Patent Application
20210205260
Various compositions for the rescue of a mp53, various pharmaceutical composition for a p53 disorder, such as cancer, and various methods for treating the p53 disorder, are disclosed herein.
This application claims priority to International Application No. PCT/CN2018070051 filed on Jan. 2, 2018, entitled “PANDA AS A NOVEL THERAPEUTIC” and International Application No. PCT/CN/2018/085190 filed on Apr. 28, 2018, entitled “PANDA AS A NOVEL THERAPEUTIC,” the content of each application is incorporated herein by reference in their entirety.
Various compounds for rescuing mp53 and treating a p53 disorder, including cancer, and various methods of treating a p53 disorder have been proposed. Because these compounds, treatments and methods of treatments are not optimal, there is a need in the field for improved mp53 rescue compounds, treatments for a p53 disorder, and methods of treating a p53 disorder.
We have described herein compounds that have one or more useful characteristic(s) and can form one or more tight association(s) with a PANDA Pocket (each compound a “PANDA Agent”). In certain embodiments, the PANDA Agent can regulate the level of one or more p53 target gene. Exemplary target genes include Apaf1, Bax, Fas, Dr5, mir-34, Noxa, TP53AIP1, Perp, Pidd, Pig3, Puma, Siva, YWHAZ, Btg2, Cdkn1a, Mdm2, Tp53i3, Gadd45a, mir-34a, mir-34b/34c, Prt3, Ptprv, Reprimo, Pai1, Pml, Ddb2, Ercc5, Fancc, Gadd45a, Ku86, Mgmt, Mih1, Msh2, P53r2, Polk, Xpc, Adora2b, Aldh4, Gamt Gs2, Gpx1, Lpin1, Parkin, Prkab1, Prkab2, Pten, Sco1, Sesn1, Sesn2, Tigar, Tp53inp1, Tsc2, Atg10, Atg2b, Atg4a, Atg4c, Atg7, Ctsd, Ddit4, Dram1, Foxo3, Laptm4a, Lkb1, Pik3r3, Prkag2, Puma, Tpp1, Tsc2, Ulk1, Ulk2, Uvrag, Vamp4, Vmp1, Bai1, Cx3cl1, Icam1, lrf5, lrf9, Isg15, Maspin, Mcp1, Ncf2, Pai1, Tlr1-Tlr10, Tsp1, Ulbp1, Ulbp2, mir-34a, mir-200c, mir-145, mir-34a, mir-34b/34c, Notch1, combinations thereof and the like. In certain embodiments, the tight association formed by PANDA Agent and PANDA Pocket substantially stabilizes p53. Preferably, the tight association increases the Tm of p53 at least by about 0.5° C., more preferably at least by about 1° C., further preferably at least by about 2° C., further preferably at least by about 5° C., further preferably at least to about 8° C. In certain embodiments, the tight association formed by PANDA Agent and PANDA Pocket increases the population of properly folded p53 at least to about 1.5 times, preferably at least to about 3 times, more preferably at least to about 5 times, more preferably at least to about 10 times, and further preferably to about 100 times. In preferred embodiments, the increase is measured to a PAb1620 immunoprecipitation assay.
In certain embodiments, the PANDA Agent includes one or more PANDA Pocket-binding groups capable of binding one or more amino acids on PANDA Pocket, preferably one or more cysteines, more preferably two or more cysteines, further preferably more than three cysteines, further preferably from about three cysteines to about 6 cysteines. The PANDA Pocket binding group is preferred to include metallic group(s), metalloid group(s), and other group(s) capable of binding to PANDA Pocket such as Michael acceptor(s) and thiol group(s). The PANDA Pocket-binding groups is further preferred to include one or more arsenic, antimony, and bismuth, including any analogue(s) thereof, and any combinations thereof. Exemplary PANDA Pocket-binding groups include compounds containing a 3-valence and/or 5-valence arsenic atom, a 3-valence and/or 5-valence antimony atom, a 3-valence and/or 5-valence bismuth atom, and/or a combination thereof.
Exemplary embodiments of a PANDA Agent can include any one of the following Formulas I-XV.
wherein:
wherein:
wherein:
In the preferred embodiment, the non-Carbon atom is selected from the group consisting of O, S, N, X, F, C, Br, I, and H.
The following Equation (1) is an reaction for PANDA Agent. A compound containing M group with a Z1 (a first group with the capacity to bind a first cysteine) and/or a Z2 (a second group with the capacity to bind a second cysteine) and/or a Z3 (a third group with the capacity to bind a third cysteine), Examples of Z1, Z2, and Z3 includes O, S, N, X, F, Cl, Br, I, OH, and H. Z1, Z2, and/or Z3 can bind to each other. M group includes for example a metal, such as an bismuth, a metalloid, such as an arsenic and an antimony, a group such as a Michael acceptor and/or a thiol, and/or any analogue with cysteine-binding ability. The PANDA Agent can undergo a hydrolysis before reacting and binding to p53 forming PANDA. In some cases, when a group cannot undergo hydrolysis, and accordingly cannot bind to a cysteine. In such cases, the remaining group(s) with cysteine binding potential binds to p53. X1 and X2 represent any groups bound to M. X1 and/or X2 can also be empty. X1 and/or X2 can also be able to bind cysteine.
The following Equations (2) and (3) is an exemplary reaction for a PANDA Agent with tri-cysteine binding potential. 3-valence ATO or KAsO2 undergoes hydrolysis, covalently binds to three PANDA Cysteines on p53.
The following equation (4) is an exemplary reaction for a PANDA Agent with tri-cysteine binding potential. 5-valence As compound undergoes hydrolysis, covalently binds to three PANDA Cysteines on p53.
The following equation (5) is an exemplary reaction for a PANDA Agent with bi-cysteine binding potential. The PANDA Agent can bind to PANDA Cysteines, or to PANDA Cysteines (Cys124, Cys135, or Cys141), or Cys275 and Cys277 or C238 and C242.
The following equation (6) is an exemplary reaction for a PANDA Agent with mono-cysteine binding potential. The PANDA Agent can bind to PANDA Cysteines, (i.e. Cys124, Cys135, or Cys141) or the other 3 cysteines on PANDA Pocket (Cys238, Cys275, or Cys277).
Exemplary PANDA Agent includes one or more of the compounds listed in Table 1-Table 6, which we predict to efficiently bind to PANDA Cysteines and efficiently rescue p53 in vitro, in vivo and/or in situ. In certain embodiments, the PANDA Agent is one or more of As2O3 (an FDA approved drug arsenic trioxide (“ATO”) for acute promyelocytic leukemia (“APL”)), As2O5, KAsO2, NaAsO2, HAsNa2O4, HAsK2O4, AsF3, AsCl3, AsBr3, AsI3, AsAc3, As(OC2H5)3, As(OCH3)3, As2(SO4)3, (CH3CO2)3As, C8H4K2O12As2.xH2O, HOC6H4COOAsO, [O2CCH2C(OH)(CO2)CH2CO2]As, Sb2O3, Sb2O5, KSbO2, NaSbO2, HSbNa2O4, HSbK2O4, SbF3, SbCl3, SbBr3, Sb3, SbAc3, Sb(OC2H5)3, Sb(OCH3)3, Sb2(SO4)3, (CH3CO2)3Sb, C8H4K2O12Sb2.xH2O, HOC6H4COOSbO, [O2CCH2C(OH)(CO2)CH2CO2]Sb, Bi2O3, Bi2O5, KBiO2, NaBiO2, HBiNa2O4, HBiK2O4, BiF3, BiCl3, BiBr3, BiI3, BiAc3, Bi(OC2H5)3, Bi(OCH3)3, Bi2(SO4)3, (CH3CO2)3Bi, C8H4K2O12Bi2.xH2O, HOC6H4COOBiO, C16H18As2N4O2 (NSC92909), C13H14As2O6 (NSC48300), C10H13NO8Sb (NSC31660), C6H12NaO8Sb+ (NSC15609), C13H21NaO9Sb+ (NSC15623), and/or combinations thereof. Further exemplar embodiments of PANDA Agent include those in Table 7, compounds that have strong p53 structural rescue capacity and p53 transcriptional activity (i.e. functional) rescue capacity, as confirmed by our experiments.
In certain embodiments, the PANDA Agent is not CP-31398; PRIMA-1; PRIMA-1-MET; SCH529074; Zinc; stictic acid, p53R3; methylene quinuclidinone; STIMA-1; 3-methylene-2-norbomanone; MIRA-1; MIRA-2; MIRA-3; NSC319725; NSC319726; SCH529074; PARP-PI3K; 5,50-(2,5-furandiyl)bis-2-thiophenemethanol; MPK-09; Zn-curc or curcumin-based Zn(II)-complex; P53R3; a (2-benzofuranyl)-quinazoline derivative; a nucleolipid derivative of 5-fluorouridine; a derivative of 2-aminoacetophenone hydrochloride; PK083; PK5174; PK7088; and other mp53 rescue compound previously identified by other groups.
A preferred mp53 has at least one mutation on p53, including any single amino acid mutation. Preferably, the mutation alters and/or partially alters the structure and/or function of p53, and more preferably the mutation is a rescuable mutation. Exemplary rescuable p53 mutations are listed in Table 8.
In certain preferred embodiments, as compared to when the PANDA Agent is not bound, the formed PANDA complex has gained one or more wtp53 structure, preferably a DNA binding structure; has gained one or more wtp53 function, preferably a transcription function; and/or has lost and/or diminishes one or more mp53 function, preferably an oncogenic function. The wildtype function can be gained in vitro and/or in vivo. Exemplary wildtype function gained can be at the molecule-level, such as association to nucleic acids, transcriptional activation or repression of target genes, association to wtp53 or mp53 partners, dissociation to wtp53 or mp53 partners, and reception to post-translational modification; at the cellular-level, such as, responsiveness to stresses such as nutrient deprivation, hypoxia, oxidative stress, hyperproliferative signals, oncogenic stress, DNA damage, ribonucleotide depletion, replicative stress, and telomere attrition, promotion of cell cycle arrest, promotion of DNA-repair, promotion of apoptosis, promotion of genomic stability, promotion of senescence, and promotion of autophagy, regulation of cell metabolic reprogramming, regulation of tumor microenvironment signaling, inhibition of cell stemness, survival, invasion and metastasis; and at the organism-level, such as delay or prevention of cancer relapse, increase of cancer treatment efficacy, increase of response ratio to cancer treatment, regulation of development, senescence, longevity, immunological processes, aging, combinations thereof, and the like. The mp53 functions can be lost, impaired and/or abrogated in vitro and/or in vivo. Exemplary mp53 function lost can include any functions, such as oncogenic functions, that promote cancer cell metastasis, genomic instability, invasion, migration, scattering, angiogenesis, stem cell expansion, survival, proliferation, tissue remodelling, resistance to therapy, mitogenic defects, combinations thereof and the like.
In certain preferred embodiments, the PANDA Agent can cause the mp53 to gain and/or lose the ability to upregulate or downregulate one or more p53 downstream targets, at an RNA level and/or protein level, in a biological system. The preferred functional change for a PANDA or a mp53 is at least to about 1.5 times, preferably to at least about 3 times, more preferably to at least about 5 times, more preferably to at least about 10 times, and further preferably to about 100 times.
In certain preferred embodiments, the PANDA Agent can be used to treat a p53 disorders in a subject with mp53 and/or without functional p53, preferably the mp53 is a rescuable mp53.
In certain preferred embodiments, PANDA Agent can suppress tumors, preferably least to a level that is statistically significant; more preferably having the ability to strongly suppress tumors at a level that is statistically significant. In certain preferred embodiments, the formed PANDA has the ability to regulate cell growth or tumor growth preferably to at least about 10% of the wtp53 level, further preferably at least about 100% of the wtp53 level, further preferably exceeding about 100% of the wtp53 level.
