WO2010099445A2 - Non-covalent inhibition of the 26s proteasome and uses thereof - Google Patents

Abstract

The present inventions relate to compositions and methods for treating inflammatory diseases and cancer by administering proteasome inhibitors. In particular, the present inventions provide a new class of orally available non-covalent proteasome inhibitors capable of reducing NF-kappaB for mediating cytokine production in vivo. Further, the use of a small molecular weight inhibitor of the 26S proteasome via a non-covalent type inhibition is contemplated for use as a means to treat NF -kB mediated diseases, including but not limited to multiple myeloma and rheumatoid arthritis.

Description

NON-COVALENT INHIBITION OF THE 26S PROTEASOME

AND USES THEREOF

FIELD OF THE INVENTION The present inventions relate to compositions and methods for treating inflammatory diseases and cancer by administering proteasome inhibitors. In particular, the present inventions provide a new class of orally available non-covalent proteasome inhibitors capable of reducing NF-kappaB for mediating cytokine production in vivo. Further, the use of a small molecular weight inhibitor of the 26S proteasome via a non-covalent type inhibition is contemplated for use as a means to treat NF-κB mediated diseases, including but not limited to multiple myeloma and rheumatoid arthritis.

BACKGROUND OF THE INVENTION The clinical benefit of proteasome inhibitors was demonstrated by the use of bortezomib in multiple myeloma and mantle cell lymphoma (in addition to multiple clinical trials with patients with advanced solid tumors, breast cancer, pancreatic cancer, colon cancer and non small lung cell cancer).

However, problems remain with host toxicity (neuropathy) and development of host resistance.

Thus more effective treatments are necessary for safer and more effective treatment of cancer such as multiple myeloma.

SUMMARY OF THE INVENTION The present inventions relate to compositions and methods for treating inflammatory diseases and cancer by administering proteasome inhibitors, including inhibitors that bind reversibly and inhibitors that bind irreversibly. In particular, the present inventions provide a new class of orally available non-covalent proteasome inhibitors capable of reducing NF-kappaB for mediating cytokine production in vivo. Further, the use of a small molecular weight inhibitor of the 26S proteasome via a non-covalent type inhibition is contemplated for use as a means to treat NF-κB mediated diseases, including but not limited to multiple myeloma and rheumatoid arthritis. While not limited to any particular mechanism, the present inventions are directed to the treatment of inflammatory and autoimmune diseases as well as cancer by administering proteasome inhibitors that, it is believed, interfere with the activation of NF-kappaB (NF-κB ) via the ubiqui tin-pro teasome pathway. The use of a small molecular weight inhibitor of the 26S proteasome via a non-covalent type inhibition is described herein. Non-covalent inhibition of the 26S proteasome is contemplated for use as a means to treat NF-κB mediated diseases, including but not limited to multiple myeloma and rheumatoid arthritis. The described and related compounds are orally available showed potent inhibition of NF-κB mediated inflammatory responses in cell culture, stimulated human blood and in vivo. Some compounds bind at a place different than the substrate binding site and are therefore classic non-competitive inhibitors.

The inventions provide a pharmaceutical composition comprising a small molecule non-covalent inhibitor of the 26S proteasome. In one embodiment, said inhibitor inhibits the 2OS catalytic core of the 26S proteasome. In one embodiment, said inhibitor is selected from the group consisting of imidizolines 1-9 (see Figure 8). In one embodiment, said compound is a therapeutically effective amount for reducing a symptom of a disease selected from the group consisting of cancer and inflammatory disease. In one embodiment, said cancer is multiple myeloma. In one embodiment, said inflammatory disease is rheumatoid arthritis.

The inventions provide a method, comprising, a) providing, i) a pharmaceutical composition comprising a small molecule non-covalent inhibitor of the catalytic region of a 26S proteosome and ii) a patient, wherein said patient is in need of treatment and b) administering said pharmaceutical composition to said patient as a therapeutically effective amount. In one embodiment, said inhibitor inhibits the 2OS catalytic core of the 26S proteasome. In one embodiment, said small molecule is selected from the group consisting of imidizolines 1-9 (see Figure 8). In one embodiment, said patient shows a symptom selected from the group consisting of a cancer and an inflammatory disease. In one embodiment, said cancer is multiple myeloma. In one embodiment, said inflammatory disease is rheumatoid arthritis.

While non-covalent inhibition has been emphasized herein, the present invention also contemplates, in one embodiment, covalent inhibitors (e.g. TCH-062) that bind irreversibly to the 2OS proteasome. DEFINITIONS

The use of the article "a" or "an" is intended to include one or more.

