WO2015023553A2 - Modification thérapeutique multi-cibles, orientée de façon hétérogène, concomitante et/ou modulation d'une maladie par administration de petites molécules acide aminé-spécifiques, contenant du soufre - Google Patents

Modification thérapeutique multi-cibles, orientée de façon hétérogène, concomitante et/ou modulation d'une maladie par administration de petites molécules acide aminé-spécifiques, contenant du soufre Download PDF

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WO2015023553A2
WO2015023553A2 PCT/US2014/050465 US2014050465W WO2015023553A2 WO 2015023553 A2 WO2015023553 A2 WO 2015023553A2 US 2014050465 W US2014050465 W US 2014050465W WO 2015023553 A2 WO2015023553 A2 WO 2015023553A2
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cancer
group
homocysteine
amino acid
sulfur
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WO2015023553A3 (fr
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Frederick H. Hausheer
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Bionumerik Pharmaceuticals, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel pharmaceutical compositions, methods, and kits used for the treatment of cancer and other medical conditions. More specifically, the present invention relates to novel pharmaceutical compositions, methods, and kits comprising medicaments used for the treatment of cellular metabolic anomalies such as cancer or other undesirable physiological conditions where the normal cellular biochemical function and/or the expression levels of various proteins (i.e., target molecules of the present invention) are abnormal and must be modified and/or modulated in order to treat these metabolic anomalies.
  • the aforementioned target molecules include: protein tyrosine kinases, DNA synthesis and repair proteins, structural proteins, oxidoreductases, and various other classes of proteins/enzymes.
  • the present invention discloses and claims methods and kits for (a) the contemporaneous modification/modulation of multiple target molecules; (b) the treatment of cancer and other undesirable physiological conditions; (c) the selection of subjects for treatment; (d) the determination of the most effective medicinal agent(s) to be administered in combination with the administration of the sulfur- containing, amino acid-specific small molecules of the present invention; (e) the dosage of the medicinal agent(s) to be administered; (f) the determination of the length and/or number of treatment cycles; (g) the adjustment of the specific medicinal agent(s) used and the dosage administered during treatment; and/or (h) ascertaining the potential treatment responsiveness of the specific abnormal metabolic condition to the medicinal agent(s) selected for administration to a subject suffering from one or more types of cancer or other undesirable physiological conditions by quantitatively determining: (i) the abnormal biochemical activity and/or (ii) the level of abnormal expression of any combination of the target molecules of the present invention.
  • the teachings in the present application take into account the concept of disease heterogeneity, in combination with new observations and data, in order to provide novel methods, pharmaceutical compositions, and kits used for the treatment of cancer and other medical conditions.
  • the present invention discloses methods and compositions to contemporaneously modulate and interact with multiple target molecules in order to provide treatment for a variety of cellular metabolic anomalies or other undesirable physiological conditions.
  • cancer is a heterogeneous disease and involves the complex interaction of numerous genes, enzymes/proteins, and metabolic pathways. Accordingly, as cancer progression becomes characterized as a genome -mediated macro-evolution (rather than a gene-centric developmental process), a change of research and drug development strategy is required in order to more fully treat the disease.
  • cancer progression is an evolutionary process where genome system replacement (rather than a common
  • genetic/epigenetic system behavior ⁇ i.e., their stability or instability
  • unpredictable replacement and switching that occurs between various pathways during cancer progression and, in particular, during subsequent medical intervention.
  • the sulfur-containing, amino acid-specific small molecules of the present invention possess the ability to contemporaneously or simultaneously modify and/or modulate: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of a multiple number of enzymes/proteins (i.e., target molecules).
  • target molecules include, but are not limited to, anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif.
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 receptor tyrosine kinase
  • EGFR epidermal growth factor receptor
  • Prx peroxiredoxin
  • ERCC1 excision repair cross-complementing protein 1
  • IGF1R insulin growth factor 1 receptor
  • RNR ribon
  • abnormal expression or increased catalytic activity, or both
  • diseases mediates a multi-component, multi-pathway mechanism which confers a survival advantage to cancer cells.
  • This abnormal expression/increased levels in cancer cells can lead to several important biological alterations including, but not limited to: (i) loss of apoptotic sensitivity to therapy (i.e., drug or ionizing radiation resistance); (ii) increased conversion of RNA into DNA (involving ribonucleotide reductase); (iii) altered gene expression; (iv) increased cellular proliferation signals and rates; and/or (v) increased angiogenic activity (i.e., increased blood supply to the tumor).
  • contemporaneous or simultaneous modification and/or modulation of the aforementioned target molecules by the administration of effective levels and schedules of the sulfur-containing, amino acid-specific small molecules of the present invention can result in substantial improvements in the effects of cancer treating agents along with substantially improved outcomes for patients.
  • non-cancer-related metabolic anomalies or other undesirable physiological conditions that exhibit evidence of: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of the same aforementioned target molecules.
  • These metabolic anomalies or other undesirable physiological conditions include, but are not limited to, heart failure, heart disease, hypertension, myocardial infarction, vascular disease, atherosclerosis, diabetes-induced heart disease, neurodegenerative diseases, Parkinson's disease, ALS, neurovascular dementia, autoimmune diseases, systemic lupus erythematosus, Graves orbitopathy, alcoholic liver disease, inflammatory bowel disease, cystic fibrosis, inflammatory diseases, diabetes, rheumatoid arthritis, progeria, Xeroderma pigementosum, Cockayne syndrome, Fanconi anemia, and cerebro-oculo-facio-skeletal syndrome.
  • the administration of the sulfur-containing, amino acid-specific small molecules of the present invention can also be effective in the treatment of these metabolic anomalies or
  • the sulfur-containing, amino acid-specific small molecules of the present invention may be administered using any combination of the following three general treatment methods in order to attain the full benefit of their contemporaneous and multi-targeted characteristics: (i) in a direct inhibitory or inactivating manner (i.e., direct chemical interactions) by, e.g., Tavocept-mediated xenobiotic modification of Cys residues that inactivates one or more of the aforementioned target molecules and/or a direct depletive manner (i.e., decreasing target molecule concentrations or production rates), thereby increasing the susceptibility of the cancer cells to any subsequent administration of any cancer treating agent or agents that may act directly or indirectly through the target molecule-mediated pathways in order to sensitize the patient's cancer and thus increase the survival of the patient; (ii) in a synergistic manner, where the target molecule-specific therapy is concurrently administered with chemotherapy administration when a cancer patient begins any chemotherapy cycle, in order to increase and optimize the pharmacological activity directed against target molecule-mediated mechanisms present while chemotherapy is
  • Cancer is a highly complex, diverse disease that is heterogeneous, rather than homogeneous.
  • the fact that some specific types of cancer are microscopically heterogeneous has been known for over a century; whereas chromosomal and molecular heterogeneity was subsequently discovered as a direct result of the more recent technological advances in cellular and molecular biology.
  • Further studies have shown that although cancer in a given individual may start in a clonal manner, subsequent mutations frequently occur due to the "pressures" exerted upon said cancer by the use of therapy (e.g., chemotherapy, radiation, and the like) and/or various other evolutionary factors that can lead to metastases or therapeutic resistance.
  • therapy e.g., chemotherapy, radiation, and the like
  • a synopsis of the aforementioned study's findings include: (i) phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63-69% of all somatic mutations not being detectable across every region of the tumor; (ii) intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating other molecular control points (e.g., other kinase activity); (iii) mutational intratumor heterogeneity was seen for multiple tumor suppressor genes converging on loss of function with multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic tumor evolution; (iv) gene expression signatures of both good and poor prognosis were detected in different regions of the same tumor; and (v) allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four different tumors all harboring divergent allelic-imbal
  • tumorigenicity the evolutionary mechanism of cancer. J. Cell Physiol. 219:288-300 (2009).
  • cancer progression is an evolutionary process where genome system replacement (rather than a common pathway) is the driving force.
  • genome system replacement rather than a common pathway
  • heterogeneity is a key inate feature of cancer and it is not practical to apply diverse genetic/epigenetic patterns to clinical usage that requires precise prediction.
  • Table 1 summarizes examples of some of the key features of heterogeneity that exist at multiple genetic and epigenetic levels. It should be noted that the list of elements enumerated in Table 1 that contribute to system heterogeneity is growing rapidly, and even more importantly, each element has the ability to interact with other elements, thus forming an almost unlimited combinational heterogeneity.
  • MicroRNAs and cancer go a longway. Cell 136:586-591 (2009).
  • heterogeneity is the reason that universal mutations can not be identified. This is illustrated by the finding that, in a majority of cases of the same type of cancer, most patients display a unique array of mutations that only have minimal inter-patient homogeneity. In a highly dynamic complex biological system, such as cancer, any given pattern might represent only a limited number of cases, as cancer cases are genetically- and environmentally-contingent. The pattern of specific gene mutations can only be used within a specific population with a similar genome, mutational composition, and a similar environment.
  • Stepwise Cancer Development Versus Stochastic Macro-Evolution As judged by drastically different karyotypes and gene mutation profiles, each tumor seems to represent one independent run of somatic evolution and does not follow a stepwise reproducible pattern. This situation is different from the natural evolution (comprised of only one run of evolution) that is familiar in non-neoplastic tissues. When tracing natural evolution, many key genes can be traced from model organisms. However, this clearly does not apply to the majority of cancers, as it would be difficult to trace the same gene mutations or ultraconserved regions among cases that evolved during different runs of evolution. See, e.g., Heng, H.H. The genome-centric concept: re-synthesis of evolutionary theory.
  • cancer progression is fundamentally different from developmental processes.
  • Developmental progression refers to the well controlled process of self-organization (both temporally and spatially) where many key genes play a crucial role; whereas in cancer evolution, even though some cases involve parts of the developmental process, in a majority of cancer cases, the dominant alterations are genome mediated stochastic system replacement, which does not follow a well controlled pattern. Accordingly, researchers argue that the terminology "cancer development” implies an incorrect concept and needs to be altered.
  • cancer progression is characterized as genome mediated macro-evolution (rather than micro-evolution or a developmental process), it requires a change of research strategy. Heavily influenced by reductionism's view, most of the molecular analyses of cancer have been focused on a particular molecule of interest, without considering the overall status of the genome system. It has been generally assumed during molecular manipulation or specific targeting that the biological system remains the same. For many exsisting approaches, this assumption has been pushed to the extreme, where genome level information has become largely ignored by most of the molecular analyses. However, this is an erroneous assumption, as when the overall karyotype changes, the role of the same gene may also be altered, as the function of genes are dependent upon their genetic network which is defined by the genome context.
  • the p53 pathway has been linked to diverse molecular mechanisms or pathways and at least 50 different enzymes can covalently modify p53 to alter its function and several thousand genes have been shown to be directly regulated by p53. See, e.g., Kruse, J., Gu, W. SnapShot: p53 posttranslational modifications. Cell 133 :930-930 (2008). Interestingly, each of these characterized functions represents one possible potential function defined by the genome context, including epigenetic regulation of the same genome but different tissue type.
  • NCCAs Non-Clonal Chromosome Aberrations
  • NCCAs non-clonal chromosome aberrations
  • chromosome aberrations See, e.g., Heng, H.H., Bremer, S.W., et al. Cancer progression by non-clonal chromosome aberrations. J. Cell Biochem. 98: 1424-1435 (2006).
  • NCCAs have previously been thought of as background "noise" with no biological significance, as there seemed to be no clear pattern according to the concept of clonal expansion.
  • the system control principle may be effectively used to study cancer systems. Specifically, it is hypothesized that by using a system control approach, the system dynamics could be measured by determining the levels of seemingly random motion within the system.
  • NCCAs When the presence of NCCAs were selected as a method to measure genome system instability, increased frequencies of NCCAs were detected from genetically unstable cell lines, including inherently genetically unstable lines, stable lines induced to be unstable by various treatments, or stable lines with over-expressed cancer genes. Moreover, numerous factors that contribute to biological system instability have also been linked to increased NCCAs. Table 2 below enumerates a number of factors that can cause an increase in NCCA frequencies.
  • NCCAs When a biological system is unstable, increased dynamics can be detected at multiple levels of the genetic and epigenetic organization. For example, increased NCCAs were observed in cell lines with increased open chromatin structure. See, e.g., Dunn, K.L., He, S., et al. Increased genomic instability and altered chromosomal protein phosphorylation timing in HRAS transformed mouse fibroblasts. Genes Chromosomes Cancer 48:397-409 (2009). Therefore, by comparing various causes and the potential biological functions of NCCAs, it is clear that NCCAs reflect increased system dynamics and indeed characterize genome system heterogeneity.
  • NCCAs The Level of NCCAs Reflects Both Internally- and Externally-Induced Instability
  • in vitro immortalization model there are two phases of karyotypic evolution that have been observed called the "punctuated” and “stepwise” phases. See, e.g., Heng, H.H., Bremer, S.W., et al. Cancer progression by non-clonal chromosome aberrations. J. Cell Biochem. 98: 1424-1435 (2006).
  • NCCAs non-clonal chromosome aberrations
  • CCAs transitional clonal chromosomal aberrations
  • a given CCA dominates with low levels of NCCAs. Since the increased level of NCCAs reflects a system's instability, it is clear that such population instability can be generated either by internal changes ⁇ e.g., shortening of telomeres, loss of system constraints, and the like) or by environmentally-induced stress ⁇ e.g., chemotherapy-treatment, culture conditions, and the like).
  • NCCAs can be used as an index to measure instability or population diversity and can be a determinant of the system's stage.
  • instability can lead to heterogeneity
  • heterogeneity can reflect levels of instability, both of them can be measured by the level of NCCAs.
  • heterogeneity might be able to further generate instability, as heterogeneity itself might function as a stress applied to a system.
  • heterogeneity are, at a minimum, very-closley linked and may even refer to essentially to the same thing.
  • the pattern of NCCAs also illustrates the difference between normal tissue and cancer tissue. In non-cancerous tissue, there is a balance between stability and heterogeneity such that the frequency of NCCAs is very low. In contrast, for cancer progression and drug resistance to occur, it is believed that a less stable status has to form, coupled with increased heterogeneity.
  • NCCAs non-clonal chromosome aberrations
  • the pre-conditions for the occurrence of cancer evolution are initiated by an unbalanced relationship between system heterogeneity and homeostasis; wherein system homeostasis can be considered an opposite force to system heterogeneity.
  • system homeostasis can be considered an opposite force to system heterogeneity.
  • the multiple levels of homeostasis is the system constraint that prevents somatic macro-evolution.
  • non-genetic features are defined by genome context, as different species display different networks and different potential responses towards stress, as well as different profiles of epigenetic patterns (i.e., most of the epigenetic landscape is determined by the genome). See, e.g., Heng, H.H. The genome-centric concept: re-synthesis of evolutionary theory. BioEssays 31 :512-525 (2009). More importantly, when examining biological evolution (e.g., cancer progression), one must remember that inheritance is a key player in the evolutionary process; as without inheritance there would be no Darwinian evolution. Lastly, many levels of homeostasis/heterogeneity are clearly linked to the genetic stability/instability of the system.
  • epigenetic alteration is an initial response when the genome system is under stress (see, e.g., Feinberg, A.P., Ohlsson, R., Henikoff, S. The epigenetic progenitor origin of human cancer. Nat. Rev. Genet. 7:21-32 (2006)), which provides an increased probability for evolution dynamics to occur within a given genome context.
  • changes are selected by the evolutionary process, these changes can be fixed either at a specific gene level or at the genome level (achieving the transition from epigenetic to genetic changes).
  • a new run of epigenetic alteration can occur from newly changed genome topology ⁇ i.e., the genome defines the epigenetic potential). Therefore, the same epigenetic changes might have different biological meaning when occurring within different genome systems.
  • the hypomethylation of DNA can have unpredictable effects in terms of promoting or inhibiting cancer formation. See, e.g., Esteller, M. Epigenetics in cancer. New Engl. J. Med. 358: 1148-1159 (2008).
  • any genome alteration will generate high levels of gene expression changes.
  • Some gene mutations that involve genome integrity can contribute to genome alteration, but only the genome level changes define a new system.
  • karyotypic changes are the point of no return for systems and both gene mutation and epigenetic alteration can contribute to this process.
  • the genome context also defines the pattern of epigenetic regulation.
  • epigenetic features are species-specific phenomena and macro-evolution acts on the genome package level with a certain stochastic feature ensured by epigenetic regulation.
  • epigenetic regulation is the alteration of system dynamics without too much specificity that can be effectively adapted by the combination of genome context and environmental stress. While it is true that inappropriate gene silencing occurs involving tumor suppressor genes, the more profound changes are the increased overall level of systems dynamics, which contributes to epigenetic heterogeneity.
  • the DNA of cancer cells is generally hypomethylated leading to higher levels of gene expression for massive numbers of genes. It is possible that at certain stages of cancer progression, some pathways become dominant, but this process is stochastic and unpredictable as there are so many pathways that could be dominant depending on the possible combinations of genome context and environment.
  • Macro-evolutionary selection mainly functions at the genome level (different genome systems are defined by different karyotypes coupled to unique gene expression profiles). See, e.g., Heng, H.H. The genome-centric concept: re-synthesis of evolutionary theory. BioEssays 31_:512-525 (2009).
  • Micro-evolution mainly involves gene mutation and epigenetic responses that are responsible for a given system's micro-evolution or adaptation.
  • an increased system complexity relies more on a layer of epigenetic regulation and copy number variation. This is important for micro-evolution and adaptation as environments are constantly changing while the framework of the genome is mainly stable.
  • heterogeneity is not as effective as monitoring genome level heterogeneity, as in order to contribute to cancer evolution, these gene level changes are significant enough to impact genome level heterogeneity.
  • Epigenetic heterogeneity has long been observed and studied. Different methylation patterns are present between and within individuals, as exemplified in, e.g., intestinal crypts and endometrial glands. See, e.g., Shibata, D., Tavare, S. Counting divisions in a human somatic cell tree: how, what and why? Cell Cycle 5:610-614 (2006). Despite average methylation increases with age, high heterogeneity is evident. It is a challenge to link age- related methylation to a specific function. The fact that epigenetic alterations may occur at different stages of tumorigenesis further complicates the issue of how to analyze epigenetic heterogeneity. See, e.g., Kurdistani, S.K.
  • Histone modifications as markers of cancer prognosis a cellular view. Br. J. Cancer 97:1-5 (2007).
  • epigenetic patterns are altered in pre-cancer tissues, and high levels of cellular epigenetic heterogeneity can be visualized in cancer tissue by monitoring the histone H3 lysine 18 acetylation.
  • epigenetic heterogeneity is an important feature of the epigenetic theory of cancer initiation. According to this concept, chronic insults repeatedly injure and transiently excite many cells in particular tissues and these excited cells undergo epigenetic response, and initiate tumorigenesis (through epigenetic heterogeneity). See, e.g., Jaffe, L.F. A calcium-based theory of carcinogenesis. Adv. Cancer Res. 94:231-263 (2005).
  • epigenetic heterogeneity few examples have been presented to measure epigenetic heterogeneity or use that data to predict the probability of tumorigenicity.
  • NCCAs non-clonal chromosome aberrations
  • the evolutionary mechanism of cancer can be explained by three main components: instability imparts heterogeneity, which is acted on by natural selection. As each component can be impacted by a great number of genetic/epigenetic and environmental factors, it would be extremely challenging to trace each of these unlimited molecular mechanisms, where each NCCA defines a system with specific pathways (and NCCAs represent the heterogeneity of cancer evolution). On the other hand, it is relatively easy to monitor patterns of evolution, by measuring population diversity, and examining the dynamic relationship between NCCAs and clonal chromosomal aberrations (CCAs).
  • CCAs clonal chromosomal aberrations
  • the predictability of cancer can be accomplished by measuring the system heterogeneity that is shared by most patients, rather than characterize each of the individual factors that contributes to cancer.
  • the multiple levels of genetic and epigenetic alteration are the key elements of cancer evolution.
  • the key is to maintain system homeostasis through system dynamics without significantly increasing heterogeneity especially at the genome level. It must be remembered that dynamics are necessary, otherwise biological processes will not function in a changing environment. However, if there is too great a dynamic interaction, the drastically increased system heterogeneity will trigger cancer evolution. Unfortunately, many factors, including but not limited to: the genetic background, the aging process, stochastic genetic and epigenetic changes, and environmental stress, will unavoidably alter the balance between heterogeneity and homeostasis favoring cancer evolution. Understanding the genome-centric concept of cancer evolution will help to develop applications, treatments, and an
  • the genome-centric concept will serve as a guide when applying genome level heterogeneity to the clinical challenges of cancer, as well as other common non-neoplastic diseases.
  • tumors Working in concert with these biological capabilities are genomic instability (which generates the genetic diversity that expedites their acquisition) and inflammation (which fosters multiple hallmark functions).
  • cancer cells themselves, tumors also exhibit another dimension of biological complexity in that they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of the aforementioned biological capabilities by creating a "tumor microenvironment".
  • Implicit in this organizing paradigm for understanding the inherent complexities of neoplastic disease is the notion that as normal cells progressively evolve to a neoplastic state, they acquire a succession of these seminal biological capabilities, and that the multistep process of human tumor pathogenesis could be rationalized by the need of incipient cancer cells to acquire the traits that enable them to become tumorigenic and ultimately malignant.
  • a long-held, but erroneous, proposition held that tumors are insular masses of proliferating cancer cells.
  • tumors are complex tissues composed of multiple distinct cell types that participate in heterotypic interactions with one another.
  • tumor-associated stroma For example, there is the recruitment of normal cells, which form tumor-associated stroma, to act as active participants in tumorigenesis rather than passive bystanders; as such, these stromal cells contribute to the development and expression of certain seminal biological capabilities. It is now understood that the biology of tumors can no longer be understood simply by enumerating the traits of the cancer cells, but instead must encompass the numerous contributions of the entire "tumor microenvironment" to process of tumorigenesis.
  • the eight seminal biological capabilities of cancer are distinctive and complementary characteristics that enable tumor growth and metastatic dissemination. Each of these eight seminal biological capabilities assist in providing a solid foundation for the understanding the biology of cancer and will be discussed individually below.
  • the apoptotic machinery is composed of both upstream regulators and downstream effector components. See, e.g., Id.
  • the regulators are divided into two major circuits: (i) one receiving and processing extracellular death-inducing signals (the extrinsic apoptotic program) involving, e.g., the Fas ligand/Fas receptor; and (ii) one sensing and integrating a variety of signals of intracellular origin (the intrinsic program).
  • the "apoptotic trigger” that conveys signals between the regulators and effectors is controlled by the counter-balancing of pro- and anti-apoptotic members of the Bcl-2 family of regulatory proteins. See, e.g., Adams, J.M., Cory, S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26: 1324-1337 (2007).
  • the archetype, Bcl-2, along with its closest relatives ⁇ i.e., Bcl-xL, Bcl-w, Mcl-1, Al) are inhibitors of apoptosis, which act in a large part by binding to and thereby suppressing two pro-apoptotic triggering proteins (Bax and Bak) embedded in the mitochondrial outer membrane.
  • Bax and Bak When relieved of inhibition by their anti-apoptotic relatives, Bax and Bak disrupt the integrity of the outer mitochondrial membrane causing the release of pro-apoptotic signaling proteins - the most important of which is cytochrome c.
  • the released cytochrome c activates, in turn, a cascade of caspases that act via their proteolytic activities to induce the multiple cellular changes associated with the apoptotic program.
  • Bax and Bak share protein-protein interaction domains ⁇ i.e., BH3 motifs) with the antiapoptotic Bcl-2-like proteins that mediate their various physical interactions.
  • TP53 induces apoptosis by up-regulating expression of the Noxa and Puma BH3-only proteins in response to substantial levels of DNA breaks and other chromosomal abnormalities.
  • Another condition leading to cell death involves hyperactive signaling by certain oncoproteins ⁇ e.g., Myc) which triggers apoptosis unless counter-balanced by anti-apoptotic factors. See, e.g., Id.
  • Tumor cells evolve a variety of strategies to limit or circumvent apoptosis. Most common is the loss of TP53 tumor suppressor function, which eliminates this critical damage sensor from the apoptosis-inducing circuitry.
  • tumors may also achieve similar results by: (i) increasing expression of anti-apoptotic regulators ⁇ e.g., Bcl-2); (ii) increasing survival signals by down-regulating pro-apoptotic factors ⁇ e.g., Bax); or (iii) short-circuiting the extrinsic ligand-induced death pathway.
  • the multiplicity of apoptosis-avoiding mechanisms presumably reflects the diversity of apoptosis-inducing signals that cancer cell populations encounter during their evolution to the malignant state.
  • the signaling pathway involving the PI3 -kinase, AKT, and mTOR kinases similarly inhibits autophagy. Conversly, when survival signals are insufficient, the PI3K signaling pathway is downregulated, with the result that autophagy and/or apoptosis may be induced. See, e.g., Sinha, S., Levine, B. The autophagy effector Beclin 1 : a novel BH3-only protein. Oncogene 27 (Suppl. 1):S137-S148 (2008).
  • cytotoxic drugs can induce elevated levels of autophagy that are apparently cytoprotective for cancer cells, impairing rather than accentuating the killing actions of these stress-inducing situations.
  • severely stressed cancer cells have been shown to shrink via autophagy to a state of reversible dormancy. This survival response may enable the persistence and eventual regrowth of some late stage tumors following treatment with potent anticancer agents.
  • TGF- ⁇ signaling which can be tumor suppressing at early stages of
  • necrotic cells In contrast to apoptosis, necrotic cells become bloated and explode, releasing their contents into the local tissue microenvironment. Research has clearly established that cell death by necrosis is clearly under genetic control in some circumstances, rather than merely being a random and undirected process. See, e.g., Galluzzi, L., Kroemer, G. Necroptosis: a specialized pathway of programmed necrosis. Cell 135 : 1161-1163 (2008).
  • necrotic cell death releases pro -inflammatory signals into the surrounding tissue microenvironment, in contrast to apoptosis and autophagy.
  • necrotic cells can recruit inflammatory cells of the immune system, whose dedicated function is to survey the extent of tissue damage and remove associated necrotic debris. See, e.g., Grivennikov, S.I., Greten, F.R., Karin, M. Immunity, inflammation, and cancer. Cell 140:883-899 (2010).
  • immune inflammatory cells can be actively tumor-promoting, given that such cells have been shown to be capable of fostering angiogenesis, cancer cell proliferation, and invasiveness.
  • necrotic cells can release bioactive regulatory factors ⁇ e.g., IL-la), which can directly stimulate neighboring viable cells to proliferate, with the potential to facilitate neoplastic progression. See, e.g., Id.
  • bioactive regulatory factors e.g., IL-la
  • IL-la bioactive regulatory factors
  • neoplasias and potentially invasive and/or metastatic tumors may gain an advantage by tolerating some degree of necrotic cell death, and in doing so recruit tumor-promoting inflammatory cells that bring growth-stimulating factors to the surviving cells within these growths.
  • cancer cells require unlimited replicative potential in order to generate macroscopic tumors. This capability stands in marked contrast to the behavior of the cells in most normal cell lineages in the body, which are able to only undergo a limited number of successive cell growth- and-di vision cycles.
  • This limitation has been associated with two distinct barriers to proliferation: (i) senescence (a typically irreversible entrance into a nonproliferative but viable state) and (ii) crisis (which involves cell death). Accordingly, when cells are propagated in vitro, repeated cycles of cell division lead first to induction of senescence and then, for those cells that succeed in circumventing this barrier, to a crisis phase, in which the great majority of cells in the population die.
  • telomeres protecting the ends of chromosomes are centrally involved in the capability for unlimited proliferation.
  • the telomeres composed of multiple tandem hexanucleotide repeats, shorten progressively in non-immortalized cells propagated in culture. This telomeric shortening eventually causes them to lose the ability to protect the ends of chromosomal DNAs from end-to-end fusions; wherein such fusions generate unstable dicentric chromosomes whose resolution results in a scrambling of karyotype that threatens cell viability.
  • telomere length dictates how many successive cell generations its progeny can pass through before telomeres are largely eroded and have consequently lost their protective functions, triggering entrance into crisis. See, e.g., Blasco, M.A. Telomeres and human disease: aging, cancer and beyond. Nat. Rev. Genet. 6: 611-622 (2005).
  • telomerase the specialized DNA polymerase that adds telomere repeat segments to the ends of telomeric DNA, is almost absent in non-immortalized cells. However, it is expressed at functionally significant levels in the vast majority (-90%) of spontaneously immortalized cells, including human cancer cells. Therefore, by extending telomeric DNA, telomerase is able to counter the progressive telomere erosion that would otherwise occur in its absence.
  • the presence of telomerase activity is correlated with a resistance to induction of both senescence and crisis/apoptosis. Conversely, suppression of telomerase activity leads to telomere shortening and to activation of one of these proliferative barriers.
  • telomeric shortening has come to be viewed as a "clocking device" that determines the limited replicative potential of normal cells and is thus, a barrier that must be overcome by cancer cells.
  • telomere maintenance has been increasingly substantiated as a condition critical to the neoplastic state
  • the concept of replication-induced senescence as a general barrier requires refinement and reformulation.
  • Differences in telomere structure and function in mouse versus human cells have also complicated investigation of the roles of telomeres and telomerase in replicative senescence.
  • Recent experiments have revealed that the induction of senescence in certain cultured cells can be delayed and possibly even eliminated by the use of improved cell culture conditions, suggesting that recently explanted primary cells may be able to proliferate unimpeded in culture up the point of crisis and the associated induction of apoptosis triggered by critically shortened telomeres. See, e.g., Ince, T.A., Richardson, A.L., et al., Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes. Cancer Cell 12: 160-170 (2007).
  • mice which were genetically engineered to lack telomerase indicate that the consequently shortened telomeres can shunt pre-malignant cells into a senescent state that contributes (along with apoptosis) to attenuated tumorigenesis in mice genetically predetermined to develop specific forms of cancer. See, e.g., Artandi, S.E., DePinho, R.A. Telomeres and telomerase in cancer. Carcinogenesis 31:9-18 (2010).
  • telomerase null mice with highly eroded telomeres exhibit multi-organ dysfunction and abnormalities that include evidence for both senescence and apoptosis, which is perhaps analogous to the senescence and apoptosis observed in cell culture.