In certain preferred embodiments, the PANDA Agent can rescue one or more wtp53 structure, preferably a DNA binding structure; rescue one or more wtp53 function, preferably a transcription function; and eliminate and/or diminish one or more mp53 function, preferably an oncogenic function. In certain preferred embodiments, this is achieved by combining PANDA Agent with a p53 to form PANDA, preferably a mp53 with at least one mutation on p53, including a single amino acid mutation. Preferably, the mutation alters and/or partially alters the structure and/or function of p53. More preferably, the mutation is a rescuable p53 mutation. Exemplary rescuable p53 mutations are listed in Table 8.
In certain preferred embodiments, one or more wtp53 structure, preferably a DNA binding structure can be rescued by adding a PANDA and/or a PANDA Agent to a cell, preferably a human cell, and/or a subject, preferably a mammal, more preferably, further preferably a human.
In certain preferred embodiments, one or more wtp53 function, preferably a transcription function can be rescued by adding a PANDA and/or a PANDA Agent to a cell, preferably a human cell, and/or a subject, preferably a human subject. In certain preferred embodiments, one or more mp53 function, preferably an oncogenic function, can be eliminated and/or diminished by adding a PANDA and/or a PANDA Agent to a cell, preferably a human cell, and/or a subject, preferably a mammal, further preferably a human subject.
We disclose herein a method of using the PANDA or PANDA Agent in vitro and/or in vivo to rescue one or more wtp53 structure, preferably a DNA binding structure; rescue one or more wtp53 function, preferably a transcription function; eliminate and/or diminish one or more mp53 function, preferably an oncogenic function, the method comprising the step of adding an effective amount of PANDA or PANDA Agent to a cell, preferably a human cell, and/or subject, preferably a human subject.
The described PANDA Agent can be used to treat a p53 disorder in a subject with mp53, the disorder is preferably cancer and/or tumor.
In certain embodiments, the PANDA Agent can be formulated in a pharmaceutical composition suitable for treating a subject with a p53 disorder. A pharmaceutical composition will typically contain a pharmaceutically acceptable carrier. Although oral administration of a compound is the preferred route of administration, other means of administration such as nasal, topical or rectal administration, or by injection or inhalation, are also contemplated. Depending on the intended mode of administration, the pharmaceutical compositions can be in the form of solid, semi-solid, or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, ointments, or lotions, preferably in unit dosage form suitable for single administration of a precise dosage. One skilled in this art may further formulate the compound in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.
In certain embodiments, the PANDA Agent can be formulated in a pharmaceutically acceptable salt or solvate. The pharmaceutically acceptable salt can be an ionizable drug that has been combined with a counter-ion to form a neutral complex. Converting a drug into a salt through this process can increase its chemical stability, render the complex easier to administer, and allow manipulation of the agent's pharmacokinetic profile (Patel, et al., 2009).
In certain embodiments, the PANDA Agent and PANDA have the following features:
Also described herein, are improved methods of diagnosing, prognosing, and treating a p53 disorder, such as cancer and methods of using the PANDA Agent, including in the diagnosis, prognosis, and treatment of a p53 disorder such as cancer are also described. The method comprises the step of administering to a subject an effective amount of a therapeutic, wherein the therapeutic comprises one or more PANDA Agent. In a preferred embodiment, the therapeutic is administered in combination with one or more additional therapeutics, preferably any known therapeutic effective at treating cancer and/or DNA damaging agent.
We further disclose a highly-efficient personalized method of treatment for a p53 disorder in a subject in need thereof. The method comprises the steps of:
We further disclose a method of identifying PANDA. The method comprising the step of: using an antibody specific for properly folded PANDA, such as PAb1620, PAb246, and/or PAb240, to perform immunoprecipitation, wherein the immunoprecipitation is performed at a temperature of greater than 4° C.; measuring increase of molecular weight by mass spectroscopy; measuring whether transcriptional activity is rescued in a luciferase assay; measuring the mRNA and protein levels of p53 targets; measuring the p53-specific DNA binding ability; co-crystalizing to construct 3-D structure; and/or measuring increase of Tm.
We disclose herein a collection of PANDA Agents having the ability to regulate the levels of p53 targets in a biological system expressing a mp53 or lacking any functional p53. We further disclose a method of controlling one or more proteins and/or RNA regulated by p53 and/or PANDA, the method comprising the step of administering a regulator to a biological system, wherein the regulator is selected from the group consisting of:
We disclose herein a collection of PANDA Agents having the ability to suppress tumors in a biological system, preferably a system that expresses a mp53. We further disclose a method of suppressing tumors, the method comprising the step(s) of administering to a subject in need thereof an effective amount of a therapeutic, where the therapeutic comprises a tumor suppressor selected from the group consisting of:
In a preferred embodiment, the suppressor is administered in combination with one or more additional suppressors, preferably any known suppressor effective at suppressing tumor growth and/or DNA damaging agent.
We disclose herein a collection of PANDA Agents having the ability to regulate cell growth or tumor growth in a biological system, preferably a system that expresses a mp53. We further disclose a method of regulating cell growth or tumor growth, the method comprising the step of administering to a subject in need thereof an effective amount of a regulator, wherein the regulator is selected from the group consisting of:
We disclose herein a method of diagnosing a p53 disorder, such as cancer, tumor, aging, developmental diseases, accelerated aging, immunological diseases, combinations thereof and the like, in a subject in need thereof. The diagnosis method comprising the steps of administering to the subject an effective amount of a therapeutic, and detecting whether PANDA is formed wherein the therapeutic is selected from the group consisting of:
In a preferred embodiment, the diagnosing method includes a treatment step wherein the therapeutic is administered in combination with one or more additional therapeutics, such as one or more additional PANDA Agent(s) and/or any other known therapeutic effective at treating cancer and/or DNA damaging agent, to effectively treat the p53 disorder in the subject.
In certain embodiments, the PANDA Agent has the potential to bind multiple cysteines and can selectively inhibit Structural mp53 expressing cells via promoting mp53 folding.
In certain embodiments, formed PANDA complex can be purified and isolated using any conventional methods, including any methods disclosed in this Application, such as by immunoprecipitation using PAb1620.
Unless otherwise indicated, this description employs conventional chemical, biochemical, molecular biology, genetics and pharmacology methods and terms that have their ordinary meaning to persons of skill in this field. All publications, references, patents and patent applications cited herein are hereby incorporated herein by reference in their entireties.
As used herein, the biological sample corresponds to any sample taken from a subject, and can include tissue samples and fluid samples such as blood, lymph or interstitial fluid and combinations thereof and the like.
As used in this specification and the appended claims, the following general rules apply. Singular forms “a,” “an” and “the” include plural references unless the content clearly indicates otherwise. General nomenclature rules for genes and proteins also apply. That is, genes are italicized or underlined (e.g.: TP53 or TP53), but gene products, such as proteins and peptides, are in standard font, not italicized or underlined (e.g.: p53). General rules for nomenclature of amino acid location also applies; that is, the amino acid abbreviation followed by number (e.g.: R175, R 175, R-175), where the amino acid name is represented by the abbreviation (e.g.: arginine by “R,” “arg,” “Arg” any other abbreviations familiar to those skilled in the art) and the location of the amino acid on the protein or peptide is represented by the number (e.g.: 175 for position 175). General rules for nomenclature of mutations also apply; for example, R175H, means arginine at location 175 is substituted by histidine. As another example mutation on p53 at location 175 from R to H can be represented by for example “p53-R175H” or “mp53-R175H.” Unless specified otherwise, any amino acid position corresponds to the amino acid location on a wildtype p53, preferably the human wtp53 isoform “a” listed in Table 14. General nomenclature rules for organism classification also apply. That is order, family, genus and species names are italicized.
As used herein, the following terms shall have the specified meaning. The term “about” takes on its plain and ordinary meaning of “approximately” as a person of skill in the art would understand, and generally plus or minus 20%, unless specified otherwise. The term “comprise,” “comprising,” “contain,” “containing,” “include,” “including,” “include but not limited to,” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements.
As used herein, the following terms shall have the specified meaning:
“expression” or “level of expression” means the level of mRNAs or proteins encoded by the referenced gene.
“PANDA” is abbreviated for 253 AND Agent complex, means a complex comprised of one or more p53s and one or more PANDA Agents.
“PANDA Agent” means a composition of matter capable of forming at least one tight association with the PANDA Pocket and has one or more useful characteristic(s). Exemplary PANDA Agent is listed in Table 1-Table 7.
“PANDA Pocket” means a region consisting essentially of an area of about 7 Å from a properly folded PANDA Triad, including, all amino acids adjacent to one or more properly folded PANDA Triad, all amino acids that contact with one or more properly folded PANDA Triad, and all PANDA Triad. It is a pocket on p53 that interacts with one or more atoms of the PANDA Agent to form PANDA. Exemplary 3D structures of a PANDA Pockets can be found
“PANDA Core” means the tertiary structure formed on the PANDA Pocket of a p53 when at least one tight association is formed between the PANDA Pocket and one or more atoms of the PANDA Agent.
“tight association” means a bond, covalent bond, a non-covalent bond (such as a hydrogen bond), and combinations thereof formed between PANDA Pocket and PANDA Agent. The tight association is preferably formed between a PANDA Agent and one or more PANDA Cysteines, preferably two or more PANDA Cysteines, and more preferably all three PANDA Cysteines.
“PANDA Cysteine” means a cysteine corresponding to one of the wtp53 positions at cysteine 124 (“C124” or “cys124”), cysteine 135 (“C135” or “cys135”), and cysteine 141 (“C141” or “cys141”) (together the “PANDA Triad”).
“p53” means any wildtype p53 (“wtp53”), including all natural and artificial p53; any mutated p53 (“mp53”), including all natural and artificial p53, combinations thereof, and the like.
“wtp53” means all wildtype p53 that is commonly considered as wildtype, or has a wildtype sequence, and includes any commonly acceptable variations, such as variations caused by single nucleotide polymorphism (“SNP”). Exemplary wtp53 includes p53α, p53β, p53γ, Δ40p53α, Δ40p53β, Δ40p53γ, and any acceptable variants, such as those with one or more single nucleotide polymorphisms (“SNP”). Exemplary wtp53 are listed in can be found in Table 14.
“SNP” means single-nucleotide polymorphism, which is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is presented to some appreciable degree within a population. An exemplary list of known SNP on p53 is Table 13.
“mp53” means mutated p53, which includes all p53 and p53 like macromolecules that is not a wtp53. mp53 includes, artificial mp53, such as recombinant p53, chimeric p53, p53 derivative, fusion p53, p53 fragment, and p53 peptide. Exemplary mp53 is a rescuable mp53.
“rescuable mp53” means a p53 with a rescuable mutation that can be rescued by a PANDA Agent (such as ATO), such that one or more of the mp53's wildtype function and/or structure can be rescued. A rescuable mp53 includes a structurally rescuable mp53 and a functionally rescuable mp53. Exemplary rescuable mp53s are provided in Table 8.
“structurally rescuable mp53” means a mp53 where one or more of the wild type structure can be rescued by a PANDA Agent (such as ATO).
“functionally rescuable mp53” means a mp53 where one or more of the wild type transcriptional function can be rescued by a PANDA Agent (such as ATO).
“hotspot mp53” means an mp53 with at least one mutation in mp53 hotspots, namely, R175, G245, R248, R249, R273, R282, combinations thereof, and the like. Examples of hotspot mp53s are listed in
“Contacting mp53” means a mp53 that loses its DNA binding ability without drastically affecting the p53 structure. Contacting mp53s are represented by, for example, p53-R273H, p53-R273C, p53-R248Q and p53-R248W.
“Structural mp53” means a mp53 that has significantly disrupted three-dimensional structure as compared to wtp53. Structural mp53s are represented by, for example, p53-R175H, p53-G245D, p53-G245S, p53-R249S, and p53-R282W.