The use of terms defined in the singular are intended to include those terms defined in the plural and vice versa. The term "proteasome" refers to a large protein complex inside eukaryote cells and archaea, and some bacteria.

The term "26S proteasome" refers to a common form of a proteasome also refered to as an "ATP-dependant proteolytic complex" that is responsible for ubiquitin-dependent protein degradation. A 26S proteasome is about 2000 kilodaltons (kDa) in molecular mass and contains one 2OS core particle complex. Additionally, this complex may further comprise two 19S regulatory subunits (caps). An alternative form of regulatory subunit called the 11 S particle can associate with the core in essentially the same manner as the 19S particle.

The term "2OS proteasome" or "2OS core particle" refers to a complex that provides the catalytic core of the 26S proteasome. The number and diversity of subunits contained in the 2OS core particle depends on the organism.

The term "inflammatory" is used to refer to pertaining, characterized by, causing, resulting from, or becoming affected by inflammation. An inflammation is a fundamental pathologic process consisting of a dynamic complex of cytologic and chemical reactions that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent; these reactions include the local reactions and resulting morphologic changes, the destruction or removal of the injurious material, and the responses that lead to repair and healing. An "inflammatory disease" refers to a disease caused by or resulting from or resulting in inflammation.

The term "anti-inflammatory" is used to refer to an effect or compound which has an effect of preventing, inhibiting, alleviating or decreasing inflammation or components of an inflammatory reaction, either completely or partially. The terms "anticancer agent" and "anticancer drug," as used herein, refer to any therapeutic agents (e.g., chemotherapeutic compounds and/or molecular therapeutic compounds), radiation therapies, or surgical interventions, used in the treatment of hyperproliferative diseases such as cancer (e.g., in mammals). The term "therapeutically effective amount," as used herein, refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder. For example, with respect to the treatment of cancer, a therapeutically effective amount preferably refers to the amount of a therapeutic agent that decreases the rate of tumor growth, decreases tumor mass, decreases the number of metastases, increases time to tumor progression, or increases survival time by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

The terms "prevent," "preventing," and "prevention," as used herein, refer to a decrease in the occurrence of pathological cells (e.g., hyperproliferative or neoplastic cells) in an animal. The prevention may be complete, e.g., the total absence of pathological cells in a subject. The prevention may also be partial, such that the occurrence of pathological cells in a subject is less than that which would have occurred without the present invention.

As used herein, the term "patient" refers to a human or animal. As used herein, the term "patient" and "subject" are used interchangeabley. As used herein, the term "administering" refers to any means of treating a patient with a composition of the present invention. In a preferred embodiment, administration is orally.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from an animal, including a human; a particular biological sample may be a fluid (for example, blood, plasma and serum), a solid (for example, stool), or a tissue; other biological samples may be obtained from other biological sources, such as food, and may be a liquid food (for example, milk), or a solid food (for example, vegetables). Environmental samples include environmental material such as surface matter, soil, water, crystals, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention. DESCRIPTION OF THE FIGURES

Figure 1 shows exemplary examples of covalent proteasome inhibitors.

Figure 2 shows exemplary examples of non-covalent proteasome inhibitors.

Figure 3 A shows exemplary Inhibition of the chymotrypsin-like activity by TCH-Ol 8, Figure 3B shows exemplary inhibition of the post-glutamyl peptidase-like activity, Figure 3 C shows exemplary inhibition of the Typsin-like activity.

Figure 4 shows an exemplary Western blot of total nuclear p65: Lane 1 : untreated; lane 2: 1% DMSO, lane 3: imidazoline 1 (20 mM), lane 4: TNF-a, lane 5: TNF-a and MG-132 (10 mM), lane 6: TNF-a and imidazoline 1 (20 mM), lane 7: TNF-a and imidazoline 1 (10 mM), lane 8: TNF-a and imidazoline 1 (1 mM), lane 9: TNF-a and imidazoline 1 (0.1 mM).