  • cell senescence is emerging as a protective barrier to neoplastic expansion that can be triggered by various proliferation-associated abnormalities (e.g., high levels of oncogenic signaling and sub-critical shortening of telomeres).
  • telomere loss-induced crisis relatively early during the course of multistep tumor progression due to their inability to express significant levels of telomerase.
  • FISH fluorescence in situ hybridization
  • telomere length and telomerase expression in atypical adenomatous hyperplasia and small bronchioloalveolar carcinoma of the lung Am. J. Clin. Pathol. 127:254-262 (2007). These results also suggest that such cells have passed through a substantial number of successive telomere-shortening cell divisions during their evolution from fully normal cells- of-origin. Accordingly, the development of some human neoplasias may be aborted by telomere -induced crisis long before they succeed in becoming macroscopic, severely neoplastic growths.
  • telomere loss may permit other incipient neoplasias to survive initial telomere erosion and attendant chromosomal breakage-fusion-bridge (BFB) cycles.
  • BFB chromosomal breakage-fusion-bridge
  • the genomic alterations resulting from these BFB cycles apparently serve to increase the mutability of the genome, thereby accelerating the acquisition of mutant oncogenes and tumor suppressor genes.
  • impaired telomere function can actually foster tumor progression has come from the study of mutant mice that lack both p53 and telomerase function. See, e.g., Artandi, S.E., DePinho, R.A. Telomeres and telomerase in cancer. Carcinogenesis 31:9-18 (2010).
  • the hypothysis that these two aforementioned defects can cooperatively enhance human tumorigenesis has not yet been quantitatively ascertained.
  • telomerase has the ability to elongate and maintain telomeric DNA.
  • telomerase also possesses novel functions that are relevant to cell proliferation, but unrelated to telomeric DNA maintenance. These novel functions, and in particular the function of its protein subunit TERT, have been demonstrated in conditions where the telomerase enzymatic activity has been eliminated.
  • telomere-independent functions of TERT/telomerase include: (i) the ability of TERT to amplify signaling by the Wnt pathway by serving as a cofactor of the ⁇ -catenin/LEF transcription factor complex; (ii) enhancement of cell proliferation and/or resistance to apoptosis; (iii) involvement in DNA-damage repair; and (iv) RNA-dependent RNA polymerase function. See, e.g., Cong, Y., Shay, J.W. Actions of human telomerase beyond telomeres. Cell Res. 18:725-732 (2008).
  • cancer cells One of the most fundamental traits of cancer cells involves their ability to sustain chronic proliferation. Normal tissues carefully control the production and release of growth- promoting signals that instruct entry into and progression through the cell growth and division cycle, thereby ensuring a homeostasis of cell number and thus maintenance of normal tissue architecture and function. Cancer cells, by deregulating these signals, obtain control of their ultimate destiny.
  • the enabling signals are conveyed, in large part, by various growth factors that bind to cell-surface receptors, which typically contain intracellular tyrosine kinase domains. The latter subsequently proceed to emit signals via branched intracellular signaling pathways that regulate progression through the cell cycle, as well as cell growth. These signals also influence numerous other cell-biological properties ⁇ e.g., cell survival, energy metabolism, and the like).
  • Cancer cells can acquire the capability to sustain proliferative signaling in a number of alternative ways. They may produce growth factor ligands themselves, to which they can respond via the expression of cognate receptors, resulting in autocrine proliferative stimulation. Alternatively, cancer cells may send signals to stimulate normal cells within the supporting tumor-associated stroma, which reciprocate by supplying the cancer cells with various growth factors. See, e.g., Cheng, N., Chytil, A., Shyr, Y., Joly, A., Moses, H.L.
  • Transforming growth factor-beta signaling-deficient fibroblasts enhance hepatocyte growth factor signaling in mammary carcinoma cells to promote scattering and invasion. Mol. Cancer Res. 6: 1521-1533 (2008).
  • Receptor signaling can also be deregulated by elevating the levels of receptor proteins displayed at the cancer cell surface, rendering such cells hyperresponsive to otherwise-limiting amounts of growth factor ligand; the same outcome can result from structural alterations in the receptor molecules that facilitate ligand-independent firing.
  • Growth factor independence may also derive from the constitutive activation of components of signaling pathways operating downstream of these receptors, obviating the need to stimulate these pathways by ligand-mediated receptor activation.
  • the activation of one or another of these downstream pathways may only recapitulate a subset of the regulatory instructions transmitted by an activated receptor.
  • PI3-kinase phosphoinositide 3- kinase
  • the prototype of this type of regulation involves the Ras oncoprotein; the oncogenic effects of Ras do not result from a hyperactivation of its signaling powers; instead, the oncogenic mutations affecting ras genes compromise Ras GTPase activity, which operates as an intrinsic negative-feedback mechanism that normally ensures that active signal transmission is transitory.
  • Analogous negative-feedback mechanisms operate at multiple nodes within the proliferative signaling circuitry.
  • a prominent example involves the PTEN phosphatase, which counteracts PI3-kinase by degrading its product, phosphatidylinositol (3,4,5) triphosphate (PIP3).
  • PTEN phosphatase which counteracts PI3-kinase by degrading its product, phosphatidylinositol (3,4,5) triphosphate (PIP3).
  • PIP3 phosphatidylinositol
  • Loss-of-function mutations in PTEN amplify PI3K signaling and promote tumorigenesis in a variety of experimental models of cancer; in human tumors, PTEN expression is often lost by promoter. See, e.g., Jiang, B.H., Liu, L.Z. PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv. Cancer Res. 102: 19-65
  • cancer cells In addition to the seminal biological capability of inducing and sustaining positively acting growth-stimulatory signals, cancer cells must also circumvent powerful programs that negatively regulate cell proliferation. Many of these programs depend on the actions of tumor suppressor genes. Dozens of tumor suppressors that operate in various ways to limit cell growth and proliferation have been discovered through their characteristic inactivation in one or another form of animal or human cancer; many of these genes have been validated as bona fide tumor suppressors through gain- or loss-of-function experiments in mice.
  • the two prototypical tumor suppressors encode the RB (retinoblastoma-associated) and TP53 proteins; they operate as central control nodes within two key complementary cellular regulatory circuits that govern the decisions of cells to proliferate or, alternatively, activate senescence and apoptotic programs.
  • the RB protein integrates signals from diverse extracellular and intracellular sources and, in response, decides whether or not a cell should proceed through its growth-and-division cycle. See, e.g., Burkhart, D.L., Sage, J. Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat. Rev. Cancer 8:671-682 (2008).
  • TP53 receives inputs from stress and abnormality sensors that function within the cell's intracellular operating systems.
  • stress and abnormality sensors that function within the cell's intracellular operating systems.
  • TP53 can trigger programmed cell death ⁇ i.e., apoptosis). It should be noted however, that the effects of activated TP53 are complex and highly context dependent, and vary by both cell type and by the severity/persistence of conditions of cell stress and genomic damage.
  • Merlin the cytoplasmic NF2 gene product
  • E-cadherin cell-surface adhesion molecules
  • transmembrane receptor tyrosine kinases e.g., the EGF receptor
  • Merlin strengthens the adhesivity of cadherin-mediated cell-to-cell attachments.
  • By sequestering growth factor receptors Merlin limits their ability to efficiently emit mitogenic signals. See, e.g., Curto, M., Cole, B.K., et al, Contact-dependent inhibition of EGFR signaling by Nf2/Merlin. J. Cell Biol. 177:893-903 (2007).
  • LKB1 epithelial polarity protein which organizes epithelial structure and helps maintain tissue integrity.
  • LKB1 can, for example, overrule the mitogenic effects of the powerful Myc oncogene when the latter is upregulated in organized, quiescent epithelial structures.
  • LKB1 expression is suppressed, epithelial integrity is destabilized, and epithelial cells become susceptible to Myc-induced transformation. See, e.g., Partanen, J. I., Nieminen, A.I., Klefstrom, J. 3-D view to tumor suppression: Lkbl, polarity and the arrest of oncogenic c-Myc. Cell Cycle 5:716- 724 (2009).
  • LKB1 has also been identified as a tumor suppressor gene that is lost in certain human malignancies, possibly reflecting its normal function as a suppressor of inappropriate proliferation. See, e.g., Shaw, R.J. Tumor suppression by LKB1 : SIK-ness prevents metastasis. Sci. Signal. 2:55 (2009). However, it yet remains to be seen how frequently these two mechanisms of contact-mediated growth suppression are compromised in human cancers.
  • TGF- ⁇ Promotion of Malignancy by Corruption of the TGF- ⁇ Pathway TGF- ⁇ is best known for its anti-proliferative effects, and the evasion of these effects by cancer cells is now known to be far more involved than just the simple shutdown of its signaling circuitry. See, e.g., Ikushima, H., Miyazono, K. TGFbeta signaling: a complex web in cancer progression. Nat. Rev. Cancer 10:415-424 (2010). In many late-stage tumors, TGF- ⁇ signaling is redirected away from suppressing cell proliferation and is found instead to activate a cellular program, termed the epithelial-to-mesenchymal transition (EMT), that confers on cancer cells traits associated with high-grade malignancy.
  • EMT epithelial-to-mesenchymal transition
  • Tumors require sustenance in the form of nutrients and oxygen as well as an ability to evacuate metabolic wastes and carbon dioxide, just as normal, non-cancerous tissues do.
  • the tumor-associated neovasculature (generated by the process of angiogenesis) addresses these metabolic needs.
  • embryogenesis the development of the vasculature involves the generation of new endothelial cells and their subsequent assembly into tubes ⁇ i.e.,
  • vasculogenesis in addition to the "sprouting" ⁇ i.e., angiogenesis) of new vessels from existing ones. Following this morphogenesis, normal vasculature becomes largely quiescent.
  • angiogenesis In the adult, as part of various physiologic processes ⁇ e.g., wound healing, female reproductive cycling, etc.) angiogenesis is transiently activated. However, in contrast, during tumor progression, an "angiogenic switch" is almost constantly activated, causing the normally quiescent vasculature to continually generate/sprout new vessels that assist in sustaining the expanding neoplastic growths. See, e.g., Baeriswyl, V., Christofori, G. The angiogenic switch in carcinogenesis. Semin. Cancer Biol. 19:329-337 (2009). A compelling body of evidence indicates that the angiogenic switch is governed by countervailing factors that either induce or oppose angiogenesis.
  • angiogenic regulators include signaling proteins that bind to stimulatory or inhibitory cell surface receptors displayed by vascular endothelial cells.
  • the well-known prototypes of angiogenesis inducers and inhibitors are vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP- 1), respectively.
  • VEGF-A vascular endothelial growth factor-A
  • TSP- 1 thrombospondin-1
  • the VEGF-A gene encodes ligands that are involved in: (i) orchestrating new blood vessel growth during embryonic and postnatal development; (ii) homeostatic survival of endothelial cells; and (iii) physiological and pathological situations in the adult.
  • VEGF signaling via three receptor tyrosine kinases (VEGFR-1-3) is regulated at multiple levels, reflecting its complex functional purpose.
  • VEGF gene expression can be up-regulated both by hypoxia and by oncogene signaling. See, e.g., Ferrara, N. Pathways mediating VEGF-independent tumor angiogenesis. Cytokine Growth Factor Rev. 21 :21-26 (2010).
  • other pro-angiogenic signals such as members of the fibroblast growth factor (FGF) family, have been implicated in sustaining tumor angiogenesis when their expression is chronically up-regulated. See, e.g., Baeriswyl, V., Christofori, G. The angiogenic switch in carcinogenesis. Semin. Cancer Biol. 19:329-337 (2009).
  • TSP-1 The primary counter-balance in angiogenic switch is TSP-1, which also binds to transmembrane receptors displayed by endothelial cells; thereby evoking suppressive signals that can counteract pro- angiogenic stimuli. See, e.g., Kazerounian, S., Yee, K.O., Lawler, J. Thrombospondins in cancer. Cell. Mol. Life Sci. 65:700-712 (2008).
  • tumor neovasculature is marked by precocious capillary sprouting, convoluted and excessive vessel branching, distorted and enlarged vessels, erratic blood flow, microhemorrhaging, leakiness, and abnormal levels of endothelial cell proliferation and apoptosis.
  • Angiogenesis is induced surprisingly early during the multistage development of invasive cancers both in animal models and in humans. Histological analyses of pre- malignant, noninvasive lesions, including dysplasias and in situ carcinomas arising in a variety of organs, have revealed the early tripping of the angiogenic switch. It had previously been thought that angiogenesis was only important when rapidly growing macroscopic tumors had formed. However, recent experimental data indicates that angiogenesis also contributes to the microscopic pre -malignant phase of neoplastic progression; thus further establishing its inclusion as a seminal biological capability of cancer. Angiogenic Switch Control
  • Tumors may exhibit diverse patterns of neo-vascularization once angiogenesis has been activated.
  • some tumors e.g., highly aggressive pancreatic ductal adenocarcinomas
  • stromal "deserts” that are largely avascular and may even be actively anti-angiogenic.
  • tumors e.g., human renal and pancreatic neuroendocrine carcinomas
  • Other tumors are highly angiogenic and consequently possess dense vascularization. See, e.g., Zee, Y.K., O'Connor, J. P., Parker, et al., Imaging angiogenesis of genitourinary tumors. Nat. Rev. Urol. 7:69-82 (2010).
  • neovascularization that is variable in intensity. This latter variability most probably being controlled by a complex biological "rheostat" that involves both the cancer cells and the associated stromal micro-environment. See, e.g., Baeriswyl, V., Christofori, G. The angiogenic switch in carcinogenesis. Semin. Cancer Biol. 19:329-337 (2009). It should be noted that the angiogenic switching mechanism can alter its form, even though the overall result is an inductive signal (e.g., VEGF). Further, in some tumors, dominant oncogenes operating within tumor cells (e.g., Ras and Myc) can upregulate expression of angiogenic factors; whereas in others, such inductive signals are produced indirectly by immune inflammatory cells.
  • inductive signal e.g., VEGF
  • endogenous angiogenic regulators A number of endogenous angiogenic regulators have been discovered. Most are proteins, and many are derived by proteolytic cleavage of structural proteins that are not themselves angiogenic regulators. These endogenous angiogeneic regulators include: TSP-1, angiostatin, and type 18 collagen (endostatin), and numerous other agents. See, e.g., Ribatti, D. Endogenous inhibitors of angiogenesis: a historical review. Leuk. Res. 33 :638-644 (2009). Interestingly, a number of these endogenous inhibitors of angiogenesis have been detected in the circulation of normal mice and humans.
  • angiogenesis inhibitors serve under normal circumstances as physiologic regulators that modulate transitory angiogenesis during tissue remodeling and wound healing; whereas they may also act as intrinsic barriers to induction and/or persistence of angiogenesis by incipient neoplasias.
  • Bone Marrow-Derived Cells to Tumor Angiogenesis
  • a number of cell types originating in the bone marrow have been shown to play a crucial role in pathological angiogenesis, including cells of the innate immune system ⁇ e.g., macrophages, neutrophils, mast cells, and myeloid progenitors) that infiltrate pre -malignant lesions and progressed tumors and assemble at the margins of such lesions.
  • These peri- tumoral inflammatory cells help to trip the angiogenic switch in previously quiescent tissue and to sustain ongoing angiogenesis associated with tumor growth and in facilitating local invasion. See, e.g., Ferrara, N. Pathways mediating VEGF-independent tumor angiogenesis. Cytokine Growth Factor Rev. 21_'.2 ⁇ -26 (2010).
  • E-cadherin was well documented as an antagonist of invasion and metastasis; whereas the reduction of its expression was known to potentiate these aforementioned phenotypes.
  • the frequently observed down-regulation and occasional mutational inactivation of E-cadherin in human carcinomas also provided strong support for its role as a key suppressor of this seminal biological capability. See, e.g., Berx, G., van Roy, F. Involvement of members of the cadherin superfamily in cancer. Cold Spring Harb. Perspect. Biol. 1 : a003129 (2009).
  • genes encoding other cell-to-cell and cell-to-ECM adhesion molecules has also been shown to be altered in some highly aggressive carcinomas, with those genes favoring cytostasis typically being markedly downregulated.
  • adhesion molecules normally associated with the cellular migrations that occur during embryogenesis and inflammation are often upregulated (e.g., N-cadherin).
  • Invasion and metastasis has typically been envisioned as a sequence of discrete steps, often termed the "invasion-metastasis cascade" (see, e.g., Talmadge, J.E., Fidler, I.J. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res.
  • EMT epithelial-mesenchymal transition
  • carcinoma cells can concomitantly acquire multiple attributes that enable invasion and metastasis.
  • This multifaceted EMT program can be activated transiently or stably, and to differing degrees, by carcinoma cells during the course of invasion and metastasis.
  • a set of pleiotropically acting transcriptional factors direct the EMT and related migratory processes during embryogenesis and are expressed in various combinations in a number of malignant tumor types.
  • Biological functions implicated in the processes of invasion and metastasis which are evoked by these transcriptional factors include: (i) a loss of adherens junctions and associated conversion from a polygonal/epithelial to a
  • IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion.
  • the EMT program regulates a particular type of invasiveness that has been termed "mesenchymal”.
  • meenchymal two other distinct modes of invasion have been identified and implicated in cancer cell invasion. See, e.g., Madsen, CD., Sahai, E. Cancer dissemination - Lessons from leukocytes. Dev. Cell 19: 13-26 (2010).
  • the second is an "amoeboid" form of invasion, in which individual cancer cells show morphological plasticity, enabling them to slither through existing interstices in the extracellular matrix rather than clearing a path for themselves, as occurs in both the mesenchymal and collective forms of invasion.
  • Another emerging concept involves the facilitation of cancer cell invasion by inflammatory cells that assemble at the boundaries of tumors, producing the extracellular matrix-degrading enzymes and other factors that enable invasive growth (see, e.g.,
  • cancer cells may secrete the chemoattractants that recruit the proinvasive inflammatory cells rather than producing the matrix-degrading enzymes themselves.
  • Metastasis can be broken down into two major phases: (i) the physical dissemination of cancer cells from the primary tumor to distant tissues, and (ii) the adaptation of these cells to foreign tissue microenvironments that results in successful colonization (i.e., the growth of micrometastases into macroscopic tumors).
  • the multiple steps of dissemination would seem to be under the aegis of the EMT and similarly acting migratory programs. It must be noted, however, that colonization is not strictly coupled with physical dissemination, as evidenced in many patients by the presence of a plethora of micrometastases that have successfully disseminated, but never progress to macroscopic metastatic tumors. See, e.g., Talmadge, J.E., Fidler, I.J. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res. 70:5649-5669 (2010).
  • the primary tumor may release systemic suppressor factors that render such micrometastases dormant, as revealed clinically by explosive metastatic growth soon after resection of the primary growth.
  • systemic suppressor factors that render such micrometastases dormant, as revealed clinically by explosive metastatic growth soon after resection of the primary growth.
  • macroscopic metastases may erupt decades after a primary tumor has been surgically removed or pharmacologically destroyed. See, e.g., Barkan, D., Green, J.E., Chambers, A.F. Extracellular matrix: a gatekeeper in the transition from dormancy to metastatic growth. Eur. J. Cancer 46: 1181-1188 (2010).
  • angiogenesis as the inability of certain experimentally generated dormant micrometastases to form macroscopic tumors has been ascribed to their failure to activate tumor angiogenesis.
  • a nutrient starvation can induce intense autophagy that causes cancer cells to shrink and adopt a state of reversible dormancy; such cells may exit this state and resume active growth and proliferation when changes in tissue micro-environment ⁇ e.g., access to more nutrients) permit.
  • disseminated cancer cells are likely to be poorly adapted, at least initially, to the microenvironment of the tissue in which they have landed. Accordingly, each type of disseminated cancer cell may need to develop its own set of ad hoc solutions to the problem of fostering in the microenvironment of one or another foreign tissue. These adaptations might require hundreds of distinct colonization programs, each dictated by the type of disseminating cancer cell and the nature of the tissue microenvironment in which colonization is proceeding. However, certain tissue microenvironments may be preordained to be intrinsically hospitable to disseminated cancer cells. See, e.g., Talmadge, J.E., Fidler, I.J. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res. 70:5649-5669 (2010).
  • Metastatic dissemination has long been depicted as the last step in multistep primary tumor progression, and indeed for many tumors that is likely the illustrated by recent genome sequencing studies that present genetic evidence for clonal evolution of pancreatic ductal adenocarcinoma to metastasis. See, e.g., Yachida, S., Jones, S., et ah, Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 467: 1114- 1117 (2010). Conversely, recent findings indicate that cells can disseminate remarkably early, dispersing from ostensibly noninvasive premalignant lesions in both mice and humans. See, e.g., Coghlin, C, Murray, G.I. Current and emerging concepts in tumour metastasis. J. Pathol. 222: 1-15 (2010).
  • cancer cells can clearly disseminate from such pre-neoplastic lesions and seed the bone marrow and other tissues, their capability to colonize these sites and develop into pathologically significant macrometastases remains to be proven.
  • Early metastatic dissemination is viewed as a demonstrable phenomenon in mice and humans whose clinical significance is yet to be established. Beyond the timing of their dissemination, it also remains unclear when and where cancer cells develop the ability to colonize foreign tissues as macroscopic tumors. This capability may arise during primary tumor formation as a result of a tumor's particular developmental path prior to any dissemination, such that primary tumor cells entering the circulation are fortuitously endowed with the ability to colonize certain distant tissue sites. See, e.g., Talmadge, J.E., Fidler, I.J.
  • AACR centennial series the biology of cancer metastasis: historical perspective. Cancer Res. 70:5649-5669 (2010).
  • the ability to colonize specific tissues may only develop in response to the selective pressure on already disseminated cancer cells to adapt to growth in foreign tissue microenvironments.
  • tissues-specific colonization programs that are evident among cells within a primary tumor may originate not from classical tumor progression occurring within the primary lesion but instead from emigrants that have returned home. Such reseeding is consistent with the aforementioned studies of human pancreatic cancer metastasis. See, e.g., Yachida, S., Jones, S., et al., Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature 467: 1114-1117 (2010).
  • the chronic and frequently uncontrolled cell proliferation that represents the essence of neoplastic disease involves not only deregulated control of cell proliferation but also corresponding adjustments of energy metabolism in order to fuel cell growth and division.
  • normal cells process glucose, first to pyruvate via glycolysis in the cytosol and thereafter to carbon dioxide in the mitochondria ⁇ i.e., mitochondrial oxidative phosphorylation).
  • glycolysis is favored and relatively little pyruvate is dispatched to the oxygen-consuming mitochondria.
  • cancer cells Even in the presence of oxygen can reprogram their glucose metabolism, and thus their energy production, by limiting their energy metabolism largely to glycolysis, leading to a state that has been termed "aerobic glycolysis".
  • Such reprogramming of energy metabolism is seemingly counter-intuitive, as the cancer cells must compensate for the 18-fold lower efficiency of ATP production afforded by glycolysis relative to mitochondrial oxidative phosphorylation.
  • the cancer cells compensate, in part, by upregulating glucose transporters ⁇ e.g., GLUT1) which substantially increases glucose import into the cytoplasm. See, e.g., Jones, R.G., Thompson, C.B. Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev.
  • Glycolytic fueling has been shown to be associated with activated oncogenes ⁇ e.g., RAS, MYC) and mutant tumor suppressors ⁇ e.g., TP53), whose alterations in tumor cells have been selected primarily for their benefits in conferring the seminal biological capabilities of cell proliferation, avoidance of cytostatic controls, and attenuation of apoptosis.
  • This reliance on glycolysis can be further accentuated under the hypoxic conditions that operate within many tumors; wherein the hypoxia response system acts pleiotropically to upregulate glucose transporters and multiple enzymes of the glycolytic pathway.
  • the hypoxia response system acts pleiotropically to upregulate glucose transporters and multiple enzymes of the glycolytic pathway.
  • both the Ras oncoprotein and hypoxia can independently increase the levels of the HIFla and HIF2a transcription factors, which in turn upregulate glycolysis. See, e.g., Id.
  • Warburg-like metabolism seems to be present in many rapidly dividing embryonic tissues, once again suggesting a role in supporting the large-scale biosynthetic programs that are required for active cell proliferation.
  • some tumors have been found to contain two distinct sub-populations of cancer cells that differ in their energy-generating pathways.
  • One sub -population consists of glucose-dependent ⁇ i.e., "Warburg effect) cells that secrete lactate; whereas cells of the second sub -population preferentially import and utilize the lactate produced by their neighbors as their main energy source, employing part of the citric acid cycle to do so.
  • Altered energy metabolism is proving to be widespread in many types of cancer cells and is becoming recognized as one of the seminal biological capabilities.
  • the redirection of energy metabolism is largely orchestrated by proteins that are involved in various ways in programming the other enumerated seminal biological capabilities.
  • aerobic glycolysis is simply another phenotype that is programmed by proliferation- inducing oncogenes.
  • activating (i.e., gain-of-function) mutations in the isocitrate dehydrogenase (IDH) enzymes have been reported in glioma and other human tumors.
  • mice genetically engineered to be deficient for various components of the immune system were assessed for the development of carcinogen-induced tumors, it was observed that tumors arose more frequently and/or grew more rapidly in the immunodeficient mice relative to immunocompetent controls.
  • immunocompetent hosts in a a process that has been referred to as
  • immunoediting leaving behind only weakly immunogenic variants to replicate and generate solid tumors; such weakly immunogenic cells can thereafter colonize both immunodeficient and immunocompetent hosts; and
  • the immunogenic cancer cells when arising in immunodeficient hosts, the immunogenic cancer cells are not selectively depleted and can, instead, prosper along with their weakly immunogenic counterparts; when cells from such nonedited tumors are serially transplanted into syngeneic recipients, the immunogenic cancer cells are rejected when they confront, for the first time, the competent immune systems of their secondary hosts.
  • cancer immunology may greatly oversimplify tumor-host immunological interactions, as highly immunogenic cancer cells may well evade immune destruction by disabling components of the immune system that have been dispatched to eliminate them.
  • cancer cells may "paralyze" infiltrating CTLs and NK cells, by secreting TGF- ⁇ or other immunosuppressive factors.
  • Tregs regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • target molecules As described below, the sulfur-containing, amino acid-specific small molecules of the present invention possess the ability to contemperaneously or simultaneously modify and/or modulate multiple target molecules.
  • kinase describes a large family of enzymes that are responsible for catalyzing the transfer of a phosphoryl group from a nucleoside triphosphate donor, such as ATP, to an acceptor molecule.
  • Tyrosine kinases catalyze the phosphorylation of tyrosine residues in proteins.
  • the phosphorylation of tyrosine residues causes a change in the function of the protein that they are contained in.
  • Phosphorylation at tyrosine residues controls a wide range of properties in proteins such as enzyme activity, subcellular localization, and interaction between molecules.
  • the MET proto-oncogene encodes for the receptor tyrosine kinase (RTK), c-MET.
  • MET encodes a protein known as hepatocyte growth factor receptor (HGFR).
  • the hepatocyte growth factor receptor protein possesses tyrosine kinase activity. See, e.g., Cooper, C.S., The MET oncogene: from detection by transfection to transmembrane receptor for hepatocyte growth factor. Oncogene 7(l):3-7 (1992).
  • c-MET is a membrane receptor that is essential for embryonic development and tissue repair ⁇ e.g., wound healing).
  • Hepatocyte growth factor (HGF) is the only known ligand of the c-MET receptor.
  • MET is normally expressed in cells of epithelial origin, although it has also been identified in endothelial cells, neurons, hepatocytes, hematopoietic cells, and melanocytes. Expression of HGF is generally restricted to cells of mesenchymal origin, although some epithelial cancer cells appear to express both HGF and MET.
  • the MET proto-oncogene has a total length of 125,982 bp and is located in the 7q31 locus of chromosome 7.
  • MET is transcribed into a 6,641 bp mature mRNA which is then translated into a 1,390 amino acid residue c-MET protein.
  • c-MET is a receptor tyrosine kinase that is produced as a primary single-chain precursor protein that is post-translationally proteolytically cleaved at a furin site to yield a highly glycosylated extracellular a-subunit and a transmembrane ⁇ -subunit, which are then covalently linked via a disulfide bond to form the mature receptor.
  • c-MET dimerizes and autophosphorylates upon ligand binding, which in turn creates active docking sites for proteins that mediate downstream signaling leading to the activation/modulation of a variety of proteins.
  • activation/modulation evokes a variety of pleiotropic biological responses leading to increased cell growth, scattering and motility, invasion, protection from apoptosis, branching morphogenesis, and angiogenesis.
  • improper activation of c-MET may confer proliferative, survival and invasive/metastatic abilities of cancer cells.
  • c-MET and HGF are highly expressed in a large number of solid and soft tumors (for a comprehensive list, see www.vai.org/met).
  • HGF is expressed ubiquitously throughout the body, showing this growth factor to be a systemically available cytokine as well as coming from the tumor stroma. See, e.g., Vuononvirta, R., Sebire, N.J., et al. Expression of hepatocyte growth factor and its receptor met in Wilms' tumors and nephrogenic rests reflects their roles in kidney development. Clin. Cancer Res. 15:2723-2730 (2009). A positive paracrine and autocrine loop of c-MET activation can therefore lead to further MET expression.
  • c-MET was first identified as the product of a chromosomal rearrangement after treatment with the carcinogen N-methyl-NO-nitro-N-nitrosoguanidine, See, e.g., Cooper, C.S., Park, M., et al., Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 311 :29-33 (1984).
  • This rearrangement results in a constitutively fused oncogene (TPR-MET) which is translated into an oncoprotein following dimerization by a leucine-zipper motif located in the TPR moiety. This provides the structural requirement for
  • c-MET kinase to be constitutively active.
  • TPR-MET has been shown to possess the ability to transform epithelial cells and to induce spontaneous mammary tumors when ubiquitously over-expressed in transgenic mice.
  • RTK receptor tyrosine kinase
  • c-MET is overexpressed in a variety of carcinomas including lung, breast, ovary, kidney, colon, thyroid, liver, and gastric carcinomas. See, e.g., Knowles, L.M., Stabile, L.P., et al. HGF and c-Met participate in paracrine tumorigenic pathways in head and neck squamous cell cancer. Clin. Cancer Res. 15:3740-3750 (2009).
  • over-expression could be the result of transcriptional activation, hypoxia-induced over- expression, or as a result of MET amplification. See, e.g., Cappuzzo, F., Marchetti, A., et al.