“artificial p53” means an artificially engineered p53. Preferred examples of an artificially engineered p53 include a p53 fusion protein, a p53 fragment, a p53 peptide, a p53-derived fusion macromolecule, a p53 recombinant protein, a p53 with second-site suppressor mutation (“SSSM”), and a super p53.
“p53 inhibiting protein” means a protein that inhibits a function of activity of p53, and includes, for example, murine double minute 2 (“MDM2”), inhibitor of apoptosis-stimulating protein of p53 (“iASPP”) and sirtuin-1 (“SIRT1”).
“useful characteristic” means an ability to efficiently and effectively rescue at least one wildtype structure, transcriptional activity, cell growth inhibition function, and/or tumor-suppressive function in a mp53. Exemplary useful characteristic includes: (a) an ability to substantially increase in the population of properly folded p53, preferably the increase is at least about 3 times more than the increase caused by PRIMA-1, more preferably the increase is at least about 5 times more than the increase caused by PRIMA-1, further preferably the increase is at least about 10 times more than the increase caused by PRIMA-1, further preferably the increase is at least about 100 times more than the increase caused by PRIMA-1; (b) an ability to substantially improve the transcription function of p53, preferably the improvement is at least about 3 times more than the improvement caused by PRIMA-1; more preferably the improvement is at least about 5 times more than the improvement caused by PRIMA-1, further preferably the improvement is at least about 10 times more than the improvement caused by PRIMA-1, further preferably the improvement is at least about 100 times than the improvement caused by PRIMA-1; and (c) an ability to substantially enhance the stability of p53 as measured by, for example, an increase p53 Tm, preferably the enhancement is at least about 3 times more than the enhancement caused by PRIMA-1, more preferably the improvement is at least about 5 times more than the improvement caused by PRIMA-1, further preferably the improvement is at least about 10 times more than the improvement caused by PRIMA-1, further preferably the improvement is at least about 100 times than the improvement caused by PRIMA-1. A preferred PANDA Agent has two or more useful characteristics, and more preferably has three or more useful characteristics. An exemplary PANDA Agent is ATO. Other exemplary PANDA Agent includes As analogs. Additional exemplary PANDA Agents are listed in Table 1-Table 7.
“efficiently” or “efficient” as used to describe the enhancement for a useful characteristic, rescue at least one wildtype structure, transcriptional activity, cell growth inhibition function, and/or tumor-suppressive function in a mp53, generally means enhancing the useful characteristic by more than about 3 times, as compared to the enhancement by PRIMA-1, preferably by more than about 5 times, more preferably by more than about 10 times, more preferably by about 100 times. For example, an efficient enhancement would be enhancing the Tm of mp53 by about 3-100 times of those of PRIMA-1, and/or folds mp53 by 3-100 times of those of PRIMA-1, and/or stimulates mp53's transcriptional activity by about 3-100 times of those of PRIMA-1.
“ATO” or “As2O3” means arsenic trioxide and compounds generally understood as arsenic trioxide.
“analog” or “analogue” means a compound obtained by varying the chemical structure of an original compound, for example, via a simple reaction or the substitution of an atom, moiety, or functional group of the original compound. Such analog may involve the insertion, deletion, or substitution of one or more atoms, moieties, or functional groups without fundamentally altering the essential scaffold of the original compound. Examples of such atoms, moieties, or functional groups include, but are not limited to, methyl, ethyl, propyl, butyl, hydroxyl, ester, ether, acyl, alkyl, carboxyl, halide, ketyl, carbonyl, aldehyde, alkenyl, azide, benzyl, fluoro, formyl, amide, imide, phenyl, nitrile, methoxy, phosphate, phosphodiester, vinyl, thiol, sulfide, or sulfoxide atoms, moieties, or functional groups. Many methods for creating a chemical analog from an original compound are known in the art.
“p53 disorder” means an abnormal physical and/or mental condition caused by a mutation in the TP53 gene and/or p53 protein. The condition can be in a human or another animal, such as a mouse, dog and other companion animals, a cattle and other livestock, a wolf or other zoo animals, and a horse or other equines. Examples of a p53 disorder include cancer, such as carcinoma (for example adenocarcinomas and squamous cell carcinoma), sarcoma, myeloma, leukemia, lymphoma, blastoma, and mixed types cancers (for example, adenosquamous carcinoma, mixed mesodermal tumor, carcinosarcoma, and teratocarcinoma); a tumor (for example, a tumor in connective tissue, endothelium and mesothelium, blood and lymphoid cells, muscle, epithelial tissues, neural, amine precursor uptake and decarboxylation system, other neural crest-derived cells, breast, renal anlage, and/or gonadal); a neurological disease, a developmental disease, an immunological disease, and aging, among others. Additional examples of known p53 disorder are listed in Section 1.2. A p53 cancer and/or tumor is a cancer and/or tumor with at least one p53 mutation. Additional examples of known p53 cancer and/or tumor are listed in Section 1.3.
“subject” means any organism. The subject is preferably an animal, such as a vertebrate; further preferably a mammal, such as a cattle, a horse, a pig, a lamb, and other livestock; further preferably a human, such as a patient, a cancer patient, an unborn child, and any un-conceived, hypothetical child of two parents.
“a person in need of” means an individual who has a p53 disorder, such as a cancer, wherein the cancer expresses a mp53, preferably a rescuable mp53.
“biological system” means a cell, bacteria, artificial system containing p53 pathway and relevant proteins.
“treatment” means the administration and/or application of the therapeutic product or method to a subject with a p53 disorder, and includes, among others, monitoring the efficacy of a type of treatment for the p53 disorder.
“diagnosis” means any method to identify a particular disease, and includes, among others, detecting the symptoms of a disease, assessing the severity of the disease, determining the stages of the disease, and monitoring the progression of the disease.
“prognosis” means any method to determine the likely course of a disease, and includes, among others, determining the predisposition of a disease, determining the likelihood a disease will onset, assessing the likely severity of the disease, determining the likely stages of the disease, and predicting the likely progression of the disease.
“a therapeutically effective amount” is an amount of a compound effective to prevent, alleviate, or ameliorate symptoms of a disorder or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. The effective dosage, level, or amount of a compound to be used in vivo can be determined by those skilled in the art, taking into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, the potency, bioavailability, and metabolic characteristics of the compound, and other factors.
“screening of effective treatments” means screening of effective therapeutic product or method for the treatment of a certain disease. It can involve in vitro and/or ex vivo screening methods, and includes, among others, both the product or composition to treat a disease and the method to prepare the composition for treatment.
“carrier” as used herein can include solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like
“pharmaceutical carrier” as used herein can include, liposomes, albumin microspheres, soluble synthetic polymers, DNA complexes, protein-drug conjugates, carrier erythrocytes, and any other substance that is incorporated to improve the delivery and the effectiveness of drugs. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
“compatible therapy for p53 disorder” means a therapy (including experimental therapies) compatible and/or synergistic with p53 treatments containing one or more PANDA Agents, The compatible therapy for p53 disorder can include surgery, chemotherapy, and radiation therapy. Experimental therapies include, but are not limited to, expression of wtp53 in tumors based on viral or viral like particle based delivery vectors.
“p53 cancer therapeutic” as used herein include, general chemotherapeutics. Examples of general chemotherapeutics include, but are not limited to, Avastin, Rituxan, Herceptin, Taxol, and Gleevec.
“DTP” means Developmental Therapeutics Program as understood by a person of ordinary skill in the art.
“DNA damaging agents” mean the anti-cancer agents in which the DNA damaging is involved when they function. Examples of a DNA damaging agent include decitabine (“DAC”), clsplatin (“CIS”), etoposide (“ETO”), adriamycin (ADM”), 5-fluorouracil (“5-FU”), cytarabine (“ARA/araC”), and azactidine (“AZA”).
The 53-kilodalton p53 protein is a transcription factor and one of the most important proteins in cell biology. p53 is the most heavily studied protein in history and it is also the most heavily studied protein in every year since 2001, yet the reusability of mp53 is still largely unknown. Wildtype p53 (“wtp53”) sequence can be found in public gene banks, such as gene bank, protein bank, and Uniport. Exemplary wtp53 sequences are listed under Table 14. Unless specified otherwise, this application uses the wtp53 sequences of human p53 isoform “a” listed under Table 14 to reference amino acid locations on p53.
The active human wtp53 is a homotetramer of 4×393 amino acids with multiple domains including an intrinsically disordered N-terminal transactivation domain (“TAD”), a proline-rich domain (“PRD”), a structured DNA-binding domain (“DBD”) and tetramerization domain (“TET”) connected via a flexible linker, and an intrinsically disordered C-terminal regulatory domain (“CTD”) (see
wtp53 plays a central part in the cells and is frequently considered as the most important tumor suppressor. Upon cellular stresses, such as DNA damage or oncogenic stress, p53 is activated and transcriptionally regulates a batch of genes to trigger events including cell-cycle arrest, DNA repair, apoptosis, cell repair, cell death, among others. Examples of genes transcriptionally regulated by p53 include Apaf1, Bax, Fas, Dr5, mir-34, Noxa, TP53AIP1, Perp, Pidd, Pig3, Puma, Siva, YWHAZ, Btg2, Cdkn1a, Mdm2, BBC3/PUMA, Tp53i3, Gadd45a, mir-34a, mir-34b/34c, Prl3, Ptprv, Reprimo, Pai1, Pml, Ddb2, Ercc5, Fancc, Gadd45a, Ku86, Mgmt, Mlh1, Msh2, P53r2, Polk, Xpc, Adora2b, Aldh4, Gamt, Gls2, Gpx1, Lpin1, Parkin, Prkab1, Prkab2, Pten, Sco1, Sesn1, Sesn2, Tigar, Tp53inp1, Tsc2, Atg10, Atg2b, Atg4a, Atg4c, Atg7, Ctsd, Ddit4, Dram1, Foxo3, Laptm4a, Lkb1, Pik3r3, Prkag2, Puma, Tpp1, Tsc2, Ulk1, Ulk2, Uvrag, Vamp4, Vmp1, Bai1, Cx3cl1, Icam1, Irf5, Irf9, Isg15, Maspin, Mcp1, Ncf2, Pai1, Tlr1-Tlr10, Tsp1, Ulbp1, Ulbp2, mir-34a, mir-200c, mir-145, mir-34a, mir-34b/34c, Notch1, combinations thereof and the like. In addition to anti-cancer role, p53 target genes also have important roles in senescence, angiogenesis, and autophagy, connecting, regulating oxidative stress, regulating metabolic homeostasis, stem cell maintenance, among others. Accordingly, a mutation in p53 (i.e. a mutant p53 or mp53) can cause a wide range of health issues, including cancer, tumor, neurological disease, developmental disease, immunological disease, and aging, among others.
