WO2019173683A1 - Discovery of novel molecules and repurposed drugs for ras family gtpases - Google Patents

Abstract

The present invention is directed to compounds, compositions and methods for modulating RAS family GTPases, in particular KRas, HRas and NRas GTPases. These GTPases are upregulated in cancer and in other tissue and represent appropriate targets for therapy. Methods for identifying the activity of compounds with respect to these and other GTPases in multiplex flow cytometry systems represents another aspect of this invention.

Description

Discovery of Novel Molecules and Repurposed Drugs for RAS Family GTPases Related Applications

This application claims the benefit of priority of provisional applications serial numbers 11862/640,162, filed March 8, 2018 and US62/689,512, filed June 25, 2018, each of said applications being incorporated by reference in their entirety herein.

This invention was made with government support under Grant Nos. P50 G. 085273 and TR.0001111 awarded by National Institutes of Health, The government has certain rights in the invention.

Field of the Invention

The present invention is directed to compounds, compositions and methods for modulating RAS family GTPases, in particular, KRas, HRas and NRas GTPases and

treatment of disease which is mediated through these G TPases or where these GTPases play a role in a disease state and/or condition. The present invention is also directed to technology for discovery of compounds, compositions, and methods for identifying

compounds/composittons which modulate RAS family GTPases, in particular KRas, HRas and NRas GTPases. These GTPases are upregulated in cancer and in other tissue and represent appropriate targets for therapy with the compounds which are identified here.

Background and Overview of the Invention

As RAS genes comprise the most frequently mutated gene family in human cancer, the validated role of mutationally activated MAS genes in driving cancer development and growth has stimulated comprehensive efforts to develop therapeutic strategies to block mutant Ras funct ion for cancer treatment. Despite more than three decades of intensive effort, no effective Ras-targeted therapies have reached the clinic while kinases have yielded dozens of approved drugs. The inventors challenge the currently held perception that all RAS mutations are“created equal” and argue that pursuit of a pan-Ras therapeutic approach will not be successful. Instead, we suggest that the recent discovery of a therapeutic approach targeting one RAS mutation (GI2C) establishes the premise that screening specific Ras mutant proteins will reveal mutation and cancer type-specific vulnerabilities for mutation-selective anti-Ras therapies. Additionally, the recent identification of unique pockets and protein-protein interaction interfaces dictate unique behaviors of individual Ras proteins (HRas, KRas and NRas) further supporting the premise that Ras selective compounds will have significant utility. We used our unique multiplexed experimental approach that ensures the stability of Ras and Ras-relaied GTPase an allows comparative assessment of target sensitivity during screening with compound libraries. The approach has demonstrated utility tor detection of hits and development of robust leads that are active against select or multiple GTPases.

Through combined testing of off-patent drugs, cheminformatics to identify the most promising scaffolds, preelioieal and clinical testing, two enantto-selective scaffolds derived tom off-patent drug libraries were shown to have clinical translational utility. The publication and patent track records of the present inventors suggest that Ras GTPases are targets useful in the treatment of disease states and/or conditions which are modulated through Ras GTPases.

Ras proteins are .molecular switches that regulate cellular metabolism an growth, toggle between inactive‘off and active‘on’ states through a process highly regulated by cellular factors^1" . Mutations in RAS commonly found in human cancer cause Ras proteins to chronically switch on, resulting in deregulate growth control⁴. Approximately one-third of all human cancers contain activating mutations in RAS genes that drive cancer development and growth^{3 6}. Still, the specific roles of particular RAS mutations in oncogenesis are poorly understood'. Additionally, information on the complex regulation through conformation, oligomerization, and membrane orientation/organization of individual Ras proteins is still emerging^{8 12}. Several small molecule and protein inhibitors of Ras have been identified through m silico , high content imaging, crystallographic, and N M -based strategies¹’"T However, despite the identification of compounds that block mutant Ras protein function, moving effective Ras-targeted therapies into the clinic remains an unmet goal⁵'''^21'2·’. Thus, further characterization an discovery_' is needed for a more knowledgeable approach to anti- Ras therapy.

Numerous drugs have been identified and developed to modulate kinases; thus part of the motivation of the present inventors was to identify drugs and other chemical entities that could be found to modulate GTPases. Like kinases, GTPases act as stimulus sensors and utilize nucleotide binding and hydrolysis to govern conformational changes membrane organization, an protein-protein interactions . Both kinases and GTPases constitute large protein families which were at one time or another dubbed Tmdruggable" due to the idea that conserved substrate binding pockets make it impossible to develop selective or specific drugs. Kinase targeted drugs are now a notable success story²⁴'^*5. The present inventors thus hypothesize that GTPases are high value, individually draggable targets, which like kinases are a large family of enzymes. Unlike kinases today, there are few inhibitors (FT! inhibitors, zoledronic acid) for GTPases that have reached the clinic an the noted examples affect multiple targets causing adverse effects. With over 500 kinases encoded in the human genome and large numbers of human diseases caused by kinase dysreguiation, kinases have a history as targets for therapeutic :inten^;ention^“6>2'. After the serendipitous discovery of staurosporine^2>\ high throughput screening identified more ATP-competitive kinase inhibitors, with optimization leading to trials and approval. Structural analyses of kinases with inhibitors bound enabled kinase drug discovery to employ structure -based rational design, using lea optimization and fragment-based strategies. Notably, compound libraries generated by combinatorial chemical synthesis have facilitated the discovery of new kinase inhibitors where the library members can be individual compounds or compound mixtures²⁹. This history·' of therapeutic targeting of kinases offers relevant perspectives for targeting GTPases that can now be exploited.

Progress into clinical trials for drugs targeting GTPases has been slo w, potentially due to several factors. First, the nucleotide binding domain is relatively small and GTPases assume a relatively smooth and globular structure⁴'**, making it more difficult to predict drug binding pockets. Second, the binding affinity of the guanine nucleotide towards GTPases is high suggesting a problem for competition^{3 !> 2}. Third, the activity of GTPases is regulated by separate proteins like GBF and GAP proteins ³. Finally, GTPases play diverse roles in cell physiology ranging from cytoskeletal changes to protein translation which suggests that toxicity from unwanted side effects could be severe, especially for compounds that are not selective or specific. Still, there has been progress. For example, virtual screening identified Rho and Rac inhibitors that block the interactions between the GTPase and its effectors*⁴"³ ', and in stiico docking has identified inhibitors of Ras and its downstream effector

Automated and efficient: screening methods now include our multiplexing strategies⁴¹. Flow cytometry based multiplex screening assay⁴² allows individual GTPases to be linked to microsphere bead sets with distinct fluorescence intensities hi the re

fluorescence channel. The extent of fluorescent GTP binding to individual GTPases in the presence of test compounds can then be analyzed in another fluorescent channel This method allows the potency and selectivity of a compound towards several GTPases to be revealed simultaneously and reduces quantities of GTPases compared to plate-based homogeneous assays. Also, the use of GST-GTPase chimeras and their immobilization on beads stabilizes GTPases against denaiuration and may mimic oligomeric stains. The inventors have bee involved with previously conducted: a) high throughput screening of multiple GTPases against - 200,000 compounds from the Molecular Libraries Small Molecule Repository to identify regulators of nucleotide bindin

quantitative analyses of cellular GTPase activities using small volume samples⁴ ; and c) small molecule mechanism of action studies using real-time kinetic measurements of ligand or effector binding^44,45. Our screening and multi-tiered analysis platforms have identified both competitive and allosteric, selective inhibitors of Rho-family GTPases with clinical applicability^{46 4}'. We also identified small molecules that potentiate GTP binding^'18.

The overall premise which le to the present invention is that different KRAS mutations drive cancer by distinct mechanisms and hence require distinct therapeutic strategies. Recent identification of small molecules that allosterically and covalently inhibit the KRas G12C mutant frequently found in lung cancer support this hypothesis^4’55. Thus . we have adapted our multiplex screening technology^42,52*55 and have performed proof-of- principle screens of small chemical libraries using wild-type KRas and its prevalent point mutant proteins (G32A, G12D, G32V, G12C, G13D, Q61R, Q61L, and Q61H). Initial experience with this screen supports the idea that we can identify Ras selective small molecules and that there is merit in identifying molecules that bind to and regulate nucleotide binding to codon-specific KRAS mutations found with high frequency in human cancers. Identifie active compounds and prevalent scaffolds can be used to confirm chemical vulnerabilities of Ras family proteins

Brief Description of the Invention

In one embodiment, the present invention is directed to discovery of selective compounds which modulate human RAS GTPases, in particular KRAS, NRAS and HRAS and methods of treating disease states and/or conditions which are modulated through human RAS GTPases. These disease states and or conditions include immune dysfunction, pigmentation or neurological disorders which occur as a consequence of impaired GTPase function and/or functional insufficiency. Additional disease states and/or conditions which may be favorably influenced by treatment with the present: compounds include cancers (e.g., leukemias, colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, lung

adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, colorectal cancer, among others, often associated with KRAS, thyroid cancer, salivar duct carcinoma, epithelial-myoepitheiial carcinoma, kidney cancer, astrocytoma, among others, often associated with ERAS and melanoma, often associated with NRAS), histiocyte disorders (e.g. Rosai-Dorftnaa disease/sinus histiocytosis with massive lymphadenopathy), Noonan syndrome (NS), Noonan syndrome with multiple lentigines, Leopard syndrome, cardiofecio- cuianeous syndrome, neurofibromatosis type 1 ( M l ! and secondary effects of same including fibromas, scoliosis, long bone dysplasmia, osteoporosis and cognitive impairment, Legius syndrome, Costello syndrome (CS), capillary·' malformation-arteriovenous malformation syndrome (CFC syndrome), congenital myopathy with excess of muscle spindles (CME S), congenita! heart dise se, hereditary gingival fibromatosis type 1 and hypertrophic

cardiomyopathy (HCM), among others. The method comprises administering an effective amount of a compound identified herein to a patient in need, optionally in combination with at least one additional bioactive agent, and further optionally, at least one pharmaceutically acceptable carrier, additive or excipient. In embodiments, the present invention is directed to the treatment of cancer. In certain embodiments, the compo und ident ified herein is combined with at least one additional bioaeiive agent in the treatment of a disease state and/or

condition. In certain embodiments, the additional bioactive agent is at least one additional anticancer agent. In other embodiments, the additional bioactive agent is an agent which is separately useful for the treatment of a disease and/or a condition, often the same disease state or condition or a related disease state or condition for which the Ras modulator may be administered.

In otter embodiments, the present invention is directed to the technology and methods used to identify pharmaceutical compositions comprising an effective amount of a compound identified herein, optionally in combination with an effective amount of an additional bioaeiive agent (often an additional anticaneer agent or other agent useful in the treatment of cancer), in combination with a pharmaceutically acceptable carrier, additive or excipient. In embodiments, the compound is an antagonist or inhibitor of KRas, NRas or HRas GTPase.

In other embodiments, the compound is an agonist of KRas. NRas or HRas GTPase. The Ras GTPase may be a wild type protein of a mutant protein as described herein.

Compounds according to the present invention are discovered employing the

technology embodied in this claim that are described as specific inhibitors or agonists of Ras GTPases (ie., KRas, NRas and HRas GTPases), pan-inhibitors or pan-activators of Ras

GTPases (te., the inhibitor or activator is active across a number of GTPases, including mutant GTPases), mixed activity modulators (he., within a family of GTPases the type of activity is the same, but outside of the family, the activity may vary such that an inhibitor may become an agonist) or other potential modulators (i.e., within the same family of GTPases such as KRas, NRas or HRas the compound exhibits categorically different activity as an inhibitor or agonist).

Thus the Ras and Ras-re!ated GTPases are important targets for the development of small molecule agonists as well as antagonists for therapy of certain disease states and/or conditions, to aid studies of disease mechanism or to serve as scaffolds or pharmacophores for future therapeutics. T he present invention identifies tec no fogy for discovery of modulators of Ras GTPases as set forth in the present application and in the examples A, B, C, D, E) which provide methods and results for: I) optimization of buffers for stability and display of KRas WT and mutants; 2) screening of multiplex KRas proteins; 3) dose-response of active compounds ; 4) selectivity o f active compounds; 5) mechanism of action of a representative active compound.

The small molecules of the present invention include antagonists, activators

(agonists), including specific (for individual proteins, including mutant versions of such proteins). Importantly, such specific- and pan-GTPase modulators, including inhibitors and activators could provide advantages over genetic methodologies in cell-based assays, for measuring initial and/or acute response of reversibly altering activities of GTPases.

Furthermore, these molec ules provide a scaffold for structure-based design of agonists and antagonists against Rho-tamily GTPases to complement existing antagonists or inhibitors.

As Ras superfamiiy GTPases gain increasing traction as viable targets for further probe and drag discovery', the present invention provides a chemical platform for the rationale design of selective activators of key Ras superfamily members that could represent a boon for expanded understanding of the biology and pharmacology of small GTPases and therapy of disease states and/or conditions which are modulated through these proteins.