  • MET activation in cancer correlates with poor prognosis, where aberrantly active MET triggers tumor growth, formation of new blood vessels (angiogenesis) that supply the tumor with nutrients, and cancer spread to other organs (metastasis).
  • MET is deregulated in many types of human malignancies, including cancers of the: kidney, liver, stomach, breast, and brain.
  • stem cells and progenitor cells express MET, which allows these cells to grow invasively in order to generate new tissues in an embryo or regenerate damaged tissues in an adult.
  • cancer stem cells are thought to hijack the ability of normal stem cells to express MET, and thus become the cause of cancer persistence and spread to other sites in the body.
  • Anaplastic Lymphoma Kinase Anaplastic lymphoma kinase (ALK) also known as ALK tyrosine kinase receptor or CD246 (cluster of differentiation 246) is an enzyme that in humans is encoded by the ALK gene. See, e.g., Cui, J.J.; Tran-Dube, M.; et ah, Structure Based Drug Design of Crizotinib (PF-02341066), a Potent and Selective Dual Inhibitor of Mesenchymal-Epithelial Transition Factor (c-MET) Kinase and Anaplastic Lymphoma Kinase (ALK). J. Med. Chem. 54:6342- 6363 (2011).
  • ALK belongs to the tyrosine kinase receptor family. By homology, ALK is most similar to leukocyte tyrosine kinase, and both belong to the insulin-receptor superfamily.
  • ALK is a single-chain transmembrane receptor comprising three structural domains. The extracellular domain contains an N-terminal signal peptide sequence and is the ligand- binding site for the putative activating ligands of ALK ⁇ i.e., pleiotrophin and midkine). This is followed by the transmembrane and juxtamembrane region which contains a binding site for phosphotyrosine-dependent interaction with insulin receptor substrate- 1. The final section has an intracellular tyrosine kinase domain with three phosphorylation sites (Y1278, Y1282, and Y1283), followed by the C-terminal domain with interaction sites for
  • phospho lipase C- ⁇ and Src homology 2 domain containing SHC are absent in the product of the transforming ALK gene.
  • binding of a ligand induces homodimerization of ALK, leading to trans-phosphorylation and kinase activation.
  • the 5 '-terminus fusion partners provide dimerization domains, enabling ligand-independent activation of the kinase.
  • native ALK which localizes to the plasma membrane
  • the majority of ALK fusion proteins localize to the cytoplasm. This difference in cellular localization may also contribute to deregulated ALK activation.
  • EML4-ALK fusion oncogene represents one of the newest molecular targets in cancer (especially in non-small cell lung carcinoma (NSCLC)).
  • EML4-ALK was identified by the screening of a cDNA library derived from a the tumor of a NSCLC (adenocarcinoma) of the lung. See, e.g., Soda, M., Choi, Y.L employ et al. Identification of the transforming EML4- ALK fusion gene in non-small cell lung cancer. Nature 448:561-566 (2007).
  • EML4-ALK echinoderm microtubule associated protein- like 4
  • ALK anaplastic large cell lymphomas
  • IMT inflammatory myofibroblastic tumors
  • neuroblastomas e.g., aplastic large cell lymphomas (ALCL), inflammatory myofibroblastic tumors (IMT), and neuroblastomas.
  • ALK anaplastic large cell lymphomas
  • IMT inflammatory myofibroblastic tumors
  • neuroblastomas e.g., aplastic large cell lymphomas (ALCL)
  • ALK translocation including EML4-ALK
  • the fusion partner has been shown to mediate ligand-independent dimerization of ALK, resulting in constitutive kinase activity.
  • EML4-ALK possesses potent oncogenic activity.
  • EML4-ALK In transgenic mouse models, lung-specific expression of EML4-ALK leads to the development of numerous lung adenocarcinomas. See, e.g., Soda, M., Takada, S., et al. A mouse model for EML4-ALK- positive lung cancer. Proc. Natl. Acad. Sci. U.S.A. 105: 19893-19897 (2008). Cancer cell lines harboring the EML4-ALK translocation can be effectively inhibited by small molecule inhibitors targeting ALK. See, e.g., Koivunen, J. P., Mermel, C, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin. Cancer Res. 14:4275-4283
  • ALK anaplastic large cell lymphoma
  • Point mutations have been found in 6-8% of primary neuroblastomas. Germ-line mutations have been identified in families with more than one sibling with neuroblastoma. Somatic mutations with wild-type ALK in matched constitutional DNAs have also been described in non- familial neuroblastoma cases. These mutations are located mainly in the TK domain; the most frequent being the gain-of- function mutations Fl 174L and R1275Q. These mutations are associated with increased expression, phosphorylation, and kinase activity of the ALK protein. Further, they have been shown to have Ba/F3 cell-transforming capacity. In some cases, these mutations coexist with an increased copy number of the ALK gene.
  • ALK ALK-selective inhibition
  • NSCLC non-small cell lung cancer
  • c-ROS gene was first discovered in 1986 when a recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ROS, of the avian sarcoma virus UR29-11 was isolated from a chicken genomic DNA library.
  • UR2 sarcoma virus is a retrovirus of chicken that encodes for a fusion protein, P68 gag"R0S ? having tyrosine - specific kinase activity. See, e.g., Feldman, R.A., Wang, L.H., et al.
  • Avian sarcoma virus UR2 encodes a transforming protein which is associated with a unique protein kinase activity. J. Virol.
  • v-ROS The oncogene, v-ROS, of UR2 carries a kinase domain that is homologous to those present in the oncogenes of the src family.
  • the c-ROS sequence appeared to be conserved in vertebrate species, from fish to mammals (including humans).
  • the comparison of the deduced amino acid sequence of c-ROS and that of v-ROS showed two differences: (i) v-ROS contains three amino acids insertion within the hydrophobic domain (TM domain), presumed to be involved in membrane association; and (ii) the twelve carboxy-terminal amino acids of
  • v-ROS are completely different from those of the deduced c-ROS sequence. See, e.g., Neckameyer, W.S., Shibuya, M., Hsu, M.T., Wang, L.H. Proto-oncogene c-ROS codes for a molecule with structural features common to those of growth factor receptors and displays tissue-specific and developmentally regulated expression. Mol. Cell Biol. 6: 1478-1486 (1986).
  • the human c-ROS gene was mapped to the human chromosome 6, region 6ql6-6q22. This region of chromosome 6 is involved in nonrandom chromosomal rearrangement in specific neoplasias, including: acute lymphoplastic leukemia, malignant melanoma, and ovarian carcinomas.
  • c-ROS gene over-expression and/or mutations were found mainly in brain and lung cancers, in addition to chemically-induced stomach cancer, breast
  • fibroadenomas liver cancer, colon cancer, and kidney cancer.
  • NSCLC Non-Small Cell Lung Cancer
  • tyrosine kinase signaling was characterized in 41 NSCLC cell lines and over 150 NSCLC tumors. See, Rikova, K., Guo, A., et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131 : 1190-1203 (2007). Profiles of phosphotyrosine signaling were generated and analyzed to identify known oncogenic kinases. Interestingly, ROS kinase was determined to be in the top-ten receptor tyrosine kinases (RTKs) found in both cell lines and tumors. RTKs in this survey were ranked according to phosphorylation rank
  • RTKs are characteristic as markers for nervous system tumors.
  • EGFR epidermal growth factor receptor
  • Erb-B oncogene Erb-B
  • 45-50% malignant gliomas show evidence for EGFR amplification.
  • c-erbB epidermal growth factor receptor gene
  • RTKs include: Neu ⁇ see, e.g., Bernstein, J.J., Anagnostopoulos, A.V., et al. Human-specific c-neu proto-oncogene protein overexpression in human malignant astrocytomas before and after xenografting. J. Neurosurg. 78:240-251 (1993)), platelet-derived growth factor (PDGF) receptor ⁇ see, e.g., Lokker, N.A., Sullivan, CM., et al. Platelet-derived growth factor (PDGF) autocrine signaling regulates survival and mitogenic pathways in glioblastoma cells. Cancer Res.
  • Neu see, e.g., Bernstein, J.J., Anagnostopoulos, A.V., et al. Human-specific c-neu proto-oncogene protein overexpression in human malignant astrocytomas before and after xenografting. J. Neurosurg. 78:240-251 (1993)
  • ROS ⁇ see, e.g., Jun, H.J., Woolfenden, S., et al. Epigenetic regulation of c-ROS receptor tyrosine kinase expression in malignant gliomas. Cancer Res. 69:2180-2184 (2009)).
  • ROS ROS was found to be expressed in 56% of glioblastoma-derived cell lines at high levels (i.e., ranging from 10 to 60 transcripts per cell), while not expressed at all or expressed minimally in the remaining cell lines. See, Birchmeier, C, Sharma, S., Wigler, M. Expression and rearrangement of the ROS gene in human glioblastoma cells. Proc. Natl. Acad. Sci. USA 84:9270-9274 (1987). Moreover, no expression of ROS gene was observed in normal, non-neoplastic brain tissues; thus, the high level of ROS expression in glioblastoma seems specific.
  • ROS may play a role in tumor progression rather than initiation.
  • c-ROS gene was found to be upregulated in gastric cancer induced by oral
  • N-methyl-NO-nitro-N-nitrosoguanidine MNNG
  • MNNG N-methyl-NO-nitro-N-nitrosoguanidine
  • ROS gene was one of six genes found to be persistently upregulated after 4 weeks from MNNG treatment. ROS gene was found also to be overexpressed (in a number of other genes) in fibroadenoma samples taken from breast tumors of five different patients. It was found to be expressed at levels more than two-fold higher than those in normal tissues.
  • the epidermal growth factor receptor is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. See, e.g., Herbst, R.S. Review of epidermal growth factor receptor biology. Int. J. Radiat. Oncol. Biol. Phys. 59:21-26 (2004).
  • EGFR is a member of the ErbB family of receptors, which comprise a subfamily of four (4) closely related receptor tyrosine kinases, which include: ErbB-1 (also known as epidermal growth factor receptor (EGFR), HER1); ErbB-2 (also know as HER 2 in humans and c-neu in rodents); ErbB-3 (also known as HER 3); and ErbB-4 (also known as HER 4). Mutations affecting EGFR expression and/or activity have been shown to be involved in many forms of cancer.
  • EGFR epidermal growth factor receptor
  • HER1 epidermal growth factor receptor
  • ErbB-2 also know as HER 2 in humans and c-neu in rodents
  • ErbB-3 also known as HER 3
  • ErbB-4 also known as HER 4
  • EGFR HER1, erbBl
  • NSCLC non- small cell lung cancer
  • breast head and neck
  • gastric colorectal
  • esophageal prostate
  • bladder renal
  • pancreatic pancreatic
  • ovarian cancers a variety of human tumors including, but not limited to: non- small cell lung cancer (NSCLC), breast, head and neck, gastric, colorectal, esophageal, prostate, bladder, renal, pancreatic, and ovarian cancers.
  • ErbB receptors (170 kDa) are comprised of an extracellular region or ectodomain that contains approximately 620 amino acid residues, a single transmembrane-spanning region, and a cytoplasmic tyrosine kinase domain.
  • the extracellular region of each ErbB family member is made up of four subdomains: LI, CR1, L2, and CR2 - wherein "L” denotes a leucine-rich repeat domain and "CR” a cysteine-rich region.
  • L denotes a leucine-rich repeat domain
  • CR cysteine-rich region
  • EGFR exists on the cell surface and is activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor a (TGF ).
  • TGF transforming growth factor a
  • ErbB2 has no known direct activating ligand, and may be in an activated state constitutively or become active upon heterodimerization with other ErbB family members.
  • EGFR Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer.
  • preformed inactive dimers may also exist before growth factor ligand binding.
  • EGFR may pair with another member of the ErbB receptor family ⁇ e.g., ErbB2/Her2/neu) to create an activated heterodimer.
  • ErbB2/Her2/neu another member of the ErbB receptor family
  • clusters of activated EGFRs form, although it remains unclear whether this clustering is important for activation itself or occurs subsequent to activation of individual dimers.
  • EGFR dimerization stimulates its intrinsic intracellular protein/tyrosine kinase activity.
  • autophosphorylation of several tyrosine amino acid residues in the carboxyl-terminal domain of EGFR occurs. These include Tyr992, Tyrl045, Tyrl068, Tyrl 148, and Tyrl 173. See, e.g., Downward, J., Parker, P., Waterfield, M.D.
  • Such proteins modulate phenotypes, including but not limited to: cell migration, cell adhesion, and cell proliferation.
  • activation of the receptor is important for the innate immune response in human skin. See, e.g., Roupe, K.M.; Nybo, M., et al. Injury is a major inducer of epidermal innate immune responses during wound healing.. J. Investigative Dermatol. 130: 1167-1177 (2010).
  • the kinase domain of EGFR can also cross-phosphorylate tyrosine residues of other receptors it is aggregated with and can itself, be activated in that same manner. See, e.g., Oda, K., Matsuoka, Y., et al. A comprehensive pathway map of epidermal growth factor receptor signaling. Mol. Syst. Biol.1:205-210 (2005).
  • EGF-EGFR protein phosphorylation and in tumorigenesis, and subsequently the EGF-EGFR signaling axis has taken an important role in developmental biology and cancer research.
  • Activated EGFR recruits a number of downstream signaling molecules, leading to the activation of several major pathways that are important for tumor growth, progression, and survival. See, e.g., Lo, H.W., Hung, M.C. Nuclear EGFR signaling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br. J. Cancer 94: 184-188 (2006).
  • the main pathways downstream of EGFR activation include those mediated by PLC-y-PKC, Ras-Raf-MEK, PI-3K-Akt-mTOR, and JAK2-STAT3.
  • the EGFRvIII variant is primarily localized on the cell- surface where it activates several signaling modules.
  • EGFRvIII is constitutively active independent of ligand stimulation, in part, due to its loss of a portion of the ligand-binding domain.
  • EGFRvIII While EGFR over-expression is found in many types of human cancers, EGFRvIII is predominantly detected in malignant gliomas. Both EGFR and EGFRvIII play critical roles in tumorigenesis and in supporting the malignant phenotypes in human cancers.
  • IGF1R Insulin Growth Factor 1 Receptor
  • IGF1R Insulin Growth Factor 1 Receptor
  • IGFIR has a high degree of structural similarity to the insulin receptor and modulates cell growth and proliferation through several key proteins including PI3K, IRS, MAPK, JAK/STAT, and others.
  • IGFIR insulin-like growth factor receptor pathway in lung cancer: problems and pitfalls. Ther. Adv. Med. Oncol. 4(2):51-60 (2012).
  • IGFIR is important in a variety of cancers including, but not limited to, lung, colon, breast, sarcoma and prostate cancer. See, e.g., Gombos, et al, Clinical Development of Insulin- Like Growth Factor Receptor- 1 (IGFIR) Inhibitors: At the Crossroad. Invest. New Drugs 30(6 :2433-2442 (2012); Gallagher and LeRoith, IGF, Insulin and Cancer.
  • IGFIR Like many receptor tyrosine kinases, IGFIR homodimerizes at the cell membrane and transduces signals through the various signaling pathways. Additionally IGFIR can form heterodimers with other receptors including, but not limited to, the insulin receptor and EGFR2 (HER-2). The heterodimerization with EGFR2 has been proposed to contribute to Trastuzumab resistance in vitro and may have important in vivo implications as well. See, e.g., Maki, Insulin-like Growth Factors and Their Role in Growth, Development, and Cancer. J. Clin. Oncol. 2803 ⁇ :4985-4995 (2011). IGFIR is the subject of many laboratory studies and more than 60 clinical trials have been initiated to evaluate agents that putatively target IGFIR.
  • IGFIR Insulin-Like Growth Factor Receptor-1
  • DNA excision repair protein ERCC-1 is a protein that in humans is encoded by the ERCC1 gene. The function of the ERCC1 protein is predominantly in nucleotide excision repair (NER) of damaged DNA. NER is one of five separate DNA repair mechanisms that also include: recombination repair, base excision repair, mismatch repair, and translesion synthesis. Nucleotide excision repair (NER) in eukaryotes is initiated by either Global Genome NER (GG-NER) or Transcription Coupled NER (TC-NER) which involve distinct protein complexes, each recognizing damaged DNA.
  • GG-NER Global Genome NER
  • TC-NER Transcription Coupled NER
  • GG- NER and TC-NER share a final common excision and repair pathway which include the following steps: (i) transcription factor II H (TFIIH) separates the abnormal strand from the normal strand; (ii) xeroderma pigmentosum group G (XPG) cuts 3' to the damaged DNA: (iii) replication protein A (RPA) protects the "normal", non-damaged strand; (iv) xeroderma pigmentosum group A (XPA) isolates the damaged segment on the strand to be cut; and (v) ERCC1 and xeroderma pigmentosum group F (XPF) cut 5' to the damaged DNA.
  • transcription factor II H TKIIH
  • XPG xeroderma pigmentosum group G
  • RPA replication protein A
  • XPA xeroderma pigmentosum group A
  • ERCC1 and xeroderma pigmentosum group F XPF
  • ERCC1 appears to have a crucial role in stabilizing and enhancing the functionality of the XPF endonuclease.
  • the excised single-stranded DNA (approximately 30 nucleotides in length) and the attached NER proteins are excised and removed. DNA polymerases and ligases then fill in the gap left by the excision of the damaged DNA strand using the normal strand as a template.
  • the ERCCl-XPF protein complex also removes non-homologous 3' tail ends in homologous recombination.
  • the ERCCl-XPF complex is a structure-specific endonuclease involved in the repair of damaged DNA.
  • ERCCl-XPF performs a critical incision step in nucleotide excision repair (NER), and is also involved in the repair of DNA interstrand crosslinks (ICLs) and some double-strand breaks (DSBs).
  • NER nucleotide excision repair
  • ICLs DNA interstrand crosslinks
  • DSBs double-strand breaks
  • ERCCl-XPF A fraction of ERCCl-XPF is localized at telomeres, where it is implicated in the recombination of telomeric sequences and loss of telomeric overhangs at deprotected chromosome ends.
  • ERCCl-XPF degrades 3' G-rich overhangs ⁇ see, e.g., Kirschner, K., Melton, D.W. Multiple roles of the ERCCl-XPF endonuclease in DNA repair and resistance to anticancer drugs. Anticancer Res. 30:3223-2332 (2010)) and various other related functions ⁇ see, e.g., Rahn, J.J., Adair, G.M., Nairn, R.S. Multiple roles of ERCCl-XPF in mammalian interstrand crosslink repair.
  • ERCC1 or XPF Deficiency of either ERCC1 or XPF in humans results in a variety of conditions, which include the skin cancer-prone disease xeroderma pigmentosum (XP), a progeroid syndrome of accelerated aging, or cerebro-oculo-facioskeletal syndrome (COFS). These diseases are extremely rare in the general population and therefore mice with low levels of either ERCC 1 or XPF have been generated and studied extensively. These murine models clearly illustrate the importance of DNA repair in preventing aging-related tissue degeneration.
  • XP skin cancer-prone disease xeroderma pigmentosum
  • COFS cerebro-oculo-facioskeletal syndrome
  • Ribonucleotide reductase is a multimeric protein that reduces the 2' hydroxyl on ribonucleotides to a 2' hydrogen yielding deoxyribonucleotides that can be utilized in DNA synthesis and DNA repair. See, e.g., Hofer, et al, DNA building blocks: Keeping control of manufacture. Crit. Rev. Biochem. Mol. Biol. 47:50-63 (2012).
  • the Ml subunit (a subunit; larger subunit) of RNR binds the ribonucleotide substrate and is catalytic while the M2 subunit ( ⁇ subunit; smaller subunit) contains the diferric tyrosyl radical that is required for catalysis.
  • M2 subunit ⁇ subunit; smaller subunit
  • the Ml subunit (a subunit; larger subunit) of RNR binds the ribonucleotide substrate and is catalytic while the M2 subunit ( ⁇ subunit; smaller subunit) contains the diferric tyrosyl radical that is required for catalysis.
  • Wan, et al Enhanced subunit interactions with gemcitabine-5 ' -diphosphate inhibit ribonucleotide reductases. Proc. Natl. Acad. Sci. U.S.A. 104(36): 14324-14329 (2010); Morandi, Biological agents and gemcitabine in the treatment of breast cancer. Annals Oncol.
  • Hydroxyurea is a classical agent targeting RNR and has been used in combination with radiation to treat head and neck cancer and cervical cancer. See, e.g., Chapman and Kinsella, Ribonucleotide reductase inhibitors: A new look at an old target for radiosensitization. Frontiers Oncol. 1: 1-6 (2009).
  • RNR has been found to be elevated in some NSCLC patients and development of agents that target and modulate RNR function would be useful in the clinic. See, e.g., Ren, et al, Individualized chemotherapy in advanced NSCLC patients based on mRNA levels of BRCA1 and RRMl. Chin. J. Cancer Res.
  • the structural proteins that comprise the microtubule arrays in vivo are critical for cell division, cell proliferation and a range of other intracellular processes. See, e.g., Harrison, et al, Beyond taxanes: A review of novel agents that target mitotic tubulin and microtubules, kinases, and kinesins. Clin. Adv. Hematol. Oncol. 7:54-64, (2009).
  • Microtubules consist primarily of a and ⁇ tubulin subunits but also contain numerous other microtubule proteins.
  • Oncology drugs that target tubulin have been developed and include drugs in the taxane, epothilone, and vinca alkaloid families. See, e.g., Gascoigne and Taylor, How do anti-mitotic drugs kill cancer cells. J. Cell. Sci. 122:2579-2585 (2009).
  • tubulin protein a well-known and highly utilized anticancer agent exerts its effect primarily by stabilizing tubulin (see, e.g., Xiao, et al, Insights into the mechanism of microtubule stabilization by Taxol, Proc. Natl. Acad. Sci, U.S.A.
  • CIPN chemotherapy-induced peripheral neuropathy
  • Amifostine, glutathione, glutamine/glutamate, calcium/magnesium infusions, neurotrophic factors, NGF, gabapentin, vitamin E, N-acetylcysteine, diethyldithiocarbamate, erythropoietin, and carbamazepine are among the many agents that have been evaluated for use as potential neuroprotective agents. See, e.g., Cavaletti, et al, Neurotoxic effects of antineoplastic drugs: The lesson of preclinical studies. Front. Biosci. 13:3506-3524 (2008).
  • CIPN chemotherapy-induced peripheral neuropathy
  • Human protein prenyltransferases include the proteins farnesyltransferase (FTase), geranylgeranyltransferase I (GGTase I), and geranylgeranyltransferase II (GGTase II). These prenyltransferases transfer lipophilic isoprene groups that enable the prenylated substrates to more avidly associate with cellular membranes.
  • the proteins that are prenylated by the human protein prenyltransferases are involved in a range of intracellular pathways and processes important for cell growth and proliferation. See, e.g., Holstein and Hohl, Is there a future for prenyltransferases inhibitors in cancer therapy? Curr.
  • prenyltransferases represent attractive targets for drug discovery especially within the area of oncology, as further discussed below. See, e.g., Holstein and Hohl, Is there a future for prenyltransferases inhibitors in cancer therapy? Curr. Opin. Pharmacol. 12:704-709 (2012). Targeting prenyltransferases requires a global cellular perspective.
  • FTase and GGTase I might not be an effective anti-cancer approach were it not for the fact that the substrates that are post-translationally modified by these prenyltransferases are essential in regulating many different cell growth and cell survival signaling pathways.
  • a specific example of FTase- and GGTase-mediated prenylation that is important and required for the regulation of cell proliferation and cell survival involves the RAS protein family.
  • RAS proteins include: KRAS, HRAS, and NRAS.
  • RAS proteins have high sequence similarity/identity and regulate proteins that have important roles in cell proliferation-related pathways, including but not limited to, MAPK, STAT, Raf, MEK, and ERK; as well as proteins that are key in anti-apoptotic pathways, including but not limited to, PI3K and Akt.
  • MAPK MAPK
  • STAT Raf
  • MEK ERK
  • ERK proteins that are key in anti-apoptotic pathways
  • PI3K and Akt proteins that are key in anti-apoptotic pathways
  • PI3K and Akt e.g., Vadakara and Borghael, Personalized medicine and treatment approaches in non-small-cell lung carcinoma. Pharmaco genomics Personalized Med. 5: 113-123 (2012); Riely, et al., KRAS mutations in non-small cell lung cancer. Proc. Am. Thorac. Soc. 6:201-205 (2009)
  • KRAS is an important oncology target that is commonly mutated in 80% of pancreatic cancer patients, 20% of all non-small cell lung cancer (NSCLC) patients, and is also often mutated in colorectal cancer patients as well. See, e.g., Adjei, Blocking
  • RAS proteins are substrates for prenyltransferases and, regardless of their mutational state, must be prenylated to be able to translocate to the cell membrane and transduce signals that regulate cell proliferation and apoptosis.
  • GTTase geranylgeranyltransferase
  • RAS proteins are important in NSCLC ⁇ see, e.g., Vadakara and Borghael, Personalized medicine and treatment approaches in non-small-cell lung carcinoma. Pharamcogen. Personalized Med. 5: 113-123 (2012); Riely, et al., KRAS mutations in non-small cell lung cancer. Proc. Am. Thorac. Soc.
  • Farnesyltransferase catalyzes the addition of a 15 carbon moiety onto key proteins, including but not limited to: (i) the RAS family of proteins; (ii) kinetochore proteins; (iii) cGMP phosphodiesterase; (iv) peroxisomal proteins; (v) nuclear lamina proteins; (vi) heat shock homologs; (vii) rhodopsin kinase; and similar proteins. See, e.g., Maurer-Stroh, et al., Protein prenyltransferases. Genome Biol. 4:212-221 (2003).
  • RAS protein family e.g., HRAS, KRAS and NRAS.
  • RAS modulates a wide range of intracellular signaling pathways the regulate cell growth, cell proliferation, and apoptosis. See,FigurQ 78; Appels, et al., Development of Farnesyl Transferase Inhibitors: A Review. 10:565-578 (2005).
  • Oxidoreductases are enzymes that catalyzes the transfer of electrons from one molecule (i.e., the reductant, also called the hydrogen or electron donor) to another (i.e., the oxidant, also called the hydrogen or electron acceptor). This group of enzymes usually utilizes NADPH or NAD + as cofactors.
  • Peroxiredoxins are a ubiquitous family of small (22-27 kDa) non-seleno peroxidases that functions as anti -oxidants and also control cytokine-induced peroxide levels and thereby mediate signal transduction in mammalian cells. Unlike Trx possessing the active double-cysteine region and forming the intramolecular disulfide bond when oxidized, Prx have no such regions; however, the easily oxidized Cys residues present in their structure can form intermolecular disulfide bonds. There are six mammalian isoforms that have been currently identified. See, e.g., Rhee, S., Chae, H., Kim, K.
  • Peroxiredoxins a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radical Biol. Med. 38: 1543-1552 (2005). Although their individual roles in cellular redox regulation and antioxidant protection are quite distinct, they all catalyze peroxide reduction of ⁇ 2 0 2 , organic hydroperoxides, and peroxynitrite. They are found to be expressed ubiquitously and in high levels, suggesting that they are both an ancient and important enzyme family.
  • Prx 1-6 Mammalian cells express six Prx isoforms (Prx 1-6), which can be divided into three subgroups as follow: (i) 2-Cys Prx proteins, which contain both the N- and C-terminal- conserved Cys residues and require both of them for catalytic function; (ii) atypical 2-Cys proteins, which contain only the N-terminal Cys but require one additional, nonconserved Cys residue for catalytic activity; and (iii) 1-Cys Prx proteins, which contain only the N- terminal Cys and require only the conserved one for catalytic function.
  • Prx 1-4 of the six mammalian Prxs belong to the 2-Cys subgroup and have the conserved N- and C-terminal Cys residues that are separated by 121 amino acid residues.
  • Prx 1 NKEF A, PAG, MSP23, OSF3, HBP23
  • Prx 2 NKEF B, Calpromotin, Torin proteins consist of 199 amino acid residues and exist in cytosol (various alternative names given without reference to peroxidase function are in parentheses).
  • Prx 3 (MER5, SP22) deduced from the cDNA sequence of MER5 is substantially larger than the 195 amino acid residue sequence of SP22, as determined directly by peptide sequencing of SP22 purified from mitochondria of bovine adrenal cortex. The additional 62 residues at the N-terminus were proved to be the mitochondrial-targeting sequence.
  • Prx 4 (AOE372, TRANK) was identified as a protein that interacts with Prx I by the yeast two-hybrid assay. See, e.g., Jin, D.Y.; Chae, H.Z.; et al. Regulatory role for a novel human thioredoxin peroxidase in NF- kappaB activation. J.
  • Prx 4 contains the N-terminal signal sequence for secretory proteins and found in culture medium.
  • the N-terminal Cys is oxidized by peroxides to cysteine sulfenic acid, which then reacts with the C-terminal-conserved cysteine of the other subunit to form an intermolecular disulfide.
  • the reduction of the intermolecular disulfide is specific to thioredoxin (Trx) and could not be achieved by glutathione (GSH) or glutaredoxin.
  • mutant 2-Cys Prx proteins that lack either the N-terminal or C-terminal Cys residues do not exhibit Trx-coupled peroxidase activity.
  • Mammalian cells contain mitochondria-specific Trx and TrxR, suggesting that Prx 3 together with the mitochondria-specific Trx and TrxR provide a primary line of defense against H 2 O 2 produced by the mitochondrial respiratory chain.
  • Prx 3 together with the mitochondria-specific Trx and TrxR provide a primary line of defense against H 2 O 2 produced by the mitochondrial respiratory chain.
  • Peroxiredoxins a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radical Biol. Med. 38: 1543-1552 (2005).
  • the amino acid sequence identity among the four mammalian 2 -Cys (Prx 1 to Prx 4) enzymes is 70%, with the homology being especially marked in the regions surrounding the conserved N- and C-terminal Cys residues.
  • the atypical 2-Cys Prx, Prx5 was identified as the result of a human EST database search with the N-terminal-conserved sequence
  • Cys 47 is the site of oxidation by peroxides, and the resulting oxidized Cys 47 reacts with the sulfhydryl group of Cys 151 to form a disulfide linkage, which was initially suggested to be intramolecular based on biochemical data.