Examples of known p53 disorders include achalasia, acinar cell carcinoma, acrofacial dysostosis, actinic cheilitis, actinic keratosis, acute lymphocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, adrenocortical carcinoma, adult hepatocellular carcinoma, adult medulloblastoma, adult t-cell leukemia, aging, agraphia, alpha-thalassemia, alpha-thalassemia/mental retardation syndrome, anal squamous cell carcinoma, anaplastic thyroid cancer, anogenital venereal wart, anterior cranial fossa meningioma, aplastic anemia, ataxia-telangiectasia, atrophic gastritis, atrophy of prostate, atypical follicular adenoma, atypical teratoid rhabdoid tumor, autonomic nervous system neoplasm, autosomal genetic disease, b cell prolymphocytic leukemia, Barrett esophagus, Barrett's adenocarcinoma, Bartholin's duct cyst, Bartholin's gland adenoma, Bartholin's gland benign neoplasm, basal cell carcinoma, basal cell carcinoma, basaloid squamous cell carcinoma, B-cell lymphomas, Beckwith-wiedemann syndrome, bile duct adenocarcinoma, bile duct carcinoma, biliary papillomatosis, biliary tract neoplasm, bladder cancer, bladder carcinoma in situ, bladder papillary transitional cell neoplasm, bladder squamous cell carcinoma, bladder transitional cell papilloma, bladder urothelial carcinoma, bone giant cell sarcoma, bone squamous cell carcinoma, brain cancer, brain ependymoma, brain glioblastoma multiforme, brain glioma, brain stem astrocytic neoplasm, brain stem cancer, brain stem glioma, breast adenocarcinoma, breast benign neoplasm, breast cancer, breast carcinoma in situ, breast disease, breast ductal carcinoma, breast malignant phyllodes tumor, breast squamous cell carcinoma, calcifying epithelial odontogenic tumor, cataract, cell type benign neoplasm, cell type cancer, cellular ependymoma, cellular neurofibroma, cellular schwannoma, central nervous system lymphoma, central nervous system organ benign neoplasm, central nervous system primitive neuroectodermal neoplasm, cerebellar angioblastoma, cerebellar astrocytoma, cerebellar liponeurocytoma, cerebellum cancer, cerebral convexity meningioma, cerebral neuroblastoma, cerebral primitive neuroectodermal tumor, cerebral ventricle cancer, cerebrum cancer, cervical adenocarcinoma, cervical cancer, cervical carcinosarcoma, cervical squamous cell carcinoma, cervix carcinoma, cervix small cell carcinoma, cervix uteri carcinoma in situ, cheilitis, childhood leukemia, cholangiocarcinoma, cholecystitis, chordoid glioma, chordoma, choroid plexus cancer, chromophobe adenoma, chronic salpingitis, clear cell adenocarcinoma, clear cell cystadenofibroma, clear cell ependymoma, clivus meningioma, cl/sll, colorectal adenocarcinoma, colorectal adenoma, colorectal cancer, conjunctival degeneration, conjunctival squamous cell carcinoma, connective tissue cancer, cystadenocarcinoma, cystic teratoma, cystitis, dedifferentiated liposarcoma, dermatofibrosarcoma protuberans, differentiated thyroid carcinoma, diffuse large B-cell lymphoma, ductal carcinoma in situ, dyskeratosis congenita autosomal recessive, dyskeratosis congenita, dyskeratosis congenita, autosomal recessive, eccrine sweat gland neoplasm, ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome, embryonal sarcoma, endocervical adenocarcinoma, endocrine gland cancer, endometrial adenocarcinoma, endometrial cancer, endometrial clear cell adenocarcinoma, endometrial stromal sarcoma, endometrium carcinoma in situ, ependymoblastoma, epidermal appendage tumor, epidural neoplasm, epithelial-myoepithelial carcinoma, esophageal basaloid squamous cell carcinoma, esophageal cancer, esophageal disease, esophagitis, esophagus adenocarcinoma, essential thrombocythemia, estrogen-receptor positive breast cancer, Ewing sarcoma, fallopian tube adenocarcinoma, fallopian tube carcinoma, familial adenomatous polyposis, familial colorectal cancer, female breast cancer, female reproductive endometrioid cancer, female reproductive organ cancer, fibrillary astrocytoma, focal cortical dysplasia, type ii, frontal convexity meningioma, gallbladder cancer, gallbladder squamous cell carcinoma, ganglioglioma, gastric adenocarcinoma, gastric adenosquamous carcinoma, gastric cancer, gastric lymphoma, gastric papillary adenocarcinoma, gastroesophageal reflux, gastrointestinal stromal tumor, gastrointestinal system benign neoplasm, gastrointestinal system cancer, germ cell and embryonal cancer, giant cell glioblastoma, glioblastoma multiforme, glioblastoma, gliofibroma, glioma susceptibility, glioma, gliomatosis cerebri, gliosarcoma, glomangiosarcoma, glomus tumor, glycogen-rich clear cell breast carcinoma, grade iii astrocytoma, granulosa cell tumor of the ovary, Helicobacter pylori infection, hematologic cancer, hepadnavirus infection, hepatoblastoma, hepatocellular carcinoma, hereditary breast ovarian cancer syndrome, hidradenocarcinoma, histiocytoma, huntington disease, hydrocephalus, hyperplastic polyposis syndrome, hypoxia, in situ carcinoma, inflammatory myofibroblastic tumor, infratentorial cancer, integumentary system cancer, intestinal benign neoplasm, intestinal disease, intracranial chondrosarcoma, intrahepatic cholangiocarcinoma, invasive bladder transitional cell carcinoma, inverted papilloma, juvenile pilocytic astrocytoma, kaposi sarcoma, keratinizing squamous cell carcinoma, keratoacanthoma, keratocystic odontogenic tumor, larynx cancer, larynx verrucous carcinoma, leiomyosarcoma, leukemia, leukemia, acute lymphoblastic, leukemia, acute myeloid, leukemia, chronic lymphocytic, lichen disease, lichen planus, lichen sclerosus, li-fraumeni syndrome, li-fraumeni syndrome, lip cancer, liposarcoma, liver angiosarcoma, lung benign neoplasm, lung cancer susceptibility, lung cancer, lung occult squamous cell carcinoma, lung papillary adenocarcinoma, lung squamous cell carcinoma, lymph node cancer, lymphoid interstitial pneumonia, lymphoma, non-hodgkin, familial, lynch syndrome, male reproductive organ cancer, malignant ependymoma, malignant giant cell tumor, malignant mesenchymoma, malignant ovarian surface epithelial-stromal neoplasm, malignant peripheral nerve sheath tumor, malignant spiradenoma, mantle cell lymphoma, Marek disease, mature B-cell neoplasm, mature teratoma, maxillary sinus squamous cell carcinoma, medulloblastoma, medullomyoblastoma, megaesophagus, megakaryocytic leukemia, melanoma, melanoma, cutaneous malignant, meningeal melanomatosis, meninges sarcoma, meningioma, familial, merkel cell carcinoma, microglandular adenosis, mixed astrocytoma-ependymoma, mixed cell type cancer, mixed glioma, mixed oligodendroglioma-astrocytoma, mucoepidermoid esophageal carcinoma, multifocal osteogenic sarcoma, multiple cranial nerve palsy, muscle cancer, mutagen sensitivity, mutyh-associated polyposis, myasthenic syndrome, myelodysplastic syndrome, myeloid leukemia, myeloma, multiple, myxoid liposarcoma, myxosarcoma, nasal cavity adenocarcinoma, nasopharyngeal carcinoma, necrotizing sialometaplasia, nervous system cancer, neuroblastoma, nevus of ota, nijmegen breakage syndrome, non-invasive bladder papillary urothelial neoplasm, non-proliferative fibrocystic change of the breast, ocular cancer, olfactory groove meningioma, oligodendroglioma, optic nerve glioma, optic nerve neoplasm, oral cancer, oral cavity cancer, oral leukoplakia, organ system benign neoplasm, oropharynx cancer, osteogenic sarcoma, ovarian cancer, ovarian cancer, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, ovary adenocarcinoma, ovary epithelial cancer, pancreas adenocarcinoma, pancreatic cancer, pancreatic ductal carcinoma, papillary adenocarcinoma, papillary serous adenocarcinoma, papilledema, papilloma of choroid plexus, papilloma, parameningeal embryonal rhabdomyosarcoma, parietal lobe neoplasm, penile cancer, penis carcinoma in situ, penis squamous cell carcinoma, periosteal osteogenic sarcoma, peripheral nervous system neoplasm, peripheral T-cell lymphoma, Peutz-jeghers syndrome, pharynx cancer, pigmented villonodular synovitis, pilocytic astrocytoma, pinguecula, plantar wart, pleomorphic adenoma carcinoma, pleomorphic adenoma, pleomorphic carcinoma, pleomorphic xanthoastrocytoma, pleuropulmonary blastoma, pre-malignant neoplasm, primary peritoneal carcinoma, prolactin producing pituitary tumor, prostate cancer, prostate squamous cell carcinoma, protoplasmic astrocytoma, pseudomyxoma peritonei, pulmonary blastoma, rare adenocarcinoma of the breast, recessive dystrophic epidermolysis bullosa, rectal neoplasm, papillary, renal cell carcinoma, respiratory system cancer, retinal cancer, retinoblastoma, rhabdomyosarcoma, Richter's syndrome, rift valley fever, ring chromosome, sarcoma, sarcomatoid squamous cell skin carcinoma, schneiderian carcinoma, sclerosing liposarcoma, scrotal carcinoma, sensory system cancer, serous cystadenocarcinoma, short-rib thoracic dysplasia with or without polydactyly, signet ring cell adenocarcinoma, skin melanoma, skin squamous cell carcinoma, small cell cancer of the lung, small cell carcinoma, small cell sarcoma, soft tissue sarcoma, spinal cancer, spinal cord astrocytoma, spinal cord glioma, spinal cord primitive neuroectodermal neoplasm, spiradenoma, spitz nevus, splenic diffuse red pulp small B-cell lymphoma, split-hand/foot malformation, sporadic breast cancer, squamous cell carcinoma, squamous cell papilloma, submandibular gland cancer, suppression of tumorigenicity, suppressor of tumorigenicity, supratentorial cancer, sweat gland cancer, synchronous bilateral breast carcinoma, teratoma, testicular germ cell tumor, testicular torsion, tetraploidy, thoracic benign neoplasm, thymus cancer, thyroid cancer, thyroid lymphoma, tongue cancer, tongue squamous cell carcinoma, transitional cell carcinoma, ulcerative stomatitis, ureteral obstruction, urinary tract papillary transitional cell benign neoplasm, uterine body mixed cancer, uterine carcinosarcoma, uterine corpus cancer, uterine corpus serous adenocarcinoma, vaccinia, vestibular gland benign neoplasm, vulva cancer, vulva squamous cell carcinoma, vulvar adenocarcinoma, vulvar intraepithelial neoplasia, vulvar sebaceous carcinoma, wilms tumor, xanthogranulomatous cholecystitis, xeroderma pigmentosum, variant type, zika virus infection, combinations thereof and the like.