The following compounds among others were identified using the technology for compound discovery as presented in the attached examples section.

Pan Activators of Ras Family G^'TP binding Salsalate, Tolfenamic acid, Dexibuprofen, Mefenamic Acid, Ibuprofen, S-(+)~

Ibuprofen, Mec!eienamic acid sodium salt monohydrate, (R)-Naproxen sodium salt,

Naproxen, Fftifenaraic Acid, Flurbiprofen, Phenmidione, Dioxybenzone, A- 7 hydrochloride, Usnic acid and lopanic acid have been found to be pan activators of Ras family GTP binding. These compounds exhibited an increase in Bodipy-GTP binding in the presence of compound.

Fan Inhibitors of Ras Family GTP binding fstradeiyllme, PR-619, N6Q22, Diflfaetaic acid. IPA 3, Fisetin, Folic acid, GSK 3787 a ve been found to be pan inhibitors o f Ras family GTP binding. These compounds exhibited a decrease in Bodipy-GTP binding in the presence of compound.

Selective Inhibitors of GTP binding to RAS proteins, but not Q6I REAS mutants

Guanabenz acetate, Chlorprothixene hydrochloride_» NSC 663284, Trifluperazine Hydrochloride have been found to be selective inhibitors of GTP binding to Ras family proeins (these compounds exhibited a decrease in Bodipy-GTP bin ing in the presence of compound), hut not: to Q6I KRas mutants.

Mixed activity modul tors of GTP binding

Ipsapiroue, GF109203X (selective activator non-RAS), Beta Lapachone (RAS aetivator/non-RAS inhibitor), SF1670 (RAS activator, non- RAS inhibitor), Darapladih

(SB480848), PD 198306, Pimethixene Maleate, Oxyqumoline Hemisulfate have been found to be mixed acti vity modulators of GTP binding. in embodiments, the present invention is directed to compositions comprising an effect i ve amount of Ras Family GTPase modulator as descr ibed herein in combination with a pharmaceutically acceptable carrier, additive or excipient and further i combination with an effective amount of at least one additional bioactive agent, often an additional anticancer agent. In embodiments, the present in vention is directed to methods of modulating a Ras Family GTPase comprising exposing said GTPase to a compound disclosed herein in effective amounts as an inhibitor or agonist of said GTPase. In embodiments, the Ras GTPase is KRas WT or a mutant for example, KRas G12v, KRas G13d, KRas G!2a. KRas G12c_> KRas G12d, KRas Q61H, KRas Q61L, KRas Q61R, HRas WT or a mutant such as HRas G12v. In preferred aspects the Ras family GTPase modulator compound is selected from the group consisting of Salsa late. Tolfenamic acid, Dexibuprofen, Mefenamic Acid, Ibuprofen, S-fFHbuprofen, Meelofenamie acid sodium salt monohydrate, (R)-Naproxen sodium salt. Naproxen, Flufenamic Acid, Flurbiprofen, Phentnidione, Dioxybenzone, A-7 hydrochloride, Usnic acid, lopanic acid, Tstradefyllme, PR-019, N6022, Diffractaic acid, IP A 3, Fisetin, Folic acid, GSR 3787, Guanahenx acetate, Chiorprothixene hydrochloride, NSC 663284, Trtfluperaztne Hydrochloride, Ipsapirone, GF109203X (selective activator non- RAS), Beta Lapaehane (RAS activator/non-RAS inhibitor), SF1670 (RAS activator, non- RAS inhibitor), Darapladib (SB480848), PD 198306, Pimettoxene Maleate, Oxyquinol e Hemisulfate, or a pharmaceutically acceptable salt, stereoisomer, alternative salt or mixture thereof In embodiments, the GTPase which is modulated (inhibited or activated/up- regulated) is CDC42 WT or a mutant such as CDC42 L61, RacI WT or a mutant such as Rad L61 and the compounds modulate one or more of the identified GTPases.

In embodiments, the present invention is directed to methods of inhibiting a Ras Family GTPase the method comprising exposing said Ras Family GTPase to an effective amount of at least one compound identified herein as an inhibitor of GTP binding to one or more Ras proteins. I n embodiments, the invention is directed to methods of up-regulating or enhancing the activity (through agonist activity) of a Ras Family GTPase, including a mutant, the method comprising exposing said Ras Family GTPase to an effective amount of an agonist of GTP binding to one or more Ras proteins in embodiments, the Ras Fami ly GTPase is KRas WT or a mutant, such as KRas G12v, KRas GI3d, KRas G12a, KRas G12c, KRas Gild, KRas Q6IH, KRas Q61L, KRas Q61R, HRas WT or a mutant such as HRas GI2v. In embodiments, the GTPase which is inhibited is CDC42 WT or a mutant such as CDC42 L61, Rael WT or a mutant such as Rad L61 and the compounds modulate one or more of the identified GTPases in embodiments, the present in vention Is directed to methods of treating a disease state or conditio which is mediated through a Ras family GTPase, the method comprising administering to a patient or subject in need thereof an effective amount of at least one modulator of GTP binding to one or more RAS proteins optionally in combination with an effective amount of at least one additional bioactive agent. Disease states or conditions which may be treated pursuant to the present invention include histiocyte disorders (e.g Rosai-Dorfinan disease/sinus histiocytosis with massive lymphadenopathy), Noonan syndrome (NS), Noonan syndrome with multiple lentigmes. Leopard syndrome, eardiofacio- cutaneous syndrome, neurofibromatosis type I ( F.1) and secondary effects of same including fibromas, scoliosis, long bone dysplasmia, osteoporosis and cognitive impairment, Legius syndrome, Costello syndrome (CS), capillary malformation-arterio venous malformation syndrome (CFC syndrome), congenital myopathy with excess of muscle spindles (CMEMS), congenital heart disease, hereditary gingival fibromatosis type 1 and hypertrophic

cardiomyopathy (flCM), cancer, other sporadic or genetic diseases or conditions and

infections, including those caused by Entamoeba histolytica, among others.

In yet another embodiment the present invention is directed to a metho of identifying a compound as a potential selective agonist, antagonist, or regulator of a protein in a flow cytometer comprising: i providing in a flow cytometer a multiplex of derivatized flow cytometer beads wherein each said derivatized bea is bound to a GST fusion protein (e g. GST-GTPase fusion protein) comprising a fused protein and a fluorescent ly labeled binding partner of said fused protein bound thereto, wherein the binding partner emits fluorescent Sight upon excitation;

if exposing the flow cytometer bead multiplex from ste i to a solution

comprising at least one compound an preferably a library of unknown activity; and

hi. identifying a compound of said solution as a potential agonist, antagonist, or regulator of said fused GTPase protein within sai mult iplex if said compound displaces or impacts the binding of said fiuorescently labeled binding partner as evidenced by a reduction or increase in the fluorescent light being emitted.

In another embodiment, the present invention is directed to a method of identifying a compound as a potential selective agonist, antagonist, or regulator of a protein in a flo cytometer comprising : a. providing in a Sow cytometer derivatized flow cytometer beads contained within a multiplex wherein the deri vat feed beads are each bound to a GST fusion protein comprising GST and a fused protein (preferably, a GST-GTPase fusion protein); b. exposing the flow cytometer bead multiplex from step a to a solution comprising at least one fluorescent i.y labeled compound having the potential for binding to said fused protein; and

c identifying a compo und of said solution as a potential binding partner of said fused protein if said compound binds to said fused protein as evidenced by an increase in the fluorescent light being emitted from said fused protein.

In yet another embodiment, the present invention is directed to a method of identifying a compound or portion of a compound as a binding partner of a protein in a flo cytometer comprising; a. providing in a flow cytometer a population ofderivatized flow cytometer beads wherein each of said derivatized beads is bound to a G ST fusion protein comprising GST and a fused protein {preferably GST-GTPase fusion protein) which is fiuorescently labeled;

b. exposing the flow cytometer beads from step a to a solution comprising at least one compound having the potential for binding to said fused protein; c. identifying the compound or a region of the compound as a potential binding partner of the fused protein if the compound binds to the fused protein as evidenced by a decrease in the fluorescent light being emitted from the fused protein;

d. determining the selectivit of said compound identified in step c with respect to individual GTPases by exposing a multiplex of individual fluorescent flow cytometer beads comprising individual GTPases to a solution comprising the compound identified in step c and comparing the binding of the compound with the individual GTPases on the individual fluorescent flow cytometer beads; and e. determining the selectivity of the compound identified in step c with respect to individual KRas mutants by exposing a multiplex of fluorescent flow cytometer beads comprising individual KRas mutant GTPases to a solution comprising the compound identified in step c and comparing the binding of the compound with the individual KRas mutant GTPases on the fluorescent flow cytometer beads, wherein the selectivity of said compound with respect to KRas mutants and other GTPases is determined by comparing the activities of said compound on said multiplexes comprising both KRas mutant and Ras GTPases with a standard.

In embodiments, the method employs a standard which is used to assess the activity of the compo und In the assay compared with the standard. la embodiments, the method employs a solution comprising a library of compounds, preferably a library of organic small molecules. In embodiments, the compound is identified as a potential agonist antagonist, or regulator of the Fused protein and is subjected to a secon assay to determine the activity of said compound as an agonist, antagonist, or regulator of fused proteins within the multiplex.

In embodiments, the fused protein comprises a KRas GTPase, preferably a wild-type or a mutant KRAS (G12D, G12A, GI2V, G12C, G13D, Q61R, Q61 L, and Q6M)

In embodiments, the fused proteins in the second assay utilizes fused proteins comprising one or more GTPase such as a Rab family ("70 mammalian GTPases), Rho family GTPase, including Rac (e.g. Raci, Rac2, Rac3) and Cdc42, Ran, or Ras family

GTPases.

In embodiments, the fused protein is a GST -GTPase fused protein.

In embodiments, the binding partner is GTP

In embodiments, the GTPase is a mutant KRas GTPase.

In embodiments, the compound is a protein or polynucleotide.

In embodiments, the compound is a small molecule.

In embodiments, the method utilizes a solution which comprises a series of fluorescent Sy labeled polypeptides or polynucleotides of varying lengths and sequences obtained from a protein or polynucleotide known to be a binding partner of the fused protein.

In embodiments, the protein or polynucleotide compound which binds to said fused protein is further identifie by sequencing. in embodiments the fused protein requires the presence of another molecule in order for the binding partner to bind to the fused protein. In embodiments, the molecule required tor the binding partner to bin is fiuoreseently labeled.

In embodiments, the method is conducted in a flow cytometer which is a high throughput flow cytometer. In embodiments, the method is conducted using multiplex high throughput flow cytometry.

Brief Description of the Figures

FIGURE 1 shows multiplex dose dependent Bodipy-GTP binding curves. (A) Dose dependent bodipy-GTP binding curves of KRas proteins (Wild type, Gi2v, GiBd, GT2a, G12c, G12d, Q61L, an Q61R) in multiplex. Binding reactions were performed as described in the examples section of the present application. Data shown represent specific binding which is derived by subtracting non-specific binding determined in the presence of excess unlabeled GTP, IQ values for Bodipy Fl-GTP binding in this experiment range from 2 3 nM to 4.6 mM. (B) Dose dependent Bodipy-GTP binding curves of non~KRas proteins (HRas WT, HRAS Gi2y, CDC42 T, CDC42 L61, Racl WT and Racl L61) in multiplex K_rf of the GTP binding in this experiment range from 12.5 to 5.3 nM

FIGURE 2 shows an analysis of kinetic experiments for both A) Protein KRas G12v and B) KRas Q61R. Kinetic binding reactions were analyzed for 42 minutes. Association equilibrium reaction was initiated by the addition of Bodipy-GTP (1 nM) and followed for 15 minutes. Dissociation equilibrium reaction was then initiated by the addition of excess GTP (30 _tuM) an fo llowed for 25 minutes.

FIGURE 3, Table 1 , show's singleplex and multiplex analyses of individual, GST - KRas proteins coupled to glutathione -beads evaluated for Bodipy-GTP binding after incubation for 1 hour at 4°C Binding affinities (EC50) for the KRas proteins were derived using Prism software. Differences between the 2 protocols are within the error of the measurements. Kd values are the average of 4-6 separate experiments.

FIGURE 4, Table 2 show's library_' screening statistics for screens which were conducted in the exam les section of the present application. FIGURE 5, Table 3, shows numerous compounds identified pursuant to the present invention and their activities against various RAS protein GTPases. Hie compounds were classified as PAN Activators, Selective Activators, Pan Inhibitors, Selective Inhibitors or Mixed Modulators.

FIGURE 6 shows the normalized dose response of activators, inhibitors and mixed modulators of RAS protein GTPases by measuring the binding of Bodipy GTP to multiplex arrays of small GTPases.