Figure 5 shows an exemplary Western blot of total IkB in THP-I cells. Lane 1 : untreated; lane 2: 1% DMSO, lane 3: 1 (20 mM), lane 4: TNF-a, lane 5: TNF-a and MG-132 (10 mM), lane 6: TNF-a and 1 (20 mM). Figure 6 shows an exemplary Imidazoline 1 induces an accumulation of modified IkBa. Lane 1) No treatment, 2) 10 ng/mL TNFa, 3) 50 mM parthenolide with 10 ng/mL TNFa, 4) 10 mM MGl 32 with 10 ng/mL TNFa, 5-7) 20, 10 and 1 mM imidazoline 1 respectively with TNFa, 8-10) 20, 10 and 1 mM imidazoline 1 respectively with 10 mM MGl 32 and TNFa. Figure 7 shows an exemplary Ubiquitinylation of IkBa. Lane 1) untreated; lane 2) TNF-a only; lane; 3) 10 mM MGl 32 with TNF-a; lane 4) 50 mM parthenolide with TNF-a; lanes 5 - 7) 20 mM, 10 mM, 1 mM imidazoline 1.

Figure 8 shows an exemplary A) dose response activity of imidazoline 1 in inhibition of luciferase production in HeLa-NF-kB-luc cells. Cells were unstimulated/stimulated with 25 ng/mL TNF-a in the absence or presence of imidazoline 1 in 1% DMSO. Fold-induction (%) of the Luciferase activity, normalized for all samples to TNF-a stimulation, is shown. All date is an average of two independent experiments (error bars included in Figure). B) Structures of imidazolines 1-9. Figure 9 shows an exemplary inhibition of IL-6. Human blood (1 :10 RPMI) was unstimulated/stimulated with 200 LVmL IL-Ib in the absence or presence of imidazoline 1 in 1% DMSO. Fold-induction of the IL-6 levels of stimulated (calculated from unstimulated) cells was determined. Data are an average of two independent experiments (error bars included in Figure). For the treatment of Multiple Myeloma

Figure 10 shows an exemplary inhibition of TNF-a. Human blood (1 :10 RPMI) was unstimulated/stimulated with 200 U/mL IL-Ib in the absence or presence of imidazoline 1 in 1% DMSO. Fold-induction of the TNF-a levels of stimulated (calculated from unstimulated) cells was determined. A contemplated use is for Rheumatoid Arthritis.

Figure 11 shows exemplary cytotoxicity of imidazoline 1. Human blood was exposed to imidazoline 1 for 24 hours at stated concentrations and the lymphocytes were evaluated for survival.

Figure 12 shows an exemplary correlation of proteasome activity with NF-kB mediated gene transcription and reduction of IL-6 levels in blood.

Figure 13 shows an exemplary induction of cell death in MM cells by imidazoline 1. RPMI-8226 cells were treated with various concentrations of imidazoline 1 in duplicate and incubated for 24 hours. Percent survival was extrapolated based on mitochondrial activity as determined by MTS assay.

Figure 14 shows an exemplary LPS challenge in BALB/C mice with and without TCH-018.

Figure 15 shows exemplary schematics of activation pathways associated with MM and RA and exemplary treatment compounds.

Figure 16 is a plot showing inhibition with TCH-018 using three substrates.

Figure 17a is a plot showing inhibition with TCH-018 is non-competitive. Figure 17b is a plot showing TCH-018 does not bind in the catalytic domains.

Figure 18 is a plot showing the addition of TCH-018 to the sulfone resulted in additive activities for CT-inhibition.

Figure 19 is a plot showing the binding of TCH-018 is reversible.

Figure 20 is a plot showing TCH-062 binds irreversibly bind to the 2OS proteasome.

Figure 21 shows the structure of TCH-062.

DESCRIPTION OF THE INVENTION

The present inventions relate to compositions and methods for treating inflammatory diseases and cancer by administering proteasome inhibitors. In particular, the present inventions provide a new class of orally available non-covalent proteasome inhibitors capable of reducing NF-kappaB for mediating cytokine production in vivo. Further, the use of a small molecular weight inhibitor of the 26S proteasome (e.g. via inhibition of the 2OS catalytic core of the 26S proteasome) via a non-covalent type inhibition is contemplated for use as a means to treat NF-κB mediated diseases, including but not limited to multiple myeloma and rheumatoid arthritis.

Host toxicity of covalent proteasome inhibitors has precluded them as clinically relevant anti-inflammatory agents. The imidazolines described in this disclosure differ from current proteasome inhibitors, it is believed, by their mechanism of proteasome inhibition. These agents have demonstrated in vivo efficacy and no apparent in vivo toxicity, making them clinically relevant for anticancer (for example multiple myeloma) as well as anti-inflammatory (rheumatoid arthritis) treatments.

As described herein a new class of orally available non-covalent proteasome inhibitors capable of reducing NF-kappaB mediate cytokine production in vivo are provided. The compounds inhibit a clinically validated target protein (for example, IL-6) via a novel mechanism of action. This may have significant advantages related to patient toxicity and drug-resistance.