  • Prx 6 The full-length cDNA (ORF06) for a human 1-Cys Prx, also termed Prx 6, was identified without any reference to peroxidase activity as the result of a sequencing project with human myeloid cell cDNA.
  • the N-terminal Cys-SH of Prx 6, which corresponds to Cys 47 of human Prx 6, is readily oxidized.
  • the resulting Cys- SOH does not form a disulfide because of the unavailability of another Cys-SH nearby.
  • some 1-Cys Prx members contain other Cys residues, such as Cys 91 of the human enzyme.
  • H 2 0 2 can rapidly transform into highly toxic reactive oxygen species (ROS), such as 0 2 ⁇ radicals, elevation of the levels of ROS can lead to development of oxidative stress causing deleterious physiological effects, including but not limited to: (i) DNA breakage; (ii) linkages in protein molecules; and (iii) activation of lipid peroxidation.
  • ROS reactive oxygen species
  • a physiological role of Prx associated with enzymatic degradation of H 2 0 2 is particularly significant in erythrocytes, in which these enzymes are ranked second or third place in overall cellular protein content.
  • Prx An important role of Prx in defense against oxidative stress was demonstrated in a series of studies with knockout of genes corresponding to Prx.
  • Hemolytic anemia characterized by hemoglobin instability developed, in PRDX1 gene knockout mice. See, e.g., Neumann, C.A., Krause, D.S., et al. Nature 424:561-565 (2003).
  • PRDX2 gene knockout mice a significant decrease of lifespan was also accompanied by development of anemia. In both cases, the knockout of the corresponding gene caused a significant elevation of ROS in erythrocytes.
  • the PRDX6 gene knockout mice were characterized by low survival, high level of protein oxidation, and significant injury of kidneys, liver, and lungs.
  • Prx 6 cannot be compensated by expression of other genes. See, e.g., Wang, X., Phelan, S.A., et al. J. Biol. Chem. 278:25179-25190 (2003).
  • the expression of genes encoding different Prx isoforms has cellular, tissue, and organ specificity. Prx 1 is the most widely represented and highly expressed member of the peroxiredoxin family in virtually all organs and tissues of mice and humans, both in normal tissues and malignant tumors.
  • the PRDX1 gene is widely expressed in various areas of the central and peripheral nervous system with expression specificity depending on the cell type.
  • High expression of the PRDX4 gene is characteristic of liver, testes, ovaries, and muscles, whereas low expression is observed in small intestine, placenta, lung, kidney, spleen, and thymus.
  • ROS reactive oxygen species
  • Prx 1 The association of Prx 1 with cell proliferation dates from early studies. In particular, it was shown that expression of the PRDX1 gene was appreciably higher in Ras-transfected epithelial cells compared with the wild-type cells. See, e.g., Prosperi, M. T., Ferbus, D., et al. J. Biol. Chem. 268: 11050-11056 (1993). Moreover, it was found that Prx 1 interacts with c- Abl and c-Myc protein kinases playing an important role in regulation of cell proliferation. See, e.g., Wen, S.-T., VanEtten, R. A. Genes Dev. 11:2456-2467 (1997).
  • Prx 1 has also been shown to be capable of regulating the tyrosine kinase activity of c-Abl (by binding with its third structural domain), which leads to restriction of the transforming ability of c-Abl. See, Id. Accordingly, it has been hypothesized that the reversible binding of Prx 1 with c-Abl can serve as a key cell cycle regulator. Prx 1 is also cpable of binding with c-Myc via the c-Myc- transactivating domain ⁇ see, e.g., Mu, Z.M., Yin, X.Y., Prochownik, E.V. J. Biol. Chem. 277:43175-43184 (2002)), with a decrease in expression of a series of genes specific for activity of c-Myc being observed in the case of over-expression of the PRDX1 gene.
  • Glutathione is the predominant nonprotein thiol in cells where it plays essential roles as an enzyme substrate and a protecting agent against xenobiotic compounds and oxidants. See, e.g., Dickinson, D.A., Forman, H.J. Cellular glutathione and thiol metabolism. Biochem. Pharmacol. 64: 1019-1026 (2002).
  • Glutathione maintained in the reduced state by glutathione reductase, is able to transfer its reducing equivalents to several enzymes, such as glutathione peroxidases (GPx), glutathione transferases (GSTs), and glutaredoxins.
  • GPx glutathione peroxidases
  • GSTs glutathione transferases
  • glutaredoxins glutaredoxins.
  • thioredoxin can interact with ribonucleotide reductase and with several other proteins involved in cellular signaling and transcription control, such as NF- ⁇ , PTP-1B, PKA, PKC, Akt, and ASK1.
  • ribonucleotide reductase can interact with ribonucleotide reductase and with several other proteins involved in cellular signaling and transcription control, such as NF- ⁇ , PTP-1B, PKA, PKC, Akt, and ASK1.
  • Mammalian cells contain a cytosolic (Grxl) and a mitochondrial (Grx2) glutaredoxin.
  • Mitochondria contain a second glutaredoxin (Grx5), which is homologous to yeast Grx5 in bearing a single cysteine residue at its active site.
  • Glutathione a tripeptide ( ⁇ -glutamyl-cysteinyl-glycine) serves a highly important role in both intracellular and extracellular redox balance. It is the main derivative of cysteine, and the most abundant intracellular non-protein thiol, with an intracellular concentration approximately 10-times higher than other intracellular thiols.
  • glutathione GSH
  • glutathione reductase NADPH
  • Glutathione functions in many diverse roles including, but not limited to, regulating antioxidant defenses, detoxification of drugs and xenobiotics, and in the redox regulation of signal transduction.
  • glutathione may serve to scavenge intracellular free radicals directly, or act as a co-factor for various other protection enzymes.
  • glutathione may also have roles in the regulation of immune response, control of cellular proliferation, and prostaglandin metabolism.
  • Glutathione is also particularly relevant to oncology treatment because of its recognized roles in tumor-mediated drug resistance to cancer treating agents and ionizing radiation.
  • Glutathione is able to conjugate electrophilic drugs such as alkylating agents and cisplatin under the action of glutathione S-transferases.
  • GSH multidrug resistance-associated protein
  • MRP multidrug resistance-associated protein
  • GSH enhances cell survival by functioning in antioxidant pathways that reduce reactive oxygen species, and maintain cellular thiols (also known as non-protein sulfhydryls (NPSH)) in their reduced states.
  • NPSH non-protein sulfhydryls
  • Cysteine another important NPSH, as well as glutathione are also able to prevent DNA damage by radicals produced by ionizing radiation or chemical agents. Cysteine concentrations are typically much lower than GSH when cells are grown in tissue culture, and the role of cysteine as an in vivo cytoprotector is less well-characterized. However, on a molar basis cysteine has been found to exhibit greater protective activity on DNA from the side-effect(s) of radiation or chemical agents. Furthermore, there is evidence that cysteine concentrations in tumor tissues can be significantly greater than those typically found in tissue culture.
  • Sensitive enzymatic cycling assay for glutathione Measurement of glutathione content and its modulation by buthionine sulfoximine in vivo and in vitro human colon cancer. Cancer Res. 54:4077-4083 (1994). Wide ranges of tumor GSH concentrations have been reported, and in general these have been greater ⁇ i.e., up to 10-fold) in tumors compared to adjacent normal tissues. Most researchers have assessed the GSH content of bulk tumor tissue using enzymatic assays, or GSH plus cysteine using HPLC.
  • NPSH cellular thiols/non-protein sulfhydryls
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • maintenance of the "normal" intracellular redox state Low levels of intracellular oxygen within tumor cells (i.e., tumor hypoxia) caused by aberrant structure and function of the associated tumor vasculature, has also been shown to be associated with chemotherapy therapy-resistance and biologically-aggressive malignant disease.
  • Oxidative stress commonly found in regions of intermittent hypoxia, has been implicated in regulation of glutathione metabolism, thus linking increased NPSH levels to tumor hypoxia. Therefore, it is also important to characterize both NPSH expression and its relationship to tumor hypoxia in tumors and other neoplastic tissues.
  • NPSH neuropeptide hydrochloride sulfate
  • the heterogeneity of NPSH levels was examined in multiple biopsies obtained from patients with cervical carcinomas who were entered into a study investigating the activity of cellular oxidation and reduction levels (specifically, hypoxia) on the response to radical radiotherapy. See, e.g., Fyles, A., et al. (Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother. Oncol. 48: 149-156 (1998).
  • the major findings from this study were that the intertumoral heterogeneity of the concentrations of GSH and cysteine exceeds the intratumoral heterogeneity, and that cysteine concentrations of approximately 21 mM were found in some samples, confirming an earlier report by
  • Radiotherapy has traditionally been a major treatment modality for cervical carcinomas. Randomized clinical trials (Rose, D., et al. Concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical carcinoma. New Engl. J. Med. 340: 1144-1153 (1999)) show that patient outcome is significantly improved when radiation therapy is combined with cisplatin-based chemotherapy, and combined modality therapy is now widely being utilized in treatment regimens.
  • cysteine concentrations greater than 1 mM in a significant number of cases suggest that these two thiols are regulated differently in tumors.
  • BSO buthionine sulfoximine
  • cysteine possesses the ability to repair radiation-induced DNA radicals and cysteine also has the potential to detoxify cisplatin; a cytotoxic agent now routinely combined with radiotherapy to treat locally- advanced cervical carcinomas.
  • Glutaredoxin like thioredoxin (Trx), are members of the thioredoxin superfamily that mediate disulfide exchange via their Cys-containing catalytic sites. While glutaredoxins mostly reduce mixed disulfides containing glutathione, thioredoxins are involved in the maintenance of protein sulfhydryls in their reduced state via disulfide bond reduction. See, e.g., Print, W.A., et al. The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J. Biol. Chem.
  • Glutaredoxins are small redox enzymes of approximately 100 amino acid residues, which use glutathione as a cofactor. Glutaredoxins are oxidized by substrates, and reduced non-enzymatically by glutathione. In contrast to thioredoxins, which are reduced by thioredoxin reductase, oxidized glutathione is regenerated by glutathione reductase. Together these components comprise the glutathione system. See, e.g., Holmgren, A. and Fernandes, A.P., Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system. Antioxid. Redox. Signal. 6:63-74 (2004).
  • Glutaredoxins basically function as electron carriers in the glutathione-dependent synthesis of deoxyribonucleotides by the enzyme ribonucleotide reductase. Like thioredoxin, which functions in a similar way, glutaredoxin possesses an active catalytic site disulfide bond. It exists in either a reduced or an oxidized form where the two cysteine residues are linked in an intramolecular disulfide bond. Human proteins containing this domain include: glutaredoxin thioltransferase (GLRX); glutaredoxin 2 (GLRX2); thioredoxin-like 2
  • glutaredoxin like thioredoxin
  • glutathione cycles between a thiol form
  • glutthione that can reduce glutaredoxin and a disulfide form (glutathione disulfide);
  • glutathione reductase enzymatically reduces glutathione disulfide to glutathione.
  • the thioredoxin system is comprised of thioredoxin reductase (TrxR) and its main protein substrate, thioredoxin (Trx), where the catalytic site disulfide of Trx is reduced to a dithiol by TrxR at the expense of NADPH.
  • the thioredoxin system together with the glutathione system (comprising NADPH, the flavoprotein glutathione reductase, glutathione, and glutaredoxin), is regarded as a main regulator of the intracellular redox environment, exercising control of the cellular redox state and antioxidant defense, as well as governing the redox regulation of several cellular processes.
  • the system is involved in direct regulation of: (i) several transcription factors; (ii) apoptosis (i.e., programmed cell death) induction; and (iii) many metabolic pathways (e.g., DNA synthesis, glucose metabolism, selenium
  • the mammalian thioredoxin reductases are enzymes belonging to the avoprotein family of pyridine nucleotide-disulfide oxidoreductases that includes lipoamide dehydrogenase, glutathione reductase, and mercuric ion reductase.
  • Members of this family are homodimeric proteins in which each monomer includes an FAD prosthetic group, an NADPH binding site and an active site containing a redox-active disulfide. Electrons are transferred from NADPH via FAD to the active-site disulfide of Trx, which then reduces the substrate. See, e.g., Williams, C.H., Chemistry and Biochemistry of Flavoenzymes (Muller, F., ed.), pp. 121-211, CRC Press, Boca Raton (1995).
  • TrxRs are named for their ability to reduce oxidized thioredoxins (Trxs), a group of small (i.e., 10-12 kDal), ubiquitous redox-active peptides that undergoes reversible oxidation/reduction of two conserved cysteine (Cys) residues within the catalytic site.
  • Trxs oxidized thioredoxins
  • Cys conserved cysteine residues
  • the mammalian TrxRs are selenium-containing flavoproteins that possess: (i) a conserved -Cys- Val-Asn-Val-GIy-Cys- catalytic site; (ii) an NADPH binding site; and (iii) a C-terminal Cys- Selenocysteine sequence that communicates with the catalytic site and is essential for its redox activity. See, e.g., Powis, G. Monofort, W.R. Properties and biological activities of thioredoxins. Ann. Rev. Pharmacol. Toxicol. 4J_:261-295 (2001). These proteins exist as homodimers and undergo reversible oxidation/reduction.
  • TrxR The activity of TrxR is regulated by NADPH, which in turn is produced by glucose-6-phosphate dehydrogenase (G6DP), the rate-limiting enzyme of the oxidative hexose monophosphate shunt (HMPS; also known as the pentose phosphate pathway).
  • G6DP glucose-6-phosphate dehydrogenase
  • HMPS oxidative hexose monophosphate shunt
  • TrxR isozyme genes Two human TrxR isozyme genes have been cloned: (i) the gene for human TrxR-1 located on chromosome 12q23-q24.1 encoding a 54 Kda enzyme that is found predominantly in the cytoplasm; and (ii) the gene for human TrxR-2 located on chromosome 22ql 1.2 encoding a 56 Kda enzyme the possesses a 33-amino-acid N-terminal extension identified as a mitochondrial import sequence. See, e.g., Powis, G. Monofort, W.R. Properties and biological activities of thioredoxins. Ann. Rev. Pharmacol. Toxicol. 41 :261- 295 (2001).
  • TrxR A third isoform of TrxR, designated (TGR) is a Trx and glutathione reductase localized mainly in the testis, has also been identified. See, e.g., Sun, Q.A., et al. Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems. Proc. Natl. Acad. Sci. USA 98:3673-3678 (2001). Additionally, both mammalian cytosolic TrxR-1 and mitochondrial TrxR-2 have alternative splice variants.
  • TrxR splice variants In humans, five different 5' cDNA variants have been reported, with one of the splice variants comprising a 67 kDa protein with an N-terminal elongation, instead of the common 55 kDa. The physiological functions of these TrxR splice variants have yet to be elucidated. See, e.g., Sun, Q.A., et al. Heterogeneity within mammalian thioredoxin reductases: evidence for alternative exon splicing. J. Biol. Chem. 276:3106-3114 (2001).
  • Trx proteins Some of the major functions of mammalian Trx proteins are to supply reducing equivalents to enzymes such as ribonucleotide reductase and thioredoxin peroxidase, as well as (through thiol-disulphide exchange) to reduce key Cys residues in certain transcription factors, resulting in their increased binding to DNA and altered gene transcription.
  • enzymes such as ribonucleotide reductase and thioredoxin peroxidase
  • TrxRs Mammalian Trxs have also been shown to function as cell growth factors and to inhibit apoptosis. Since TrxRs are the only class of enzymes known to reduce oxidized Trx, it is possible that alterations in TrxR activity may regulate some of the activities of Trxs. In addition to Trxs, other endogenous substrates have been demonstrated for TrxRs, including, but not limited to: lipoic acid, lipid hydroperoxides, the cytotoxic peptide NK-lysin, vitamin K 3 , dehydro ascorbic acid, the ascorbyl free radical, and the tumor-suppressor protein p53. See, e.g., Mustacich, D., Powis, G. Thyrodoxin Reductase. Biochem. J. 346:1-8 (2000). However, the physiological role that TrxRs play in the reduction of most of these substrates has not been fully elucidated.
  • Mammalian TrxRs are promiscuous enzymes capable of reducing Trxs of different species, proteins such as NK lysin and p53, a variety of physiological substrates (see, e.g., May, J.M., Cobb, C.E., et al. J. Biol. Chem. 273 :23039-23045 (1998), as well as several exogenous compounds (see, e.g., Kumar, S., Bjornstedt, M., Holmgren, A. Eur. J. Biochem. 207:435-439 (1992).
  • TrxR One suggested catalytic mechanism for human TrxR is that the C- terminal end of the protein is flexible, allowing the -Cys-SeCys-Gly moiety to carry reducing equivalents from the conserved active-site Cys residues to the substrate. See, e.g., Gromer, S., Wissing, J., et al. Biochem. J. 332:591-592 (1998).
  • TrxR The involvement of TrxR in biological functions such as cell growth and protection from oxidative stress has, to date, centred around its role as a reductant for Trx. Further studies are needed to determine whether TrxR has biological functions that are not directly mediated by reduction of Trx.
  • Trx a physiological substrate of TrxRs
  • TrxRs has been shown to play an important role in regulating cell growth and inhibiting apoptosis. See, e.g., Baker, A., Payne, CM., Briehl, M.M., Powis, G. Cancer Res. 57:5162-5167 (1997). Trx has to be in a reduced form in order to exert these effects, and mutant redox-inactive forms of Trx are unable to stimulate cell growth or inhibit apoptosis. The only known mechanism for the reduction of Trx is through NADPH-dependent reduction by TrxR.
  • TrxR activity is associated with inhibited cell growth.
  • Several in vitro inhibitors of TrxR have been reported and, although many of these compounds only inhibit the reduced form of TrxR, it is likely that TrxR will be sensitive to these inhibitors in vivo, since TrxR is expected to exist predominantly in the reduced form due to the presence of cytosolic NADPH concentrations that are greater than the K m of TrxR for NADPH. See, e.g., Gromer, S., Arscott, L.D., et al. J. Biol. Chem. 273 :20096-20101 (1998).
  • TrxR Two such inhibitors of TrxR are the anti-tumour quinones doxorubicin and diaziquone; wherein treatment of cells with either of these compounds leads to secondary inhibition of ribonucleotide reductase and inhibition of cell growth. See, e.g., Hofman, E.R., Boyanapalli, M., et al. Mol. Cell. Biol. 18:6493-6504 (1998).
  • ROS reactive oxygen species
  • Trx has been shown to prevent apoptosis in cells treated with agents known to produce ROS.
  • the levels of TrxR- 1 mRNA and Trx mRNA are increased in the lungs of newborn baboons exposed to air or 0 2 breathing, and increases in TrxR-1 and Trx mRNA are also observed in adult baboon lung explants in response to 95% 0 2 .
  • TrxRl and Trx play a protective role against 0 2 breathing in the mammalian lung.
  • TrxR is highly expressed on the surface of human keratinocytes and melanocytes, where it has been suggested to provide the skin's first line of defence against free radicals generated in response to UV light. See, e.g., Schallreuter, K.U., Wood, J.M. Cancer Lett. 36:297-305 (1997). Cancer Involvement
  • TrxR has been suggested, based on purification yields, that the level of TrxR in tumor cells is 10-fold or more greater than in normal tissues. See, e.g., Tamura, T., Stadtman, T.C. Proc. Natl. Acad. Sci. U.S.A. 93: 1006-1011 (1996). TrxR has also been reported to be elevated in human primary melanoma and to show a correlation with invasiveness. See, e.g., Fuchs, J. Arch. Dermatol. 124:849-850 (1998).
  • the Trx system is as an electron donor for ribonucleotide reducatse, which is frequently greatly over-expressed in cancer cells potentially leading to expanded and inbalanced deoxynucletide pools which are mutagenic, which may accelerate the development of the malignant phenotype by major genetic rearrangements, gene amplifications, total loss of growth control and therapy resistance. It is clearly evident that the Trx system plays a central role in established cancers particularly for distant metastasis and angiogenesis. A recent study utilizing TrxR-1 knock-down in tumor cells interestingly demonstrated a necessity of TrxR-1 expression for cancer cell growth and tumor development. See, e.g., Yoo, M.H., Xu, X.M., et al. Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells. J. Biol. Chem. 281 : 13005-13008 (2006).
  • Thioredoxins are proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. While glutaredoxins mostly reduce mixed disulfides containing glutathione, thioredoxins are involved in the maintenance of protein sulfhydryls in their reduced state via disulfide bond reduction. Thiol-disulfide exchange is a chemical reaction in which a thiolate group (S ) attacks a sulfur atom of a disulfide bond (-S-S-). The original disulfide bond is broken, and its other sulfur atom is released as a new thiolate, thus carrying away the negative charge.
  • S thiolate group
  • a new disulfide bond forms between the attacking thiolate and the original sulfur atom.
  • the transition state of the reaction is a linear arrangement of the three sulfur atoms, in which the charge of the attacking thiolate is shared equally.
  • the protonated thiol form (-SH) is unreactive (i.e., thiols cannot attack disulfide bonds, only thiolates).
  • thiol-disulfide exchange is inhibited at low pH (typically, ⁇ 8) where the protonated thiol form is favored relative to the deprotonated thiolate form.
  • the pK a of a typical thiol group is approximately 8.3, although this value can vary as a function of the environment.
  • Thiol-disulfide exchange is the principal reaction by which disulfide bonds are formed and rearranged within a protein.
  • the rearrangement of disulfide bonds within a protein generally occurs via intra-protein thiol-disulfide exchange reactions; a thiolate group of a cysteine residue attacks one of the protein's own disulfide bonds.
  • This process of disulfide rearrangement (known as disulfide shuffling) does not change the number of disulfide bonds within a protein, merely their location (i.e., which cysteines are actually bonded).
  • Disulfide reshuffling is generally much faster than oxidation/reduction reactions, which actually change the total number of disulfide bonds within a protein.
  • the oxidation and reduction of protein disulfide bonds in vitro also generally occurs via thiol-disulfide exchange reactions.
  • a redox reagent such as glutathione or dithiothreitol (DTT) attacks the disulfide bond on a protein forming a mixed disulfide bond between the protein and the reagent.
  • This mixed disulfide bond when attacked by another thiolate from the reagent, leaves the cysteine oxidized. In effect, the disulfide bond is transferred from the protein to the reagent in two steps, both thiol-disulfide exchange reactions.
  • the mammalian thioredoxins are a family of 10-12 kDa proteins that contain a highly conserved -Trp-Cys-Gly-Pro-Cys-Lys- catalytic site. See, e.g., Nishinaka, Y., et al. Redox control of cellular functions by thioredoxin: A new therapeutic direction in host defense. Arch. Immunol. Ther. Exp. 49:285-292 (2001).
  • the active site sequences is conserved from Escherichia coli to humans.
  • Thioredoxins in mammalian cells possess >90% homology and have approximately 27% overall homology to the E. coli protein.
  • Trx-1 is a 105-amino acid protein. In almost all (>99%) of the human form of Trx-1, the first methionine (Met) residue is removed by an N-terminus excision process (see, e.g., Giglione, C, et al. Protein N-terminal methionine excision. Cell. Mol. Life Sci. 61: 1455-1474 (2004), and therefore the mature protein is comprised of a total of 104-amino acid residues from the N-terminal valine (Val) residue. Trx-1 is typically localized in the cytoplasm, but it has also been identified in the nucleus of normal endometrial stromal cells, tumor cells, and primary solid tumors.
  • Trx -2 is a 166-amino acid residue protein that contains a 60-amino acid residue N-terminal translocation sequence that directs it to the mitochondria. See, e.g., Spyroung, M., et al. Cloning and expression of a novel mammalian thioredoxin. J. Biol. Chem. 272: 2936-2941 (1997). Trx-2 is expressed uniquely in mitochondria, where it regulates the mitochondrial redox state and plays an important role in cell proliferation. Trx-2-deficient cells fall into apoptosis via the mitochondria-mediated apoptosis signaling pathway. See, e.g., Noon, L., et al.
  • Trx-2 The absence of mitochondrial thioredoxin-2 causes massive apoptosis and early embryonic lethality in homozygous mice. Mol. Cell. Biol. 23:916-922 (2003). Trx- 2 was found to form a complex with cytochrome c localized in the mitochondrial matrix, and the release of cytochrome c from the mitochondria was significantly enhanced when expression of Trx-2 was inhibited. The overexpression of Trx-2 produced resistance to oxidant-induced apoptosis in human osteosarcoma cells, indicating a critical role for the protein in protection against apoptosis in mitochondria. See, e.g., Chen, Y., et al.
  • Trx-1 and Trx-2 are known regulators of the manifestation of apoptosis under redox-sensitive capases, their actions may be coordinated.
  • Trx-1 and Trx-2 do not seem to be capable of compensating for each other completely, since Trx-2 knockout mice were found be embryonically lethal. See, e.g., Noon, L., et al.
  • the absence of mitochondrial thioredoxin-2 causes massive apoptosis and early embryonic lethality in homozygous mice. Mol. Cell. Biol. 23:916-922 (2003).
  • TrxR thioredoxin reductase
  • Trx thioredoxin
  • Trx itself is not mutagenic
  • the Trx system is involved in antioxidant defense and probably in prevention of cancer via the removal of carcinogenic oxidants or by repair of oxidized proteins.
  • repair of mutagenic DNA lesions by Trx system-dependent nucleotide excision repair and ribonucleotide reductase may protect from cancer.
  • the Trx system as an electron donor for ribonucleotide reducatse, which is often greatly over- expressed in cancer cells.
  • Thioredoxin (Trx) expression is frequently markedly increased in a variety of human malignancies including, but not limited to, lung cancer, colorectal cancer, cervical cancer, hepatic cancer, pancreatic cancer, and adenocarcinoma. See, e.g., Arne, E.S.J., Holmgren, A. The thirodoxin system in cancer. Sem. Cancer Biol. 16:420-426 (2006).
  • Trx over-expression has also been associated with aggressive tumor growth. See, e.g., Id.
  • This increase in expression level is likely related to changes in the Trx protein structure and function.
  • Trx levels were found to be elevated in 24 of 32 cases, as compared to normal pancreatic tissue; whereas glutaredoxin levels were increased in 29 of 32 of the cases.
  • glutaredoxin levels were increased in 29 of 32 of the cases. See, e.g., Nakamura, H., et al. Expression of thioredoxin and glutaredoxin, redox-regulating proteins, in pancreatic cancer. Cancer Detect. Prev. 24:53-60 (2000).
  • tissue samples of primary colorectal cancer or lymph node metastases had significantly higher Trx-1 levels than normal colonic mucosa or colorectal adenomatous polyps. See, e.g., Raffel, J., et al. Increased expression of thioredoxin-1 in human colorectal cancer is associated with decreased patient survival. J. Lab. Clin. Med. 142:46-51 (2003).
  • Trx expression was associated with aggressive tumor growth and poorer prognosis.
  • tumor cell Trx expression was measured by immunohistochemistry of formalin-fixed, paraffin- embedded tissue specimens. See, e.g., Kakolyris, S., et al. Thioredoxin expression is associated with lymph node status and prognosis in early operable non-small cell lung cancer. Clin. Cancer Res. 7:3087-3091 (2001).
  • TrxR activity is less clear. Tumor cells may not need to increase expression of the TrxR enzyme, although its catalytic activity may be increased functionally.
  • human colorectal tumors were found to have 2-times higher TrxR activity than normal colonic mucosa. See, e.g., Mustacich, D. and Powis, G., Thioredoxin reductase. Biochem. J. 346: 1-8 (2000).
  • TrxR has also been reported to be elevated in human primary melanoma and to show a correlation with invasiveness. See, e.g., Schallreuter, K.U., et al.
  • Thioredoxin reductase levels are elevated in human primary melanoma cells. Int. J. Cancer 48: 15-19 (1991). Further evaluations relating TrxR enzyme levels and catalytic activity with cancer stage and outcome are required to fully elucidate this relationship.
  • Trx thioredoxin
  • This evidence includes, but is not limited to: (i) the resistance of adult T-cell leukemia cell lines to doxorubicin and ovarian cancer cell lines to cisplatin has been associated with increased intracellular Trx-1 levels; (ii) hepatocellular carcinoma cells with increased Trx-1 levels were less sensitive to cisplatin (but not less sensitive to doxorubicin or mitomycin C); (iii) Trx-1 mRNA and protein levels were increased by 4- to 6- fold in bladder and prostate cancer cells made resistant to cisplatin, but lowering Trx-1 levels with an antisense plasmid restored sensitivity to cisplatin and increased sensitivity to several other cytotoxic drugs; (iv) Trx-1 levels were elevated in cisplatin-resistant gastric and colon cancer cells; and (v) stable transfection of
  • Glutathione may also play a role in resistance to the effects of cancer drugs.
  • Glutathione-S-transferases catalyze the conjugation of glutathione to many electrophilic compounds, and can be upregulated by a variety of cancer drugs. Glutathione-S-transferases possess selenium-independent peroxidase activity. ⁇ also has been shown to possess glutaredoxin activity. Some agents are substrates for glutathione-S-transferase and are directly inactivated by glutathione conjugation, thus leading to resistance. Examples of enzyme substrates include melphalan, carmustine (BCNU), and nitrogen mustard. In a panel of cancer cell lines, glutathione-S-transferase expression was correlated inversely with sensitivity to alkylating agents.
  • the sulfur-containing, amino-acid specific small molecules of the present invention include the following molecules: (i) 2,2'-dithio-bis-ethane sulfonate; (ii) the metabolite of 2,2'-dithio-bis-ethane sulfonate, known as 2-mercapto ethane sulfonate; and (iii) additional molecules comprising 2-mercapto-ethane sulfonate conjugated as a disulfide with a substituent group selected from the group consisting of: -Cys, -Homocysteine, -Cys-Gly, - Cys-Glu, -Cys-Glu-Gly, -Cys-Homocysteine, -Homocysteine-Gly, -Homocysteine-Glu, - Homocysteine-Glu-Gly, and
  • Tavocept also know in the literature as 2,2'-dithiobis ethane sulfonate; BNP7787, dimesna
  • 2-mercapto ethane sulfonate act to selectively reduce the toxicity of certain antineoplastic agents in vivo.
  • 2-mercapto-ethane sulfonate conjugated as a disulfide with substituent group comprising of one or more amino acid residues are known herein as Tavocept-derived heteroconjugates.
  • Tavocept is the physiological auto -oxidation dimer of mesna.