FIGURE 7 shows (A) the chemical structure of Guanabenz acetate. (B) shows normalized dose response of Guanabenz acetate from 0 1 mM to 100 mM with KRas proteins (WT, G12v, G13d, G12a, GT2c, l2d, Q61H, Q6 L and Q61R) in multiplex with Bodipy- GTP at 10 MB. (C) shows normalized dose response of Guanabenz acetate from 0 1 mM to 100 mM with non-KRas proteins (HRas WT, HRas Gl 2v, CDC42 WT, CDC42 L6L Racl WT and Racl 1,61) in multiplex with Bodipy-GTP at 10 nm.

FIGURE 8 shows Tables 4a-4d and Umax and KΪ comparison for compounds according to the present invention. The Cmax row provides the maximum concentration of the compound in blood serum. For each compound the KI values were calculated tor each protein tested with that compound. Tables 4a and 4b pro vide all the KI values calculated while Tables 4c and 4d only show the values where the Ki has a lower value than the Cmax. ND stands for not determined

at the beginning of the number signifies that the calculated number was ambiguous.

FIGURE 9 shows that RAS genes encode proteins of 18 amino acids containing a highly conserved guanine nucleotide binding domain (G domain) and a hypervariable carboxyl terminal region RAS is frequently mutated in human cancer, with most point mutations occurring at positions 12, 13 and 61 in the G-domain.

FIGURE 10 shows assays tor multiplex screening, and ibllow-up measurements of compound mechanism of action on nucleotide binding or effector protein interactions are in hand and are part of the work-flow. Shown are results for GTPase inhibitor (C!Dl 067700) that acts as a competitive inhibitor of nucleotide binding and prevents adoption of active conformation in vitro and in ceils (8, I I, 13-14). Detailed Description of the Invention

T he following terms shall be used throughout the specification to describe the present invention. Where a term is not specifically defined herein, that term shall be understood to be used in a manner consistent with its use by those of ordinary skill in the art.

Where a range of values is provided, it is understood that each intervening va lue, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervenin value in that stated range is encompassed withi the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically exclude limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also inc luded in the in vention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skil l in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used In the practice or test ing of the present in vention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms ’ ^tand" and "the" include plural references unless the context clearly dictates otherwise.

Furthermore, the following temis shall have the definitions set out below.

The term“patient’’ or“subject” is used throughout the specification within context to describe an animal, generally a mammal, especially including a domesticated animal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific lor a specific animal such as a human patient, the term patient refers to that specific animal In most instances, the patient or subject of the present invention is a human patient of either or both genders. The ter “effective” is used herein, unless otherwise indicated, to describe an.

amo unt of a compound, composition or component which, when used within the cont ext of its use, produces or effects an intended resuit, whether that result relates to the prophylaxis and/or therapy of an infection and/or disease state or as otherwise described herein. The term effective subsumes all other effective amount or effective concentration terms

(including the term“therapeutically effective^' ,⁾ which are otherwise described or used in the present application.

The term“compound” is used herein to describe an specific compound or bioactive agent disclosed herein, .including any an all stereoisomers, individual optical isomers or racemic mixtures, pharmaceutically acceptable salts and prodrug forms. Within its use in context, the term compound may refer to a single compound or a mixture of compounds as otherwise described herein.

The term“modulator” as used herein refers to a compound that serves as an agonist, antagonist or regulator of a GTPase as described herein.

The ten» ''agonist", as used herein, is meant to refer to a compound or agent that mimics or upregulates (e.g , potentiate or supplements) the activity of GTPase.

The term "antagonist" as used herein is meant to refer to a compound that

downregulates (e.g , suppresses or inhibits} at least one activit of a compound, e.g , a protein. An antagonist can be a compound which inhibits or decreases the interaction between a protein and another molecule, e.g., a target peptide or enzyme substrate. An antagonist can also be a compound that downregulates expression of a gene or which reduces the amount of expressed protein present. The term ^t4bioactive agent” refers to any biologicall active compound or drug which may be formulated for use in the present invention. Exemplary bioactive agents include the compounds according to the present invention which are used to modulate GTPases and to treat cancer as well as other disease states and/or conditions which are otherwise described herein.

The terms“treat”,“treating’, and“treatment”, are used synonymously to refer to any action providing a benefit to a patient at risk tor or a icted with a disease including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease or delay in the onset of the disease, etc. Treatment, as used herein, encompasses prophylactic and therapeutic treatment, depending on the context of the treatment used. Compounds according to the present in vention can, for example, be admin istered prophylactica!ly to a mammal in advance of the occurrence of disease to reduce the like lihood of that disease. Prophylactic administration is effective to reduce or decrease the likelihood of the subsequent occurrence of disease in the mammal or decrease the severity of disease that subsequently occurs. Alternatively, compounds according to the present invention can, for example, be administered therapeutically to a mammal that is already afflicted by disease to one embodiment of therapeutic administration, administration of the present compounds is effective to eliminate the disease and produce a remission or substant ially eliminate the symptoms of a disease s tate and/or condition : in another embodiment, administration of the compounds according to the present invention is effective to decrease the severity of the disease or lengthen the lifespan of the mammal so afflicted, in the case of cancer, as well as other diseases and conditions that are Ras GTPase driven, including for example, histiocyte disorders (e.g. Rosai-Dorfh n disease/sinus histiocytosis wit massive Symphadenopaihy), Noonan syndrome (NS). Noonan syndrome with multiple lentigmes, Leopard syndrome, cardfofacio -cutaneous syndrome, neuro fibromatosis type I (NF1) and secondary effects of same including fibromas, scoliosis, long bone dysplasmia, osteoporosis and cognitive impairment, Legius syndrome, Costello syndrome (CS), capillary inalfbrmation-aiteriovenous malformation syndrome (CFC syndrome), congenital myopathy with excess of muscle spindles (CMEMS), congenital heart disease, hereditary gingival fibromatosis type 1 and hypertrophic cardiomyopathy (HCM), among others. The term“pharmaceutically acceptable⁵⁵ a used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the sever ity of the disease and necessity of the treatment .

The term“inhibit’⁵ as used herein refers to the partial or complete elimination of a potential effect such as a symptom or a secondary condition of a disease state, while inhibitors are compounds that have the ability to inhibit.

The term“prevention⁵ when used in context shall mean“reducing the likelihood ⁵ or preventing a condition or disease state from occurring as a consequence of administration or concurrent administration of one or more compounds or compositions according to the present invention, alone or in combination with another agent. It is noted that prophylaxis will rarely be 100% effective; consequently the terms prevention and reducing the likelihood are used to denote the feci that within a given population of patients of subjects,

administration with compounds according to the present invention will reduce the likelihood or inhibit a particular condition or disease state (in particular, the worsening of a disease state such as the metastasis of cancer or other accepte indicators of disease progression in the case of inflammatory and neurologic diseases) from occurring.

The term "bapeef” shall refer to a proliferation of tumor cells having the unique trait of loss of normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis. Examples of cancers from which the compounds of the present invention may be used to treat include, without limitation, carcinomas (e.g., squamous-cell carcinomas, basal cell carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas), partic ularly those of the bladder, bone, bo wel, breast, cervix, colon

(colorectal), esophagus, head, kidney, liver, lung, nasopharyngeal neck, ovary, pancreas, prostate, and stomach; hematologic cancers, including leukemias, such as acute myelogenous leukemia, acute lymphocytic leukemia, acute promye!ocytie leukemia (APL), acute T-cell lymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaxyoeytie leukemia,

mierorayeioblastic leukemia, monocytic leukemia, neutrophilic leukemia and stem cell leukemia; benign and malignant lymphomas, particularly Burkitfs lymphoma, Non- Hodgkin’s lymphoma and B-eell lymphoma; benign and malignant melanomas;

myeloproliferative diseases; sarcomas, particularly Ewing’s sarcoma, hemangtosatcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; blastemas, Including glioblastoma and medulloblastoma (brain tumors),

hepatoblastoma (liver tumor), nephroblastoma (kidney tumor), neuroblastoma (neural tumor), osteoblastoma (bone tumor) and retinoblastoma (retinal tumor In the eye), tumors of the central nervous system (e.g., gliomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ- line (germ cell) tumors (e.g., bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer (e.g., small cell lung cancer, mixed small cell and non-small cell cancer, pleural mesothelioma, including metastatic pleural mesothelioma small cell lung cancer and non-small cell lung cancer), ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixed types of neoplasias, particularly carcinosarcoma and

Hodgkin's disease; and tumors of mixed origin, such as Wilms’ tumor and teratocarcmomas, among others. It is noted that certai cancers such as leukemias, colorectal cancer, pancreatic cancer, lung cancer, lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, colorectal cancer, thyroid cancer, sali vary' duet carcinoma, epithelial -myoepithelial carcinoma, kidney cancer, astrocytoma and melanoma, have been shown are believed to be associated with RAS GTPase modulation (often KRas, HRas or NRas are upregulated or hyperexpressed in the cancer tissue and are principal target cancers for compounds and therapies according to the present invention. The term cancer includes naive cancers, recurrent cancers, drug resistant cancers and metastatic cancers, including cancer stem cells. In embodiments, the compounds according to the present invention are effective to treat recurrent cancers and/or metastatic cancers and to inhibit and/or reduce the likelihood that a cancer stem cell will grow and elaborate into a more advanced form of cancer.

In addition to the treatment of principally ectopic cancers as described above, the present invention also may be used preferably to treat eutopic cancers such as choriocarcinoma, testicular choriocarcinoma, non-seminomatons germ cell testicular cancer, placental cancer (trophoblastic tumor) and embryonal cancer, among others.

The term "neoplasia" refers to the uncontrolled and progressive multiplication of tumor cells, under conditions that would not elicit, or would cause cessation of multiplication of normal cells. Neoplasia results in a "neoplasm", which is defined herein to mean any new and abnormal growth, particularly a new growth of tissue, in which the growth of cells is uncontrolled and progressive. Thus, neoplasia subsumes "cancer", which here refers to a proliferation of tumor cells having the unique trait of loss of normal controls resulting in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis.

As used herein, neoplasms include, without limitation, morphological irregularities in cells in tissue of a subject or host, as well as pathologic proliferation of cells in tissue of a subject, as compared with norma! proliferation in the same type of tissue. Additionally;

neoplasms include benign tumors and malignant tumors (e.g., colon tumors, among numerous others as described herein) that are either invasive or nonravasive. Malignant neoplasms are distinguished from benign neoplasms in that the former show a greater degree of anaplasia, or loss of differentiation and or ientat ion of cells, and ha ve the properties of invasion and metastasis. Examples of neoplasms (many of which or more are identified above as‘cancer”) inc lude neoplasms or neoplasias from which the target cell of the present invention may be derived including without limitation, carcinomas (e.g., squamous-cell carcinomas, basal cell carinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas),

particularly those of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and on- Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases: sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, i!posarcoma, myosarcomas, peripheral

neuroepithelioma, and synovial sarcoma; tumors of the central nervous system (e g., gliomas, astrocytomas , oligodendrogliomas, ependymomas, g!io bastomas, neuroblastomas, ganglioneuromas, gangllogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovaria cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease; and t umors of mixed origin, such as Wilms^* tumor and teratocarcinomas, among others. See, Beers and Berkow (eds.), The Merck Manual of Diagnosis and Therapy, 17.sup.th ed. (Whstehouse Station, NJ.r Merck Research Laboratories, 1999} 973-74, 976, 986, 988, 991.