It has been estimated that the direct medical expenses for mp53 patients in 2017 alone amounts to approximately 65 billion USD.
p53 is the most frequently mutated cancer protein (
Examples of cancers and/or tumors reported to harbor one or more p53 mutations include carcinoma, acinar cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, apocrine adenocarcinoma, basal cell carcinoma, basaloid carcinoma, basosquamous carcinoma, bronchiolo-alveolar adenocarcinoma, carcinoma in pleomorphic adenoma, cholangiocarcinoma, choriocarcinoma, choroid plexus carcinoma, clear cell adenocarcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, comedocarcinoma, cribriform carcinoma, ductal carcinoma, solid type, eccrine adenocarcinoma, endometrioid adenocarcinoma, follicular adenocarcinoma, giant cell and spindle cell carcinoma, giant cell carcinoma, hepatocellular carcinoma, hepatoid adecarcinoma, infiltrating basal cell carcinoma, infiltrating duct carcinoma, infiltrating ductular carcinoma, inflammatory carcinoma, intraductal carcinoma, intraductal carcinoma and lobular carcinoma, intraductal papillary adenocarcinoma, intraductal papillary-mucinous carcinoma, large cell carcinoma, large cell neuroendocrine carcinoma, leiomyosarcoma, lobular carcinoma, medullary carcinoma, merkel cell carcinoma, metaplastic carcinoma, mixed cell adenocarcinoma, mucinous adenocarcinoma, mucinous cystadenocarcinoma, mucoepidermoid carcinoma, multifocal superficial basal cell carcinoma, neuroendocrine carcinoma, non-small cell carcinoma, oat cell carcinoma, papillary adenocarcinoma, papillary carcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, papillary transitional cell carcinoma, pituitary carcinoma, plasmacytoid carcinoma, pleomorphic carcinoma, pseudosarcomatous carcinoma, renal cell carcinoma, sebaceous adenocarcinoma, secretory carcinoma of breast, serous cystadenocarcinoma, serous surface papillary carcinoma, signet ring cell carcinoma, small cell carcinoma, solid carcinoma, spindle cell carcinoma, squamous cell carcinoma, sweat gland adenocarcinoma, teratocarcinoma, thymic carcinoma, transitional cell carcinoma, trichilemmocarcinoma, tubular adenocarcinoma, sarcoma, alveolar rhabdomyosarcoma, carcinosarcoma, chondroblastic osteosarcoma, chondrosarcoma, clear cell sarcoma of kidney, dedifferentiated chondrosarcoma, dermatofibrosarcoma, embryonal rhabdomyosarcoma, embryonal sarcoma, Ewing sarcoma, fibrosarcoma, gastrointestinal stromal sarcoma, gliosarcoma, hemangiosarcoma, kaposi sarcoma, liposarcoma, mixed liposarcoma, myxoid liposarcoma, osteosarcoma, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, rhabdomyosarcoma, sarcoma, synovial sarcoma, undifferentiated sarcoma, myeloma, multiple myeloma, leukemia, acute leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, Burkitt cell leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, hairy cell leukemia, lymphoid leukemia, myeloid leukemia, plasma cell leukemia, precursor B-cell lymphoblastic leukemia, precursor cell lymphoblastic leukemia, precursor T-cell lymphoblastic leukemia, prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, undifferentiated leukemia, lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, malignant lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, mature T-cell lymphoma, NK/T-cell lymphoma, precursor cell lymphoblastic lymphoma, primary effusion lymphoma, splenic marginal zone-B-cell lymphoma, ameloblastoma, giant cell glioblastoma, glioblastoma, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pleuropulmonary blastoma, and pulmonary blastoma, and retinoblastoma, tumor, adenocarcinoid tumor, atypical carcinoid tumor, Brenner tumor, carcinoid tumor, epithelial tumor, gastrointestinal stromal tumor, giant cell tumor of soft parts, glomus tumor, granulosa cell tumor, Klatskin tumor, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, mesodermal mixed tumor, mixed tumor, mucinous cystic tumor of borderline malignancy, Mullerian mixed tumor, myofibroblastic tumor, peripheral neuroectodermal tumor, phyllodes tumor, phyllodes tumor, primitive neuroectodermal tumor, serous surface papillary tumor, solitary fibrous tumor, tumor cells, yolk sac tumor, adenoma, adrenal cortical adenoma, atypical adenoma, cystadenoma, atypical adenoma, cystadenoma, fibroadenoma, follicular adenoma, hepatocellular adenoma, intraductal papillary-mucinous adenoma, pleomorphic adenoma, serous cystadenoma, serrated adenoma, tubular adenoma, tubulovillous adenoma, villous adenoma, mixed tumors, angiomyolipoma, astrocytoma, atypical fibrous histiocytoma, Barrett's esophagus, Bowen disease, central neurocytoma, clear cell adenocarcinofibroma, dysgerminoma, dysplasia, embryo fibroblasts, endometriosis, ependymoma, esophagitis, essential thrombocythemia, fibrillary astrocytoma, fibrosis, gemistocytic astrocytoma, germinoma, glandular intraepithelial neoplasia, glioma, gliomatosis cerebri, glucagonoma, goblet cell carcinoid, hemangioendothelioma, hemangiopericytoma, hidrocystoma, hydatidiform mole, hyperplasia, insulinoma, keloid, keratoacanthoma, keratosis, Langerhans cell histiocytosis, lentigo maligna melanoma, leucoplakia, lipoma, malignant fibrous histiocytoma, malignant histiocytosis, malignant melanoma, malignant myoepithelioma, meningioma, mesothelioma, metaplasia, mixed glioma, mycosis fungoides, myelodysplastic syndrome, myelosclerosis with myeloid metaplasia, myoepithelioma, neoplasm, neurilemoma, nodular melanoma, oligodendroglioma, osteochondroma, pheochromocytoma, pigmented nevus, pilocytic astrocytoma, plasmacytoma, pleomorphic xanthoastrocytoma, polycythemia vera, polyp, pterygium, pulmonary sclerosing hemangioma, refractory anemia, seminoma, serous adenocarcinofibroma, Sezary syndrome, squamous intraepithelial neoplasia, superficial spreading melanoma, teratoma, thymoma, urothelial papilloma, Waldenstrom macroglobulinemia, aggressive fibromatosis, lymphomatoid papulosis, combinations thereof and the like.
Approximately one-third of the p53 mutations are located on one of six mp53 hotspots: R175, G245, R248, R249, R273, and R282, (each a “mp53 hotspot”) (Freed-Pastor and Prives, 2012). Mutated p53 (or mp53) falls roughly into two categories. Contacting mp53 has lost its DNA binding ability without drastically affecting the p53 structure (“Contacting mp53”). Examples of Contacting mp53s include p53-R273H (3.0% mutation frequency), p53-R273C (2.5% mutation frequency), p53-R248Q (3.3% mutation frequency) and p53-R248W (2.7% mutation frequency). See also
As seen in its representative member, the R282W mutation disrupts the hydrogen-bond network in the local loop-sheet-helix motif, reducing the melting temperature (“T”, an index for thermally stability of protein) and cause global, structural destabilization. A broad-spectrum rescue agent would thus need to increase the Tm. We further discovered that four pairs of the 10 mp53 cysteines (C176/C182, C238/C242, C135/C141, and C275/C277) are in close proximity to the Structural mp53 hotspots (
PANDA also regains transcriptional activities on most of the p53 target genes as shown in the heatmap of RNA expression level of a set of 127 p53 targets. RNA sequencing (RNA-seq) data also shows that among the reported 116 genes p53-activated targets, the majority of the target genes were up-regulated by PANDA-R282W, including the well-known p53 targets BBC3, BAX, TP5313, CDKN1A, and MDM2.
We solved the 3D structure of at least one mp53 at a resolution of approximately 1-3 Å (see
In certain embodiments, the PANDA Core is produced by a reaction between the PANDA Pocket and the PANDA Agent. Preferably, the reaction is mediated by an As, Sb, and/or Bi group oxidizing one or more thiol groups of PANDA Cysteines (PANDA Cysteines lose between one to three hydrogens) and the As, Sb, and/or Bi group of PANDA Agent is reduced (PANDA Agent loses oxygen). In certain embodiments, the PANDA Agent is the reduzate formed from having tightly associated with p53. In certain embodiments, the PANDA Agent is an arsenic atom, an antimony atom, a bismuth atom, any analogue thereof, combinations thereof, and the like.
In certain embodiments, the PANDA Agent transforms cancer-promoting mp53 to tumor suppressive PANDA and have significant advantages over existing therapeutic strategies such as by reintroducing wtp53 or promoting degradation/inactivation of endogenous mp53 in the patient. The PANDA Agent mediated mp53 rescue through PANDA, high rescue efficiency and mp53 selectivity are the two superior characteristics over previously-reported compounds. In certain embodiments, the PANDA Agent ATO can provide a near complete rescue of p53-R175H, from a level equivalent to about 1% of that of wtp53 to about 97% of that of wtp53 using the robust PAb1620 (also for PAb246) IP assay. In certain embodiments, the PANDA Agent ATO also provides a near complete rescue of the transcriptional activity of p53-G245S and p53-R282W on some pro-apoptotic targets, from a level equivalent to about 4% of that of wtp53 to about 80% of that of wtp53, using a standard luciferase reporter assay. In other embodiments, the PANDA Agent ATO can rescue the function of mp53s to a level that exceeds that of the wtp53, as shown, for example, in our luciferase assay for p53-1254T and p53-V272M. We have robustly reproduced these superior results, as compared to existing compounds, in numerous contexts and know no existing compound that can rescue the structure or transcriptional activity of a hotspot mp53 by a level equivalent to about 5% of that of wtp53 in our assays.
In certain embodiments, the PANDA Agent ATO and PANDA can selectively target Structural mp53 with strikingly high efficiency. In addition, Contracting mp53s can also be rescued with moderate efficiency. For example, we found a wide range of Structural mpS3s, including a large percentage of hotspot mp53s, can be efficiently rescued by the PANDA Agent ATO through the formation of PANDA. In addition, we also found that the Contacting mp53s can be rescued by ATO through PANDA with a limited efficiency. This remarkable property is not only superior but is conceptually different from most of the reported compounds, including CP-31398 (Foster et al., 1999), PRIMA-1 (Bykov et al., 2002), SCH529074 (Demma et al., 2010), Zinc (Puca et al., 2011), stictic acid (Wassman et al., 2013), p53R3 (Weinmann et al., 2008), and others that are reported to be able to rescue both types of mp53.
As used in this application, “PANDA” refers to the p53 and arsenic analogue complex. “PANDA Cysteine” refers to one of C124, C135, or C141. “PANDA Triad” refers to the C124, C135, C141 together. “PANDA Pocket” refers to the three-dimensional structure centered around PANDA Triad. The PANDA Pocket includes PANDA Triad and directly contacting residues (S116 contacts C124, C275 and R273 contact C135, Y234 contacts C141), residues adjacent to PANDA Triad (V122, T123, T125, and Y126; M133, F134, Q136, and L137; K139, T140, P142, and V143), and residues in 7-A distance to PANDA Triad (L114, H115, G117, T118, A119, K120, S121, A138, 1232, H233, N235, Y236, M237, C238, N239, F270, E271, V272, V274, A276, C277, P278, G279, R280, D281, and R282) (
We discovered that in general, compounds with one or more cysteine-binding potentials on p53, preferably two or more cysteine-binding potential on p53, and more preferably three cysteine-binding potential on p53 are good rescue compounds for a broad spectrum of mp53s. Some of these compounds can rescue mp53 to near wildtype-like conditions (see
We further suggest other non-As, Sb, and Bi compounds can also serve as efficient a PANDA Agent as long as they can bind PANDA pocket which leads to mp53 stability. These compounds can contain group of thiols (e.g.: 1,4-Benzenedithiol), Michael acceptor (e.g.: (1E,6E)-1,7-Diphenyihepta-1,6-diene-3,5-dione), and others which can bind cysteine. These compounds can also lack of cysteine-binding ability, however, they bind other residues of PANDA pocket to stabilize mp53.
We further discovered that the preferred rescue compounds for mp53 can (i) upon hydroxylation, simultaneously bind to one or more mp53 cysteines, preferably two or more mp53 cysteines, more preferably three mp53 cysteines; (ii) can form at least one tight bond to PANDA Pocket; (iii) can increase the ratio of folded p53 to unfolded p53 and/or refold mp53 with high efficiency, at levels comparable to that of wtp53 in some cases (as measured by immunoprecipitation with, for example, PAb1620 and/or PAb246); (iv) can rescue the transcriptional activity of mp53s at levels comparable to that of wtp53 in some cases (as measured by, for example, luciferase report assay); (v) can stabilize p53 and increase the melting temperature of mp53; (vi) can selectively inhibit mp53 expressing cell lines, such as the NCI60 cell lines that expresses the Structural hotspot mp53; (vii) can inhibit mouse xenografts dependent on Structural mp53s; and/or (viii) can be used to treat mp53 harboring cancer patients in combination with DNA-damaging agents.