  • Tavocept (II) have the following molecular structures:
  • the pharmaceutical chemistry of the aforementioned compounds indicates that the terminal sulfhydryl group of mesna (and to a lesser extent the disulfide linkage in dimesna) acts as a substitution group for the terminal hydroxy- or aquo- moiety in the active metabolites of, e.g., platinum complexes.
  • Dimesna requires a metabolic activation, such as by glutathione reductase, to exert its biologically efficacious results. Dimesna also exhibits significantly lower toxicity than mesna.
  • the putative mechanisms of the sulfur-containing, amino-acid specific small molecules of the present invention which function to increase the cytotoxic or cytostatic activity of cancer treating agents may involve one or more of several novel pharmacological and physiological factors.
  • Preferred doses of the sulfur-containing, amino-acid specific small molecules of the present invention range from about 1 g/m 2 to about 50 g/m 2 , preferably about 5 g/m 2 to about
  • 40 g/m 2 (for example, about 10 g/m 2 to about 30 g/m 2 ), more preferably about 14 g/m 2 to about 22 g/m 2 , with a most preferred dose of 18.4 g/m 2.
  • Tavocept is a sulfur-containing, amino acid-specific, small molecule that possesses the ability to function as a multi-target modifier and/or modulator of the function of the target molecules of the present invention.
  • Tavocept mediates the non-enzymatic xenobiotic modification of sulfur-containing amino acid residues (e.g., cysteine) on proteins.
  • sulfur-containing amino acid residues e.g., cysteine
  • Tavocept is autocatalytic and requires no protein cofactor to cause the xenobiotic modification of cysteine, but appears to be specific for cysteine residues located within a particular structural context (i.e., not all cysteines in a protein are so modified).
  • Tavocept-mediated, xenobiotic modification represents a novel mechanism of action for a cancer treating agent and can be compared to a degree with post-translational modifications of cysteine residues in proteins (see, Table 3, below).
  • an important element of Tavocept's effectiveness as a compound in the treatment of cancer is its selectivity for normal cells versus cancer cells and its absence of interference with the anti-cancer activity of cancer treating agents.
  • In vitro studies demonstrated that Tavocept does not interfere with paclitaxel induced apoptosis, as assessed by PARP cleavage, Bcl-2 phosphorylation, and DNA laddering in human breast, ovarian and lymphoma cancer cell lines. Additionally, Tavocept was shown not to interfere with paclitaxel- and platinum-induced cytotoxicity in human cancer cell lines, which are discussed herein, infra.
  • Tavocept The believed mechanisms underlying the absence of interference with anti-cancer activity by Tavocept are multifactorial and, as previously discussed, may involve its selectivity for normal cells versus cancer cells, inherent chemical properties that have minimal impact in normal cells on critical plasma and cellular thiol-disulfide balances, and its interactions with cellular oxidoreductases, which are key in the cellular oxidative/reduction (redox) maintenance systems.
  • Tavocept may elicit the restoration of apoptotic sensitivity in tumor cells through, e.g., thioredoxin- and glutaredoxin-mediated mechanisms and this may be an important element of its effectiveness as a chemotherapeutic agent.
  • cysteine residue requires certain physico-chemical characteristics, these include: (i) accessibility; (ii) proximity to a hydrogen bond donor (facilitating thiolate formation); (iii) a shielded or hydrophobic microenvironment (to stabilize the thiolate); and (iv) location within or near a-helix (cysteines within ⁇ -strands do not appear to react).
  • a number of important target molecules contain Tavocept-reactive cysteine moieties.
  • molecular targets include, but are not limited to, anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS 1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif comprising the physicochemical characteristics described above and in subsequent paragraphs.
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS 1 receptor tyrosine kinase
  • EGFR epidermal growth factor receptor
  • Prx peroxiredoxin
  • ERCC1 excision repair cross-complementing protein 1
  • Tavocept-mediated xenobiotic modification on molecular targets that are involved in regulating cell growth and cell survival, and thereby impact cancer and other diseases, manifests itself in distinct, target-specific ways that are correlated to the role of the cysteine residue that undergoes xenobiotic modification, including:
  • the sulfur-containing, amino acid-specific small molecules of the present invention represent novel, first in class, cancer treating agents that are shown herein, specifically and unequivocally, to work through a mechanism of action involving cysteine modification of proteins that directly translates to impaired, inhibited, or altered protein function.
  • rheumatoid arthritis In rheumatoid arthritis (RA), the synovial membrane exhibits molecular features common to cancer including hyperplasia and a tendency towards invasiveness.
  • Swanson et al at Stanford's Division of Immunology and Rheumatology, studied a mouse model of human rheumatoid arthritis (the murine collagen-induced arthritis model) and conducted studies on human cell and tissue samples. They observed that rheumatoid arthritis patients highly express activated EGFR in their synovial tissue. They also found that vascular endothelial and fibroblast cells from rheumatoid arthritis patients express the epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • the EGFR targeted inhibitor, Erlotinib was show to inhibit proliferation of the human endothelial cell line HUVEC in vitro. This finding is important because in rheumatoid arthritis, endothelial cells line the blood vessels found in the synovial membrane. As rheumatoid arthritis progresses, neovascularization or neoangiogenesis occurs providing nourishment for the continued growth of the synovial membrane (synovium). Compounds that inhibit EGFR are expected to inhibit the formation of new blood vessels in the synovium and to serve as effective anti -rheumatoid arthritis treatment agents.
  • thioredoxin levels of thioredoxin are elevated in human immunodeficiency virus 1 (HIV-1) infected patients that have the acquired immune deficiency syndrome (AIDS) phenotype.
  • thioredoxin cleaves the Cys296-Cys331 disulfide bond present in the HIV envelope glycoprotein (gpl20), resulting in gpl20 refolding/reorganization, a process which activates the protein and facilitates infection. Subsequent to the thioredoxin-mediated disulfide bond cleavage, gpl20 fuses with the cell membrane and infects the cell.
  • thioredoxin inhibition of chemotaxis and curtailment of life expectancy in AIDS, Proc. Natl. Acad. Sci. U.S.A. 980 ⁇ :2688-2693(2OOl).
  • amyloid ⁇ ( ⁇ ) peptide which is mediated by the secretase proteins (a-secretase, ⁇ -secretase, and ⁇ -secretase).
  • secretase proteins a-secretase, ⁇ -secretase, and ⁇ -secretase.
  • elevated amyloid ⁇ ( ⁇ ) peptide has been reported to be correlated to peroxiredoxin 1 levels, and
  • peroxiredoxin appears to modulate ⁇ -secretase expression. See, e.g., Lee, et al,
  • Peroxiredoxin 1 regulates the component expression of ⁇ -secretase complex causing the Alzheimer's disease. Lab. Anim. Res. 27(4):293-299 (2011); De Strooper, et al, The secretases: enzymes with therapeutic potential in Alzheimer disease. Nat. Rev. Neurol. 6(2):99-107 (2010).
  • a post-mortem study also indicated that peroxiredoxin 1 is elevated in the brains of Alzheimer's disease patients. See e.g., Cumming et al, Protein Synthesis, Post- Translational Modification and Degradation: Increase in Expression Levels and Resistance to Sulfhydryl Oxidation of Peroxiredoxin Isoforms in Amyloid ⁇ -Resistant Nerve Cells. J. Biol. Chem.
  • ALS amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • cysteine residues ⁇ e.g., by glutathione and nitric oxide
  • glutathione and nitric oxide The post-translational modification of cysteine residues ⁇ e.g., by glutathione and nitric oxide) on key proteins important in neurodegenerative processes appears to be important and may impact disease progression ⁇ see, e.g., Liedhegner ,et al, Mechanisms of Altered Redox Regulation in Neurodegenerative Diseases - Focus on S- Glutathionylation. Antiox. Redox. Signal. 16(6):543-566 (2012); Mieyal, et al, Molecular Mechanisms and Clinical Implications of Reversible Protein S-Glutathionylation. Antiox. Redox. Signal. 10(11): 1941-1988 (2008)); therefore, the development of small molecules that can modulate cysteine function is believed to have clinically important potential for the treatment of these diseases.
  • ACS acute Coronary Syndrome
  • PCM Dilated Cardiomyopathy
  • Trx thioredoxin
  • Trx Serum Thioredoxin
  • Progerin is a key protein that is mutated in progeria and is a truncated variant of prelamin A. Progerin contains a famesylated cysteine residue at its carboxy-terminus that prevents the protein from dissociating from the nuclear membrane. See, e.g., Capell, et al., Inhibiting famesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome. Proc. Natl. Acad. Sci. U.S.A.
  • Inhibitors of famesyltransferase improve the phenotypic symptoms associated with progeria in mouse models, inhibit typical nuclear malformations seen in progeria patients, and restored gene expression in cells from HGPS patients to a normal profile. See, e.g., Capell, et al, Inhibiting famesylation of progerin prevents the
  • the teachings in the present application take into account the concept of disease heterogeneity, in combination with new observations and data, in order to provide novel pharmaceutical compositions, methods, and kits used for the treatment of cancer and other medical conditions.
  • One embodiment of the present invention discloses a contemporaneous,
  • heterogeneously-oriented, multi-targeted method comprising the therapeutic modification and/or modulation of one or more types of disease (including cancer) for purposes of minimizing or overcoming the deleterious physiological ramifications of, e.g., cancer heterogeneity, where the method is comprised of the modification and/or modulation of: (i) the expression level and/or (ii) the biochemical function of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; by
  • Another embodiment of the present invention discloses a contemporaneous, heterogeneously-oriented, multi-targeted method comprising the therapeutic modification and/or modulation of one or more types of disease (including cancer) for purposes of minimizing or overcoming the deleterious physiological ramifications of, e.g., cancer heterogeneity, where the method is comprised of the modification and/or modulation of: (i) the expression level and/or (ii) the biochemical function of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other
  • One embodiment of the present invention discloses a method for the metabolic modification and/or modulation of the expression level of multiple target molecules; where the target molecules are selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase
  • ROS1 epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif ; and where the method is comprised of the administration of the sulfur-containing, amino acid-specific small molecules of the present invention in an amount sufficient to provide a therapeutic benefit to a subject suffering from one or more types of cellular metabolic anomalies or other pathophysiological conditions where the expression levels of one or more of the target molecules is abnormally elevated and metabolic modification and/or modulation of the target molecule(s) is used to treat the cellular metabolic anomalies or other pathophysiological conditions.
  • Another embodiment of the present invention discloses a method for the metabolic modification and/or modulation of the biochemical activity of multiple target molecules; where the target molecules are selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif; and where the method is comprised of the administration of the sulfur-containing, amino acid-specific small molecules of the present invention in an amount sufficient to provide a therapeutic benefit to a subject suffering from one or more types of cellular metabolic anomalies or other pathophysiological conditions where the biochemical activities of the multiple
  • the sulfur-containing, amino acid- specific small molecules are selected from the group consisting of: (i) 2,2'-dithio-bis-ethane sulfonate; (ii) the metabolite of 2,2'-dithio-bis-ethane sulfonate, known as 2-mercapto ethane sulfonate; and (iii) 2-mercapto -ethane sulfonate conjugated as a disulfide with a substituent group selected from the group consisting of: -Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -
  • cancers selected from the group consisting of: lung cancer, colorectal cancer, gastric cancer, esophageal cancer, cancer of the biliary tract, gallbladder cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, myeloma, uterine cancer, prostate cancer, hepatic cancer,
  • adenocarcinoma pancreatic cancer
  • brain cancer various types of skin cancer, including melanoma, are disclosed.
  • the cellular metabolic anomalies or other pathophysiological conditions are non-cancerous diseases selected from the group consisting of: heart failure, heart disease, hypertension, myocardial infarction, vascular disease, atherosclerosis, diabetes-induced heart disease, neurodegenerative diseases,
  • Parkinson's disease ALS, neurovascular dementia, autoimmune diseases, systemic lupus erythematosus, Graves orbitopathy, alcoholic liver disease, inflammatory bowel disease, cystic fibrosis, inflammatory diseases, diabetes, rheumatoid arthritis, progeria, Xeroderma pigmentosum, Cockayne syndrome, Fanconi anemia, and cerebro-oculo-facio-skeletal syndrome.
  • One embodiment of the present invention discloses a method to modify and/or modulate the intracellular environment of cancer cells in a subject suffering from cancer such that the intracellular environment of said cancer cells is made more amenable to the pharmacological activity of cancer treating agent(s) administered to treat the subject's cancer; where the method is comprised of the administration of an amount of the sulfur-containing, amino acid-specific small molecules of the present invention sufficient to modify and/or modulate the intracellular environment of cancer cells in the subject suffering from cancer; and where the cancer involves: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF
  • Another embodiment of the present invention discloses a method to modify and/or modulate the intracellular environment of cells in a subject suffering from cellular metabolic anomalies or other pathophysiological conditions such that the intracellular environment of the cells is made more amenable to the pharmacological activity of medicinal agent(s) administered to treat the subject's cellular metabolic anomalies or other pathophysiological conditions; where the method is comprised of the administration of an amount of the sulfur- containing, amino acid-specific small molecules of the present invention sufficient to modify and/or modulate the intracellular environment of cells in the subject suffering from the cellular metabolic anomalies or other pathophysiological conditions where the cellular metabolic anomalies or other pathophysiological conditions involve: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS 1), epidermal
  • cancers selected from the group consisting of: colorectal cancer, gastric cancer, esophageal cancer, cancer of the biliary tract, gallbladder cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, lung cancer, vaginal cancer, uterine cancer, prostate cancer, hepatic cancer, adenocarcinoma, pancreatic cancer, brain cancer, lung cancer, various types of skin cancer (e.g., melanoma), and myeloma, lymphoma and other cancers of the blood are disclosed.
  • skin cancer e.g., melanoma
  • cellular metabolic anomalies or other pathophysiological conditions of non-cancerous diseases selected from the group consisting of: heart failure, heart disease, hypertension, myocardial infarction, vascular disease, atherosclerosis, diabetes-induced heart disease, neurodegenerative diseases,
  • Parkinson's disease ALS, neurovascular dementia, autoimmune diseases, systemic lupus erythematosus, Graves orbitopathy, alcoholic liver disease, inflammatory bowel disease, cystic fibrosis, inflammatory diseases, diabetes, rheumatoid arthritis, progeria, Xeroderma pigmentosum, Cockayne syndrome, Fanconi anemia, and cerebro-oculo-facio-skeletal syndrome are disclosed.
  • One embodiment of the present invention discloses a method for treating a subject suffering from cancer where a multi-targeted, molecular-directed treatment regimen is beneficial in overcoming cellular metabolic resistance to treatment in a subject with cancer that is heterogeneous; where the cellular metabolic resistance to treatment is associated with: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif; and where the method is comprised of the administration of an amount of the sulfur-containing, amino acid-specific small molecules of the present invention sufficient to overcome the cellular metabolic resistance to treatment in said subject with cancer that is heterogeneous.
  • Another embodiment of the present invention discloses a method for treating a subject suffering from cellular metabolic anomalies or other pathophysiological conditions where a heterogeneous, multiple targeted, molecular-directed treatment regimen is beneficial in overcoming cellular metabolic resistance to treatment in the subject with cellular metabolic anomalies or other pathophysiological conditions; wherei the cellular metabolic resistance to treatment is associated with: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules
  • a method to determine the amount of the sulfur-containing, amino acid-specific small molecules of the present invention required to be administered to provide a therapeutic benefit to a subject with cancer that involves: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the method is comprised of determining: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of the target molecules and then
  • the method of determining the amount of the sulfur-containing, amino acid- specific small molecules of the present invention required to be administered to provide a therapeutic benefit to a subject with cancer that involves: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of target molecules is selected from the group consisting of: (i) fluorescence in situ hybridization (FISH), nucleic acid microarray analysis, immunohistochemistry (IHC), radioimmunoassay (RIA), quantitative immunofluorescence and/or automated quantitative analysis ⁇ e.g., Genoptix's AQUA); (ii) ELISA approaches including, but not limited to, high-throughput ELISA, InCell ELISAs, or quantitative western analyses ⁇ e.g., Licor and related systems), and related ELISA methodologies, and flow cytometry-based analyses ⁇ e.g., Affymetrix's Luminex assay and related approaches); (iii) PCR coupled with MS approaches including, but not limited to, MALDI-TOF MS ⁇ e
  • a method to determine the amount of the sulfur-containing, amino acid-specific small molecules of the present invention required to be administered to provide a therapeutic benefit to a subject with cellular metabolic anomalies or other undesirable physiological conditions that involve: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS 1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the method is comprised of determining (i) the abnormal biochemical activity and/or (ii)
  • a method for use in: (a) the selection of subjects for treatment; (b) the determination of the most effective chemotherapeutic agent(s) to be administered in combination with the administration of the sulfur-containing, amino acid-specific small molecules of the present invention; (c) the dosage of the chemotherapeutic agent(s) to be administered; (d) the determination of the length and/or number of treatment cycles; and/or (e) adjustment of the specific chemotherapeutic agent(s) used and the dosage administered during treatment of a subject having cancer is disclosed; where the method is comprised of quantitatively determining the levels of expression of target molecules selected from the group consisting of: and other target molecules possessing a similar active site or structural motif, and then using these expression levels in determining: (i) the specific subjects to be treated; (ii) the chemotherapeutic agent(s) to be administered in combination with the administration of the sulfur-containing, amino acid-specific small molecules of the present invention; (iii) the dosage of the chemotherapeutic agent(s
  • cancers selected from the group consisting of: colorectal cancer, gastric cancer, esophageal cancer, cancer of the biliary tract, gallbladder cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, uterine cancer, prostate cancer, hepatic cancer, adenocarcinoma, pancreatic cancer, brain cancer, lung cancer, various types of skin cancer (e.g., melanoma), and lymphoma and other cancers of the blood are disclosed.
  • colorectal cancer gastric cancer, esophageal cancer, cancer of the biliary tract, gallbladder cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, uterine cancer, prostate cancer, hepatic cancer, adenocarcinoma, pancreatic cancer, brain cancer, lung cancer, various types of skin cancer (e.g., melanoma), and lymphoma and other cancers of the blood are disclosed.
  • cancer treating agent(s) are selected from the groups consisting of: (i) fluropyrimidines; pyrimidine nucleosides; purine nucleosides; anti-folates, platinum agents; anthracyclines/anthracenediones;
  • epipodophyllotoxins camptothecins; vinca alkaloids; taxanes; epothilones; antimicrotubule agents; alkylating agents; antimetabolites; topoisomerase inhibitors; aziridine-containing compounds, and various other cytotoxic and cytostatic agents;
  • hormones, hormonal complexes, and antihormones selected from the group comprising: interleukins, interferons, leuprolide, and pegasparaginase;
  • enzymes, proteins, peptides, and antivirals including enzymes, proteins, peptides, and antivirals selected from the group consisting of: acyclovir and zidovudine;
  • cytotoxic agents and cytostatic agents; polyclonal and monoclonal antibodies, including agents selected from the group consisting of: crizotinib, gefitinib, erlotinib, cetuximab, afatinib, dacomitini
  • the subjects selected for treatment are further categorized into various subtypes for more beneficial treatment which include, but are not limited to, female subjects; non-smoker subjects; female, non-smoker subjects; male, non-smoker subjects; non-smoker subjects with expression of ALK and/or MET; subjects over 65 years of age; subjects whose cancer has been categorized as Stage Mia or Mlb; subjects who are currently being or have previously been treated with paclitaxel and/or cisplatin, and various combinations of the foregoing.
  • a method for use in: (a) the selection of specific subjects for treatment; (b) the determination of the most effective medicinal agent(s) in combination with the administration of the sulfur-containing, amino acid-specific small molecules of the present invention; (c) the dosage of the medicinal agent(s) to be administered; (d) the determination of the length and/or number of treatment cycles to be administered; and/or (e) adjustment of the specific medicinal agent(s) used and the dosages administered during treatment of a subject with non-cancerous, cellular metabolic anomalies or other pathophysiological conditions is disclosed; where the method is comprised of quantitatively determining the levels of expression of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ALK), mesenchy
  • a method for maximizing the length of time before there is cancer progression in a subject who has cancer that involves: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the method comprises the administration of the sulfur-containing, amino acid-specific small molecules of the present invention which function to delay the reoccurrence and/or progression of the cancer in the subject by modifying
  • kits for use in the treatment of a subject having cancer that is resistant to the chemotherapeutic agent(s) being used to treat the subject with cancer where the cancer is any cancer which: (i) abnormally overexpresses anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), ribonucleotide reductase (RNR), tubulin, farnesyltransferase, and/or other target protein (possessing a similar active site or structural motif) and/or (ii) exhibits evidence of anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kina
  • ALK abnormally overexpresse
  • a further embodiment of the present invention discloses a kit for use in the treatment of a subject having cancer that is resistant to the chemotherapeutic agent(s) being used to treat the subject with cancer, where the cancer is any cancer which: (i) possesses abnormal biochemical activity in anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, and/or other target protein (possessing a similar active site or structural motif) and/or (ii) exhibits evidence of anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor
  • kits comprising: (a) one or more medicinal agents; (b) the sulfur-containing, amino acid-specific small molecules of the present invention; and (c) instructions for the administration of said medicinal agents and the sulfur-containing, amino acid-specific small molecules of the present invention to a subject having cellular metabolic anomalies or other undesirable physiological conditions that cause: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the sulfur-containing, amino acid-specific small molecules of the present invention function to modify and/or modulate the abnormal biochemical activity and/or abnormal expression of the target molecules in the subject having cellular metabolic anomalies or other undesirable physiological conditions.
  • kits comprising: (a) one or more chemotherapy agents; (b) the sulfur-containing, amino acid-specific small molecules of the present invention; and (c) instructions for administering said chemotherapy agent(s) and the sulfur-containing, amino acid-specific small molecules of the present invention to a subject with a type of cancer that is generally less responsive to particular types of chemotherapeutic treatments; where the sulfur-containing, amino acid-specific small molecules of the present invention are administered in an amount sufficient to cause an increase in the cytotoxic or cytostatic activity of the administered chemotherapeutic agent(s) whose cytotoxic or cytostatic activity was heretofore adversely affected by: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of the target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor
  • ALK anaplastic lymphom
  • One embodiment of the present invention discloses a medicament which modifies and/or modulates the expression levels of the target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the medicament is the sulfur-containing, amino acid-specific small molecules of the present invention administered in an amount sufficient to provide a therapeutic benefit to a subject having a type of cellular metabolic anomaly or other undesirable physiological condition where the expression levels of said target molecules are abnormally elevated and must be modified and/or modulated in order to treat said
  • a further embodiment of the present invention discloses a medicament which modifies and/or modulates the biochemical activity of the target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; wherein said medicament is the sulfur-containing, amino acid-specific small molecules of the present invention administered in an amount sufficient to provide a therapeutic benefit to a subject having a cellular metabolic anomaly or other undesirable physiological condition where the biochemical activities of the target molecules are abnormal and must be modified and/or modulated in order to treat the
  • a method for the prophylactic use of the sulfur-containing, amino acid-specific small molecules of the present invention administered in an amount sufficient to provide a prophylactic benefit to a subject who has previously suffered from a form of cancer that involves: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the sulfur-containing, amino acid-specific small molecules of the present invention function to
  • a method for the prophylactic use of the sulfur-containing, amino acid-specific small molecules of the present invention administered in an amount sufficient to provide a prophylactic benefit to a subject who has previously suffered from a type of cellular metabolic anomaly or other undesirable physiological condition that involves: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the sulfur-
  • a further embodiment of the present invention discloses a method to restore normal cellular biochemical function and/or normal expression levels of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross- complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the method is comprised of the administration of the sulfur-containing, amino acid-specific small molecules of the present invention in an amount sufficient to provide a therapeutic benefit to a subject having cancer where the normal cellular biochemical function and/or the expression levels of the target molecules are abnormal and must be modified and/or modulated in
  • One embodiment of the present invention discloses a method to restore the normal cellular biochemical function and/or the expression level of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the method is comprised of the administration of the sulfur-containing, amino acid-specific small molecules of the present invention in an amount sufficient to provide a therapeutic benefit to a subject having a cellular metabolic anomaly or other undesirable physiological condition, including cancer, where the normal cellular biochemical function and/or the expression levels of the target molecules
  • Another embodiment of the present invention discloses a method for the maintenance of a subject having cancer; where the method is comprised of the modification and/or modulation of: (i) the expression level and/or (ii) the biochemical function of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross- complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif ; and where the method is comprised of the method is comprised of the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition
  • Another embodiment of the present invention discloses a treatment method which comprises the administration of one or more cancer treating agents and an amount of the sulfur-containing, amino acid-specific small molecules of the present invention sufficient to provide a therapeutic benefit to a subject with lymphoma, acute lymphocytic leukemia (ALL), or acute myelogenous leukemia (AML) cancers that involve: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of the tyrosine kinase enzyme, anaplastic lymphoma kinase (ALK) or epidermal growth factor receptor (EGFR).
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • adduct formation comprising the covalent-binding of one or more sulfur-containing, amino acid-specific small molecules of the present invention to cysteine amino acid residues within a target molecule selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; where the adduct formation has the ability to modify and/or modulate abnormal expression and/or biochemical activity of said target molecule(s) so as to provide a therapeutic
  • ALK anaplastic lymphoma kinase
  • Another embodiment discloses a method for quantitatively ascertaining the level of DNA, mRNA, and/or protein of a target molecule selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif or in other target molecules possessing cysteine residues with similar functional or structural characteristics, in cells which have been isolated from a patient who is suspected of having cancer or has already been diagnosed with cancer; where the method used to identify levels of the DNA, mR A, and/or protein of a target molecule(s
  • IHC immunohistochemistry
  • RIA radioimmunoassay
  • quantitative immunofluorescence and/or automated quantitative analysis ⁇ e.g., Genoptix's AQUA
  • ELISA approaches including, but not limited to, high-throughput ELISA, InCell ELISAs, or quantitative western analyses ⁇ e.g., Licor and related systems), and related ELISA methodologies, and flow cytometry-based analyses ⁇ e.g., Affymetrix's Luminex assay and related approaches
  • PCR coupled with MS approaches including, but not limited to, MALDI-TOF MS ⁇ e.g., Sequenom's MassARRAY system and related approaches
  • mass spectroscopy based methods including, but not limited to, NanoLC coupled with ESI-MS ⁇ e.g., Bruker
  • RNAs corresponding to the protein targets described above have been determined in a range of biological samples (cell lines, biological fluids, patient samples, and the like) and some examples are summarized in Table 4. Additionally, the amount of up- or down- regulation of the RNAs corresponding to the protein targets described above has been determined and some examples are summarized in Table 5. Additionally, the Human Protein Atlas ⁇ see, www.proteinatlas.org) provides information on mRNA and protein expression patterns for all of the targets cited herein across a range of biological samples although the data on this site is not exhaustive.
  • a further embodiment discloses a method for quantitatively ascertaining the level of DNA, mRNA, and/or protein of a target molecule for the purpose of providing treatment with the sulfur-containing, amino acid-specific small molecules of the present invention, where the target molecule is selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross- complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif or in other target molecules possessing cysteine residues with similar functional or structural characteristics, in cells which have been isolated from a patient who is suspected of having a non-cancerous
  • a further embodiment of the present invention discloses a method for improving biological system stability in a subject with one or more types of cancer, where the system stability is impacted by: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; and where the method is comprised of the administration of the sulfur-containing, amino acid-specific small molecules of the present invention in an amount sufficient to provide a therapeutic benefit by improving biological system
  • Another embodiment of the present invention discloses a method for improving biological system stability in a subject having one or more types of cellular metabolic anomalies or other pathophysiological conditions, where the system stability is impacted by: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 epidermal growth factor receptor
  • Prx peroxiredoxin
  • ERCC1 excision repair cross-complementing protein 1
  • IGF1R insulin growth factor 1 receptor
  • tubulin tubulin
  • RNR ribonucleotide reductase
  • farnesyltransferase and other target molecules possessing a similar active site or structural motif
  • a further embodiment of the present invention discloses a method for improving biological system stability by altering the relative level of non-clonal chromosomal aberrations (NCCAs) in a subject with one or more types of cancer, where the relative level of non-clonal chromosomal aberrations (NCCAs) is impacted by: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross- complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or
  • NCCAs non-clonal chromosomal aberrations
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 receptor tyrosine kinase
  • EGFR epidermal growth factor receptor
  • Poxiredoxin P
  • One embodiment of the present invention discloses a method for improving biological system stability by altering the elevated levels of non-clonal chromosomal aberrations (NCCAs) in a subject having one or more types of cancer, where the method is comprised of the administration of the sulfur-containing, amino acid-specific small molecules of the present invention in an amount sufficient to provide a therapeutic benefit by modifying and/or modulating the elevated level of non-clonal chromosomal aberrations (NCCAs) in the subject having one or more types of cancer.
  • NCCAs non-clonal chromosomal aberrations
  • Another embodiment of the present invention discloses a method for improving biological system stability by altering the elevated levels of non-clonal chromosomal aberrations (NCCAs) in a subject having one or more types of non-cancerous cellular metabolic anomalies or other pathophysiological conditions, where the method is comprised of the administration of the sulfur-containing, amino acid-specific small molecules of the present invention in an amount sufficient to provide a therapeutic benefit by modifying and/or modulating the elevated level of non-clonal chromosomal aberrations (NCCAs) in the subject having one or more types of non-cancerous cellular metabolic anomalies or other pathophysiological conditions.
  • NCCAs non-clonal chromosomal aberrations
  • a method for the prophylactic use of the sulfur-containing, amino acid-specific small molecules of the present invention administered in an amount sufficient to provide a prophylactic benefit to a subject who has previously suffered from a type of cellular metabolic anomaly or other undesirable physiological condition that involves: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif; and where the method is
  • a method for the treatment of a subject who has one or more types of cancer that involve: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif, is disclosed; where the method is comprised of the administration of the sulfur- containing, amino acid-specific small molecules of the present invention in combination with: (a) the chemotherapeutic agent cisplatin; and
  • a method for the neo-adjuvant treatment of a subject who has one or more types of cancer that involve: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif, is disclosed; where the method is comprised of the administration of the sulfur- containing, amino acid-specific small molecules of the present invention prior to the subsequent administration of the primary chemotherapeutic
  • target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif.