The term“additional anti-cancer agent’^" is used to describe an additional compound which may be coadministered with one or more compounds of the present invention in the treatment of cancer. Such agents include, for example, everolimus, trabec tedin, abraxane ILK 286, AV-299, DM- 101 , paxopa h, GSK690693, ETA 744, ON 09!0.Na, AZD 6244 (ARRY- 142886), A N-107, TK1-25S GSK461364. AZD 1 152. enzastaorin, vandeianih, ARQ-197, MK-0457, MLN8054. PHA-739358, R-763, AΪ-9263, a FLT-3 inhibitor, a VEGPR. inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-I modulator, a Bd-2 inhibitor, an I1DAC inhbitor, a c~MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an 1GFR-TK inhibitor, an anti-HGF antibody, a RΪ3 kinase inhibitors, an AKT inhibitor, a JAR/STAT inhibitor, a checkpoint- ! or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, er!otinib, dasaianlh, nilotinib, decatanib, pankumumab, atnrubicin, oregovomab, Lep-etu, oolatrexed, azd2! 7j , batabuhn, oihtntnnmab, zanolimnmab, edotecar n, tetrandrine, rnbk.eeaa, tesmilifene, oblimersen, ticilimnmab, ipilimumab, gossypol. Bio i l l , 131 -l-TM-601 , ALT- 1 10, BIO 140, CC 8490, dlengitide, gimatecan, IL13-PE38QQR, INO 1001 , IPdRt KRX- 0402, iueanthone, LY 3176.15, neoradiab, vitespan. Rta 744, Sdx 02, a!ampanel, atrasentan, Xr 31 1 , romMepsis, ADS- 100380, susMatb, 5-Suorouraeil vorinostat, etoposide, genidtabine, doxorubicin, irinotecan, liposomal doxorubicin, S'-deoxy-S-fiuorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901 , AZD-6244, eapeeitabine, L- Glutamic acid, N -[4-[2~(2-amino-4.7-dihyd.ro-4-oxo~i H - pym>lo[2,3- d ]pyri.midln-5- yi)etbyi|benaoyi]-, disodinm salt, heptahydrate, earnptotheda, PEG-laheled irinotecan, tamoxifen, toremitene citrate, anastrazole, exemesiane, teirozole, DES(dietbyIsiilbestroi) estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C1 1 , CHIR-258,}· 3-[5~ (methyisnlfonylpiperadiuemethyi)- kfoolylj-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D- Ser(Bu t } 6 ,Azgly 10 j (pyro~Gl«~His~Trp~$er-Tyr-D~Ser(Bu t )-Leu- Arg-Pro- Azgly-NH 2 acetate [C^H^N sOL -(CsHUOsJx where x ~ 1 to 2.4], goserelin acetate, leuprolide acetate, txiptorelin pamoate, medroxyprogesterone acetate,

hydroxyprogesferone caproate, megestrol acetate, raloxifene, hicalutamide, fluiamide, nilutamide, megestrol acetate, CP-724714; TAK-165, IIKΪ-272, erlorinib, lapatanib, caneriti.it>, ABX-BGF antibody, erbitux, BKB-569, PKI- 166, GW-57201 , lonafemih, SMS- 214662. tiptfarnib; amitostme, NVP-LA.Q824, siiheroyl analide hydroxamie add, valproic acid, triehostatm A, FK-228, SU 1 1248, sorafenib, RN951 , aminogkiethjmide, arnsacrine, anagrelide, L-asparagkase, Bacillus Cakiette-Guerin (BCG) vaccine, bleomycin, buserel , husidian. carhoplatm, carmnsiine, chlorambucil. eisplatk. cladriblne, elodronate, cyproterone, cytarabrae, dacarbazine, dactin myc in, daunombicra, diethylstilbestrol, epimbidn, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemdtabine, lee vac, hydroxyurea, idarubie , iibsiamide, imaiimb, leaprotide, levaraisoie, lomustine,

mechlorethamine, melp halau, 6-mercaptopurine, esua, methotrexate, mitomycin, mltotane, mitpxantrone, ni ta ide, octreotide, oxaiiplatin, pantidronaie, pentostaiin, plicamyck, porfimer, procarbazine, raititrexed, rituximab, streptozoc , teniposide, testosterone, thalidomide, fhioguanme, thiotepa, tretinoin, vkdeske, 13-ds-retkoic acid, phenylalanine mustard, uracil mustard, estramustme, altretamine, floxuridke, 5~deooxy«ridine, cytosine arabinoside, 6 niecaptopurine, deoxycoformyck, calcitriol, vairabieiu, mithramycin, vinblastine, vinoreibme, topotecan, razoxin, marimastat, COL-3, neovastat, B MS-275291 , squaiam e, endostadn, SU3416 SU6668, EMD121974, interleukin- 12, ΊM862 angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, deniieukk diiiitoxygelitinib, hostezinhb, paclitaxel, ir otecan, topotecan, doxorubicin, docetaxei, vkorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, BRA- 923, axzoxlfene, felvestrant, acolbifene, lasofexifene, idoxifene, TSB- 424, HMR- 3339, ZK 186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-0- (2-hydroxyethyl)-rapamycin, temsirolimus, AP -23573, RADO01 , ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L- 779,450, PEG-filgrasiim, darbepoetln, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor histrelk, pegylated interferon aSfa~2a, interferon alfa-2a, pegylated interferon alfa~2b, interferon alfe-2b, azacitidke, PEG-L-asparagmase, lenalidomide, gemtuzumab,

hydrocortisone, interleukin-1 1 , dexrazoxane, alemruzomab, all-transretinoic acid, ketoconazo!e, kterleukk-2, raegestrol, immune globulin, nitrogen mustard,

methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, edltronate, mltotane, cyclosporine, liposomal daunorubicin, Edwina-asparag ase, strontium 89, casopitant, netupitant, air NK-1 receptor antagonists, pakmosetroo, aprepkant, dipbenhydramine, hydroxyzine. metoclofiramide, lorazepam, alprazolam, haloperid i. droperidol, dronabinol, dexamethasone, meihylprednisotone, prochlorperazine, granisetron, ondansetron doksetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa, darbepoetin alfa, ipilivmiimab, veinnrafenib among others among others, including immunotherapy agents such as IDO inhibitors (an inhibitor of indoieamine 2,3 -dioxygenase ί 1 DO ) pathway) such as indoximo (NLG-8187), Navoxlmod (GDC-0919) and NLG802, FDLl inhibitors (an inhibitor of programmed death-ligand 1) including, for example, nivolumab, durvafnmab and aiezolmrmab PD1 inhibitors such as pembroiizumab (Merck) and CTLA-4 inhibitors (an inhibitor of cytotoxic T-lymphocyte associated protein 4/cluster of differentiation 152), including ipitimumab and tremelimumab, among others.

The terra‘GTPase” is used to describe the RAS GTPases, which is a fam ly of GTPases related to RAS family. These include the KRas GTPases, NRas GTPases and the HRas GTPases, including wild-type (WT) an re lated prevalent mutant forms of t hese GTPases such as G12A, G12D, G12V, G12C, G13D, Q61R, Q61L, and Q61 H Ras (KRas,NRas and liras) mutant forms. Together, these GTPase proteins are intimate to processes which are related to cancer and its elaboration and are targets for cancer treatment through modulation, in more particular aspects, inhibition of these GTPase targets. GTPase mediates a number of disease states, including cancer, as otherwise disclosed herein, as well as a number of sporadic and genetic diseases including, histiocyte disorders (e.g. Rosai-Dorfiman disease/sinns histiocytosis with massive lymphadenopathy), Noonan syndrome ( NS k Noonan syndrome with multiple lentigines. Leopard syndrome, cardiofacio -cutaneous syndrome, neurofibromatosis type 1 (NF1) and secondary effects of same including fibromas, scoliosis, long bone dysplasmia, osteoporosis and cognitive impairment, Legius syndrome, Costello syndrome (CS), capillary malformation-arteriovenous malformation syndrome (CPC syndrome), congenital myopathy with excess of muscle spindles (CMBMS), congenital heart disease, hereditary gingival fibromatosis type 1 and hypertrophic cardiomyopathy (HCM) among others, including infections such as Entamoeba histolytica , among others.

The term“non-RAS GTPases”, me Judes the Rho family of GTPases, which is a family of small signaling GTPases, of which Rad, Cdc42 and RhoA are the most well studied members. These G TPases have been shown to regulate many aspects of intracellular dynamics, and play a role in cell proliferation, apoptosis, gene expression, and other common cellular functions. They consequentl have utility in the treatment of sporadic and genetic diseases, as well as cancers in certain embodiments according to the present invention.

The term“co^dnilmslration’ or "'adjunct therapy” shall mean that at least two compounds or compositions are administered to the patient at the same time, such that effec tive amounts or concentrations of eac h of the two or more compounds may he fo und in the patient at a given point in time. Although compounds according to the present invention may be co-administered to a patient at the same time, the term embraces both administration of two Of more agents at the same time or at dtilei itt times, including sequential

administration. Preferably, effective concentrations of ail co-administered compounds or compositions are found i the subject at a given time. The term co-administration or adjunct therapy also contemplates other bioactive agents being coadministered with pharmaceutical compositions according to the present invention, especially where a cancer has metastasized or is at risk for metastasis.

The term“sequenc ing” refer to the process of determining the sequence of a polynucleotide or protein compound which binds to a target protein in the assays according to the present invention. Such polynucleotide or protein to any polynucleotide or protein, including, e.g., a cDNA, a cDNA fragment, a genomic DNA, a genomic DNA fragment, and a synthetic DNA, among numerous others. Moreover, certain differences in nucleotide sequences may exist between individual organisms, of the same or different species, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a polypeptide with the same biological activity.

The term“Auoreseently labeled” is used to describe a protein (e.g. a fused protein), a binding partner of a protein (e.g , a ligand of a protein such as GTP for GTPase) or a compound fluorophore label that is selected such that its emitted fluorescent energy can be detected by fiuorimetry, especially including by flow cytometry (e.g. high throughput flow cytometry). The fluorophore label may be a fluorescent protein or dye, e.g. , a fluorescent protein as described in Matz et al , Nature Biotechnology (October 1999) 17:969-973, a green fluorescent protein from Aequoria victoria or fluorescent mutant thereof e.g., as described in US. Pat. No. 6,066,476; 6,020,3 2; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5, 19,445; 5,874,304, the disclosures of which are herein incorporated by reference.

Fluorescent dyes whic may be used to fluorescently label the protein, binding partner or compound other fluorescent dyes, e.g., coumarin and its derivatives, e.g. 7-amino-4- methylcoumarm, aminocouraarin, bodipy dyes, such as Bodipy FL, cascade blue, fluorescein and its derivatives, e.g. fluorescein isothioeyanate, Oregon green, rhodamlne dyes. e.g. texas red, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye, etc., chemitnmescent dyes, e.g., luciferases, including those described in U.S. Pat. Nos. 5,843,746; 5,700,673; 5,674,713; 5,618,722; 5,418,155; 5,330,906; 5,229,285; 5,221,623; 5,182,202; the disclosures of which are herein incorporated by reference.

The term“standard” is used to describe binding measurements of kno wn

agonists/antagonists/regulaiors or other ligands with a target protein (e.g. a fused protein or receptor) in an assay such that the binding measurements of the known

agonist/antagonist/regulator in the assay may be compared with binding measurements of a compound of unknown activity in the same assay. By comparing the binding measurements of the compound of unknown activity to the target protein with the binding measurements of the known compound to the target protein, a determination may be made as to the activity of the compound of unknown activity as an agonist/antagonist/regulator or a compoun which does not bind to the target protein.

Compounds according to the present invention may be readily formulated into pharmaceutical compositions, useful m the treatment of disease states and/or conditions as otherwise described herein. These disease states and/or conditions include immune

dysfunction, pigmentation or neurological disorders which occur as a consequence of impaired GTPase function and/or functional insufficiency. Additional disease states and/or conditions which may be favorably influenced by treatment with the present compounds include cancers (e.g., leukemias, colorectal cancer, pancreatic cancer, hmg cancer, lung adenocarcinoma, muc inous adenoma, ductal carcinoma of t he pancreas, colorectal cancer , among others, often associated with KRAS, thyroid cancer, salivary duct carcinoma, epithelial-myoepithelial carcinoma, kidney cancer astrocytoma, among others, often associated with BRAS and melanoma, often associated with NR AS), histiocyte disorders (e.g. Rosai-Dorf an disease/simts histiocytosis wit massive Symphadersopathy), Noonan syndrome (NS). Noonan syndrome with multiple lontigines. Leopard syndrome, cardtofacio- cutaneoos syndrome, neurofibromatosis type 1 (NF l ) and secondary effects of same including fibromas, scoliosis, long bone dysplasmla, osteoporosis and cognitive impairment, Legiits syndrome, Costello syndrome (CS), capillary malformation-arteriovenous malformation syndrome (CFC syndrome), congenital myopathy with excess of muscle spindles (CMBMS), congenital heart disease, hereditary gingival fibromatosis type l and hypertrophic

cardiomyopathy (HCM), among others, including infections caused by Entamoeba histolytica, among others.

Pharmaceutical compositions comprise art effective amount of one or more compounds according to the present invention in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with at least one additional anticancer agent.

As noted above, the compounds and method of the in ventio modulate GTPase as otherwise described herein, and are useful for the inhibition (including prophylaxis) and/or treatment of cancer, sporadic or genetic diseases or conditions aid infections, including those caused b Entamoeba histolytica

methods according to the present invention, subjects or patients in need are treated with the present compounds, pharmaceutical compositions in order to inhibit, reduce the likelihood or treat a disease state, condition and/or infection as otherwise described herein. The disease states, conditions and infections treated by the present compounds and

compositions are readily recognized and diagnosed by those of ordinary skill in the art and treated by administering to the patient an effective amount of one Or more compounds according to the present invention.

