasive cancers.
Generation of Fn14-NF-κB-luciferase Cell Line and Assay Optimization
Human embryonic kidney cells (HEK293) express low levels of the Fn14 receptor. We developed a cell-based assay for the Fn14-TWEAK pathway signaling events using HEK293 cells that stably co-expresses high levels of full length Fn14 receptor and a firefly luciferase reporter driven by NF-KB-response elements (Fn14-NF-KB-Luc). A schematic model of the cell-based TWEAK-Fn14-NF-KB luciferase reporter is shown in
We optimized HEK293 Fn14-NF-KB Luc cell line for HTS by testing TWEAK-stimulated Luc-reporting as a function of growth medium, TWEAK concentration, induction time, assay duration, cell density, and DMSO concentration. For example, we seeded HEK293 Fn14-NF-KB-Luc and control cells (HEK293 NFKB-Luc) into a 96-well plate at a density of 10,000 cells/well, in DMEM or Opti-MEM® (Reduced serum media, protein concentration 15 μg/mL) and incubated at 37 C for 24 or 48 h. We then induced them with various concentrations of TWEAK in buffer or buffer alone (PBS+1 mg/mL BSA) for 5 hr and then measured firefly luciferase levels by chemiluminescence (Bright-Glo™ Promega). Maximum signal and fold-induction of firefly luciferase was observed with Opti-MEM® at 48 h of cell incubation (
We achieved the same conclusion from both 96-well and 384-well format that signal-to-background and fold-inductions are increased with increasing TWEAK concentration, peaking between 40-80 ng/mL and decreased at 100 ng/mL (
In the majority of seeding density conditions and two different TWEAK concentrations, the assay performance improved with increasing TWEAK induction time. At 8 hours of TWEAK stimulation, the fold induction (S/B) and Z′ factor was approximately 8-fold and 0.8, respectively, and the average % CV was 6.8%. Among all of the induction times and cell seeding densities, TWEAK stimulation at 40 ng/mL consistently yielded better fold induction, Z′ factors, and % CVs than TWEAK stimulation at 30 ng/mL. Additionally, with all cell seeding densities tested, 1000-1500 cells/well appeared to yield the highest acceptable performance, considering the minimum amount of cells, time, and TWEAK needed to support a full screen of >365,000 compounds. Indeed, two additional experiments refined these conditions and set the optimal conditions to be 1000 cells/well with 8 hours induction of 40 ng/mL TWEAK.
Moreover, we explored pre-incubation of compounds or DMSO control at 1 or 2 hours prior to TWEAK induction, and showed that there was no significant difference of the assay performance between pre-incubation times (
Furthermore, determination of the DMSO tolerance (
Potency Estimations (IC50) from Drug Dose-response Curves
We had three control molecules to test if the TWEAK induced Fn14-NF-KB-Luc assay was suitable to obtain inhibitor potency data to support structure activity relationship (SAR) studies: (1) LY294002, a PI3K inhibitor which should inhibit the pathway downstream of TWEAK-Fn14 binding; (2) BAY11-7082, an irreversible inhibitor of TNFα-stimulated IKBα phosphorylation and subsequent inhibition of NF-KB activity; and (3) a “decoy” receptor comprising the extracellular Fn14 domain lacking the cell-anchoring transmembrane domain, which ligates TWEAK thereby aborting Fn14 signaling. Additionally, dexamethasone is used as a negative signaling control. The structures of these compounds are show in
Pilot HTS on the Prestwick Collection and Counterscreens to Confirm TWEAK Dependence
Using the optimized conditions, we performed replicate screens of the 1,120 member Prestwick Collection of FDA-approved drugs comprising 4×384-well plates at final concentrations of 10 and 20 μM [final 0.1% and 0.2% DMSO (v/v)] for a total of four runs. The hit statistics for these four runs are summarized in Table 6.
In general, the compounds scoring as hits at 10 μM were more inhibitory at 20 μM and thus reported as hits. We noted that the concordance between runs 1 and 2 were good for each condition. Our data showed that 35 compounds were prioritized as hits in all 4 runs (both concentrations at both runs), thus representing a hit rate of 3.1%, which is somewhat high; however, cheminformatic filtering and counterscreens rapidly reduced this number.
TNFα induction of the HEK293 NF-κB cell line was used as a counterscreen. In
Furthermore, we tested the two control compounds, LY294002 and BAY11-7082, in a drug dose-response format. There was no significant difference in drug inhibition of TNFα activation in the Fn14 and control cell lines (
In order to eliminate the false positive hits from the pilot screen, we further performed the counterscreen by testing 35 “positives” from the Prestwick collection against HEK293 NF-KB-Luc control cell lines induced by TNFα. From this experiment we eliminated 28 compounds that inhibited luciferase expression after TNFα induction of HEK293 NF-KB Luc cells. Thus, after counterscreen only 7 compounds appeared to be specific inhibitors to the TWEAK-Fn14 signaling pathway. One “hit” example was Indoprofen, which demonstrated drug sensitivity in a dose-dependent manner in Fn14 NF-KB-Luc cell line induced with TWEAK, whereas no significant effect was observed in NF-KB Luc cells induced with TNFα (
Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.F
This application is the U.S. National Stage of International Application No. PCT/US2014/046047, filed on Jul. 9, 2014, which is a continuation of U.S. patent application Ser. No. 14/327,448 (published as US 2015/0017263), filed Jul. 9, 2014, and claims the benefit of U.S. Provisional Application Ser. No. 61/844,336, filed on Jul. 9, 2013, and U.S. Provisional Application Ser. No. 61/880,544, filed on Sep. 20, 2013, the contents of each of which are hereby incorporated by reference in their entirety.
This invention was made with governmental support under grant number CA130940 awarded by the National Institutes of Health and grant number MH090878 from the National Institute of Mental Health. The United States government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/046047 | 7/9/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/006508 | 1/15/2015 | WO | A |
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| Number | Date | Country | |
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| 20160146784 A1 | May 2016 | US |
| Number | Date | Country | |
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| 61844336 | Jul 2013 | US | |
| 61880544 | Sep 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14327448 | Jul 2014 | US |
| Child | 14904363 | US |