We further discovered that elemental arsenic, elemental bismuth, elemental antimony, and compounds containing elemental arsenic, bismuth, and/or antimony are good rescue compounds for mp53. We showed that arsenic, bismuth, and antimony containing compounds can stabilize the structure of mp53s and/or rescue its transcriptional activities (see Table 7). The arsenic-, bismuth-, and antimony-mediated mp53 rescue is achieved by binding of the released arsenic, bismuth, and antimony to mp53. For example, mass spectroscopy data showed arsenic, bismuth, and antimony atom binds to mp53 directly and covalently (see
We further discovered that the following six classes of compounds are preferred mp53 rescue compounds: a three-valence arsenic containing compound, preferably the compound can be hydrolyzed, further preferably the compound does not have a carbon-arsenic bond, further preferably the compound is one that is listed in Table 1; a five-valence arsenic containing compounds, preferably the compound can be hydrolyzed, further preferably the compound does not have a carbon-arsenic bond, further preferably the compound is one that is listed in Table 2); a three-valence bismuth containing compounds, preferably the compound can be hydrolyzed, further preferably the compound does not have a carbon-bismuth bond, further preferably the compound is one that is listed in Table 3; a five-valence bismuth containing compounds, preferably the compound can be hydrolyzed, further preferably the compound does not have a carbon-bismuth bond, further preferably the compound is one that is listed in Table 4; a three-valence antimony containing compounds, preferably the compound can be hydrolyzed, further preferably the compound does not have a carbon-antimony bond, further preferably the compound is one that is listed in Table 5; and five-valence antimony containing compounds, preferably the compound can be hydrolyzed, further preferably the compound does not have a carbon-antimony bond, further preferably the compound is one that is listed in Table 6. We arrived at the lists of compounds in Table 1-Table 6 by analyzing, in silico, approximately 94.2 million compounds derived from PubChem (https://pubchem.ncbi.nlm.nih.gov/), using the selection criteria of (i) compounds containing elemental arsenic or its analogues, such as antimony, and bismuth and (ii) the capacity to simultaneously bind to 3 cysteines (our compounds listed in Table 1-Table 6 are predicted to rescue mp53 with very high efficiency because they can simultaneously bind 3 cysteines of PANDA triad). These rescue compounds include three-valence and five-valence arsenic, three-valence and five-valence antimony, and three-valence and five-valence bismuth. The discovery of compounds containing Bi and/or Sb, and As, Sb, and/or Bi compounds with mp53 rescue capacity has tremendous clinical value because these compounds generally have lower toxicities than inorganic As compounds in the body.
Exemplary embodiments of the rescue compound can include any one of the Formulas I-XV.
wherein:
wherein:
wherein:
In the preferred embodiment, the non-Carbon atom is selected from the group consisting of O, S, N, X, F, Cl, Br, I, and H.
Exemplary rescue compound with the structure of Formula I includes
Exemplary rescue compound with the structure of Formula II includes
Exemplary rescue compound with the structure of Formula III includes As+(OH)2.
Exemplary rescue compound with the structure of Formula V includes
Exemplary rescue compound with the structure of Formula V includes
and.
Exemplary rescue compound with the structure of Formula VI includes
Exemplary rescue compound with the structure of Formula VIII includes
Exemplary rescue compound with the structure of Formula IX includes.
Exemplary rescue compound with the structure of Formula X includes
Exemplary rescue compound with the structure of Formula XII includes
Exemplary rescue compound with the structure of Formula XIII includes
Exemplary rescue compound with the structure of Formula XV includes
The following Equation (1) is an reaction for PANDA Agent. A compound containing M group with a Z1 (a first group with the capacity to bind a first cysteine) and/or a Z2 (a second group with the capacity to bind a second cysteine) and/or a Z3 (a third group with the capacity to bind a third cysteine), Examples of Z1, Z2, and Z3 includes O, S, N, X, F, Cl, Br, I, OH, and H. Z1, Z2, and/or Z3 can bind to each other. M group includes for example a metal, such as an bismuth, a metalloid, such as an arsenic and an antimony, a group such as a Michael acceptor and/or a thiol, and/or any analogue with cysteine-binding ability. The PANDA Agent can undergo a hydrolysis before reacting and binding to p53 forming PANDA. In some cases, when a group cannot undergo hydrolysis, and accordingly cannot bind to a cysteine. In such cases, the remaining group(s) with cysteine binding potential binds to p53. X1 and X2 represent any groups bound to M. X1 and/or X2 can also be empty. X1 and/or X2 can also be able to bind cysteine.
The following Equations (2) and (3) is an exemplary reaction for a PANDA Agent with tri-cysteine binding potential. 3-valence ATO or KAsO2 undergoes hydrolysis, covalently binds to three PANDA Cysteines on p53.
The following equation (4) is an exemplary reaction for a PANDA Agent with tri-cysteine binding potential. 5-valence As compound undergoes hydrolysis, covalently binds to three PANDA Cysteines on p53.
The following equation (5) is an exemplary reaction for a PANDA Agent with bi-cysteine binding potential. The PANDA Agent can bind to PANDA Cysteines, or to PANDA Cysteines (Cys124, Cys135, or Cys141), or Cys275 and Cys277 or C238 and C242.
The following equation (6) is an exemplary reaction for a PANDA Agent with mono-cysteine binding potential. The PANDA Agent can bind to PANDA Cysteines, (i.e. Cys124, Cys135, or Cys141) or the other 3 cysteines on PANDA Pocket (Cys236, Cys275, or Cys277).
We further discover that KAsO2, AsCl3, HAsNa2O4, NaAsO2, AsI3, As2O3, As2O5, KAsF6, LiAsF6, SbCl3, SbF3, SbAc3, Sb2O3, Sb(OC2H5)3, Sb(OCH3)3, SbI3, Sb2O5, Sb2(SO4)3, BiI3, C1H1As2N4O2, C13H14As2O6, C17H28AsClN4O6S, C1OH13NO6Sb, C6H12NaO6Sb+, (CH3CO2)3Sb, C6H4K2O12Sb2.xH2O, C13H21NaO9Sb+, HOC6H4COOBiO, [O2CCH2C(OH)(CO2)CH2CO2]Bi, (CH3CO2)3Bi, As2S2, As2S3, and As2S5 are remarkable mp53 rescue compounds, capable of rescuing both the structure and transcriptional function of mp53 in experimental assays (see Table 7). For example, we tested some structural mp53s for their abilities to refold protein, increase Tm, and stimulate transcriptional activity. Among these preferred mp53 rescue compounds, We discovered that As2O3 was previously approved by the U.S. Food and Drug Administration to treat acute promyelocytic leukemia (“APL”) in 2000 as NDA 21-248, but was not approved to treat other cancer types yet, because it did not provide any statistically significant efficacy. Additionally, the PANDA Agent Fowler's solution (KAsO2) has significant side-effects and are not used in clinical settings any more in past decades, but this may now be overcome by selecting and treating a patient with rescuable mp53, as disclosed in this Application. The PANDA Agent As4S4 has been shown to be as effective as conventional intravenous ATO in treating APL patients, but unlike ATO, As4S4 can be conveniently orally administrated (Zhu et al., 2013), making particularly attractive cancer therapy. Furthermore, we also discover that PANDA Agents As2S3, As2S2, and As2S5, which have strong ability to rescue mp53, can also be formulated for oral administration.
We further discovered that arsenic trioxide (ATO: NSC92859 & NSC759274) and potassium arsenite (KAsO2: NSC3060) are two wide-spectrum mp53 rescuing agents with remarkably high rescue efficiency (Table 7, Table 9 and
The PANDA Agent comprising a three and/or five valence arsenic is generally effective in treating cancer in a subject, including an animal, at a dose at a wide range of dosages by intravenous injection and oral administration. In certain embodiments, the daily dosage is from about 0.5 mg/kg to about 50 mg/kg, preferably from about 0.5 mg/kg to about 25 mg/kg, more preferably from about 1 mg/kg to about 25 mg/kg, more preferably from about 1 mg/kg to about 15 mg/kg, more preferably from about 1.7 mg/kg to about 15 mg/kg, and more preferably from about 1.7 mg/kg to about 5 mg/kg. In certain embodiments, the dose is about 5 mg/kg. In certain embodiments, the PANDA Agent ATO is administered by intravenous injection or by oral administration at 1 mg/ml concentration, at a dose of 5 mg/kg per day.
In other embodiments, the daily dosage is from about 10 mg/kg to about 1000 mg/kg, preferably from about 10 mg/kg to about 500 mg/kg, more preferably from about 20 mg/kg to about 500 mg/kg, more preferably from about 20 mg/kg to about 300 mg/kg, more preferably from about 33 mg/kg to about 300 mg/kg, and more preferably from about 33 mg/kg to about 100 mg/kg. In certain embodiments, the dose is about 100 mg/kg. In certain embodiments, the PANDA Agent As2S2, As2S3, As2S5, and As4S4 is administered by oral administration at 15 mg/L concentration, at a dose of 100 mg/kg
The PANDA Agent comprising a three valence and/or five valence antimony is generally effective in treating cancer in a subject, including an animal, at a dose at a wide range of dosages by intravenous injection and oral administration. In certain embodiments, dosage is from about 60 mg/kg to about 6000 mg/kg, preferably from about 60 mg/kg to about 3000 mg/kg, more preferably from about 120 mg/kg to about 3000 mg/kg, more preferably from about 120 mg/kg to about 1500 mg/kg, more preferably from about 150 mg/kg to about 1200 mg/kg, and more preferably from about 300 mg/kg to about 1200 mg/kg. In certain embodiments, the dose is about 600 mg/kg. In certain embodiments, the PANDA Agent is administered by intravenous or oral administration at 100 mg/ml concentration, at a dose of 600 mg/kg per day.
The PANDA Agent comprising a three valence and/or five valence bismuth is generally effective in treating cancer in a subject, including an animal, at a dose at a wide range of dosages by intravenous injection and oral administration. In certain embodiments, the daily dosage is from about 60 mg/kg to about 6000 mg/kg, preferably from about 60 mg/kg to about 3000 mg/kg, more preferably from about 120 mg/kg to about 3000 mg/kg, more preferably from about 120 mg/kg to about 1500 mg/kg, more preferably from about 150 mg/kg to about 1200 mg/kg, and more preferably from about 300 mg/kg to about 1200 mg/kg. In certain embodiments, the dose is about 600 mg/kg. In certain embodiments, the PANDA Agent is administered by intravenous or oral administration at 100 mg/ml concentration, at a dose of 600 mg/kg per day.
The PANDA Agent comprising a three and/or five valence arsenic is generally effective in treating cancer in a human at a wide range of dosages by intravenous injection and oral administration. In certain embodiments, the effective dose results in a maximum As concentration in the patient's blood (plasma) from about 0.094 mg/L to about 9.4 mg/L, preferably from about 0.094 mg/L to about 4.7 mg/L, more preferably from about 0.19 mg/L to about 4.7 mg/L, more preferably from about 0.31 mg/L to about 2.82 mg/L, more preferably from about 0.31 mg/L to about 1.31 mg/L, more preferably from about 0.57 to about 1.31 mg/L. In certain embodiments, the daily dose is from about 0.67 mg/kg to about 12 mg/kg, more preferably from about 0.2 to about 4.05 mg/kg, wherein the maximum As concentration is about 0.57 mg/L to about 1.31 mg/L, and wherein the platform As concentration in blood (plasma) is from about 0.03 mg/L to about 0.07 mg/L. In certain embodiments, the PANDA Agent is ATO, As2S2, As2S3, As2S5, and As4S4.
The PANDA Agent comprising a three and/or five valence antimony is generally effective in treating cancer in a human at a wide range of dosages by intravenous injection and oral administration. In certain embodiments, the effective dose results in a maximum Sb concentration in the patient's blood (plasma) from about 3.58 mg/L to about 357.5 mg/L, preferably from about 3.58 mg/L to about 179 mg/L, more preferably from about 7.15 mg/L to about 179 mg/L, more preferably from about 7.15 mg/L to about 107 mg/L, more preferably from about 12 mg/L to about 107 mg/L, more preferably from about 32.7 to about 38.8 mg/L. In certain embodiments, the daily dose is from about 20 mg/kg, wherein the maximum Sb concentration is from about 32.7 mg/L to about 38.8 mg/L, and wherein the platform Sb concentration in blood (plasma) is about 3.5 mg/L.