Generally, dosages and routes of administration of the compound are determined according to the size and condition of the subject, according to standard pharmaceutical practices. Dose levels of compounds employed can vary widely, and can readily be

determined by those of skill in the art. Typical ly, amo unt s in the milligram up to gram quantities are employed, although in certain instances, amounts above or below that range may also be used. The composition may be administered to a subject by various routes, e.g. orally, transdermaily, perhieumily or parentera!!y, that is, by intravenous, subcutaneous, intraperitonea!, intrathecally or by intramuscular injection, among others, including buccal, rectal, and transdermai administration. Subjects contemplated for treatment according to the method of the in vention include hutnans, companion animals, laboratory animals, and the like.

Formulations containing the compounds according to the present invention may take the -form of solid semi-solid, lyophilked powder, or liquid dosage forms, such as, lor example, tablets capsules, powders, sustained -release formulations, solutions, suspensions, emulsions, suppositories, creams, ointments, lotions, aerosols, patches or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

Pharmaceutical compositions according to the present invention typically include a conventional harmaceutical carrier or excipient and may additionally include other med icinal agents, carriers, adjuvants, additives and the like. Preferably, the composition is about 0.1 % to about 85%, about 0.5% to about. 75% by weight of a compound or compounds of the in vention, with the remainder consisting essentially of suitable pharmaceutical excipients.

For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or

Liquid compositions can be prepared by dissolving or dispersing the compounds (about 0.5% to about 20% by weight or more), and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension. For use in oral liquid preparation, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.

When the composition is employed in the form of solid preparations for oral administration, the preparations may be tablets, granules, powders, capsules or the like, in a tablet formulation, the composition is typically formulated with additives, e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filer, a disintegrator, and other additives typically used In the manufacture of medical preparations.

An injectable composition for parenteral administration will typically contain the compound in a suitable i.v. solution, such as sterile physiological sail solution. The composition may also he formulated as a suspension in a lipi or phospholipid in a liposomal suspension, or in an aqueous emulsion.

Methods for preparing such dosage forms are known or is apparent to those skille in the art; for example, see Remington’s Pharmaceutical Sciences (17th Ed., Mack Pub. Co., 1985). The composition to be administered will contain a quantit y of the selected compound in a pharmaceutically effective amount for modulating GTPase in a subject according to the present invention in a subject.

Examples

The present inventor’s view is that the recent discovery of a therapeutic approach targeting one RAS .mutation (G1 C) establishes the premise that screening specific Ras mutant proteins will reveal mutation- and cancer type-specific vulnerabilities for mutation- selective anti-Ras therapies. Additionally, the recent identification of unique pockets and protein-protein interaction interfaces dictate unique behaviors of individual Ras proteins (HRas, KRas and NRas) further supporting the premise that Ras selective compounds will have significant utility. The present invention relates further to our unique multiplexed experimental approach that ensures the stability of Ras and Ras-related GTPase and allows comparative assessment of target sensitivity during screening with compound libraries (methods). The approach has demonstrated utility for detection of hits and development of robust leads that are active against select or multiple GTPases. Through combined testing of off-patent drugs, cheminformatics to identify the most promising scaffolds, and preelmieal and clinical testing, two enantio-selective scaffolds derived from off-patent drug libraries were shown to have clinical translational utility. Our publication and patent track records suggest that GTPases are directly draggable targets.

Through a pilot inter-institutional CTSC collaboration, we tested repurposed drugs in a multiplex screen against wild-type KRas and prevalent point mutant proteins (G! 2 A,

G12D, G 1 V, G12C, Gl 3D, Q61R, Q61L, and QbUi) The screen identified KRas and HRas protein selective compounds in primary and dose response secondary screens.

In order to identify and validate mutant KRas proteins as targets for drug repurposing, the inventors prepared 9 distinct, highly purifie oncogenic Ras proteins for display on microspheres (See methods and results).

PanActivators of GTP binding

• Salsalate, Tolfenamic acid, Dexibuprofen, Mefenamic Acid, Ihuprofen, S-D i-ibuproien, Meciofenamic acid sodium salt monohydrate, (R) -Naproxen sodium salt, Naproxen, Flitfenamic Acid, Flurbiprofen, Pheninidione, Dioxybenzone, A-? hydrochloride, tJsnic acid, lopantc acid, Meniudione, lopaftic acid.

Fan inhibitors of GTP binding

* Lstradefylilne, PR-619, N6022, Dlflfactarc acid, IPA 3, Pisetin, Folic acid, GSR 3787

Selective Inhibitors of GTP binding to RAS proteins, but not Q61 KRAS mutants • Guanabenz acetate, Chlorprothixene hydrochloride, NSC 663284.

Mixed activity modulators of GTP binding

* Ipsapirone, GF1092G3X (selective activator non-RAS)_s Beta Lapachone (RAS activator/non- RAS inhibitor), SFI670 (RAS activator, non-RAS inhibitor), Dafapladib (SB4S0848), PD 1 8306, Pimeth ene Maleate, Oxyquiftolme Hemisuliate. Materials and Methods:

Reagents: All reagents were from Sigma (St Louis, MO) unless otherwise indicated. Plastic-ware was from V WR (Radnor, PA) and Greiner Bio-One (Monroe, NC). Bead sets for multiplex assays were provided by Duke Scientific (Fremont, CA) following protocols developed by the NMMLSC⁵⁴'⁵⁶. Guanosine 5'-Triphosphate, BODIPY™ FL 2’-(or-3’)-0-(N- (2-Aminoethyl) Urethane), Trisodium Salt (BODIPY FL GTP) and anhydrous DMSG were from ThermoFisher Scientific (Waltham, MA). GST-fosion proteins were either from

Cytoskeleton, Inc. (Denver, CO) or were purified from E, co!i as described below. Ail solutions were prepared with ultra-pure 18 MW water or anhydrous DMSO. Flow cytometric calibration beads were from Bangs Laboratories Inc., (Fishers. IN) and Spheroiech, Inc.,

(Lake Forest IL). Off patent commercial libraries were purchased from Prestwick Chemical (Olkirch-Graffeiistadeit France), SelleckCbem (Houston, TX), Spectrum Chemical {New Brunswick, NJ), and Tocris Bio-Science (Bristol, UK). We also purchased a collection of on patent drags from MedChem Express (Monmouth Junction, NJ) that was specifically assembled byU M collaborators. All purchased libraries were provided as 10 mM stock solutions in 96-well matrix plates except the MedChe Express library which was provided as individual powders that were subsequently solubilized in DMSO. All libraries were reformatte using a Biomek FX^P laboratory automated workstation into 384-well plates for storage (Greiner #784201; Labcyte #PP-0200). Low volume dispensing plates (Labcyte #LR- 0200) were assembled using an Agilent BtoCeil wor station (Santa Clara. CA).

Expression and purification of GST-fusion proteins: Human KRAS4B cDNA sequences encoding the G domains of wild-type and mutant KRAS (G12D, G1 A, 012 V,

G 12C, G13D. Q61 R. Q61 L, and Q61H) fused with glutathione S-transferase were generously provided by the National Cancer Institute through the Ras initiative. The constructs have a Tev protease cleavage site that leaves an extra t lis on the KRas amino terminus after cleavage. The presence of this additional glycine does not alter activity, structure, or other properties measured b NCI Through an VITA, NCI offers a complete suite of KRAS, HRAS and NRAS clones bearing mutations that can be assessed as needed⁵'. Vector options include a T7 promoter based GST- fusion or a T7 promoter based HSis6~GST-fusion that allows testing of purified proteins with both bead types.

Proteins (residues 1 -169) were subcloned into a pET21 vector that adds an N-terminal 6-histidine tag and a TEV protease clea vage site for expression of recombinant protein in Escherichia coll BL21 (DE3) cells (Novagen). The mutations were subsequent ly verified by DMA sequencing. E. coil BL21 (DE3) cells were grown at 37' Lttr -Bertani (LB) medium supplemente with ampieiilin and chloramphenicol until A6O0 of ~0.5. The temperature was then lowered t

and GST-KRAS expression was induced with 0.5 iriM isopropyl-P-D- l-thiogalaciopyranoside (IPTG) after 30 min. The cells were grown for an additional 15 at 18°C. The cells were then harvested and pelleted at 4000 rpm, resuspended in a lysis buffer (20 inM HEPES, 500 M NaCl, 1 raM MgC12, 20 M Imidazole, 5% glycerol (pH 7 75), and protease inhibitor phenylmethanesulfony! fluoride (ACROS

Organics), and sonicated. The cell lysate was centrifuged at 15,000 rpm, and the supernatant was isolated. KRAS proteins were purified using glutathione-agarose affinity

chromatography (Qiagen), and were collected by application glutathione to the column. If needed, KRAS proteins were further purified by size exclusion chromatography using a Sephadex G-75 column. Protein purity of >95% was obtained and verified by SDS-PAGE analysis. GST-fusion proteins were stored at 80°C in 20 mM HEPES, pH 7.4; 50mM NaCL; 5mM MgC12; 1.0% glycerol; 10-lOOuM GDP. After their initial use, protein preparations were snap- frozen with liquid nitrogen (LN2). Subsequently, single-use aliquots were stored at -30°C in buffer containing 40% glycerol and 20 uM GDP.

Bead Coupling: 4 pm diameter, glutathione-beads (GSH-beads), distinguishe by ten different intensities of red fluorescence (varying by several orders of magnitude of emission at 665±I0 nm with excitation at 635 nm) were obtained by special order from Duke Scientific Corp. Each polystyrene bead set is supplied at 1.4 * 10^s beads/ml with approximately 1.2 ^c 10* glutathione sites per bead (determined by using GST- green fluorescent protein⁴² ) Prior to incubation with individual GST-GTPase fusion proteins, an appropriate volume of each bead slurr was incubated for 30 minutes at room temperature in assay buffer (20 mM

HEPES pH 7.5, 125 mM (NH^SOa, 1 raM MgCB, 0.5 raM EDTA pH 8.0, 0.01% NP-40) supplemented with 1 mM dithio thre it o I and 0.1% BSA. Following this initial incubation, passivated beads were collected by centrifugation and resuspended in assay buffer with individual GST-GTPase fission proteins at a final concentration of 1 mM. Overnight coupling reac tions were c arried out at 4°C with rotation. The amount of eac h bead type used for coupling and the final volume of the coupling reaction was determined by the experimental protocol. Typically, bead sets are used at a final concentration of 200 beads/mE. To remove unbound GST-GTPase, bead sets were washed twice by centrifugation with ice-cold assay buffer. Individually coated beads were pooled together and kept cold in an appropriate volume before use in binding assays. Bead-bound proteins are sufficiently stable to study secondary interactions between GTPase-GST chimera’s and Bodipy-FL GTP⁵⁴ Mote that an extra GSH-bead set is included that is not protein coupled. This set serves as a‘scavenger’ for proteins that might dissociate during the binding assay.

Dose Dependent Bodipy FL-GTP Binding Assays: For dose dependence binding- assays, GTPase-coupied bead sets were incubated on a rotator for 1 hoar at 4°C with varying concentrations of Bodipy FL-GTP (0.3 - 300 nM). Total assay vo lume was 10 pL when performed in microtiter plates and 50 mT when performed in tubes. At these concentrations of beads, the concentration of each GTPase in a given reaction is approximately 300 pM. Nonspecific binding of Bodipy-FL GTP was assessed by incubating coupled beads with excess GTP prior to the addition of the Bodipy analogue. Dose dependent binding assays were performed in both single-and multiplex format. When assays -were performed in plates, the plate assembly was completed as described below.

Assay Plate Assembly: Plate assays were performed in 384-well microtiter plates (Greiner Bio-one, #784101); plates were assembled using a Bio^'Tek Multiple Microplate Dispenser For dose-dependent Bodipy FL-GTP binding assays, total and non-specific binding were assessed by pre- incubation (30 min./4°€) in the absence or presence of 30 mM GTP, respectively. This was followed by the addition of varying concentrations of Bodipy FL-GTP Plates were protected from light and incubated on a rotator for 60 min at 4°C

For assay screening plates, compound libraries were first dispensed into columns 3 22 using a Labcyte 555 Echo Acoustic Dispenser (San Jose, CA) for a final concentration of 10 mM An equal volume (10 nL) ofDMSO was added to the vehicle control wells (column 2). Following the addition of library compounds, 2 mΐ of assay buffer was added an the plates were mixed before add ition of 5 L of the protein-coupled bead mixtures; 10 pL of assay buffer was added to empty wells in columns 1 and 24. Empty wells serve as wash wells between compound wells and control wells. Plates were mixed and incubated on a rotator for 30 minutes at 4°C before the addition of 3 tiL of Bodipy FL-GTP resulting in a final

concentration 10 nM Bodipy FL-GTP Plates were mixed and incubated for 1 hour at 4°C. Negative controls, containing bead mixtures, Bodipy FL-GTP, and 30 mM unlabeled GTP were assayed separately. Dose response plates were assembled similarly. In this instance, test compounds were added to dose response plates using a dilution protocol of the acoustic dispenser that resulted in a final concentration range of 100 - 0.01.5 mM Data Acquisition: Assay plates were sampled using the HyperCyf^, high throughput flow cytometry platform (Intellicyt; Albuquerque, NM). During sampling the probe moves from well to well and samples 1 - 2 pL from each well with 0.4 sec transit time in the air before sampling the next well The resulting sample stream consisting of 384 bubble- separated samples is delivered to an Aceuri€6 flow cytometer ( BD Blosciences: San Jose, CA). Bodtpy FL-GTP fluorescence is excited at 488 ran and detected with a 533/30 bandpass filter. Plate data are acquired as time-resolved files that are parsed by software-based well identification algorithms, segregating individual well data that Is merged with compound library files to determine compound activity in each well. Gating based on red fluorescence emission distinguishes the separately coated beads Plate performance was validated using the Z-prime calculation⁵⁸.