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 receptor tyrosine kinase
  • EGFR epidermal growth factor receptor
  • Prx peroxiredoxin
  • ERCCl excision repair cross-complementing protein 1
  • IGFIR insulin growth factor 1 receptor
  • tubulin tubulin
  • a method for the adjuvant treatment of a subject who has one or more types of cancer that involve: (i) the abnormal biochemical activity and/or (ii) the abnormal expression of any combination of target molecules selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross-complementing protein 1 (ERCCl), insulin growth factor 1 receptor (IGFIR), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif, is disclosed; where the method is comprised of the administration of the sulfur- containing, amino acid-specific small molecules of the present invention subsequent to the administration of the initial, primary chemotherapeutic regimen in an amount sufficient
  • cancer with a T790 mutation in the epidermal growth factor receptor (EGFR) gene is disclosed.
  • active site refers to a specific region of an enzyme where a substrate binds and catalysis takes place (binding site/active site). This is the region of an enzyme where the chemical reaction occurs.
  • the active site is usually found in a 3- Dimensional groove or pocket of the enzyme, lined with amino acid residues (or nucleotides in RNA enzymes). These residues are involved in recognition of the substrate. Residues that directly participate in the catalytic reaction mechanism are called active site residues. After an active site has completed the specific chemical reaction, it can then bind another substrate molecule and catalyze another chemical reaction. Substrates bind to the active site of the enzyme through hydrogen bonds, hydrophobic interactions, temporary covalent interactions (van der Waals) or a combination of all of these to form the enzyme-substrate complex.
  • Residues of the active site will act as donors or acceptors of protons or other groups on the substrate to facilitate the reaction. Therefore, the active site modifies the reaction mechanism in order to change the activation energy of the reaction.
  • An enzyme binding to a substrate will lower the energy barrier that normally stops the reaction from occuring.
  • the product of the chemical reaction is usually unstable in the active site due to steric hinderances that force it to be released, thus returning the enzyme to its initial unbound state.
  • the induced fit theory of enzyme-substrate binding states that the active site and the binding portion of the substrate are not exactly complementary.
  • the induced fit model is a development of the lock-and-key model and assumes that an active site is "flexible" and it changes shape until the substrate is completely bound.
  • the substrate is thought to induce a change in the shape of the active site.
  • the hypothesis also predicts that the presence of certain amino acid residues in the active site will encourage the enzyme to locate the correct substrate. Conformational changes may then occur as the substrate is bound. After the products of the reaction move away from the enzyme, the active site returns to its initial conformational shape. Active sites which possess similar conformational shapes and/or amino acid sequences frequently catalyze similar substrates, e.g., kinases catalyze the phosphorylation of proteins and enzymes.
  • adenocarcinoma refers to a cancer that originates in glandular tissue.
  • Glandular tissue comprises organs that synthesize a substance for release such as hormones. Glands can be divided into two general groups: (i) endocrine glands - glands that secrete their product directly onto a surface rather than through a duct, often into the blood stream and (ii) exocrine glands - glands that secrete their products via a duct, often into cavities inside the body or its outer surface.
  • the tissues or cells do not necessarily need to be part of a gland, as long as they have secretory properties.
  • Adenocarcinoma may be derived from various tissues including, but not limited to, breast, colon, lung, prostate, salivary gland, stomach, liver, gall bladder, pancreas ⁇ e.g., 99% of pancreatic cancers are ductal adenocarcinomas), cervix, vagina, and uterus, as well as unknown primary adenocarcinomas.
  • Adenocarcinoma is a neoplasm which frequently presents marked difficulty in differentiating from where and from which type of glandular tissue the tumor(s) arose. Thus, an adenocarcinoma identified in the lung may have had its origins (or may have metastasized) from an ovarian
  • Adjuvant therapy means additional treatment of a subject with cancergiven after the primary treatment or surgery to lower the risk that the cancer will come back.
  • Adjuvant therapy may include treatment with cancer treating agents such as chemotherapeutic agents, radiation therapy, hormones, cytotoxic or cytostatic agents, antibodies and/or sulfur-containing, amino acid-specific small molecules of the present invention.
  • an amount sufficient to provide a therapeutic benefit” or “a therapeutically-effective” amount” in reference to the medicaments, compounds, or compositions of the instant invention refers to the administered dosage that is sufficient to induce a desired biological, pharmacological, or therapeutic outcome(s) in a subject suffering from one or more types of cellular metabolic anomalies or other pathophysiological conditions, including cancer.
  • such outcome(s) can include: (i) cure or remission of previously observed cancer(s); (ii) shrinkage of tumor size; (iii) reduction in the number of tumors; (iv) delay or prevention in the growth or reappearance of cancer; (v) selectively sensitizing cancer cells to the activity of the anti-cancer agents; (vi) restoring or increasing apoptotic effects or sensitivity in tumor cells; and/or (vii) increasing the time of survival of the subject, alone or while concurrently experiencing reduction, prevention, mitigation, delay, shortening the time to resolution of, alleviation of the signs or symptoms of the incidence or occurrence of an expected side- effect(s), toxicity, disorder or condition, or any other untoward alteration in the subject.
  • cancer refers to all known forms of cancer including, solid forms of cancer (e.g., tumors), lymphomas, and leukemias.
  • cancer treating agent refers to medicament(s) that reduces, prevents, mitigates, limits, and/or delays the growth of metastases or neoplasms, or kills neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used, in a pharmaceutically- effective amount, to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease.
  • the cancer treating agents of the present invention include, but are not limited to: (i) chemotherapeutic agents (e.g., fluropyrimidines, pyrimidine nucleosides, purine nucleosides, anti-folates, platinum agents,
  • chemotherapeutic agents e.g., fluropyrimidines, pyrimidine nucleosides, purine nucleosides, anti-folates, platinum agents
  • anthracyclines/anthracenediones epipodophyllotoxins, camptothecins, vinca alkaloids, taxanes, epothilones, antimicrotubule agents, alkylating agents, antimetabolites,
  • hormones e.g., interleukins, interferons, leuprolide, pegasparaginase, and the like
  • antihormonals e.g., interleukins, interferons, leuprolide, pegasparaginase, and the like
  • enzymes, proteins, and peptides enzymes, proteins, and peptides; antivirals (e.g., acyclovir, zidovudine, and the like);
  • cytotoxic agents cytostatic agents
  • polyclonal and monoclonal antibodies e.g., crizotinib, geiitmib, erlotinib, cetuximab, afatinib, dacomitinib, ramucirumab, necitumumab, lenvatinib, palbociclib, alectinib, zybrestat, tecemotide, obinutuzumab (GA101), AZD9291 , CO-1686, vintafolide, CRLX101 , ipilimumab, yervoy, nivolumab, ibrutinib, selumetinib, olaparib, trastuzumab, lucitanib, rucaparib, NOV-002, MPDL3280A, pembrolizumamb, lambrolizumab (MK-3475), MEDI4736, tremelimum
  • cancer treating agent cycle(s) or “cancer treating agent regimen(s)” or “chemotherapeutic regimen(s)” or “chemotherapy cycle(s)” or “treatment cycle(s)” refer to treatment using one or more of the cancer treating agents, mentioned above, with or without the use of the sulfur-containing small molecules of the present invention.
  • cancer treating agent(s) or “cancer treating drug(s)”or “cancer treating compositions” refer to a medicament or medicaments that reduces, prevents, mitigates, limits, and/or delays the growth of metastases or neoplasms, or kills neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used, in a pharmaceutically-effective amount, to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease.
  • Cancer treating agents of the present invention include, but are not limited to: (i)
  • chemotherapeutic agents e.g., fluropyrimidines, pyrimidine nucleosides, purine nucleosides, anti-folates, platinum agents, anthracyclines/anthracenediones, epipodophyllotoxins, camptothecins, vinca alkaloids, taxanes, epothilones, antimicrotubule agents, alkylating agents, antimetabolites, topoisomerase inhibitors, and the like); (ii) hormones, hormonal complexes, and antihormonals (e.g., interleukins, interferons, leuprolide, pegasparaginase, and the like); (iii) enzymes, proteins, and peptides; antivirals (e.g., acyclovir, zidovudine, and the like); (iv) cytotoxic agents and cytostatic agents; (v) polyclonal and monoclonal antibodies (e.g., crizotinib, ge
  • cancer treating agent effect or “cancer treating agent effects” or “chemotherapeutic effect” or “cytotoxic or cytostatic activities” refer to the ability of an agent/medicament/composition to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms, or kill neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease.
  • the term “contemporaneous” refers to, e.g., an event existing, occurring, or originating during approximately the same period of time.
  • “contemporaneous” could refer to the sulfur-containing, amino acid-specific small molecules of the present invention interacting with and acting upon numerous target molecules in a contemporaneous manner.
  • the term contemporaneous includes, without limitation, an event occurring, or originating
  • cycle refers to the administration of a complete regimen of medicaments to the patient in need thereof in a defined time period.
  • cytostatic agents are mechanism-based agents that slow the progression of neoplastic disease and include drugs, biological agents, and radiation.
  • cytotoxic agents are any agents or processes that kill neoplastic cells and include drugs, biological agents, and radiation.
  • cytotoxic is inclusive of the term “cytostatic”.
  • the term "evidence of as it applies to the exhibition of abnormal expression and/or abnormal biochemical activity of the target molecules of the present invention selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross- complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), farnesyltransferase, and other target molecules possessing a similar active site or structural motif, means that it is probable or likely that abnormal expression and/or abnormal biochemical activity of the target molecule(s) has occurred or will occur.
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 receptor tyrosine
  • FISH florescence in situ hybridization
  • IHC immunohistochemistry
  • RIA radioimmunoassay
  • Such expectation of a therapeutic response is not 100% certain, and is related to many factors, not the least of which is the diagnostic accuracy of the test utilized which, in turn, is also limited by the sampling of the tumor and various other factors ⁇ e.g., laboratory methodology/technique, reagent quality, and the like).
  • the terms "Hazard Ratio”, “HR”, and “hazard ratio” refer to the chance of an event occurring with treatment "A” divided by the chance of the event occurring with treatment "B”.
  • the hazard ratio is an expression of the hazard or chance of events occurring in one treatment arm as a ratio of the hazard of the events occurring in the other treatment arm.
  • a hazard ratio less than 1.0 means that treatment "A" is more favorable than treatment "B” in terms of the result being measured.
  • treatment “A” refers to treatment with Tavocept (together with either paclitaxel or docetaxel and cisplatin) and treatment “B” refers to treatment with placebo (together with either paclitaxel or docetaxel and cisplatin).
  • a hazard ratio less than 1.0 relating to Tavocept treatment refers to a more favorable outcome in the result being measured for Tavocept treatment in comparison to the result being measured for the treatment other than Tavocept.
  • References to an "improvement” or “reduction” in the hazard ratio in favor of Tavocept refer to a more favorable outcome in the result being measured for Tavocept treatment in comparison to the result being measured for the treatment other than Tavocept.
  • heterogeneous refers to something that is not of uniform composition, quality, or structure.
  • maintenance therapy means the ongoing or chronic use of an agent to help lower the risk of recurrence (i.e., the return of cancer) after it has disappeared or been substantially reduced or diminished or not detectable following initial therapy or surgery. Maintenance therapy also may be used for patients with advanced cancer (cancer that cannot be cured) to help keep it from growing and spreading farther.
  • modulates or “modulation” or “metabolic modification and/or modulation” refer to any biological molecule, pharmacological medicament, or process that regulates the frequency, rate, or extent of any biological process, quality, or function. Modulation may either be "positive” (e.g., the initiation or start up of an inactive process, the maintenance of a process already occurring, the increase in rate of an existing process, and the like) or “negative” (e.g., the cessation or halting of a process with the concomitant decrease in rate, the prevention of an inactive process from becoming active, and the like).
  • Neoadjuvant therapy means treatment given as a first step to shrink a tumor before the main treatment or surgery is conducted.
  • Neoadjuvant therapy may include treatment with cancer treating agents such as chemotherapeutic agents, radiation therapy, hormones, cytotoxic or cytostatic agents, antibodies and/or sulfur- containing, amino acid-specific small molecules of the present invention.
  • Neoadjuvant therapy is intended to make later treatment or surgery easier and more likely to succeed, and reduce the consequences of a more extensive treatment or surgical technique that would be required if the tumor wasn't reduced in size or extent.
  • pathophysiological condition or "undesirable
  • physiological condition refers to abnormal anatomical or physiological conditions and their objective or subjective manifestations of disease.
  • pathophysiology refers to the study of the biologic and physical manifestations of disease as they correlate with the underlying abnormalities and physiologic disturbances. Pathophysiology explains the processes within the body that result in the signs and symptoms of a disease.
  • an "effective amount” or a “pharmaceutically-effective amount” in reference to the compounds or compositions of the instant invention refers to the amount that is sufficient to induce a desired biological, pharmacological, or therapeutic outcome in a subject with neoplastic disease. That result can be reduction, prevention, mitigation, delay, shortening the time to resolution of, alleviation of the signs or symptoms of, or exert a medically-beneficial effect upon the underlying pathophysiology or pathogenesis of an expected or observed side-effect, toxicity, disorder or condition, or any other desired alteration of a biological system.
  • the result will generally include the reduction, prevention, mitigation, delay in the onset of, attenuation of the severity of, and/or a hastening in the resolution of, or reversal of chemotherapy-associated toxicity; an increase in the frequency and/or number of treatments; an increase in duration of
  • chemotherapeutic therapy an increase or improvement in Progression Free Survival (PFS); and/or Complete Remission (CR).
  • PFS Progression Free Survival
  • CR Complete Remission
  • the term "pharmaceutically-acceptable salt” means salt derivatives of drugs which are accepted as safe for human administration.
  • the sulfur-containing, amino acid-specific small molecules of the present invention include pharmaceutically-acceptable salts, which include but are not limited to: (i) a monosodium salt; (ii) a disodium salt; (iii) a sodium potassium salt; (iv) a dipotassium salt; (v) a calcium salt; (vi) a magnesium salt; (vii) a manganese salt; (viii) an ammonium salt; and (ix) a monopotassium salt.
  • the term “Quality of Life” or “QOL” refers, in a non-limiting manner, to a maintenance or increase in a cancer subject's overall physical and mental state (e.g., cognitive ability, ability to communicate and interact with others, decreased dependence upon analgesics for pain control, maintenance of ambulatory ability, maintenance of appetite and body weight (lack of cachexia), lack of or diminished feeling of "hopelessness”; continued interest in playing a role in their treatment, and other similar mental and physical states).
  • the terms “target molecule” or “target molecules” or “molecular target” or “molecular targets” of the present invention refer to one or more
  • proteins/enzymes selected from the group consisting of: anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition (MET) kinase, the receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), peroxiredoxin (Prx), excision repair cross- complementing protein 1 (ERCC1), insulin growth factor 1 receptor (IGF1R), tubulin, ribonucleotide reductase (RNR), famesyltransferase, and other target molecules possessing a similar active site or structural motif.
  • ALK anaplastic lymphoma kinase
  • MET mesenchymal epithelial transition
  • ROS1 receptor tyrosine kinase
  • EGFR epidermal growth factor receptor
  • Prx peroxiredoxin
  • ERCC1 excision repair cross- complementing protein 1
  • IGF1R insulin growth factor 1 receptor
  • tubulin
  • the term “multiple” refers to one or more of, including by way of non- limiting example, the target molecules of the present invention which are contemporaneously modified/modulated by the sulfur-containing, amino acid-specific small molecules of the present invention.
  • the term “non-small cell lung cancer (NSCLC)” accounts for approximately 75% of all primary lung cancers. NSCLC is pathologically characterized further into adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and various other less common forms.
  • ELISA fluorescence in situ hybridization
  • IHC immunohistochemistry
  • RIA radioimmunoassay
  • quantitative immunofluorescence and/or automated quantitative analysis e.g., Genoptix's AQUA
  • ELISA approaches including, but not limited to, high-throughput ELISA, InCell ELISAs, or quantitative western analyses (e.g., Licor and related systems), and related ELISA
  • PCR coupled with MS approaches including, but not limited to, MALDI-TOF MS (e.g., Sequenom's MassARRAY system and related approaches);
  • mass spectroscopy based methods including, but not limited to, NanoLC coupled with ESI-MS (e.g., Bruker Daltonics/Eksigent Technologies system and related approaches), LC-MS, LC- MS/MS, and other MS systems designed to generate accurate-mass, high-resolution data on heterogeneous samples; and
  • the term "reducing” includes preventing, attenuating or mitigating the overall severity of, delaying the initial onset of, and/or expediting the resolution of the acute and/or chronic condition in a subject suffering from one or more types of cellular metabolic anomalies or other pathophysiological conditions.
  • the phrase "seminal biological capabilities" refers to the eight (8) characteristics which are acquired by the constituent cancer during the multistep development of human tumors. These biological capabilities constitute an organizing paradigm for understanding the inherent complexities of neoplastic disease and include: (i) resisting cell death (apoptosis); (ii) enabling replicative immortality; (iii) sustaining proliferative signaling; (iv) evading growth suppressors; (v) inducing angiogenesis; (vi) activating invasion and metastasis; (vii) reprogramming of energy metabolism; and (viii) evading immune destruction. See, Hanahan, D. and Weinberg, R.A., Hallmarks of cancer: the next generation. Cell 144:646-674 (2011).
  • structural motif refers to a supersecondary structure found in a chain-like biological molecule, such as a protein, nucleic acid, and a variety of other molecules. Motifs do not allow the prediction of the biological functions, as they are found in proteins and enzymes with dissimilar functions. Because the relationship between the primary structure and tertiary structure is not straightforward, two biopolymers may share the same structual motif, yet lack appreciable primary structure similarity. In other words, a structural motif does not have to be associated with a sequence motif. Also, the existence of a sequence motif does not necessarily imply a distinctive structure.
  • Structural motif elements include: (i) Beta Hairpin: two anti-parallel ⁇ -strands connected by a tight turn of a few amino acids between them; (ii) Greek Key: four ⁇ -strands folded over into a "sandwich shape"; (iii) Omega Loop: a loop in which the residues that make up the beginning and end of the loop are very close together; (iv) Helix-Loop-Helix: consists of a-helices bound by a looping stretch of amino acids; and (v) Zinc Finger: two ⁇ -strands with an a-helix end folded over to bind a zinc ion.
  • system stability or “biological system stability” refer to the maintenance of the normative physiological state or genome level stability of the organism.
  • sulfur-containing, amino acid-specific small molecules of the present invention include: (i) 2,2'-dithio-bis-ethane sulfonate; (ii) the metabolite of 2,2'- dithio-bis-ethane sulfonate, known as 2-mercapto ethane sulfonate; and (iii) 2-mercapto- ethane sulfonate conjugated as a disulfide with a substituent group selected from the group consisting of: -Cys, -Homocysteine, -Cys-Gly, -Cys-Glu, -Cys-Glu-Gly, -Cys-Homocysteine, -Homocysteine-Gly, -Homocysteine-Gly, -Homocysteine-
  • Tavocept- or BNP7787-derived metabolite or “Tavocept- or BNP7787-derived heteroconjugate” or “Tavocept- or BNP7787-derived adduct” represent the metabolite of disodium 2,2'-dithio-bis-ethane sulfonate, 2-mercapto ethane sulfonate sodium as a disulfide form which is conjugated with a substituent group consisting of: -Cys, - Homocysteine, -Cys-Gly, -Cys-Glu, -Cys-Glu-Gly, -Cys-Homocysteine, -Homocysteine-Gly, -Homocysteine-Glu, -Homocysteine-Glu-Gly, -Homocysteine—Glu; and
  • BNP7787-derived heteroconjugate compounds are included in the sulfur-containing, amino acid-specific small molecules of the present invention and may be synthesized as described in U.S. Patent Nos. 7,829,117; 7,829,538; 7,829,539; 7,829,540; and 7,829,541, the disclosures of which are incorporated herein, by reference, in their entirety.
  • Tavocept refers to disodium 2,2'-dithio-bis-ethane sulfonate, and is also referred to in the literature as dimesna and BNP7787.
  • the term "treat” or “treated”, with respect to a subject without cancer refers to a subject, who is in need thereof, and who has received, is currently receiving, or will receive the sulfur-containing, amino acid-specific small molecules of the present invention.
  • the term "treat” or “treated”, with respect to a subject with cancer refers to a subject, who is in need thereof, and who has received, is currently receiving, or will receive one or more cancer treating agents and/or the sulfur-containing, amino acid- specific small molecules of the present invention and/or other treatment agents.
  • xenobiotic refers to or denotes a substance, typically a synthetic chemical, which is found in an organism but which is not normally produced or expected to be present in said organism.
  • the term can also refer to substances which are present in much higher concentrations than are usual.
  • xenobiotic modification As used herein, the terms “xenobiotic modification”, “Tavocept-mediated xenobiotic modification”, or a “Tavocept mediated, non-enzymatic xenobiotic modification” refer to the covalent binding of a “Tavocept- or BNP7787-derived metabolite” or "Tavocept- or
  • BNP7787-derived heteroconjugate or "Tavocept- or BNP7787-derived adduct” to one or more sulfur-containing amino acids of a protein or enzyme.
  • Figure 1 illustrates the ability of Tavocept to undergo thiol disulfide exchange reactions intracellularly and/or within the interstitial space.
  • Figure 2 illustrates the chemical structures of Tavocept, 2-mercaptoethene sulfonate, glutathione, and selected Tavocept-derived heteroconjugates. It should be noted that in the structures of the Tavocept-derived heteroconjugates, the portion of the heteroconjugates comprising the Tavocept metabolite, 2-mercaptoethene sulfonate, is shaded.
  • FIG. 3 Panel A: Glutaredoxin (Grx) catalyzes the reduction of disulfide bonds in proteins converting glutathione (GSH) to glutathione disulfide (GSSG). GSSG is, in turn, recycled to GSH by the enzyme glutathione reductase at the expense of NADPH. During the reaction cycle it is thought that a cysteine pair in the active site of glutaredoxin is converted to a disulfide. Panel A: illustrates the conversion of glutaredoxin from the disulfide form (oxidized) to the dithiol (reduced) form, as catalyzed non-enzymatically by glutathione.
  • GSH glutathione
  • GSSG glutathione disulfide
  • Panel A illustrates the conversion of glutaredoxin from the disulfide form (oxidized) to the dithiol (reduced) form, as catalyzed non-enzymatically by glutathione.
  • glutaredoxin is also thought to be important for deglutathionylation of protein thiols. In this reaction only a single cysteine is required. Indeed, many naturally occurring glutaredoxins contain only one cysteine in the active site. It should be noted that the direction of the glutaredoxin-catalyzed cycle depends on the relative concentrations of GSH and GSSG. High concentrations in the cell of GSSG relative to GSH will drive
  • Figure 4 illustrates an example of the Xenobiotic Metabolism Pathway. Tavocept is thought to react with cisplatin resulting in the formation of a mesna-cisplatin adduct that is not a substrate for the xenobiotic metabolism pathway.
  • Figure 5 illustrates the domains of cMet; the kinase domain of MET (residues 956-end) was used in these experiments.
  • Figure 6 illustrates the domain organization of MET.
  • Panel A illustrates the domain organization and structure.
  • Panel B illustrates a slightly modified MET showing sites for tyrosine phosphorylation in the intracellular kinase portion of MET.
  • Figure 7 illustrates increasing concentrations of MET result in increasing ADP production (reflected in increasing RLU).
  • Assay volume was 10 ⁇ ; therefore, for the assay represented by the 0.78 ng bar above, MET Kinase was 0.078 ng/ ⁇ and this corresponded to a molar concentration of 0.96 nM .
  • Figure 8 illustrates the effect of Tavocept (BNP7787) on MET (0.1 ng/ ⁇ ) activity in assays with 10 ⁇ ATP; Determination of IC 50 value.
  • Figure 9 illustrates the effect of Tavocept (BNP7787) on MET (0.1 ng/ ⁇ ) activity in assays with 100 ⁇ ATP; Determination of IC 50 value.
  • Figure 10 illustrates the effect of Tavocept (BNP7787) on MET (2.5 ng/ ⁇ ) activity in assays with 100 ⁇ ATP; Determination of IC 50 value.
  • Figure 11 illustrates the effect of Tavocept (BNP7787) on MET (2.5 ng/ ⁇ ) activity in assays with 10 ⁇ ATP; Determination of IC 50 value.
  • Figure 12 illustrates the effect of Crizotinib on MET (0.1 ng/ ⁇ ) activity in assays with 10 ⁇ ATP; Determination of IC 50 value.
  • Figure 13 illustrates the effect of Crizotinib effect on MET (2.5 ng/ ⁇ ) activity in assays with 100 ⁇ ATP; Determination of IC 50 value.
  • Figure 14 illustrates the effect of Tavocept (BNP7787) on Crizotinib-mediated inhibition of MET (0.1 ng ⁇ L) activity under 10 ⁇ ATP conditions at 20 nM and 40 nM Crizotinib.
  • Figurel5 illustrates the effect of Tavocept (BNP7787) on Crizotinib-mediated inhibition of MET (2.5 ng/ ⁇ ) activity under 100 ⁇ ATP conditions at 45 nM and 90 nM Crizotinib.
  • Figure 16 illustrates the effect of Staurosporine on MET (0.1 ng/ ⁇ ) activity in assays with 10 ⁇ ATP; Determination of IC 50 value.
  • Figurel7 Illustrates the effect of Tavocept (BNP7787) on Staurosporine -mediated inhibition of MET (0.1 ng/ ⁇ ) activity under 10 ⁇ ATP conditions at a 100 nM and 300 nM concentration of Staurosporine.
  • FIG 18 Panel A: illustrates a ribbon diagram of ALK with covalently bound Tavocept (BNP7787)-derived mesna adducts. Tavocept (BNP7787)-derived mesna adducts were observed at Cys 1235 and Cys 1156. Panel B: illustrates an overlay of region of apo-ALK with Tavocept (BNP7787) xenobiotically-modified ALK that has a Cys-1156-mesna adduct. The Tavocept (BNP7787)-derived mesna adduct occupies the same pocket at Phel 127 of the P-loop.
  • Figure 19 illustrates a Fo-Fc electron density map contoured at 1 sigma showing Tavocept (BNP7787)-derived mesna adducts on ALK.
  • Panel A at Cys 1235.
  • Panel B at Cys 1156.
  • Panel C Binding site of the Tavocept (BNP7787)-derived mesna adduct at Cys 1235. There are no obvious interactions with the protein other than the covalent bond with Cys 1235.
  • Panel D Molecular surface of ALK with the Tavocept (BNP7787)-derived mesna at Cys 1156 removed to show the interaction of the adduct with the protein.
  • a water mediated hydrogen bond is present between the mesna sulfonate and Asp 1 160 carbonyl.
  • Figure 20 illustrates the X-ray crystallographic structure of ALK with Tavocept (BNP7787)-derived mesna adducts on cysl 156 and cysl235.
  • Figure 21 illustrates the kinase domain of ALK (residues 1058-1623) which was used in these experiments.
  • Figure 22 illustrates increasing concentrations of ALK result in increasing ADP production (reflected in increasing RLU).
  • Figure 23 Panel A: illustrates Crizotinib's effect on ALK activity in assays with 100 ⁇ ATP; Panel B: illustrates a summary of IC 50 value determination.
  • Figure 24 Panel A: illustrates Crizotinib's effect on ALK activity in assays with 500 ⁇ ATP; Panel B: illustrates a summary of IC 50 value determination.
  • Figure 25 illustrates the effect of Tavocept (BNP7787) on Crizotinib-mediated inhibition of ALK activity under 100 ⁇ ATP conditions and 15 nM Crizotinib (Panel A) or 30 nM Crizotinib (Panel B).
  • Figure 26 illustrates the effect of Tavocept (BNP7787) on Crizotinib-mediated inhibition of ALK activity under 500 ⁇ ATP conditions at 30 nM Crizotinib (Panel A) or 65 nM Crizotinib (Panel B).
  • Figure 27 illustrates a homology model of human ROSl overlaid with the
  • FIG. 28 illustrates the domain organization of ROSl .
  • Panel A Domain organization of ROSl compared to other kinase receptors. Below each kinase, genes are listed that can fuse with the kinase (fused products may be involved in cancer or disease).
  • Panel B Panel B:
  • Intracellular kinase region of ROSl including residues 1883-2347 with tyrosine (Y) and serine (S) phosphorylation sites identified.
  • Figure 29 illustrates that increasing concentrations of ROSl results in increasing ADP production (reflected in increasing RLU). It should be noted that above a concentration of 1 ng ⁇ L assay, ATP becomes rate limiting; therefore a lower ROSl concentration of 0.5 ng/ ⁇ assay was utilized with an ATP concentration of 100 ⁇ .
  • Figure 30 illustrates the effects of crizotinib on ROSl activity.
  • Assays with 100 ⁇ ATP Determination of IC 50 value assays contained 100 ⁇ ATP and 0.5 ng ROSl/ ⁇ assay volume, the data is shown to two decimal places.
  • Figure 31 illustrates the time-dependent decrease in ROS 1 activity when Tavocept (BNP7787) and ROSl are incubated together prior to initiating the kinase assay.
  • Panel A ROS 1 and Tavocept (BNP7787) assayed immediately in the presence of assay mixture containing ATP and polyGT.
  • Panel B ROSl and Tavocept (BNP7787) incubated together for 3 hours, then added to assay mixture containing ATP and polyGT and assayed.
  • Panel C ROSl and Tavocept (BNP7787) preincubated together for 24 hours, then added to assay mixture containing ATP and polyGT and assayed.
  • Figure 32 illustrates the time-dependent decrease in ROSl activity when Tavocept (BNP7787) and ROSl are incubated together prior to initiating the kinase assay.
  • Panel A ROSl and Tavocept (BNP7787) assayed immediately in the presence of assay mixture containing ATP and polyGT.
  • Panel B ROSl and Tavocept (BNP7787) incubated together for 3 hours, then added to assay mixture containing ATP and polyGT and assayed.
  • Panel C ROSl and Tavocept (BNP7787) preincubated together for 24 hours, then added to assay mixture containing ATP and polyGT and assayed.
  • Figure 33 illustrates the effect of Tavocept (BNP7787) on Crizotinib-mediated inhibition of ROSl kinase activity in assays with simultaneous addition of all assay components Tavocept (BNP7787), ATP, and polyGT were added to ROSl kinase simultaneously.