The PANDA Agent comprising a three and/or five valence bismuth is generally effective in treating cancer in a human at a wide range of dosages by intravenous injection and oral administration. In certain embodiments, the effective dose results in a maximum Bi concentration in the patient's blood (plasma) from about 3 mg/L to about 300 mg/L, preferably from about 3 mg/L to about 150 mg/L, more preferably from about 6 mg/L to about 150 mg/L, more preferably from about 6 mg/L to about 90 mg/L, more preferably from about 10 mg/L to about 90 mg/L, more preferably from about 30 mg/mL. In certain embodiments, the daily dose is from about 20 mg/kg, wherein the maximum Bi concentration is from about 32.7 mg/L to about 38.8 mg/L, and wherein the platform Bi concentration in blood (plasma) is about 3.5 mg/L.
We further discovered that combining ATO and other approved drugs can be effective to treating cancer. For example, we found the combination therapy of ATO and a DNA-damaging agents can treat patients with AML and MDS. Results from our phase I Decitabine (“DAC”)—ATO combination therapy trial for Myelodysplastic Syndrome (DMS) showed complete remission for the two patients that harbored rescuable mp53s (Table 11 and
We also identified patient #19, who harbored wtp53 during initial screening, but later developed DAC treatment related rescuable p53-Q038H and p53-Q375X on the 8th month of the DAC mono-treatment (see
Taken together, we have discovered that ATO is effective in treating cancer patients, such as MDS patients, particularly those harboring mp53s rescuable mutation. We further discovered that the efficacy of treatment can be improved by (1) obtaining a sample from the patient and sequencing patient's p53, (2) determining whether the mp53 is rescuable or not, and (3) administering an effective amount of one or more PANDA Agent, such as ATO and/or other drug candidates alone or in combination with other effective cancer drugs to the patient; selecting patients with p53 mutations on residues most responsive to ATO, such as mutations on S241C and S241F. Importantly, we have determined that the ATO rescuable mp53 includes: R175H, R245S, R248Q, R249S, R282W, I232T, F270C, Y220H, I254T, C176F, H179R, Y220C, V143A, S033P, D057G, D061G, Y126C, L130H, K132M, A138V, G154S, R156P, A159V, A159P, Y163H, Y163C, R174L, C176Y, H179Y, C238Y, G245A, G245D, R248W, G266R, F270S, D281H, D281Y, R283H, F054Y, SO90P, Q375X, Q038H, R156P, A159V, A159P, Y163H, Y163C, R174L, C176Y, H179Y, H179Q, P190L, H193R, R209K, V216E, Y234H, M2371, V272M, S241A, S241C, S241D, S241E, S241F, S241G, S241H, S2411, S241L, S241M, S241N, S241P, S241Q, S241R, S241T, S241V, S241W, and S241Y (see Table 8, mp53s that are indicated as either structurally rescuable or functionally rescuable). Additionally, we have determined that the ATO non-rescuable mp53s includes: R273H, R273C, R278S, S006P, L014P, Q052R, P072A, P080S, T081P, S094P, S095F, R273S, R273L, P278H, L383P, M384T, S241K (see Table 8 mp53s that are indicated as neither structurally rescuable nor functionally rescuable).
mp53 is associated with considerably poor overall survival and prognosis of a wide range of cancers, including myeloid leukemia (AML/MDS) patients (Cancer Genome Atlas Research et al., 2013; Lindsley et al., 2017). Under NCCN guidelines, the majority of recommended AMLMDS treatments, aside from APL, are DNA-damaging agents. These DNA-damaging agents are known to activate wtp53 function to kill cancer cells through p53 post-translational modifications (“PTM”s) (Murray-Zmijewski et al., 2008). These PTMs include, for example, phosphorylation, acetylation, sumoylation, neddylation, methylation, and ubiquitylation.
Our discovery further shows that PANDA Agent ATO can be used for a wide range of ATO-responsive cancers in clinical trials. It is preferred that patient recruitment follow a specific, highly precise, recruitment prerequisite, in order to achieve maximum efficacy. While ATO was approved by FDA to treat acute promyelocytic leukemia (APL), a subtype of leukemia and intensively trialed, with the aim to broaden its application to non-APL cancer types over the past two decades, it has not yet been approved for this purpose. This is largely attributed to a failure to reveal an ATO-affecting cancer spectrum. Indeed, no mp53 dependency can be observed in the sensitivity profile of ATO on the NCI60 cell panel simply by differentiating lines into a mp53 group and a wtp53 group. Non-ATO rescue compounds were also extensively researched and some were identified, including, CP-31398; PRIMA-1; PRIMA-1-MET; SCH529074; Zinc; stictic acid, p53R3; methylene quinuclidinone; STIMA-1; 3-methylene-2-norbomanone; MIRA-1; MIRA-2; MIRA-3; NSC319725; NSC319726; SCH529074; PARP-PI3K; 5,50-(2,5-furandiyl)bis-2-thiophenemethanol; MPK-09; Zn-curc or curcumin-based Zn(II)-complex; P53R3; a (2-benzofuranyl)-quinazoline derivative; a nucleolipid derivative of 5-fluorouridine; a derivative of 2-aminoacetophenone hydrochloride; PK083; PK5174; and PK7088. However, they have low rescue efficacy.
The PANDA Agents we identified and described herein, including the PANDA Agents with Formulation I-XV, the PANDA Agents listed in Table 1-Table 6, and PANDA Agents listed in Table 7 show exceptional efficacy in rescuing mp53 with rescuable mutations (for example, those listed in Table 8) in vitro and in vivo, among others. Many of them have structures that are significantly different from ATO and have not previously been proposed for use in treating a p53 disorder. By separating rescuable mp53s from in a pool of patients with a p53 disorder, we have, for the first time, discovered a compound and method to effectively treat multiple types of p53 disorders, including multiple classes of cancers. The size of the class is considerably large, covering an estimated amount of 15%-30% cancer cases. As discussed, this is partly because p53 is one of the most important protein in cell biology and is implicated in wide range of disorders. For example, we have identified at least 4 of the 6 hotspot mp53s and a large number of non-hotspot mp53s to be efficiently rescuable by ATO and PANDA.
Our personalized treatment separates those patients suitable for treatments with PANDA Agent and those who are not. By selecting those patients with rescuable mp53, we can begin to treat patients based on p53 mutation rather than cancer type. It is known that different missense mutations will confer different activities to mp53 (Freed-Pastor and Prives, 2012), which can lead to different treatment outcomes in patients harboring different mp53s. Accordingly, others like us advocate tailoring treatments to the types of p53 mutations present rather than simply whether mp53 or wtp53 is present (Muller and Vousden, 2013, 2014). However, a compound that can effectively treat and rescue mp53 was not identified until now. Remarkably, our discoveries on the MDS patient-derived p53-S241F, p53-S241C as well as the other artificially generated p53 mutants on S241 support that PANDA Agents rescuing efficiency is determined not only by the p53 mutation site but also by the new residue generated (
We have previously described a method of determining whether a mp53 is rescuable or not by IP or functional assays. However, these procedures must be done in a professional laboratory, and is time consuming and costly. The method of determining whether a mp53 is rescuable by determining whether a rescuable mp53 is present in the subject, as described herein, greatly improves the efficiency and financial burden for the subject.
In addition to use in humans, results from our animal studies also support using PANDA agent to treat a p53 disorder, such as cancer, for veterinary use, for example, in such as a mouse, dog, a cat, and other companion animals, a cattle and other livestock, a wolf, a panda bear, or other zoo animals, and a horse or other equines
Additionally, we discovered that mp53 (for example, p53-R175H) and PANDA (for example, PANDA-R175H) responded differently to the DNA-damaging agents, such as Cisplatin, Etoposide, Adriamycin/Doxorubicin, 5-Fluorouracil, Cytarabine (ara-C), Azacitidine, and Decitabine(DAC), suggesting they may trigger distinctly treatment outcomes. We discovered Ser15, Ser37, and Lys382 were inertly modified on p53-R175H upon DNA-damaging treatment; however, they behave like wtp53 in that they are actively modified on PANDA-R175H upon DNA-damaging treatment (
The characteristics of PANDA-forming reactions include the following:
The characteristics of ATO mediated folding include:
As disclosed herein, we discovered that (1) that ATO can function synergistically with other cancer inhibition therapies, (2) that combination anticancer therapy containing ATO has significant promises, and (3) that ATO may increase the efficacy of the wtp53-reactivating agents, such as MDM2 inhibitors, many of which are currently under clinical trials (
pcDNA3.1 expressing human full length p53 was gift from Prof. Xin Lu (the University of Oxford), pGEX-2TK expressing fusion protein of GST and human full length p53 was purchased from Addgene (#24860), pET28a expressing p53 core was cloned for crystallization experiment without introducing any tag.
Primary antibodies were purchased from the following companies: DO1 (ab1101, Abcam), PAb1620 (MABE339, EMD Millipore), PAb240 (OP29, EMD Millipore), PAb246 (sc-100, Santa Cruz), PUMA (4976, Cell signaling), PIG3 (ab96819, Abcam), BAX (sc-493, Santa Cruz), p21 (sc-817, Santa Cruz), MDM2 (OP46-100UG, EMD Millipore), Biotin (ab19221, Abcam), Tubulin (ab11308, Abcam), β-actin (A00702, Genscript), p53-S15 (9284, Cell signaling), p53-S20 (9287, Cell signaling), p53-S37 (9289, Cell signaling), p53-S392 (9281, Cell signaling), p53-K382 (ab75754, Abcam), KU80 (2753, Cell signaling). CM5 antibody was gift from Prof. Xin Lu. HRP conjugated secondary antibody specifically reacts with light chain was from Abcam (ab99632).
H1299 and Saos-2 cell lines expressing null p53 was gift from Prof. Xin Lu. H1299 cell lines expressing tet-off regulated p53-R175H or tet-on regulated wtp53 were prepared as reported previously (Fogal et al., 2005). MEFs were prepared from E13.5 TP53-/- and TP53-R172H/R172H embryos. The other cell lines were obtained from ATCC.
Compounds were purchased from the following companies: DMSO (D2650, sigma), CP31398 (PZ0115, sigma), Arsenic trioxide (202673, sigma), STIMA-1 (506168, Merck Biosciences), SCH 529074 (4240, Tocris Bioscience), PhiKan 083 (4326, Tocris Bioscience), MiRA-1 (3362, Tocris Bioscience), Ellipticine (3357, Tocris Bioscience), NSC 319726 (S7149, selleck), PRIMA-1 (S7723, selleck), RITA (NSC 652287, S2781, selleck), Cycloheximide (C7698, sigma), Biotin (A600078, Sangon Biotech), Doxycycline hyclate (D9891, sigma), Cisplatin (CIS, P4394, sigma), Etoposide (ETO, E1383, sigma), Adriamycin (ADM, S1208, selleck), 5-Fluorouracil (5-FU, F6627, sigma), Cytarabine (ARA, S1648, selleck), Azacitidine (AZA, A2385, sigma), Decitabine (DAC, A3656, sigma). Bio-As and Bio-Dithi-As were gift from Kenneth L. Kirk (NIH; PMID: 18396406).
The TP53 wild-type mice, female nude mice and NOD/SCID mice were obtained from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. TP53-R172H/R172H mice were generated from the parent mice (026283) purchased from Jackson Lab. TP53−/− mice (002101) were purchased from National Resource Center of Model Mice of China.
DNA samples were sequenced in rainbow-genome technique Ltd (Shanghai) and Shanghai Biotechnology corporation (Shanghai).