Compounds that satisfied t fee hit selection criteria in the primary screen (change to % binding of 50% from baseline) were cherry-picked from compound storage plates and tested to confirm activity and determine potency. Dose response data points were fitted by Prism® software (GtaphPad Software Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose-response model with variable slope, also known as the 4-parameter logistic equation. Curve fit statistics were used to determine the concentration of test compound that resulted in 50% of the maximal effect (BC50), the confidence interval of the EC50 estimate, the Hill slope, and the curve fit correlation coefficient

Equilibrium Kinetics Assays GTPase-co pled beads were prepared as described above and were kept on ice until used. Reactions were performed at room temperature in amber miero-centriluge tubes in an appropriate volume of assay buffer. Bead mixtures were initially incubated on a rotator with Guanabenz acetate (100 mM) or DMSO for 30 mm at 4°C prior to the addition of 1 nM Bodipy FL-GTP. Real-time binding kinetics was recorded using an Accuri C6 flow cytometer. Binding association was followed for 15 minutes at which time excess GTP was added Disassociation kinetics was followed for an additional 25 minutes. Data was analyzed using GraphPa Prism soft ware. The associatio time course was fitted to a two phase exponential association and the dissociation time course was fitted to a two phase decay (exponential). Results

The success of previous studies set the stage for identifying new chemical entities as well as repurposed drugs that inhibit oncogenic KRas proteins to support the notion of Ras GTPases as draggable targets. In order to identify and validate mutant KRas proteins as targets for drug repurposing, we have: 1) prepared 9 distinct, highly purified oncogenic Ras proteins for display on mierospheres for multiplex analysis; 2) undertaken semi-quantitative studies for optimizing bead-based display and small molecule screening; 3 } identified approved drags that regulate nucleotide binding to these proteins using multiplex screening and dose- response follow-up technologies; 4) performed proof-of-principle mechanism of action studies as well as initial validation of cellular activities. Multiplexing used UNM’s patented GSH bead-GST fusion protei technology³⁹. The NCI RAS initiative provided subcloned Entry clones into GST E. coti vectors.

Construction and performance of multiplex display of GTPases:

a) Optimization of buffer conditions.

The assay for GTP binding uses a GTP analogue tagged with Bodipy. Previous studies with these conjugates have determined that their fluorescence yield is significantly enhanced when bound within the GTP pocket^60,61. Our initial experiments with GST-KRas proteins foiled to detect significant binding of Bodipy FL-GTP to KRas WT, G12d, and G13d whereas binding to the G12 V mutant was marginally detectable in a magnesium- free buffer used previously in a high throughput campaign to screen non- KRas proteins⁴². We have noted similar changes in detectable binding of Bodipy FL-GTP by other Rho family GTPases when Mg^* was replaced by EDTA . This prompted a review of 5 buffer conditions based on team experience and literature resulting in the use of a buffer that contained both EDTA and

Mg³ . Similar observations regarding Mg and EDTA have been repotted by

Korlacli et aL^!yt b) Stability; The initial set of proteins (KRas WT, Gl2v„ and G13d) was tested under different storage conditions (-80°C vs a glyeerol/GDP storage at -30°C) with the 30% glycerol/20 uM GDP storage condition chosen to minimize loss of function din ing storage and freeze-thaw cycles. Stability ofHRas is significantly improved when stored in the presence of GDP’². It. should be noted, however, that useful lifetimes for these GST chimeric proteins was still limited to several weeks after the initial thaw of stock material . The inventors were not able to establish satisfactory conditions tor storage of the G13D mutant. This ma be due to the particular attributes of this fast eye ling’ KRas mutant⁶". Once these conditions were chosen, we evaluated dose-dependent binding of Bodipy FL-GTP using established GTP binding procedures.³⁵

Over the course of the screening the library plates the KRas proteins remained relatively stable. The KRas proteins were thawed and aliquoted and stored in the -30°€ freezer with glycerol and GDP. After the initial thaw of the proteins, at the beginning of the library screens, there was a 20-30% decrease in the acti vity of the proteins. For the next 5 weeks the proteins remaine consistent at this activity while running the rest of the compound libraries. After this point the KRas proteins decrease down to around 40 % when running the Cherry Pick screens.

Initial tests were earned out in 50 uL volumes before transitioning to a plate-based, multiplex format where the remaining 8 proteins could be successfully tested. Previous reports determined that the GTPase-coupIed glutathione-heads used here express

approximately 1.2 x 10⁶ GST-GTPase molecules/bead⁴² and the amount of beads used represents an approximate concentration of 0.3 nM/GTPase. Examples of dose dependent Bodipy FL-GTP binding in multiplex to the various immobilize GTPase chimeric proteins is shown in FIGURES 1 A and IB. Briefly, 8 (FIGURE 1 A) or 6 (FIGURE 1 B) sets of beads, individually coated with GST-GTPase chimeric proteins, were incubated with increasing concentrations of Bodipy FL-GTP for 1 hour at 4°C Non- specific binding was deter mined by pre~ incubating bead mixtures with 30 pM unlabeled GTP. The nucleotide binding pocket of these GST-GTPase chimeric proteins is likely occupie by GDP, so it is reasonable to assume that the observed binding of Bodipy FL-GTP to the bead-bound GTPases involves an exchange reaction.³⁴'⁶⁵ As expected increasing concentrations of fluorescent ligand leads to increased bead fluorescence with maximal signals occurring between 100 - 300 nM Bodipy FL-GTP Data analysis of the separate KRas proteins yielde binding affinities of 1 - 50 nM which is within the range published previously for these GTP analogues when testing other GTPase family members.³⁴'⁶⁵ . Cumulative affinity calculations from 3— 6 binding experiments performed in both single and multiplex formats are given in FIGURE 3, Table 1. These data demonstrate stable nucleotide binding over a 1 ··· 2 hour time period which would allow for tbe use of bigb throughput screening capabilities of commercial sets of small molecules representing 5000 unique compounds. c. Screens. Five commercial libraries comprising greater than 5000 unique

compounds that include FDA-approved drugs, natural products, and bioactive small molecules (7073 compounds in total) were screened against 8 KRas proteins. Each well of an assay plate con tained 10 sets of beads with variable red fluorescent intensities. Nine protein- couple bead sets, carrying the different GST-KRas proteins were combined with an

uncoupled GSM-bead that serves as a scavenger for GST-proteins that might dissociate during the assay. Bead mixtures were dispensed into individual wells of a 384-well assay plate and incubated with fluorescent GXP in the presence of library compounds. The final concentration of reagents in each assay well was 10 mM compound, 10 nM Bodipy FL-GTP, and 0.1 % DMSO. Each bead set was added at 200 heads/pL. Screening statistics and performance are given in FIGURE 4, Table 2. Each plate was analyzed both in forward

(starting with Al) and reverse direction to account for fluorescent compounds that can carryover during sampling and effect subsequent sample values. The initial hit selection criteria for the primary screen of small molecules was as follows; for any given protein- coupled bead, a hit was defined as a compound well that resulted in a 50% deviation in the Bodipy FL-GTP binding signal compared to the average signal calculated from the DMSO control wells that was also greater than 3 standard deviations from the DMSO controls included on every compound plate. Excess GTP containing wells were evaluated separately and were used, along with the DMSO control wells, to calculate a Z’ value for each bead set V values serve as an indicator of assay plate reliability.⁵⁸ Over the course of the screen the average Z’⁴ⁱ value for each bead set ranged from 0 769 - 0.880, indicative of a robust assay. Using these selection criteria more than 300 small molecules were chosen for further

evaluation. A secondary single point evaluation was performed on these identified primary hits. Identified active compounds were tested at three concentrations (2, I Q, and 20 mM). This secondary analysis led to the identificatio of 61 compounds that were further tested In dose dependen assays.

The KRas multiplex performed in multi-point dose-response confirmed ~50 KRas modulators (50/5000 -1%). The top 64 compounds were examined for concentration

dependent effects on GTP binding using a range chose for complete dose-response from the cherry pick. The dose response was run as an 8 point multiplex with a well with no

compound added for each compound as a control. For visualization of the bindin g, MFI signals were scaled to 100% binding in the absence of compound. d. Selectivity. To assess selectivity of the compounds with respect to GTPase families we compared the dose-response to the multiplex described previously. Activators showed an increase in the MFI for proteins while inhibitors showed a decrease in the MFI for the proteins. For analysis all data was normalized to 100% using the vehicle control (DMSO) wells. Compounds were identified as Activators, Pan Inhibitors, Selective Inhibitors, and Mixed Activity Modulators as shown in FIGURE 5, Table 3 and FIGURE 6.

Pan activators increased the binding of BODIPY -FL GTP to essentially all of the G TPases tested. They include NSAIDS, as previously reported⁴⁷. In approximate rank order, these include: tolfenamic acid, salsalate dexibuprofen, mefenamic acid, Ibuprofen, $-(+)- ibuprofen, meclofenamic acid sodium salt monohydrate, fufenamic acid, (R)-naproxen sodium salt, naproxen and tlurihuprofen. It is worth noting the variation in the binding increase. Because a single concentrat io of the BODIPY -FL GTP was used, and the EC50 varies among proteins (Table 1), the increased binding is larger when the concentration of BODIPY-FL GTP is lower (Le., lower fractional occupancy). It is worth noting that (R)~ naproxen sodium salt showed greater activity than did the naproxen sample. This may be due to naproxen having both R and S enantiomers while the (R)-naproxen sodium salt only has the R enantiomer. Similarly, S -ibuprofen was lower affinity than the mixture.

The inventors had previously identified“canonical” activator probes’³'^4® with two aromatic rings, one carboxylate, and a bridging chain, analogous to fenainates (flufeiiamie acid, raelcfenamics acid, mefanamk acid, and tolfenamic add), as distinct from the propionic acid NSAIDS (naproxen, ibuprofen, and dexiprolen but not flurbiprofen) A recent report identified bis-pheoo!s as activators with -1000 fold less potency than the most active of those we have described ^6’’ It is interesting that the fenamate PD 198306 appears to show mixed activity with mostly activation, but some inhibition as well. The NS AID ketorolac and

NSAID-hke su! dac sulfide have been reported as inhibitors/^7,64 In addition, the NSAID- !ike iopanic acid (ra iocontrast agent) and the aromatic phenidone and dioxybenzone, with acidic PKa, were also activators. In contrast, the orthoqumones 8-lapachone and SF1760 with acid PKa exhibited mixed activity (see Mixed modulators below).

Pan inhibitors that decrease the binding of BODIPY -FL GXP to GTPases include; Istr&defyiline, PR-619, Diffiactaic Acid, ΪRA 3, Fisetin, Folic acid, GSK37S7 (HRas), N6022, and NSC 663284. The comparison of 1C, and C_m;¾ suggests that istradefylline and the polypheno!ic coloring agent fisetin (3 could have physiological activity. The structural relationships among these molecules, our pan inhibitor ML2S2, an those previously described⁵ are worthy of further study. The physiological relationshi between the extent of inhibition, the mechanism, and celt physiology also remain to be studied.

Selective Inhibitors decrease the binding ofBODlPY-FL GTP to a subset of the proteins. The most active include guanabenz acetate, an antihypertensive o2 adrenergic agonist, an ehiorprothixene hydrochloride, an antiemetic as compared to NSC663284 and trifluoperazine. Guanabenz acetate and ehiorprothixene hydrochloride appeal- to he selective for KRas WT, the K.R.as G12 mutants, and the HRas proteins. Based on C_mm and Kj, both drugs have the potential for in vivo physiological activity^65'06. Our earlier screening

previously identified a CDC42 selective inhibitor characterized in some detail, and a RHO family selective inhibitor.⁴²⁴⁵ Guanabenz, based on its upstream activity against EIf4 as a regulator ofRacl has bee of interest i a recently closed bone resorption/metasiasis trial The tricyclic antidepressant ehiorprothixene is related to a series of molecules identified by Burns et al as selective for Ras/SOS and similar to spiclomazine.