  • Tavocept BNP7787
  • ATP ATP
  • polyGT polyGT
  • BNP7787 was found to have an additive effect on crizotinib-induced inhibition of ROSl kinase activity.
  • BNP7787 was preincubated with ROS 1 kinase for: Panel A: 0 hours; Panel B: 3 hours; or Panel C: 24 hours prior to addition of crizotinib, ATP, and polyGT.
  • Figure 35 Panel A: Tavocept effect on Wild Type EGFR activity in assays with
  • Figure 36 illustrates Tavocept effect on T790M EGFR activity in assays with 10 ⁇ ATP concentrations.
  • Figure 37 illustrates the structures of the Tavocept-derived heteroconjugates evaluated in EGFR Kinase assays.
  • Figure 38 illustrates Tavocept potentiation of Erlotinib-mediated inhibition of WT EGFR Kinase activity (10 ⁇ ATP).
  • Figure 39 illustrates Tavocept potentiation of Erlotinib-mediated inhibition of T790M EGFR Kinase activity ( 10 ⁇ ATP) .
  • Figure 40 illustrates the effect of Tavocept-derived heteroconjugates on Erlotinib- mediated inhibition of WT EGFR activity under 10 ⁇ ATP conditions.
  • Panel A Effect of mesna-cysteine
  • Panel B Effect of mesna-glutathione
  • Panel C Effect of mesna- cysteinylglutamate
  • Panel D Effect of mesna-cysteinylglycine.
  • Figure 41 illustrates the effect of Tavocept-derived heteroconjugates on Erlotinib- mediated inhibition of WT EGFR activity under 100 ⁇ ATP conditions.
  • Panel A Effect of mesna-cysteine
  • Panel B Effect of mesna-glutathione
  • Panel C Effect of mesna-cysteinyl glutamate
  • Panel D Effect of mesna-cysteinyl glycine.
  • Figure 42 illustrates the effect of Tavocept-derived heteroconjugates to potentiate the inhibitory effect of Erlotinib on T790M EGFR activity (10 ⁇ ATP).
  • Panel A Mesna- cysteine and Mesna-glutathione inhibition of T790M EGFR and potentiation of Erlotinib inhibition (10 ⁇ ATP);
  • Panel B Mesna-cysteinylglycine and mesna-cysteinylglutamate inhibition of T790M EGFR and potentiation of Erlotinib inhibition (10 ⁇ ATP).
  • Figure 43 illustrates the effect of Tavocept-derived heteroconjugates potentiating the inhibitory effect of Erlotinib on T790M EGFR activity (100 ⁇ ATP).
  • Panel A MSSC potentiates Erlotinib-mediated inhibition of T790M EGFR kinase activity (100 ⁇ ATP);
  • Panel B MSSGlutathione potentiates Erlotinib-mediated inhibition of T790M EGFR kinase activity (100 ⁇ ATP);
  • Panel C MSSCysteinylglycine potentiates Erlotinib-mediated inhibition of T790M EGFR kinase activity (100 ⁇ ATP);
  • Panel D MSSCystemylglutamate potentiates Erlotinib-mediated inhibition of T790M EGFR kinase activity (100 ⁇ ATP).
  • Figure 44 A graphic illustration of the intracellular pathways related to IGF1R (as adopted from Fidler, et al. Targeting the insulin-like growth factor receptor pathway in lung cancer: Problems and pitfalls. Ther. Adv. Med. Oncol. 4(2 :51-60 (2012)).
  • Figure 45 illustrates the effect of Tavocept on IGF1R Kinase activity.
  • Figure 46 illustrates the amino acid sequence of human ERCCl (cysteines are underlined).
  • the N-terminal 6-histidine tag (HHHHHH) used to express human ERCCl in E. coli is not shown.
  • the ERCCl sequence was obtained from
  • FIG. 47 Panel A: Positive ion ESI mass spectrum of ERCCl control sample corresponding to tryptic ERCCl fragment VTECLTTVK containing Cys238.
  • Figure 48 Panel A: Positive ion ESI mass spectrum of ERCCl control unmodified sample corresponding to tryptic ERCCl fragment EDLALCPGLGPQK containing Cys274.
  • Panel B Fragment from ERCCl control contains EDLALCPGLGPQK fragment that contains Tavocept modification on Cys274 (predicted 1478.6; observed 1480.8).
  • Figure 49 Whole Protein MS data showing - Panel A: Apo-RNRl .
  • Panel B RNR1 with Tavocept-derived mesna adducts.
  • Figure 50 (A) Tavocept structure; (B) Paclitaxel structure; (C) Cisplatin structure and subsequent aquation leading to formation of cisplatin-DNA adducts that interfere with DNA replication and damage DNA.
  • Figure 51 Panel A: Example of time dependent decay of microtubule protein's ability to polymerize into microtubules (control sample with no drug treatment). Percent
  • polymerization values are OD350 readings 30 minutes after polymerization was initiated and are normalized relative to the sample that was not preincubated prior to initiation of the MTP polymerization assay (the 0 hour sample). Note: individual microtubule protein preparations vary slightly in terms of decay profiles). Panel B: Bar graph comparison of percent polymerization of microtubule protein after preincubation with mesna only,
  • Figure 52 Panel A: Postulated S N 2 route of non-enzymatic reduction of Tavocept to mesna in the kidney (see, e.g., Verschraagen M, Boven E, Torun E, Hausheer FH, Bast A, van dV. Possible enzymatic routes and biological sites for metabolic reduction of BNP7787, a new protector against cisplatin-induced side-effects. Biochem. Pharmacol. 68:493-502 (2004)).
  • Panel B Mesna may displace the aquo group of monoaquocisplatin and the formation of a possible sulfur-platinum adduct could prevent monoaquoplatin from forming an adduct with surface cysteine residues on tubulin.
  • FIG. 53 Effects of Tavocept and mesna on microtubule protein polymerization under various assay conditions.
  • Panel A Tavocept has a dose-dependent inhibitory effect on GTP driven microtubule protein polymerization (the data in Panel A was obtained using a Caryl 00 UV-vis cuvette based spectrometer).
  • Panel B Mesna does not affect GTP Promoted
  • Panel B Microtubule Protein Polymerization (the data in Panel B was obtained using a SpectraMax Plus microtiter plate UV-Vis spectrophotometer).
  • Panel C Tavocept modulates
  • GTP/paclitaxel-promoted microtubule protein polymerization (note - the line with open squares is a microtubule protein polymerization assay promoted only by GTP, a GTP-only control).
  • Panel D Tavocept modulates paclitaxel-promoted microtubule protein polymerization (no GTP present) and this effect is not due to the two sodium counterions of Tavocept, since 32 mM NaCl alone was shown to have no effect.
  • Figure 54 Electron micrographs of microtubule polymerization - Panel A: initiated with GTP; Panel B: initiated with GTP with Tavocept (10 mM) present; Panel C: initiated with GTP with paclitaxel (6 ⁇ ); and Panel D: initiated with GTP with paclitaxel (6 ⁇ ) and with Tavocept (10 mM) present.
  • Figure 55 illustrates Liquid Chromatography data of peroxiredoxin fragment
  • HGEVCPAGWK containing Cysl73.
  • Panel A peroxiredoxin incubated with Tavocept ion at m/z 1245.5 corresponding to fragment [HGEVCPAGWK + H]+;
  • Panel B peroxiredoxin incubated without Tavocept does not have ion at m/z 1245.5 and exhibits no peaks corresponding to this mass.
  • Figure 56 positive -ion mass spectra for peroxiredoxin (Prx) fragments containing Tavocept-derived mesna adduct at Cysl73 in fragment TDKHGEVCPAGW.
  • Panel A
  • Figure 57 positive -ion mass spectra for peroxiredoxin (Prx) fragment VCPTEIIAF containing Cys52.
  • Panel A peroxiredoxin incubated with Tavocept; ion at m/z 1132.8 corresponding to fragment [VCPTEIIAF + H]+;
  • Panel B peroxiredoxin incubated without Tavocept does not have ion at m/z 1132.8 but contains "parent" unmodified peak at 992.0.
  • Figure 58 illustrates Prx assay that was coupled to thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH.
  • Figure 59 illustrates that Tavocept-modified Prx (Prx -mesna) is less active than Apo-Prx in assays monitoring initial velocity.
  • Figure 60 crystal structure of Prx4 complexed with Tavocept-derived mensa moiety showing analogue formation at Cysl24 but not Cysl48. Cys245 was not visible in the electron density map.
  • Figure 61 illustrates Prx apo structure showing multimer interface. The C-terminal "tail" of molecule A wraps around molecule B such that Cysl24 of Molecule B is in close proximity to Cys245 of Molecule A.
  • Figure 62 close-up of Tavocept-derived mesna binding site showing partial unwinding of helix 124 and unwinding of helix 165 to accommodate the Tavocept-derived mesna moiety at C124.
  • Figure 63 illustrates the Mass Spectroscopy analysis of Prx Protein after reaction with Tavocept. Peak at 25572 corresponds to Prx monomer containing two Tavocept-derived mesna adducts (apo-protein is approximately 25292; the peak at 25751 corresponds to Prx with three Tavocept-derived mesna adducts); the peak at 51041 corresponds to dimer containing up to three Tavocept-derived mesna adducts.
  • Figure 64 illustrates the Prx protein sample after crystallization; crystals were dissolved for Mass Spectroscopy analysis to confirm the C-terminal tail was intact. Observed peak at 25574 corresponds extremely well to the initially observed peak at 25572, suggesting no C- terminal proteolysis has occurred.
  • Figure 65 illustrates the sequence alignment of Prx 1 and Prx 4 (Prx IV) showing position of the conserved, catalytic cysteine residues in boxes.
  • Figure 66 Electrophoretic profile of Trx incubated with and without Tavocept.
  • Panel A TrisGlycine SDS PAGE, reducing (DTT) conditions.
  • Panel B TrisGlycine Native PAGE, non-reducing conditions.
  • Panel C TrisGlycine Native PAGE, reducing (DTT) conditions.
  • Lane 1 contains SeeBlue Plus Two Standards (Panel A) NativeMarks (Panels B and C). Due to steps required in the technical manipulation of samples as they are prepared for PAGE, 0 hrs samples are actually closer to a 15 to 30 minute time-point. Freezing samples for up to 3 weeks gave identical PAGE profiles as when samples were analyzed immediately; therefore, summary gels showing the various time points are presented here.
  • Panel A IEF gel.
  • Panel B IEF gel where all samples were incubated with Trx reductase and NADPH prior to loading.
  • Lane 1 contains IEF protein standards (Panels A and B). Due to steps required in the technical manipulation of samples as they are prepared for PAGE, 0 hour samples are actually closer to a 15 to 30 minute time-point. Freezing samples for up to 3 weeks gave identical PAGE profiles as when samples were analyzed immediately as they were generated; therefore, summary gels showing the various time-points are presented here.
  • Figure 68 Liquid Chromatography data of thioredoxin incubations.
  • Panel D Thioredoxin incubated with mesna; ion at m/z 1288.3 with retention time of 13.26 minutes corresponding to fragment [CMPTFQFFK + Mesna + H ] + with Cys73 -mesna adduct.
  • Figure 69 Mass Chromatography data of thioredoxin incubations.
  • Panel A The positive -ion mass spectrum for thioredoxin fragments containing covalent mesna adducts from reactions where thioredoxin was incubated with Tavocept for Cys73 -mesna adduct at ions of m/z 1288.3 [CMPTFQFFK + Mesna + H] + and m/z 1310.2 [CMPTFQFFK + Mesna + Na + H] + , respectively.
  • Panel B The negative-ion mass spectrum for thioredoxin fragments containing covalent mesna adducts from reactions where thioredoxin was incubated with Tavocept for identification of Cys73-mesna adducts at the ion of m/z 1286.3 [CMPTFQFFK + Mesna - H].
  • Panel C The positive-ion mass spectrum for thioredoxin fragments containing covalent mesna adducts from reactions where thioredoxin was incubated with Tavocept for identification of a mesna adduct in Trypsin digested thioredoxin fragment containing Cys62 and Cys69 at ions of m/z 2718.6 [YSNVIFLEVDVDDCQDVASECEVK + H] + and m/z 2860.4 [YSNVIFLEVDVDDCQDVASECEVK + Mesna + H] + , respectively.
  • FIG 70 Summary of effect of Tavocept and Tavocept-derived mesna-disulfide heteroconjugates on Trx activity.
  • Panel A Tavocept and Tavocept-derived mesna-disulfide heteroconjugates are alternative substrates for the TrxR/Trx system, and as such can act as competitive inhibitors of the Trx.
  • NADPH oxidation by TrxR and Trx increases in the presence of increasing concentrations of Tavocept, MSSG, MSSC, and MSSH. (Note: error bars are small and may be obscured by the symbols).
  • Panel B Tavocept has a discernable effect on the rate of NADPH oxidation in the TrxR/Trx catalyzed reduction of the insulin AB chain disulfide and more notably increasing Tavocept concentrations prevent most of the precipitation of the insulin B chain when the AB chain disulfide is cleaved. (Note: error bars were omitted but assays were run in quadruplicate with typical errors of 5-8% between individual replicates).
  • Panel C Progress curves of assays where Trx-mesna and Trx-GSH were purified away from unreacted Tavocept and glutathione disulfide, respectively, and then compared to apo-Trx in the Trx reductase insulin disulfide reduction assay.
  • Panel D Initial velocity reaction rates corresponding to progress curves shown in Panel C. With time, Trx-mesna and Trx-GSH are converted back to apo-Trx by Trx reductase (see also, Figure 69, Panel E).
  • Figure 71 Molecular assembly of the Trx tetramer. All three Trx crystals were highly similar in overall fold conformation, the Trx pH 9.0/8.5 tetramer is shown in this figure (i.e., adduct formation at pH 9.0; crystal grown at pH 8.5).
  • Panel A Ribbon diagram of tetramer with the site of conformational change (light color) for molecules A and B and (darker color) for C and D, respectively.
  • Panel B The intra- and intermolecular disulfide pattern is illustrated.
  • Figure 72 Molecular interface of the Trx structure containing Tavocept-derived mesna adduct.
  • Panel A Close-up of the tetramer interface showing the formation of a six stranded ⁇ -barrel at the dimer interface of molecules C and D.
  • Panel B View down the "barrel" of the dimer interface showing the newly formed disulfides.
  • Panel C Representative 2Fo-Fc electron density map contoured at 1 sigma at the ⁇ -barrel motif. (Nomenclature note: Cys69- D indicates Cys69 of molecule D, etc.). Geometry of tetramer interface was similar in all three crystal structures; here crystals obtained at pH 8.5 with adduct formed at pH 9.0 are shown.
  • Figure 73 Scheme showing Tavocept-derived modification of proteinaceous cysteine residues resulting in formation of mixed mesna-cysteine disulfides on protein targets. This process is called Tavocept-mediated xenobiotic modification or modulation.
  • Figure 75 Atomic resolution map of Tavocept-derived mesna adduct formation on Cys7 (Panel A) and Cys82 (Panel B).
  • the adduct on Cys7 is in one conformation.
  • the adduct on Cys82 is shown in two conformations for S-CP bond of the adduct.
  • Figure 76 Ribbon diagram showing the binding site of the two Tavocept-derived adducts to Grxl .
  • Panel A Tavocept-derived mesna adduct at Cys82.
  • the sulfur and carbon atoms of the mesna are in two orientations.
  • the sulfate is in a single orientation and making a hydrogen bond with Ser83 (also in two orientations).
  • Panel B Tavocept-derived mesna adduct at Cys7. This adduct is solvent exposed and not making any interactions with the protein. Both adducts are located at a crystal contact.
  • FIG 77 Overlay of BNPI proprietary Grx structure with PDB entry 1KTE (rms 0.562). Tavocept adducts are depicted as sticks on the Grx structure.
  • Figure 78 Overview of some intracellular pathways regulated or modulated by RAS. Adapted from Appels, et al., Development of Farnesyl Transferase Inhibitors: A Review. 10:565-578 (2005).
  • Figure 79 Inhibition of FTase activity by Tavocept.
  • Panel A Progress curve showing FTase-mediated famesylation of Dansyl-GCVLS peptide.
  • Panel B Relative rates from reactions shown in Panel A.
  • Figure 80 FTase assay summary.
  • Figure 81 Scheme showing possible interactions between Tavocept and Dansyl-GCVLS peptide.
  • Figure 82 Summary of Mass Spectroscopy data showing that Tavocept reacts with the Dansyl-GCVLS peptide resulting in the formation of a xenobiotically modified Dansyl- GCVLS substrate.
  • kinase describes a large family of enzymes that are responsible for catalyzing the transfer of a phosphoryl group from a nucleoside triphosphate donor, such as ATP, to an acceptor molecule.
  • Tyrosine kinases catalyze the phosphorylation of tyrosine residues in proteins. The phosphorylation of tyrosine residues, in turn, cause a change in the function of the protein that they are contained in. Phosphorylation at tyrosine residues controls a wide range of properties in proteins such as enzyme activity, subcellular localization, and interaction between molecules.
  • Tyrosine kinases function in a variety of processes, pathways, and actions, and is responsible for key events in the body.
  • the receptor tyrosine kinases function in
  • tyrosine kinases within the cell function in signal transduction to the nucleus.
  • Tyrosine kinase activity in the nucleus involves cell-cycle control and properties of transcription factors. In this way, in fact, tyrosine kinase activity is involved in mitogenesis, or the induction of mitosis in a cell; proteins in the cytosol and proteins in the nucleus are phosphorylated at tyrosine residues during this process. Cellular growth and reproduction may rely in some part on tyrosine kinase.
  • Tyrosine kinase function has been observed in the nuclear matrix, which is comprised not of chromatin, but of the nuclear envelope and a "fibrous web" that serves to physically stabilize DNA.
  • the transmission of mechanical force, regulatory signaling, and cellular proliferation are fundamental in the normal survival of a living organism and protein tyrosine kinases also play a role in these functions.
  • Tyrosine kinases also function in many signal transduction cascades wherein extracellular signals are transmitted through the cell membrane to the cytoplasm and often to the nucleus where gene expression may be modified. See, e.g., Cox, Michael; Nelson, David R. (2008). Lehninger: Principles of Biochemistry (5 th edition). W. H. Freeman & Co. Signals in the surroundings received by receptors in the membranes of cells are transmitted into the cell cytoplasm. Transmembrane signaling due to receptor tyrosine kinases relies heavily upon interactions, for example, mediated by the SH2 protein domain; it has been determined via experimentation that the SH2 protein domain selectivity is functional in mediating cellular processes involving tyrosine kinase.
  • Receptor tyrosine kinases may, by this method, influence growth factor receptor signaling. Finally mutations can cause some tyrosine kinases to become constitutively active, a nonstop functional state that may contribute to initiation or progression of cancer.
  • Tyrosine kinases are divided into two main families: (i) transmembrane receptor- linked kinases and (ii) cytplasmic proteins. Approximately 2000 kinases are known, and more than 90 protein tyrosine kinases (PTKs) have been identified in the human genome. They are divided into two classes, receptor and non-receptor PTKs. At present, 58 receptor tyrosine kinases (RTKs) are known, and grouped into 20 subfamilies. RTKs play pivotal roles in diverse cellular activities including growth, differentiation, metabolism, adhesion, motility, and cellular death.
  • PTKs protein tyrosine kinases
  • RTKs 58 receptor tyrosine kinases
  • RTKs are composed of an extracellular domain, which is able to bind a specific ligand, a transmembrane domain, and an intracellular catalytic domain, which is able to bind and phosphorylate selected substrates. Binding of a ligand to the extracellular region causes a series of structural rearrangements in the RTK that lead to its enzymatic activation. In particular, movement of some parts of the kinase domain gives free access to adenosine triphosphate (ATP) and the substrate to the active site. This triggers a cascade of events through phosphorylation of intracellular proteins that ultimately transmit (i.e.,
  • transduce the extracellular signal to the nucleus, causing changes in gene expression.
  • Many RTKs are involved in oncogenesis, either by gene mutation, or chromosome translocation, or simply by over-expression. In every case, the result is a hyper-active kinase, that confers an aberrant, ligand-independent, non-regulated growth stimulus to the cancer cells.
  • cytoplasmic/non-receptor protein tyrosine kinases In humans, a total of 32 cytoplasmic/non-receptor protein tyrosine kinases have been identified. The first non-receptor tyrosine kinase identified was the v-src oncogenci protein. Most animal cells contain one or more members of the Src family of tyrosine kinases. A chicken sarcoma virus was found to carry mutated versions of the normal cellular Src gene. The mutated v-src gene has lost the normal built-in inhibition of enzyme activity that is characteristic of cellular Src (c-src) genes. Src family members have been found to regulate many cellular processes. For example, the T-cell antigen receptor leads to intracellular signalling by activation of Lck and Fyn, two proteins that are structurally similar to Src.
  • tyrosine kinase enzyme Major changes are sometimes induced when the tyrosine kinase enzyme is affected by other factors.
  • One of the factors is a molecule that is bound reversibly by a protein, called a ligand.
  • a number of receptor tyrosine kinases though certainly not all, do not perform protein-kinase activity until they are occupied, or activated, by one of these ligands. It is interesting to note that, although many more recent cases of research indicate that receptors remain active within endosomes, it was once thought that endocytosis caused by ligands was the event responsible for the process in which receptors are inactivated.
  • Activated receptor tyrosine kinase receptors are internalized in short time and are ultimately delivered to lysosomes, where they become work adjacent to the catabolic acid hydrolases that partake in digestion. Internalized signaling complexes are involved in different roles in different receptor tyrosine kinase systems, the specifics of which have been examined. See, e.g., Wiley H.S., Burke, P.M. Regulation of receptor tyrosine kinase signaling by endocytic trafficking. Traffic 2(1 ⁇ : 12- 18 (2001).
  • ligands participate in reversible binding, a term that describes those inhibitors that bind non-covalently (inhibition of different types are effected depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both).
  • Multivalency which is an attribute that bears particular interest to some people involved in related scientific research, is a phenomenon characterized by the concurrent binding of several ligands positioned on one unit to several coinciding receptors on another. In any case, the binding of the ligand to its partner is apparent owing to the effects that it can have on the functionality of many proteins.
  • Ligand-activated receptor tyrosine kinases demonstrate a unique attribute. Once a receptor tyrosine kinase is bonded to its ligand, it is able to bind to tyrosine kinase residing in the cytosol of the cell.
  • the MET proto-oncogene encodes for the receptor tyrosine kinase (RTK), c-MET.
  • MET encodes a protein known as hepatocyte growth factor receptor (HGFR).
  • HGFR hepatocyte growth factor receptor
  • the hepatocyte growth factor receptor protein possesses tryrosine kinase activity. See, e.g.,
  • HGF Hepatocyte growth factor
  • MET is normally expressed in cells of epithelial origin, although it has also been identified in endothelial cells, neurons, hepatocytes, hematopoietic cells, and melanocytes.. Expression of HGF is generally restricted to cells of mesenchymal origin, although some epithelial cancer cells appear to express both HGF and MET.
  • the MET proto-oncogene has a total length of 125,982 bp and is located in the 7q31 locus of chromosome 7.
  • MET is transcribed into a 6,641 bp mature mRNA which is then translated into a 1,390 amino acid residue c-MET protein.
  • c-MET is a receptor tyrosine kinase that is produced as a primary single-chain precursor protein that is post-translationally proteolytically cleaved at a furin site to yield a highly glycosylated extracellular a-subunit and a transmembrane ⁇ -subunit, which are then covalently linked via a disulfide bond to form the mature receptor.
  • c-MET dimerizes and autophosphorylates upon ligand binding, which in turn creates active docking sites for proteins that mediate downstream signaling leading to the activation/modulation of a variety of proteins.
  • activation/modulation evokes a variety of pleiotropic biological responses leading to increased cell growth, scattering and motility, invasion, protection from apoptosis, branching morphogenesis, and angiogenesis.
  • improper activation of c-MET may confer proliferative, survival and invasive/metastatic abilities of cancer cells.
  • MET kinase proto-oncogene The mesenchymal epithelial transition (MET) kinase proto-oncogene has been know for almost 30 years, and MET kinase activity is dysregulated and/or upregulated in a range of cancers including, but not limited to, lung, breast, ovarian, kidney, colorectal, stomach and head and neck cancer.
  • This receptor tyrosine kinase is activated by hepatic growth factor (HGF) but is also structurally related to the insulin growth factor receptor family. See, e.g., Lawrence and Salgia, MET molecular mechanisms and therapies in lung cancer. Cell Adhes. Migrat.
  • Met kinase is heterodimer and contains numerous cysteine residues that form disulfide bonds between the heterodimeric subunits.
  • MET kinase undergoes autophosphorylation and is coupled to a range of intracellular signaling pathways that regulate cell growth including, but not limited, to FAK, RAS, RAC, PI3K, CAS-CR and other pathways. See, e.g., Eder, et al., Novel therapeutic inhibitors of the c-MET signaling pathway in cancer. Clin. Cancer Res. 15(7 :2207-2214 (2012).
  • HGF-MET cascade a key target for inhibiting cancer metastasis: The impact of NK4 discovery on cancer biology and therapeutics. Int. J. Mol. Sci. 14:888-919 (2013).
  • MET kinase has been shown to be overexpressed in up to 40% of lung cancer tissue samples and has been a focal target for small molecule development. See, e.g., Villaflor and Salgia, Targeted agents in non-small cell lung cancer therapy: What is there on the horizon. J.
  • MET kinase amplification upregulation
  • this amplification is associated with resistance to the important NSCLC drugs Erlotinib and/or Gefitinib.
  • MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc. Natl. Acad. Sci. U.S.A. 26;104(52):20932- 20937 (2007).
  • MET kinase is coupled to FAK, RAS, RAC, PI3K, CAS-CRK and other pathways. These pathways are central to cell growth and also regulate various physiological processes in cancer (invasion, metastasis, and the like).
  • the extracellular region possess the following characteristics: (i) a region of homology to semaphorins (Sema domain), which includes the full a-chain and the N-terminal part of the ⁇ -chain; (ii) a cysteine -rich MET -related sequence (MRS domain); (iii) glycine -proline -rich repeats (G-P repeats); and (iv) four
  • immunoglobulin-like structures Ig domains
  • the intercellular, juxtamembrane region possesses the following characteristics: (a) a serine residue (Ser 985), which inhibits the receptor kinase activity upon phosphorylation; (b) a tyrosine (Tyr 1003), which is responsible for c-MET polyubiquitination, endocytosis, , and degradation upon interaction with the ubiquitin ligase CBL; (c) a tyrosine kinase domain, which mediates c-MET biological activity (following c-MET activation,
  • a C-terminal region contains two crucial tyrosines (Tyr 1349 and Tyr 1356), which are inserted into the multisubstrate docking site, capable of recruiting downstream adapter proteins with Src homology (SH2) domains.
  • the two tyrosines of the docking site have been reported to be necessary and sufficient for the signal transduction both in vitro. See, generally, Trusolino, L., Bertotti, A. and Comoglio, P.M., MET signaling: principles and functions in development, organ regeneration and cancer. Nat. Rev. Mol. Cell Biol. 11:834-848 (2010).
  • c-MET activation by its ligand HGF induces c-MET kinase catalytic activity, which triggers transphosphorylation of the tyrosines - Tyr 1234 and Tyr 1235 .
  • tyrosines engage various signal transducers, thus initiating a whole spectrum of biological activities driven by MET, collectively known as the invasive growth program.
  • the transducers interact with the intracellular multisubstrate docking site of c-MET either directly, such as GRB2, SHC, SRC, and the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI3K), or indirectly through the scaffolding protein Gabl .
  • PI3K phosphatidylinositol-3 kinase
  • Tyr 1349 and Tyr 1356 of the multisubstrate docking site are both involved in the interaction with GABl , SRC, and SHC, while only Tyr 1356 is involved in the recruitment of GRB2, phospholipase Cy (PLC- ⁇ ), p85, and SHP2.
  • GABl is a key coordinator of the cellular responses to MET and binds the MET intracellular region with high avidity, but low affinity. Upon interaction with MET, GABl becomes phosphorylated on several tyrosine residues which, in turn, recruit a number of signaling effectors, including PI3K, SHP2, and PLC- ⁇ . GABl phosphorylation by MET results in a sustained signal that mediates most of the downstream signaling pathways.
  • c-MET engagement activates multiple signal transduction pathways including: (i) the RAS pathway mediates HGF-induced scattering and proliferation signals, which lead to branching morphogenesis. HGF is different from most mitogens in that it induces sustained RAS activation, and thus prolonged MAPK activity; (ii) the phosphatidylinositol 3 -kinase (PI3K) pathway is activated in two ways - PI3K can be either downstream of RAS, or it can be recruited directly through the multifunctional docking site. Activation of the PI3K pathway is currently associated with cell motility through remodeling of adhesion to the extracellular matrix as well as localized recruitment of transducers involved in cytoskeletal reorganization, such as RAC1 and PAK.
  • PI3K phosphatidylinositol 3 -kinase pathway
  • PI3K activation also triggers a survival signal due to activation of the AKT pathway; (iii) the STAT pathway, together with the sustained MAPK activation, is necessary for the HGF-induced branching morphogenesis.
  • MET activates the STAT 3 transcription factor directly, through an SH2 domain: (iv) the ⁇ -catenin pathway, a key component of the Wnt signaling pathway, translocates into the nucleus following MET activation and participates in transcriptional regulation of numerous genes; and (v) the Notch pathway, through transcriptional activation of Delta ligand (DLL3).
  • DLL3 Delta ligand
  • MET mediates a complex program known as invasive growth. Activation of c-MET triggers mitogenesis and morphogenesis.
  • transformation of the flat, two-layer germinal disc into a three-dimensional body depends on transition of some cells from an epithelial phenotype to spindle-shaped cells with motile behavior (i.e., a mesenchymal phenotype). This process is referred to as epithelial -mesnenchymal phenotype (EMT).
  • EMT epithelial -mesnenchymal phenotype
  • MET is crucial for gastrulation, angeogenesis, myoblast migration, bone remodeling, and nerve sprouting, embryogenesis, among others. See, e.g., Birchmeier C, Gherardi, E.
  • c-MET was first identified as the product of a chromosomal rearrangement after treatment with the carcinogen N-methyl-NO-nitro-N-nitrosoguanidine, See, e.g., Cooper, C.S., Park, M., et al., Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 311 :29-33 (1984).