Constructions expressing recombinant TP53 core domain were transformed into E. coli strain BL21-Gold. Cells were cultured in either LB or M9 medium at 37° C. to mid-log phase. 0.5 mM isopropyl-p-D-thiogalactopyranoside (IPTG) was added in presence/absence of 50 μM As/Sb/Bi and 1 mM ZnCl2 at 25° C. for overnight. Cells were harvested by centrifugation at 4 000 RPM for 20 minutes (˜10 g cell paste yielded from 1 liter of medium) and then sonicated in lysate buffer (50 mM Tris, pH 7.0, 50 mM NaCl, 10 mM DTT and 1 mM phenylmethylsulfonyl fluoride) in presence/absence of 50 μM As/Sb/Bi. Soluble lysate was loaded onto a SP-Sepharose cation exchange column (Pharmacia) and eluted with a NaCl gradient (0-1 M) then, if necessary, additionally purified by affinity chromatography with a heparin-Sepharose column (Pharmacia) in Tris.HCl, pH 7.0, 10 mM DTT with a NaCl gradient (0-1 M) for elution. Future purification was performed by gel-filtration using Superdex 75 column using standard procedure.
Processes after cell lysing are done at 4° C. Protein concentration was measured spectrophotometrically by using an extinction coefficient of 16 530 cm−1M−1 at 280 nm. All protein purification steps were monitored by 4-20% gradient SDS-PAGE to ensure they were virtually homogeneous.
Constructions expressing GST-p53 (or GST-mp53) were transformed into E. coli strain BL21-Gold. Cells were grown in 800 ml LB medium at 37° C. to mid-log phase. 0.3 mM IPTG with/without 50 μM As/Sb/Bi was added at 16° C. for 24 h. Cells were harvested by centrifugation at 4 000 RPM for 20 minutes and then sonicated in 30 ml lysate buffer (58 mM Na2HPO4.12H2O, 17 mM NaH2 PO4.12H2O, 68 mM NaCl, 1% Triton X-100) in presence/absence of 50 μM As/Sb/Bi. Cell supernatant after 9000 RMP for 1 hour was added with 400 μl glutathione beads (Pharmacia) and incubated overnight. Beads were washed with lysate buffer for 3 times. Recombinant protein was then eluted by 300 μl elution buffer (10 mM GSH, 100 mM NaCl, 5 mM DTT and 50 mM Tris-HCl, pH 8.0). Processes after cell lysing are done at 4° C. All protein purification steps were monitored by 4-20% gradient SDS-PAGE to ensure they were virtually homogeneous.
Baculovirus infected Sf9 cells expressing recombinant human full-length p53 or p53 core in presence/absence of 50 μM As/Sb/Bi were harvested. They lysed in lysate buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% NP-40.5 mM DTT, 1 mM PMSF, and 0.15 M NaCl) in presence/absence of 50 μM As/Sb/Bi. The lysates were then incubated on ice for 30 min, followed by centrifuging at 13000 rpm for 30 min. The supernatant was diluted 4-fold using 15% glycerol, 25 mM HEPES, pH 7.6, 0.1% Triton X-100, 5 mM DTT and 1 mM Benzamidine. They were further filtered using a 0.45 mm filter, and purified by Heparin-Sepharose column (Pharmacia). Purified protein was then concentrated using YM30 Centricon (EMD, Millipore). All protein purification steps were monitored by 4-20% gradient SDS-PAGE to ensure they were virtually homogeneous.
PANDA can be efficiently formed by mixing p53, either purified p53 or p53 in cell lysate, with one or more PANDA Agent. For example, in reaction buffer (20 mM HEPES, 150 mM NaCl, pH 7.5), we mixed purified recombinant p53 core and As/Sb/Bi compounds in a ratio ranging from 10:1-1:100 at 4° C. for overnight. The formed PANDA was then purified using dialysis to eliminate compounds.
50 μM purified recombinant protein GST-p53-R175H in reaction buffer (10 mM GSH, 100 mM NaCl, 5 mM DTT and 50 mM Tris-HCl, pH 8.0) was added with Biotin-As to obtain arsenic to p53 molar ratio of either 10:1 or 1:1. The mixture solution was incubated at 4° C. for overnight and then divided into three parts. Each part was subjected to SDS-PAGE, followed by Coomassie blue staining (5 μg GST-p53-R175H applied), p53 immunoblotting (0.9 μg GST-p53-R175H applied) or Biotin immunoblotting (5 μg GST-p53-R175H applied), respectively.
For immunoprecipitation, mammalian cells or bacteria cells were harvested and lysed in NP40 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40) with cocktail of protease inhibitors (Roche Diagnostics). Cell lysates were then sonicated for 3 times, followed by spinning at 13,000 RPM for 20 min. Supernatant was adjusted to a final concentration of 1 mg/ml total protein using 450 μl NP40 buffer and incubated with 20 μl protein G beads and 1-3 μg corresponding primary antibody for 2 hr at 4° C. The beads were washed for three times with 20-25° C. NP40 buffer at room temperature. After spinning down, the beads were boiled for 5 min in 2×SDS loading buffer, followed by Western blotting.
Cells were treated with 4 μg/ml Bio-As or Bio-dithi-As for 2 hours. Cells were lysed in NP40 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40) with cocktail of protease inhibitors (Roche Diagnostics). Cell lysates were then sonicated for 3 times, followed by spinning at 13,000 RPM for 1 hr. Supernatant was adjusted to a final concentration of 1 mg/ml total protein using 450 μl NP40 buffer and incubated with 20 μl streptavidin beads for 2 hr at 4° C., followed by bead washing and Western blotting.
To prepare double-stranded oligonucleotides, equal amount of complementary single stranded oligonucleotides were heated at 80° C. for 5 min in 0.25 M NaCl, followed by slow cooling to room temperature. Sequences of single stranded oligonucleotides were followed:
Cells were harvested and lysed in NP40 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40) with cocktail of protease inhibitors (Roche Diagnostics). Cell lysates were then sonicated for 3 times, followed by spinning at 13,000 RPM for 1 hr. Supernatant was adjusted to a final concentration of 1 mg/ml total protein using 450 μl NP40 buffer and incubated with 20 μl streptavidin beads (s-951, Invitrogen), 20 pmoles of biotinylated double-stranded oligonucleotides, and 2 μg of poly(dl-dC) (sc-286691, Santaz cruz). Lysates were incubated for 2 hr at 4° C., followed by bead washing and immunoblotting.
Immunoblotting was performed as reported previously (Lu et al., 2013).
Cells were plated at a concentration of 2×104 cells/well in 24-well plates, followed by transfection of luciferase reporter plasmids for 24 hr. All transfection contained 300 ng p53 expressing plasmid, 100 ng of luciferase reporter plasmid and 5 ng of renilla plasmid per well. After agent treatment, cells were lysed in luciferase reporter assay buffer and determined using a luciferase assay kit (Promega). Activities of luciferase were divided by that of renilla to normalize the transfection efficiency. For more details, see (Lu et al., 2013).
Treated cells were digested with trypsin. 100, 1000 or 10,000 cells/well were seeded in 12-well plates and kept in culture for 2-3 weeks. Fresh medium was replaced every three days.
Cells were lysed in either CHAPS buffer (18 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid in TBS) or M-PER buffer (78501, Invitrogen) containing DNase and protease inhibitors for 15 min at 4° C. or 37° C. Cell lysate was added with 20% glycerol and 5 mM Coomassie G-250 before loading into 3-12% Novex Bis-Tris gradient gels. The electrophoresis was performed at 4° C. according to the manufacturer's instructions. Proteins were transferred onto the polyvinylidene fluoride membranes and fixed with 8% acetic acid for 20 min. The fixed membranes were then air dried and destained with 100% methanol. Membranes were blocked for overnight with 4% BSA in TBS at 4° C. before immunoblotting.
Total RNA was isolated from cells using Total RNA Purification Kit (B518651, Sangon Biotech). 1 μg total RNA was reverse-transcribed using the GoScript®™ Reverse Transcriptase System (A5001, Promega) following manufacturer's protocol. PCR was performed in triplicate using SYBR green mix (Applied Biosystems), and a ViiA™ 7 Real-Time PCR System (Applied Biosystems) under the following conditions: 10 min at 95° C. followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. Specificity of the PCR product was checked for each primer set and samples from the melting curve analysis. Expression levels of targeted genes were normalized relative to levels of β-actin adopting comparative Ct method. The primer sequences are as follows: MDM2 forward 5′-CCAGGGCAGCTACGGTTTC-3′, reverse 5′-CTCCGTCATGTGCTGTGACTG-3′; PIG3 forward 5′-CGCTGAAATTCACCAAAGGTG-3′, reverse 5′-AACCCATCGACCATCAAGAG-3′; PUMA forward 5′-ACGACCTCAACGCACAGTACG-3′, reverse 5′-TCCCATGATGAGATTGTACAGGAC-3′; p21 forward 5′-GTCTTGTACCCTTGTGCCTC-3′, reverse 5′-GGTAGAAATCTGTCATGCTGG-3′; Bax forward 5′-GATGCGTCCACCAAGAAGCT-3′, reverse 5′-CGGCCCCAGTTGAAGTTG-3′; #-actin forward 5′-ACTTAGTTGCGTTACACCCTTTCT-3′, reverse 5′-GACTGCTGTCACCTTCACCGT-3′.
H1299 xenograft. H1299 cells expressing tet-off regulated p53-R175H (1*106 cells) suspended in 100 μl saline solution were subcutaneously injected into the flanks of 8-9 weeks old female nude mice. When the tumor area reached 0.1 cm (day 1), 5 mg/kg ATO were intraperitoneally injected 6 consecutive days per week. In DOX groups, 0.2 mg/ml doxycycline was added to drinking water. Tumor size was measured every 3 days with vernier callipers. Tumor volumes were calculated using the following formula: (L*W* W)/2, in which L represents the large diameter of the tumor, and W represents the small diameter. When tumor area reached −1 cm diameter in any group, mice were sacrificed and isolated tumors were weighed. The analysis of the differences between the groups was performed by Two-way RM ANOVA with Bonferroni correction.
CEM-C1 xenograft. 8-9 week old NOD/SCID mice were intravenously injected through the tail vein with 1*107 cells of CEM-C1 T-ALL cells (day 1). After engraftment, peripheral blood samples were obtained from the mice retro-orbital sinus every 3 or 4 days from day 16 to day 26. Residual red blood cells were removed using erythrocyte lysis buffer (NH4Cl 1.5 mM, NaHCO3 10 Mm, EDTA-2Na 1 mM). The isolated cells were double stained with PerCP-Cy5.5-conjugated anti-mouse CD45 (mCD45) (BD Pharmigen™, San Diego, Calif.) and FITC-conjugated anti-human CD45 (hCD45) (BD Pharmigen™, San Diego, Calif.) antibodies before flow cytometric analysis conducted. When the percent of hCD45+ cells in peripheral blood reached 0.1% one mice (day 22), ATO was prepared for injection. On day 23, 5 mg/kg ATO were intravenously injected via tail-vein in 0.1 ml saline solution 6 consecutive days per week. The comparison of the hCD45+ cells percent between the groups was performed by unpaired t test. The life-span of mice was analyzed by Log-rank (Mantel-Cox) test.
All statistical analysis was performed using GraphPad Prism 6.00 for Windows (La Jolla Calif., USA). The animals were housed in specific pathogen-free conditions. Experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Statistical analysis was carried out using Fisher's exact test (two-tailed) unless otherwise indicated. p values less than 0.05 were considered statistically significant unless otherwise indicated.
aPatient No. 19 was DAC-resistant and AML -prone
The following publications, references, patents and patent applications are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/070117 | 1/2/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2018/085190 | Apr 2018 | US |
| Child | 16959894 | US | |
| Parent | PCT/CN2018/070051 | Jan 2018 | US |
| Child | PCT/CN2018/085190 | US |