Moreover, for the first time to our knowledge, our screens Identified number of modulators with mixed activity, including the orihoquinolones SF1670 (activates Q61 KRas mutants) and Beta-lapachone mentioned above, the fenamate-hke FD 198306, Ipsapirone (selective 5-HTla agonist), GF109203X, darap!adib, pimethixene maleate, and oxyquionline hemis lfate. It is noteable that as a class the trifluorperazine/tricyc lie antidepressants exhibit weak inhibition (trifluoperazine), selective inhibition (chlorproxithene), and mixe activity (pimetiiixine maleate). A potential role ofmaieate as a divalent chelator has not been further investigated with respect to GTPase.

Overall the following observations are worthy of further consideration: I ) a structural progression from pan to selective inhibitors within small molecule ehemotypes; 2) the potential role of the aromatic acids as modulators of divalent cation sites with respect to nucleotide binding; 3) inhibition of nucleotide binding by sulindac sulfide and ketorolac ; 4} the potential for a single drug to exhibi mixed activity for GTPases through allosteric divalent cation site modulation. Mechanism of Action.

To determine mechani sm of action, we evaluated bind ing and dissociation o f Bodipy F^'L-GTP in real-time kinet ic experiments in the presence of Guana benz acetate or GTP. Tests included pre-binding of Bodipy-FL GTP at 4°C or RT in the presence ofMg^{^}T In general the compound was not able to displace Bodipy FL-GTP whereas it could be displaced by GTP. Since the KRas mutants were not stable in the presence of Mg, the inventors also tested stable GTPases in the absence of Mg, in which case GB also did not induce dissociation. i t was t hen elected to perform association rate ana lysis, using t he order of addition of reagents used in screening, where guanahenz was added first, incubated at 4°C, then Bodipy FL-GTP was added at room temperature. Dramatic differences in association were noted between the GTPases identified previously as selective for guanabenz action (KRas GI2v and HRas G12V vs Kras Q61R, Rad L61 and CD C42) (FIGURE 7).

Discussion

Three MAS genes (ERAS, KRAS and NRAS) comprise the most frequently mutated oncogene family in cancer, with single point mutations (99%) predominately localized to codons 12, 13 and 61 (FIGURE 9). A common feature of these point mutations is that they render Ras insensitive to down regulation by GTPase activating proteins (GAPs) that catalyze hydrolysis of GTP, resulting in constitutive signaling As such, individual mutants have historically been considered oncogenic equivalents. However, recent observations suggest that codon-spec i tic missense mutations result in mutant Ras proteins with different biochemical and tutnorigenic properties that exhibit varying abilities to engage signaling effectors. ⁶² Differences have also been observed in response and resistance to specific anti- cancer therapies. Delineating these differences has important clinical and biological implications. In particular, KRAS mutations are most prevalent in pancreas (G12D) followed by colon and lung (G12C).³'’

To identify molecules active on and selective for KRas, the inventors took advantage of a multiplex HTS platform ( FIGURE 10) that was previously described for Rho, Rab, an liras families, but not KRas . These studies led to the identification of pan activators, pan inhibitors, selective inhibitors, and repurposed drags. Assay performance was robust for all of the GTPases based on the Zf screening reliability statistic ^'4. Approximatel 1000 compounds including FD A approved drugs were selected and tested in secondary dose-response assays (Fig 6B leading to the identification of several novel GTPase inhibitors, one evidencing utility in human cancer treatment based o Racl and Cdc42 inhibitory activity^{5442 45 4}' ⁶1 One competitive guanine nucleotide binding inhibitor (001067700} showed inhibitory activity against H-Ras and H-RasG12V but also functioned as a broad spectrum inhibitor of the Rab and Rho subfamilies (FIGURE

A selective inhibitor of the Rho-famMy protein Cdc42 (cell division control protein 42), that acts as a noncompetitive allosteric inhibitor^'45 and a Rho family selective inhibitor were also identified⁴ . The combination of high throughput screening leads and cheminloroiatic analyses predicted the FDA-approved drug Toradol™ ([R, S] ketorolac) as a Racl. and/or Cdc42 inhibitor^{'w 47}'ⁱ"'^€,8.Taken together, we have identified new chemical entities selective for class, family, and individual GTPases.

Cell-based assay assays. Inhibitors identified in these earlier studies were evaluated in cells to determine whether GTPase activities were impaired, as quantified using a flow based effector binding G-TRAP assay (FIGURE

For example, die assay was able to distinguish individual ketorolac isomers and reveale that the IC50 values for R -ketorolac inhibitory activity against the GTPases (0.5-1. mM) are 2-3 orders of magnitude less than S- ketorolac. The reverse is true for the enantiomer-selective inhibitory activities against cyclooxygenase (COX) enzymes (not shown).

Translation of re urposed drug. The inventors have translated repurposed drugs targeting Rho GTPases as a novel intervention for ovarian cancer*’^9'71 with detailed

biochemical cellular, and human data demonstrating that the R-enantiomer of an FDA approved NSAID, [R,S] -ketorolac, possesses a previously unrecognized pharmacologic property as a selective inhibitor of the Ras -related, Racl and Cdc42 GTPase with anti-tumor activity . in summary, the present invention developed a innovative toolset that includes multiplexing with color-coded microspheres for: a) simultaneous high throughput screening of multiple GTPases to identify regulators of nucleotide bmding^{> 42 53,55>5} ; b) quantitative analyses of cellular GTPase activities using small volume samples^’*5' ⁴; and c) small molecule mechanism of action studies through real-time kinetic measurements of ligand or effector ^_>

binding ^*. The inventors have guided production of multiplexed beads from commercial vendors i sourced mutant KRas constructs and cell- lines horn NCI Frederick/Leidos. This screening and multi-tiered analysis platform previously identified allosteric, selective inhibitors of Rbo-family GTPases with clinical applicabiiity^{5 >} as well as bioactives and repurposed drugs for KRas. This toolkit can be deployed to uncover novel KRas selective compounds, an area that remain relatively undere plored, which should help define the principles of KRas druggability and identify leads for therapeutic development.

One of the active molecules, guanabenz acetate has comparable activity to a recently described KRas inhibitor with low mM affinity⁵⁹, but does not appear to limit selectivit to Gi 2D. To our knowledge, this is the first report of an approved drug selective for lias family GTPases.

All references cited herein are incorporated by reference herein.

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Claims

Claims i

I. A method of identifying a compound as a potential selective agonist, antagonist, or regulator of a protein in a flo w cytometer comprising a. providing i a flow cytometer a multiplex comprising derivatized flow cytometer beads wherein each said erivati/ed bead is bound to a GST fusion protein comprising a fused protein and a fl core seemly labeled binding partner of said fused protein bound thereto, wherein said binding partner emits fluorescent light upon excitation;

b. exposing said flow cytometer bead multiple from step a to a solution comprising at least one compound of unknown activity; and

c. identifying said at least one compound of said solution as a potential agonist, antagonist, or regulator of said fused protein within said multiplex if said compound displaces or impacts the binding of said fluorescently labele binding partner as evidenced by a reduction or increase in the fluorescent light being emitted.

2. The method according to claim 1 wherein said solution comprises a library of compounds.

3 The method according to claim 2 wherein said compounds are organic small molecules.

4. The method according to claim 1 wherein said compound identified as a potential agonist, antagonist, or regulator of said fused protein in said assay (first assay) is subjected to a second assay comprising derivatized flow cytometer beads comprising fused proteins to determine the activity of said compound as an agonist, antagonist, or regulator of fused proteins, wherein said second assay comprises a multiplex of derivatized flow cytometer beads wherein each of said derivatized beads is bound to a GST fission protein comprising at least one fused protein an a fluorescent Sy labeled binding partner of said fused protein bound thereto, wherein said derivatized beads of said second assay comprise fused proteins other than the fused proteins in said first assay wherein sai flow cytometer bead multiplex in said second assay is exposed to a solution comprising at least one compound identified as an agonist, antagonist, or regulator of fused protein in said first assa and identifying said compound Rom said first assay as a potential agonist, antagonist, or regulator of said fused protein within said multiplex of said second assay if said compound displaces or impacts the binding of said fiuorescenti labeled binding partner in said second assay as evidenced by a reduction or increase in the fluorescent light being emitted compared to a standard.

5. The method according to any of claims I -4 wherein said fused protein is a GTPase.

6. The method according to any of claims l~4 wherein said fused protein is a GST- GTPase.

7. The method according to claim 5 wherein said GTPase is a Rah, Rae, Rho, Cdc42,

Ran, or Ras GTPase.

8. The method of any of claims 5-7 wherein said binding partner is GXP

9. The method according to any of c laims 5-8 wherein said GTPase is a mutant KRas GTPase.

10. A method of identifying a compound or portion of a compound as a binding partner of a protein in a flow cytometer comprising:

d providing in a flow' cytometer derivatized flow cytometer beads contained within a multiplex wherein said derivatized beads are each bound to a GST fusion protein comprising GS T and a fused protein;

e. exposing said flow cytometer bead multiplex from ste a to a solution comprising at least one fiuorescentiy labeled compound having the potential for binding to said fused protein; and

£ identifying a compoun of said solution as a potential binding partner of said fused protein if said compound binds to said fused protein as evidenced by an increase in the fluorescent light being emitted from said fused protein.

1 1. The method according to claim 10 wherein said compound is a protein or polynucleotide.

12. The method according to claim 10 or 11 wherein said solution comprises a series of fiuorescently labeled polypeptides or polynucleotides of varying lengths and sequences obtained fr om a protein or polynucleotide known to be a binding partner of said fused protein.

13 Tiie method according to claim 1 1 or 12 wherein said compound which hinds to said fused protein is further identified by sequencing.

1.4. The method according to any of claims 1-13 wherein said fused protein requires the presence of another molecule in order tor said binding partner to bind to said fused protein,

15. A method of identifying a compound or portion of a compound as a binding partner of a prot ein in a flo w cytometer comprising

f providing in a flow cytometer a population of derivatized flow cytometer beads wherein each of said derivatized beads is bound to a GST fus ion protein comprising GST and a fused protein which is fiuorescently labeled; g, exposing said flow cytometer beads t orn step a to a solution comprising at least one compound having the potential for binding to said fused protein; h. identifying said compound or a region of said compound as a potential binding partner of said fused protein if said compound binds to said fused protein as evidenced by a decrease in the fluorescent light being emitted from said fused protein;

i. determining the selectivity of said compound identi fied in step c with respect to individual GTPases by exposing a mult iplex of individual fluorescent flow cytometer beads comprising individual GTPases to a solution comprising said compound identified in step c and comparing the binding of said compound with said individual GTPases o said individual fluorescent flo cytometer beads; and j determining the selectivit y of said compound identified in step c with respect to individual KRas mutants by exposing a multiplex of fluorescent flow cytometer beads comprising individual KRas mutant GTPases to a solution comprising said compound identified in step c and comparing the binding of said compound with said individual KRas mutant GTPases on said fluorescent flow cytometer beads, wherein the selectivity of said compound with respect to KRas mutants and other GTPases is determined by comparing the activities of said compound on said multiplexes comprising both KRas mutant and Ras GTPases with a standard.

16. Tire method according to claim 15 wherein said solutio comprises a series of polypeptides or polynucleotides of varying lengths and sequences obtained from a protein or polynucleotide known to be a binding partner of said fused protein.

17. The method according to claim 16 wherein said compound which hinds to said fused protein is further identified by sequencing.

18. The method according to any of claims 1 -1? wherein said flow cytometer is a high throughput flow cytometer.

19. The method according to any of claims 10-18 wherein said fused protein is a GTPase.

20. The method according to claim 19 wherein said GTPase is a Rah. Rho, Ran or Ras GTPase.

21. The method according to claim 19 or 20 wherein said GTPase is a Rac or Cdc42 GTPase.

22. The metho of any of claims 15-21 wherein said binding partner of GTPase is GXP.

23. The method according to any of claims 15-20 wherein said GTPase is a mutant KRas GTPase.

24. A method according to any of claims 1-23 wherein said flow cytometer is a high throughput flow cytometer.

25. A pharmaceutical composition comprising an effective amount of a GTPase modulator ia combination with a pharmaceutically acceptable carrier, additive or excipient

26. The composition according to claim 25 wherein said modulator is an inhibitor of GTPase.

27. The composition according to claim 25 wherein said modulator is an agonist of

GTPase.

28. The composition according to claim 25 wherein said modulator is a regulator of GTPase.

29. The composition according to eitherof claims 25 or 26 wherein said modulator is an inhibitor of Ras GTPase.

30. The compositio according to either of claims 25 or 27 wherein said modulator is an agonist of Ras G Pase.

31. T he composition according to claim 25 or 26 wherein said modulator is a pan inhibitor of GTPase

32. The composition according to claim 25 or 27 wherein said modulator is a pan agonist of GTPase.

33. The composition according to clai 25 or 26 wherein said modulator Is a selective inhibitor of Ras GTPase.

34. The composition according to claim 25 or 27 wherein said modulator is a selective agonist of Ras GTPase

35. The composition according to claim 25 wherein said modulator is a mixed activity modulator

36. The composition according to claim 25 wherein said modulator is a modulator with different activities within family of GTPases.