  • This rearrangement results in a constitutively fused oncogene (TPR-MET) which is translated into an oncoprotein following dimerization by a leucine-zipper motif located in the TPR moiety. This provides the structural requirement for c-MET kinase to be constitutively active.
  • TPR-MET has been shown to possess the ability to transform epithelial cells and to induce spontaneous mammary tumors when ubiquitously over-expressed in transgenic mice.
  • c-MET is overexpressed in a variety of carcinomas including lung, breast, ovary, kidney, colon, thyroid, liver, and gastric carcinomas. See, e.g., Knowles, L.M., Stabile, L.P., et al. HGF and c-Met participate in paracrine tumorigenic pathways in head and neck squamous cell cancer. Clin. Cancer Res. 15:3740-3750 (2009).
  • Such over-expression could be the result of transcriptional activation, hypoxia-induced over- expression, or as a result of MET amplification. See, e.g., Cappuzzo, F., Marchetti, A., et al. Increased MET gene copy number negatively affects survival of surgically resected non- small-cell lung cancer patients. J. Clin. Oncol. 27: 1667-1674 (2009); Cappuzzo, F., Janne, P. A., et al. MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients. Ann. Oncol. 20:298-304 (2009).
  • transgenic mice overexpressing c-MET have been reported to spontaneously develop hepatocellular carcinoma, and when the transgene was inactivated, tumor regression was reported even in large tumors. See, e.g., Wang, R., Ferrell, L.D., et al. Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice. J. Cell. Biol. 153: 1023-1034 (2001).
  • MET activation in cancer correlates with poor prognosis, where aberrantly active MET triggers tumor growth, formation of new blood vessels (angeo genesis) that supply the tumor with nutrients, and cancer spread to other organs (metastasis).
  • MET is deregulated in many types of human malignancies, including cancers of the: kidney, liver, stomach, breast, and brain.
  • cancer stem cells are thought to hijack the ability of normal stem cells to express MET, and thus become the cause of cancer persistence and spread to other sites in the body.
  • HGF-MET cascade a key target for inhibiting cancer metastasis: The impact of NK4 discovery on cancer biology and therapeutics. Int. J. Mol. Sci. 14:888-919 (2013).
  • MET kinase has been shown to be overexpressed in up to 40% of lung cancer tissue samples and has been a focal target for small molecule development. See, e.g., Villaflor and Salgia, Targeted agents in non-small cell lung cancer therapy: What is there on the horizon. J.
  • MET kinase amplification upregulation
  • this amplification is associated with resistance to the important NSCLC drugs Erlotinib and/or Gefitinib.
  • MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc. Natl. Acad. Sci. U.S.A. 26;104(52):20932- 20937 (2007).
  • MET kinase is coupled to FAK, RAS, RAC, PI3K, CAS-CRK and other pathways. These pathways are central to cell growth and also regulate various physiological processes in cancer (invasion, metastasis, and the like).
  • Tavocept might interact with and modify human mesenchymal epithelial transition (MET) kinase.
  • MET mesenchymal epithelial transition
  • Studies in the specific example of MET kinase described herein were designed to evaluate the effect of Tavocept on MET kinase activity in the presence and absence of the known ATP-competitive MET kinase inhibitor, Crizotinib and Staurosporine.
  • Tavocept may xenobiotically modify Cysl091 from the Phosphate-loop (P-loop). Since the P-loop is located on top of the ATP substrate binding site, Tavocept-mediated xenobiotic modification at this site may impact MET kinase activity.
  • Other modifications may be possible and an X- ray structure would be needed to unequivocally verify this hypothesis. See, Figure 5.
  • N-terminal GST tagged recombinant human MET expressed in Sf9 cells was purchased from SignalChem ( Figure 1 ; Cat. #M52- 18G- 10, lots V273-2, MW 81.0 kDa and aliquoted to 10 fractions when it was used for the first time (so as to avoid multiple freeze/thaw cycles for subsequent experiments).
  • Tavocept BNP7787 was prepared by a proprietary method (lots #205001 or 450002-2, > 97%, no mesna was detected by Mass Spectroscopy).
  • Kinase inhibitor, PF-02341066 also known as Crizotinib
  • Staurosporine was purchased from Calbiochem, LLC (Cat. #569396, lot #D00127851).
  • Kinase assay buffer was purchased from SignalChem (Cat. # K03-09, Lot # R301- 3W) and consisted of 40 mM Tris, pH 7.5, 20 mM MgCl 2 , 0.1 mg/mL bovine serum albumin (BSA) and 150 ⁇ dithiothreitol (DTT).
  • BSA bovine serum albumin
  • DTT dithiothreitol
  • Microplates were purchased directly from VWR or Corning and initial assay optimization was performed using whole area 96-well white microplates (Corning 3912, lot 29011050) but to save reagents and costs, most IC 5 o determinations and subsequent experiments were conducted in half area 96 well white microplates (Corning 3642, lot 05312045).
  • ADP-glo reagents were purchased from Promega and consisted of ADP (V916A, lot
  • V915A 32551702
  • ADP-glo V912A, lot 32559601 or V912B, lot 0000010953
  • kinase detection reagent buffer V913A, lot 32179101 or V913B, lot
  • the 10 volume assays in half area 96-well microtiter plates contained MET (2.5 ng/ ⁇ ) with ATP (100 ⁇ ) or MET (0.1 ng ⁇ L) with ATP (10 ⁇ ), PolyGT substrate (0.1 ⁇ / ⁇ ) and the concentrations of Tavocept (BNP7787)) and/or Crizotinib and/or Staurosporine as indicated (final assay volume was 10 ⁇ ).
  • MET 2.5 ng/ ⁇
  • ATP 100 ⁇
  • MET 0.1 ng ⁇ L
  • ATP 10 ⁇
  • PolyGT substrate 0.1 ⁇ / ⁇
  • concentrations of Tavocept BNP7787
  • Crizotinib and Staurosporine as indicated
  • Staurosporine was dissolved as a 5 mM and 1 mM stock in DMSO, respectively, and then further diluted in kinase assay buffer (DMSO only controls were always run to ensure that DMSO did not interfere with the assay).
  • the reactions, in microplate, were incubated for 60 minutes at 25°C on a heat block. Following this 60 minute incubation, the kinase activity was evaluated using the ADP-Glo system from Promega that monitored ADP produced when MET phosphorylated the PolyGT substrate.
  • Kinase assays were run in triplicate or quadruplicate in microplates. Following this, the ADP-Glo detection system (Promega) was used to determine how much ADP had been produced. For 10 ⁇ , volume assays, to each microplate well containing 10 ⁇ , of kinase reaction was added ADP-Glo reagent (10 ⁇ ), plates were spun in a table top centrifuge (1000 rpm (123 x g) for 1 minute) to ensure no reagent remained on the well walls, and then agitated for 1 minute to ensure optimal mixing. Plates were incubated at 25 C on a heat block for 40 minutes. Next, kinase detection reagent (20 ⁇ ) was added and, as above,
  • Tecan Ultra contained a built-in plate definition file for the whole area 96-well white Corning plates but a plate definition file for the half area 96-well Corning plates was created using the RdrOle component of the Tecan Ultra software.
  • Kinases vary in their ability to turnover ATP in vitro; therefore, the activity of the MET over a concentration range from 0.78 ng to 20 ng was evaluated in assay volumes of 10 ⁇ ⁇ . See, Figure 7. These values represent a molar range of 0.96 to 247 nM MET.
  • PolyGT Polyglutamate tyrosine
  • each glu-glu-glu-glu-tyr "subpolymer' in this polymer has a mass of approximately 698 g/mole. Therefore, each mole of polymer of 20,000 g/mole would contain approximately 28 moles of the glu-glu-glu-glu-tyr "subpolymer" in a 10 assay volume containing 1 ⁇ g of the polyGT substrate. Assuming the lower polymer mass of 20,000 g/mol mass, this translates to approximately 5 ⁇ polyGT per assay and 140 ⁇ glu-glu-glu-glu-tyr
  • Tavocept (BNP7787) inhibited MET with an IC 50 of 17.36 mM (single experiment) under assay conditions of 10 ⁇ ATP (with 2.5 ng/ ⁇ MET per assay) and with an IC 50 of 15.21 ⁇ 0.36 mM under assay conditions of 10 ⁇ ATP (with 0.1 ng/ ⁇ MET per assay).
  • Tavocept (BNP7787) had an IC 50 > 40 mM and 37.12 mM (single experiment), respectively, in assays containing 2.5 or 0.1 ng/ ⁇ MET per assay, respectively. See, Figure 8, Figure 9, Figure 10, and Figure 1 1.
  • ATP concentration increases. It was observed in the studies disclosed herein that as the ATP concentration was increased, the IC 50 for Tavocept (BNP7787) also increased. Table 6, below, illustrates the IC 50 of Tavocept (BNP7787) under varying concentrations of MET and ATP. Consequently, while the inhibition of MET by Tavocept (BNP7787) is not "classic" competitive inhibition (i.e., where ATP and Tavocept (BNP7787) have nearly identical or at least significantly overlapping binding sites and only one molecule, either ATP or Tavocept (BNP7787), can occupy that site at a time), it is "competitive-like" based upon the increasing IC 50 as the ATP concentration is increased.
  • Tavocept (BNP7787) concentrations of Tavocept (BNP7787) as high as 18 mM have been achieved in the clinic.
  • Tavocept (BNP7787) has been administered at a dose of 18.4 g/m and this translates to C max values in plasma of 10 mM and higher.
  • the concentration of Tavocept (BNP7787) required to see an effect in vitro on MET activity under the lower ATP assay conditions (10 ⁇ ) are physiologically relevant.
  • the concentration of Tavocept (BNP7787) required to observe an effect on MET activity under the higher ATP assay conditions (100 ⁇ ) are not physiologically relevant.
  • Tavocept (BNP7787) is used in combination with Crizotinib or staurosporine, notable potentiation occurs under both lower and higher ATP assay conditions.
  • ATP is often in the millimolar range in vivo, and the human body is reported to contain no more than 0.5 moles (-250 g) of ATP at any time, but this supply is constantly and efficiently recycled. See, e.g., Lu X, Errington J, Chen VJ, Curtin NJ, Boddy AV, Newell DR. Cellular ATP depletion by LY309887 as a predictor of growth inhibition in human tumor cell lines. Clin. Cancer Res. 6(l):271-277 (2000).
  • ATP - dependent enzymes that compete for ATP binding, including kinases, synthetases, helicases, membrane transporters and pumps, chaperones, motor proteins, and large protein complexes like the proteasome; therefore, the concentrations of 10 and 100 ⁇ ATP used herein are approximations for ATP concentrations that may be available to MET in vivo as it competes for ATP with various other enzymes and proteins that utilize ATP.
  • Crizotinib is a reported ATP-competitive inhibitor of MET. See, e.g., Bang YJ. The potential for Crizotinib in non-small cell lung cancer: a perspective review. Ther. Adv. Med. Oncol. 3(6):279-291 (2011). In the in vitro kinase studies reported herein, we observed that Crizotinib inhibited MET with an IC 50 of 38.39 nM (see, Figure 12) under assay
  • Crizotinib has previously been characterized as a competitive inhibitor of MET, with respect to ATP, and our data are consistent with this previously reported observation. It should be noted that in clinical trials where Crizotinib was administered orally at doses of 250 mg twice daily, concentrations of Crizotinib of 57 nM were reported.
  • Crizotinib near the IC 50 value of Crizotinib resulted in 6% greater inhibition than 40 nM Crizotinib alone whereas 10 mM Tavocept (BNP7787) in combination with 40 nM Crizotinib resulted in 11% greater inhibition than 40 nM Crizotinib alone.
  • These assays near the IC 50 value for Crizotinib i.e., 40 nM, when ATP is 10 ⁇
  • have similar stimulation compared to 10 ⁇ ATP and 20 nM Crizotinib conditions see, Figure 14.
  • Tavocept (BNP7787) alone or Crizotinib alone were both effective at inhibiting MET in vitro.
  • Tavocept (2.5 ng/uL) Activity In Vitro Under 100 uM ATP Conditions
  • concentrations of Crizotinib near the IC 25 and IC 50 concentrations of Crizotinib under assay conditions with either 100 ⁇ or 10 ⁇ ATP were examined.
  • Concentrations of Crizotinib of 57 nM have been reported in clinical trials; therefore, concentrations of Crizotinib used in these studies are within physiologically relevant ranges.
  • Tavocept (BNP7787) is administered at a dose of 18.4 g/m and this translates to C max values in plasma of 10 mM and higher. Tavocept
  • BNP7787 notably potentiates the inhibitory effect of Crizotinib on MET at physiologically relevant concentrations of both Tavocept (BNP7787) and Crizotinib.
  • 5 mM Tavocept (BNP7787) in combination with 45 nM Crizotinib resulted in 15% greater inhibition than 45 nM Crizotinib alone
  • 10 mM Tavocept (BNP7787) in combination with 45 nM Crizotinib resulted in 14% greater inhibition than 45 nM Crizotinib alone.
  • Tavocept (BNP7787) in combination with 90 nM Crizotinib resulted in 10% greater inhibition than 90 nM Crizotinib alone; whereas 10 mM Tavocept (BNP7787) in combination with 90 nM Crizotinib resulted in 10% greater inhibition than 90 nM Crizotinib alone.
  • These assays near the IC 50 value for Crizotinib ⁇ i.e., 90 nM, when ATP is 100 ⁇ ) have similar stimulation compared to 100 ⁇ ATP and 45 nM Crizotinib conditions. See, Figure 15.
  • Tavocept (BNP7787) alone or Crizotinib alone or Tavocept (BNP7787) in combination with Crizotinib were effective at inhibiting MET in vitro.
  • Staurosporine is a reported ATP-competitive inhibitor of many kinases. See, e.g., Tanramlu D, Schreyer A, Pitt WR, Blundell TL. On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinases binding to staurosporine. Chem. Biol. Drug Des. 74(1): 16-24 (2009). In the in vitro kinase studies reported herein, we observed that Staurosporine inhibited MET (0.1 ng/ ⁇ ) with an IC 50 of 340 nM ⁇ see, Figure 16) under assay concentrations using ATP at 10 ⁇ .
  • Tavocept (BNP7787)
  • BNP7787 The effect of physiologically achievable concentrations of Tavocept (BNP7787) near the IC 25 and IC 50 concentrations of Staurosporine under assay conditions with 10 ⁇ ATP were evaluated.
  • Tavocept (BNP7787) is administered at a dose of 18.4 g/m and this translates to C max values in plasma of 10 mM and higher.
  • Tavocept (BNP7787) notably potentiates the inhibitory effect of Staurosporine on MET at the tested concentrations of both Tavocept (BNP7787) and Staurosporine.
  • Tavocept BNP7787 inhibits MET (2.5 ng/ ⁇ ) with an IC 50 value > 40 mM.
  • Tavocept (BNP7787) inhibits MET (0.1 ng ⁇ L) with an IC 50 value of 15.21 ⁇ 0.36 mM.
  • Staurosporine inhibits MET (0.1 ng/ ⁇ ) with an IC 50 value of 340 nM.
  • Tavocept modulates the activity of MET kinase in vitro, if this occurs in vivo, a potential survival benefit could accompany this MET kinase modulation in NSCLC patients bearing MET kinase fusions or mutations.
  • Anaplastic lymphoma kinase also known as ALK tyrosine kinase receptor or CD246 (cluster of differentiation 246) is an enzyme that in humans is encoded by the ALK gene. See, e.g., Cui, J.J.; Tran-Dube, M.; et al, Structure Based Drug Design of Crizotinib (PF-02341066), a Potent and Selective Dual Inhibitor of Mesenchymal-Epithelial Transition Factor (c-MET) Kinase and Anaplastic Lymphoma Kinase (ALK). J. Med. Chem. 54:6342- 6363 (2011).
  • ALK belongs to the family of insulin growth factor receptor kinases and fusions of ALK with other genes are common in several diseases and cancers. See, e.g., Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: Signalling in development and disease. Biochem. J. 420(3):345-361 (2009); Kruczynski, et al, Anaplastic lymphoma kinase as a therapeutic target. Expert Opin. Ther. Targets 16: 1127-1138 (2012). Fusions in ALK result in constitutively active protein that results in stimulation of a variety of intracellular pathways critical for cell growth and proliferation.
  • TRK- fused gene is a new partner of ALK in anaplastic large cell lymphoma producing two structurally different TFG-ALK translocations.
  • EML4-ALK fusions are thought to account for approximately 2-7% of NSCLC cases. See, e.g., Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: signalling in development and disease. Biochem. J.
  • nucleophosmin-ALK (including the nucleophosmin-ALK (NPM-ALK)) fusion, are found in a range of other cancers including, but not limited to, breast cancer, colorectal cancer, esophageal cancer, anaplastic large cell lymphoma, chronic myelogenous leukemia, and acute leukemias. See, e.g., Grande, et al., Targeting oncogenic ALK: A promising strategy for cancer treatment. Mol. Cancer Ther. 10:569-579 (2011); Ok, et al, Aberrant activation of the hedgehog signaling pathway in malignant hematological neoplasms. Am. J. Path. 180:2-11 (2012).
  • ALK is coupled to numerous signaling pathways that regulate cell proliferation including Ras-ERK, JAK3-STAT3, PLCy and PI3K and, therefore, represents an important target for anti-cancer drug development. See, e.g., Chiarle, R. et al, The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat. Rev. Cancer 8(1): 11-23 (2008); Ou,
  • Crizotinib A novel and first-in-class multitargeted tyrosine kinase inhibitor for the treatment of anaplastic lymphoma kinase rearranged non-small cell lung cancer and beyond. Drug Design, Devel. Therap. 4:471-485 (2011).
  • ALK provides us with one of the most recognized examples of personalized medicine success in Crizotinib, which effectively modulates ALK function in NSCLC patients harboring ALK fusions despite being initially developed to target MET kinase. See, e.g., Ong, et al, Personalized medicine and pharmacogenetic biomarkers: Progress in molecular oncology testing, Expert Rev. Mol. Diagnosis 12(6):593-602 (2012).
  • ALK ALK-like ligands
  • pleiotrophin and midkine are the sole ALK ligands in vivo or if other ligands exist (these molecules activate other receptors and are certainly not exclusive to ALK).
  • ALK fusions are known to be important in a number of cancers
  • point mutations in ALK resulting in gain-of-function mutants are also known and are associated with increases in ALK kinase activity, ALK-mediated phosphorylation of downstream targets, and ALK expression levels. See, e.g., See, e.g., Grande, et al., Targeting oncogenic ALK: A promising strategy for cancer treatment. Mol. Cancer Ther. 10:569-579 (2011); Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: Signalling in development and disease. Biochem. J. 420(3):345-361 (2009).
  • ALK point mutations are also thought to be important in one of the leading causes of cancer deaths in children,
  • neuroblastoma See, e.g., Carpenter and Mosse, Targeting ALK in neuroblastoma:
  • ALK represents an important target for anti-cancer drug development across a range of cancers and agents that modulate ALK, as single agents or in combination with other ALK agents, may have widespread clinical utility.
  • ALK belongs to the tyrosine kinase receptor family. By homology, ALK is most similar to leukocyte tyrosine kinase, and both belong to the insulin-receptor superfamily.
  • ALK is a single-chain transmembrane receptor comprising three structural domains. The extracellular domain contains an N-terminal signal peptide sequence and is the ligand- binding site for the putative activating ligands of ALK ⁇ i.e., pleiotrophin and midkine). This is followed by the transmembrane and juxtamembrane region which contains a binding site for phosphotyrosine-dependent interaction with insulin receptor substrate- 1. The final section has an intracellular tyrosine kinase domain with three phosphorylation sites (Y1278, Y1282, and Y1283), followed by the C-terminal domain with interaction sites for
  • phospho lipase C- ⁇ and Src homology 2 domain containing SHC are absent in the product of the transforming ALK gene.
  • binding of a ligand induces homodimerization of ALK, leading to trans-phosphorylation and kinase activation.
  • the 5 '-terminus fusion partners provide dimerization domains, enabling ligand-independent activation of the kinase.
  • ALK which localizes to the plasma membrane
  • ALK fusion proteins localize to the cytoplasm. This difference in cellular localization may also contribute to deregulated ALK activation.
  • EML4-ALK fusion oncogene represents one of the newest molecular targets in cancer (especially in non-small cell lung carcinoma (NSCLC)).
  • EML4-ALK was identified by the screening of a cDNA library derived from a the tumor of a NSCLC (adenocarcinoma) of the lung. See, e.g., Soda, M., Choi, Y.L employ et al. Identification of the transforming EML4- ALK fusion gene in non-small cell lung cancer. Nature 448:561-566 (2007).
  • EML4-ALK echinoderm microtubule associated protein- like 4
  • ALK anaplastic large cell lymphomas
  • IMT inflammatory myo fibroblastic tumors
  • neuroblastomas e.g., aplastic large cell lymphomas (ALCL), inflammatory myo fibroblastic tumors (IMT), and neuroblastomas.
  • ALK anaplastic large cell lymphomas
  • IMT inflammatory myo fibroblastic tumors
  • neuroblastomas e.g., aplastic large cell lymphomas
  • EML4-ALK the fusion partner has been shown to mediate ligand-independent dimerization of ALK, resulting in constitutive kinase activity.
  • EML4-ALK possesses potent oncogenic activity.
  • EML4-ALK In transgenic mouse models, lung-specific expression of EML4-ALK leads to the development of numerous lung adenocarcinomas. See, e.g., Soda, M., Takada, S., et al. A mouse model for EML4-ALK- positive lung cancer. Proc. Natl. Acad. Sci. U.S.A. 105: 19893-19897 (2008). Cancer cell lines harboring the EML4-ALK translocation can be effectively inhibited by small molecule inhibitors targeting ALK. See, e.g., Koivunen, J. P., Mermel, C, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin. Cancer Res. 14:4275-4283
  • ALK is believed to play a role in the development and function of the nervous system. Studies using ALK knockout mice have reported an increase in
  • ALK adenosine-binding protein
  • PTN pleiotrophin
  • midkine small, heparin-binding growth factors, implicated in neuron development as well as neurodegenerative diseases. See, e.g., Palmer, R.H.,
  • Pleiotrophin and midkine have a similar distribution to ALK, mainly in the nervous system during fetal development followed by downregulation at birth. These ligands display neurotrophic functions on receptor binding.
  • PTN may also activate ALK indirectly by binding to and inactivating the receptor protein tyrosine phosphatase Zl .
  • the receptor protein tyrosine phosphatase Zl See, e.g., Perez-Pinera P., Zhang, W., et al. Anaplastic lymphoma kinase is activated through the pleiotrophin/receptor protein-tyrosine phosphatase beta/zeta signaling pathway: an alternative mechanism of receptor tyrosine kinase activation. J. Biol. Chem. 282:28683-28690 (2007). Whether there are other ALK ligands or other mechanisms of ALK activation remains to be determined.
  • the oncogenic fusion protein promotes the activation of, primarily, three key signaling pathways: (i) the Janus-activated kinase (JAK3)-STAT3 intracellular pathway ; (ii) phosphoinositide 3-kinase (PI3K)-Akt pathway; and (iii) the Ras/mitogen activated protein/extracellular signal regulated kinase (ERK) kinase (Mek)/Erk pathway to promote cell cycle progression, survival, and proliferation.
  • JK3K Janus-activated kinase
  • PI3K phosphoinositide 3-kinase
  • ERK Ras/mitogen activated protein/extracellular signal regulated kinase
  • Mek extracellular signal regulated kinase
  • Erk Erk pathway
  • NIP A anaplastic lymphoma kinase
  • Ras/Mek/Erk pathway is important for driving cell proliferation; whereas the PI3K/Akt and JAK3-STAT3 pathways are important for cell survival and cytoskeletal changes.
  • EML4-ALK differentially activate downstream signaling pathways
  • NPM-ALK nuclear factor-activated protein
  • PI3K/Akt nuclear factor-activated protein
  • Pharmacologic inhibition of EML4-ALK using TKIs leads to downregulation of Ras/Mek/Erk and PI3K/Akt and apoptosis , consistent with the notion that activation of these two pathways is critical for EML4-ALK-mediated transformation.
  • both Ras/Mek/Erk and PI3K/Akt pathways are reactivated despite the continued presence of the TKI.
  • ALK anaplastic large cell lymphoma
  • DLBCL diffuse large B-cell lymphomas
  • the novel fusion transcript with transforming activity formed by the translocation of echinoderm microtubule associated protein like 4 (EML4) located at 2p21, and the ALK located at 2p23, has been described in a subset of patients with non-small cell lung cancer (NSCLC; see, e.g., Soda, M., Choi, Y.L shadow et al. Identification of the transforming EML4-ALK fusion gene in non-small cell lung cancer. Nature 448:561-566 (2007)) and several additional variants of the rearranged gene have also been identified.
  • NSCLC non-small cell lung cancer
  • ALK partners such as the kinesin family member 5B (KIF5B) located at lOpl 1.22 and TRK fused gene (TFG) located at 3ql2.2
  • KIF5B kinesin family member 5B
  • TRK fused gene TRK fused gene
  • ALK chimeric proteins This biological property results in ligand-independent dimerization and, thus, in constitutive activation of the kinase.
  • the oncogenic role of ALK chimeric proteins has also been shown by pre-clinical studies and mouse models with forced expression of ALK.
  • NSCLC the ALK translocation seems to define a subgroup of patients with specific clinical, pathologic, and molecular characteristics. This alteration is more frequent in younger patients, who are non- or light-smokers, with adenocarcinoma histology with presence of signet ring-type cells, EGFR, and/or KRAS wildtype tumors ⁇ see, e.g., Wong, D.W., Leung, EX., et al.
  • the EML4- ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS. Cancer 115: 1723-1733 (2009)), together with non-c-MET copy number increase ⁇ see, e.g., Varella-Garcia, M., Cho, Y., Lu, X. ALK gene
  • Point mutations have been found in 6-8% of primary neuroblastomas. Germ-line mutations have been identified in families with more than one sibling with neuroblastoma. Somatic mutations with wild-type ALK in matched constitutional DNAs have also been described in non-familial neuroblastoma cases. These mutations are located mainly in the TK domain; the most frequent being the gain-of- function mutations Fl 174L and R1275Q. These mutations are associated with increased expression, phosphorylation, and kinase activity of the ALK protein. Further, they have been shown to have Ba/F3 cell-transforming capacity. In some cases, these mutations coexist with an increased copy number of the ALK gene.
  • ALK ALK-selective inhibition
  • NSCLC non-small cell lung cancer

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Abstract

La présente invention concerne de nouvelles compositions pharmaceutiques, de nouveaux procédés, et de nouveau kits utilisés pour la modification thérapeutique multi-cibles, orientée de façon hétérogène, concomitante et/ou la modulation d'anomalies métaboliques cellulaires ou d'autres affections physiologiques indésirables, comprenant un cancer, dans lesquelles la fonction biochimique cellulaire normale et/ou les taux d'expression de différentes protéines/enzymes (c'est-à-dire, les molécules ciblent) sont anormaux et doivent être modifiés et/ou modulés afin de traiter ces anomalies métaboliques ou autres affections physiologiques indésirables, comprenant un cancer. Les molécules cibles mentionnées ci-dessus, à titre d'exemple non limitatif, comprennent : la kinase de lymphome anaplasique (ALK), la kinase de transition mésenchymateuse-épithéliale (MET), la récepteur tyrosine kinase (ROS1), le récepteur de facteur de croissance épidermique (EGFR), la peroxirédoxine (Prx), la protéine de complémentation croisée de réparation 1 (ERCC1), le récepteur de facteur de croissance insulinomimétique 1 (IGF1R), la ribonucléotide réductase (RNR), la tubuline, la farnésyltransférase, et différentes autres classes de protéines/enzymes. De plus, la présente invention concerne des procédés et des kits pour (a) la sélection de sujets pour traitement ; (b) la détermination du/des agent(s) médicinaux les plus efficaces à administrer en combinaison avec l'administration des petites molécules acide aminé-spécifiques contenant du soufre de la présente invention ; (c) la dose du/des agent(s) médicinaux à administrer ; (d) la détermination de la durée et/ou du nombre de cycles de traitement ; (e) l'ajustement du/des agent(s) médicinaux spécifiques utilisés et de la dose administrée au cours du traitement ; et/ou (f) la détermination de la réponse potentielle au traitement de la maladie spécifique au(x) agent(s) médicinaux sélectionnés pour administration à un sujet souffrant d'un ou plusieurs types de : (i) cancer ou (ii) d'anomalies métaboliques cellulaires ou autres affections physiologiques indésirables par détermination quantitative du taux d'activité biochimique anormale et/ou d'expression anormale d'une combinaison quelconque des molécules cibles mentionnées ci-dessus ; par utilisation de méthodologies de mesure quantitative comprenant, mais non limitées à : l'hybridation par fluorescence in situ (FISH), l'analyse sur puce à acide nucléique, l'immunohistochimie (IHC), un radio-immunodosage (RIA), l'immunofluorescence quantitative et/ou l'analyse quantitative automatisée ; des analyses à base d'ELISA et de cytométrie en flux ; la PCR couplée à des approches MS ; les procédés à base de spectrométrie de masse ; et la cristallographie à rayons X, et d'autres méthodologies analytiques associées.
PCT/US2014/050465 2013-08-13 2014-08-10 Modification thérapeutique multi-cibles, orientée de façon hétérogène, concomitante et/ou modulation d'une maladie par administration de petites molécules acide aminé-spécifiques, contenant du soufre WO2015023553A2 (fr)

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WO2017129753A1 (fr) * 2016-01-27 2017-08-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédés de stadification, typage et traitement d'une maladie cancéreuse
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JP2019534711A (ja) * 2016-12-08 2019-12-05 上海優▲か▼迪生物医薬科技有限公司Ｓｈａｎｇｈａｉ Ｕｎｉｃａｒ−Ｔｈｅｒａｐｙ Ｂｉｏ−Ｍｅｄｉｃｉｎｅ Ｔｅｃｈｎｏｌｏｇｙ Ｃｏ．，Ｌｔｄ ヒトＰＤ−１をノックダウンしたｓｉＲＮＡ、組換え発現ＣＡＲ−Ｔベクターおよびその構築方法と使用
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