37 The composition according to claim 36 wherein said family of G f^'Pases is the Mas GTPases.

38 The composition according to claim 25 wherein said GTPase modulator is selected from the group consisting of Sa!sa!ate, Tolfenamic acid, Dexibuprofeo, Mefenamic Acid, Ibuprofen, S-(+)~ibuprofen, Meclofenamic acid sodium salt monohydrate, (R)_~

NapiOxen sodium sali, Naproxen, Flufenamic Acid, Flurbiprofen, Pheninidione,

Dioxybenzone, A-7 hydrochloride, Usnic acid, lopanic acid, Menindione, lopanic acid, Istradefyllhie, PR-619, N6022, Diffractaic acid, 1PA 3, Fisetin, Folic acid, GSK 3787,

Guanabenz acetate, Cblorprothixene hydrochloride, NSC 663284, Ipsapirons, GF109203X, Beta Lapaclione, SF1 70, Darapladib (SB480848), PD 198306, Pmiethixene Maleate,

Oxyquinoline Heraisulfate, a pharmaceutically acceptable salt or alternative salt thereof, a stereoisomer thereof or a mixtures thereof.

39 The composition according to an of claims 25-38 further comprising an additional bioactive agent.

40. A modulator of GTPase which is selected from the group consisting of Salsahde, Tolfenamic acid, Dexibuprofeo, Mefenamic Acid, Ibuprofen, S-(+)-ftmprofen, Meclofenamic acid sodium salt monohydrate, (R) -Naproxen sodium salt, Naproxen, Flufenamic Acid, Flurbiprofen, Pheninidione, Dioxybenzone, A-7 hydrochloride, Usnic acid, lopanic acid, Menindione, lopanic acid, Istradefyllhie, PR-619, N6022, Diffractaic acid, IP A 3, Fisetin, Folic acid, GSK 3787, Guanabenz acetate, Chlorprothixene hydrochloride, NSC 663284, Ipsapirone, GF1O9203X, Beta Lapaclione, SF1670, DarapSadib (SS480848), PD 198306, Pimethixene Maleate, Oxyqufeoline Hemisul&te, a pharmaceutically acceptable salt or alternative salt thereof, a stereoisomer thereof or a mixtures thereof.

41. A met ho of treatin a disease state or condit ion which is mediated through a GTPase in a patient in need comprising administering to sai patient an effective amount of a composition selected from the group consisting of Salsalate, Tolfenamic acid, Dexibuprofen, Mefenamic Acid, ibuprofen, S-(÷)-Ibuprofen, Meclofenamic acid sodium salt monohydrate, (R)_-Naproxen_sodiurajsaIt, Naproxen, Flufenamic Acid, Flurbiprofen, Pheninidione, Dioxybenzone, A-7 hydrochloride, Usnic acid, lopanic acid, Menindione, lopanic acid. !stradefylline, PR-619, N6022, Dtfimciatc acid, IPA 3, Fisetin, Folic acid, GSK 3787,

Goanabenz acetate, Chlorprothixene hydrochloride, NSC 663284,

Ipsapirone, GF109203X, Beta Lapachone, SFI670, Darapladib (SB480848), PD 198306. Pimethixene Maleate, Oxyquinoline Hemisulfate, a pharmaceutically acceptable salt or alternative salt thereof, a stereoisomer thereof or a mixtures thereof

42. The method according to claim 41 wherein said disease state or condition is cancer, a histiocyte disorder, Noonan syndrome (NS), Noonan syndrome with multiple lentigtnes, Leopard syndrome, cardiofacio-cutaneous syndrome, neurofibromatosis type I (NF1) and secondary effects of neurofibromatosis type I, Legius syndrome, Costello syndrome (CS), capillary malformation-arteriovenous malformation syndrome (CFC syndrome), congenital myopathy with excess of muscle spindles (C EMS), congenital heart disease, hereditary gingival fibromatosis type 1 or hypertrophic cardiomyopathy (HCM).

43. The method according to claim 42 wherein said disease state or condition is cancer.

44. The method according to claim 42 or 43 wherein said cancer is a naive, recurrent, drug resistant or metastatic cancer.

45. The method according to claim 43 or 44 wherein said treatment further comprising co-administering an additional anticancer agent.

46. The method according to any of c laims 43-45 wherein said cancer is selected from the group consisting of carcinomas (e.g., squamous-cell carcinomas, basal cell carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas), particularly those of the bladder, bone, bowel, breast, cervix, colon (colorectal), esophagus, head, kidney, liver, lung, nasopharyngeal, neck, ovary, pancreas, prostate, and stomach; hematologic cancers, including leukemias, such as acute myelogenous leukemia, acute lymphocytic leukemia, acute promye!ocytie leukemia (APL), acute T-cel! lymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, raegakaryoeytic leukemia, microrayelohlastic leukemia, monocytic leukemia, neutrophilic leukemia and stem cell leukemia; benign and malignant lymphomas, particularly Burkitt’s lymphoma, Non-Hodgkin’s lymphoma and B-cell lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma,

hernangiosarcoma, Kaposi’s sarcoma, !iposarcoma, myosarcomas, peripheral

neuroepithelioma, and synovial sarcoma; blastemas, including glioblastoma and

medulloblastoma (brain tumors), hepatoblastoma (liver tumor), nephroblastoma (kidney tumor), neuroblastoma (neural tumor), osteoblastoma (bone tumor) and retinoblastoma (retinal tumor in the eye), tumors of the central nervous system (e.g., gliomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ-line (germ cell) tumors (e.g , bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer (e.g., small ceil lung cancer, mixed small cell and non-small cell cancer, pleural mesothelioma, including metastatic pleural mesothelioma small cel! lung cancer and non-small cell lung cancer), ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas.

47. The method according to any of claims 43-45 wherein said cancer is thyroid cancer, salivary duct carcinoma, epithelial-myoepithelial carcinoma, kidney cancer, astrocytoma, and melanoma.

48. The method according to claim 43 or 44 wherein said cancer is choriocarcinoma, testicular choriocarcinoma, non-seminomatous germ cell testicular cancer, placental cancer (trophoblastic tumor) or embryonal cancer.

49. The method according to any of claims 45-48 wherein said additional anticancer agent is selected from the group consisting of everolinms, trabeeted , abraxane, TLK 236, AY- 299, DN-I01 , pazopanib, GS 690693, ETA 744, ON 09i 0. a, AZD 6244 (ARRY- 142886), AM -107, TK!-258, GSK46B64, AZD 1152, enxasiaurin, vandetanib, ARQ-197, MK-0457, LN8054, PBA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK. inhibitor, an aurora kinase inhibitor, a PIN-1 modulator, a Bel-2 inhibitor, an l!BAC mhbiior, a c-MET inhibitor, a PAR? inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an 1GFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a 3AK/STAT inhibitor, a checkpoint- 1 or 2 inhibitor, a focal adhe-non kinase inhibitor, a Map kinase kinase (raek) inhibitor, a VEG trap antibody, pemetrexed, erfotinib, dasatanib, mloti b, decatanib, panitomomafe, amrubicm, oregovomab, Lep-etu, nolatrexed, azxl21?l₅ feata.bu1in, ofatinmmiab, zanoiimumab, edotecarin, tetraudrine, rubitecan, tesmilifene, oblimersen, tieili u ab, ipilimimiab, gossypol, Bio I I ! , I3I -I-TM-601 , ALT-1 10, BIO 140, CC 8490, ciiengifkie, ginateean, IL13-PE33QQR, INO 1001 , Ml: KRX-0402, locanthone, LY 31761 $, neuradkb, vitespan, Rta 744, Sdx 102, talampanel atrasentam Xr 31 1 , mnhdepsin, ADS- 100380, sunitiuib, 5~finorouraeiL vorinost at, etoposide, gemciiabine, doxorubicin, irinotecan, liposomal doxorubicin, $ ~deoxy 5-i¾orooridine, vincristine, temozolomide, ZK-304709, seilciclib; PDO325901 , AZD-6244, eapecitabme, L~Gi«tamic acid, N -£4-£2-{2-ami«o~4,7~dihydro-4~oxo-1 H - pyrrole^; 2,3- d Jpyrimidio-S- y!)eihyl]benzoyI]~, disodiom salt, heptahydrate, eamptotbecin, PEG- labeled irinotecan, tamoxifen, toreraiiene citrate, anasirazoie, exe esiane, Isirozole, BES(diethyIstilbestroI}, estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1CU , CHIR-258,); 3-[5~ (methyisulfenylpiperadmemethyl)- indolyij-quinolone, vatalanib, AG-013736. AVE-0005, the acetate salt of [D- Ser(Bu } 6 ,Azgly 10 ] (pyro-Gl«-His~Trp-Ser~Tyr-D-Ser(Bu t )-Leu~ Arg-Pro- Azgly-NH ₂ acetate [Cs^H_&tNigOu -(CfefROslx where x = .1 to 2.4], goserehn acetate, leuprohde acetate, triptoreHn pamoate, medroxyprogesterone acetate,

hydroxyprogesterone caproate, megestrol acetate raloxifene, bicaiula ide, llotamkie niiutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, eriotinih, lapatanib, canertinib, ABX-EGF antibody, erfeitux, EKB-569, PK1-166, GW-572016, lonafemib, 8MS- 214662, tipifamife; amifostine, NVP-LAQS24, sofeeroy! analide hydroxamic acid, valproic acid, trichostatin A, FK-228, $141 1248, sorafenife, RN9S1 , aminogiutethimkte, arasaerinc, anagreiide, L-asparagiaase, Bacillus Cabnette-Guerin (BCG) vaccine, bleomycin, busereiln, bustdfan, carhop iat in, carmnsline, chlorambucil, cisplatin, dadribine, ciodronate,

eyproterone, eytarabine, daearbaxine, dactlnoinycin, daunomblc in, dletbylstilbestrol epirnbicin, fludarabine, fludrocortisone, fluoxymesteroue, flu tamide, gemcitafeine, glee vac, hydroxyurea, idambidn._, ifosiamide, ioiatimb, leuprolide, levamisole, iomustiue,

meehlorethamine, melphafan, b-mercaptoput e, rnesna, methotrexate, mitomycin, mitoiane, mitoxamrooe, ndutamide, octreotide, oxaliplatin. pamklronate, pentostat , plicamycin, porflmer, procarbazine, raltitrexed, rsiuximah, streptozocln, teniposk!e, testosterone, thalidomide, thioguanmc, thiotepa, tretinoin, vindesine, O-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramnstioe, ahretarnioe, ffoxorkline, 5-deooxymkiine, cytosine arahinoside, 6-mecaptopurine, deoxycoiormycin, calcitriol, valrtsbicio, miihramycio, vinblastine, v orelbiae topoiecan, razoxln, marimastat, COL-3, neovastat BMS-275291 , sqna!atmne, endostatm, SI G4 I6, SU6668, EMD12 I974, sftierle.ukiii-l2, IM862, angiostatin, vitaxln, dfoloxiiene, idoxylene, spironolactone, finasteride, cimitidlne, trastuzumafe, denileukin dift ox,gefttmib, bortezimib, paciitaxel, irinoteean, topotecan, doxorubicin, docetaxel, vinoreibme. bevacizuraab (monoclonal antibody) and erbitux, cremophor-ifee paciitaxel, epithi!one B, BMS- 247550, BMS-310705, droloxifbfte, 4-hydroxytamoxifen, pipendoxifeae, ERA- 923, arzoxifene, fulvestranl, aeolbifene, lasofoxifene, idoxifene, TSE- 424, HMR- 3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycm, 40-0- (24iydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD0O1 , ABT-57S, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L- 779, 45(1, PEG- filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon a!fa~2a, pegylated interferon aifa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,

hydrocortisone, interleukin- 1 1 , dexrazoxane, alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard,

methylprednisolone, ibriigumomab tiuxetan, androgens, dedtabine, hexamethylmeiamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunombicin, Ed wina -asparaginase, strontium 89, casopitani, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine,

metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, doksetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alia, darbepoetin alia, spilumnmab, vemurafenib among others among others, including immunotherapy agents such as IDO inhibitors (an inhibitor of indoieamine 2, 3 -dioxygenase (IDO) pathway) such as Indoximod (NLG-8187), Navoximod (GDC-091 ) and LG802, PDL1 inhibitors (an inhibitor of programmed death- ligand 1) including, for example, nivolumab, durvalumab and atezolizumab, PD1 inhibitors such as pembrolmmiab (Merck) and CTLA-4 inhibitors (an inhibitor of cytotoxic T~lymphocyte associated protein 4/cluster of differentiation 1 2), inc luding ipilimumab, tre elimumab and mixtures thereof