A clinical benefit of proteasome inhibitors was demonstrated by the use of bortezomib in multiple myeloma and mantle cell lymphoma (in addition to multiple clinical trials with patients with advanced solid tumors, breast cancer, pancreatic cancer, colon cancer and non small lung cell cancer). However, host toxicity of covalent proteasome inhibitors has precluded them as clinically relevant antiinflammatory agents. The imidazolines described in this disclosure and in U.S. Patents including

U.S. patent No. 6,878,735 B2 and U.S. applications related to U.S. Patent No. 6,878,735 B2, herein incorporated by reference in their entirety, differ from current proteasome inhibitors by their mechanism of proteasome inhibition and thus are contemplated to overcome either host toxicity or other adverse clinical outcomes in order to provide significant benefits to patients. These agents have demonstrated in vivo efficacy and no apparent in vivo toxicity, making them clinically relevant for anticancer (for example multiple myeloma) as well as anti-inflammatory (rheumatoid arthritis) treatments. The mammalian nuclear transcription factor NF-κB is responsible for the transcription of multiple cytokines, including the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-R) and interleukin 6 (IL-6). Elevated levels of proinflammatory cytokines play an important role in the pathogenesis of inflammatory disorders such as rheumatoid arthritis (RA). Inhibition of the pro-inflammatory transcription factor NF -KB has therefore been identified as a possible therapeutic treatment for RA. We describe herein the synthesis and biological activity of a series of imidazoline-based scaffolds as potent inhibitors of NF-κB mediated gene transcription in cell culture as well as inhibitors of TNF-R and IL-6 production in interleukin 1 beta (IL-IB) stimulated human blood (Kahlon, et al., Nuclear Factor-KB Mediated Inhibition of Cytokine Production by Imidazoline Scaffolds, J. Med. Chem., Article ASAP • DOI: 10.1021/jm8013162 • Publication Date (Web): 16 February 2009, herein incorporated by reference in its entirety).

The 2OS proteasome. The 2OS proteasome is the catalytic core of the 26S proteasome. This threonine protease exhibits three distinct peptidase-like hydrolytic activities: Chymotrypsin-like , Trypsin-like and Post-glutamyl-like.

Covalent proteasome inhibitors: Since the ATP-dependent degradation of many regulatory proteins is required to regulate multiple key intracellular regulatory cascades, inhibition of the proteasome has been identified as a means to treat inflammatory diseases and cancer.¹ Inhibition of the 2OS proteasome has been validated in the clinic approval of the proteasome inhibitor, bortezomib, for the treatment of multiple myeloma. One rational is that the proteins that regulate cell cycle and apoptotic events have short half-lives, thus will be more affected by modulation of the proteasome. Tumor types relying on NF-DB mediated cytokes, such as IL-6 and TNF-a, should be particularly sensitive to proteasome inhibition, while normal cells would remain unresponsive.

Nearly all proteasome inhibitors reported, inhibit the chymotrypsin-like activity of the 2OS proteasome via the formation of a covalent adduct to the N- terminal threonine on the active site of the beta-subunit. Representative examples among the known covalent proteasome inhibitors are: peptide aldehydes (MG-132, ALLN), peptide vinyl sulfones (Ac-YLLN-vs), peptide boronates (bortezomib), peptide epoxyketones (epoxomicin) and beta-lactones (lactacystine, salinosporamide A).¹ Although many of these agents are very potent, inherent drawbacks include a lack of target specificity and the highly reactive and often unstable nature of the agent. Applications of these covalently-linked inhibitors in vivo often induce apoptosis and cell death. By far the most successful clinical example of inhibitors of the 2OS proteasome is bortezomib (also known as velcade and formally PS-341, Figure 1). Bortezomib represents the first selective and reversible covalent proteasome inhibitor to demonstrate both significant preclinical activity in several tumor models and significant efficacy in patients with refractory or relapsed MM.¹⁴ Bortezomib is a covalent proteasome inhibitor and thus exerts significant cell death as well as toxicity via the modulation of multiple signaling pathways.

Non-covalent proteasome inhibitors: It can be reasoned that the toxicity of a reversible and time-limited proteasome inhibitor may be reduced. ^]β' ¹⁷ Unfortunately, examples of non-covalent inhibitors are scares, with TMC-95 being one of the most cited examples.¹⁸ The natural product and cyclic peptide, TMC-95, is structurally unrelated to the aforementioned covalent inhibitors and binds to all three proteolytically active binding sites in the beta-subunits of the 2OS proteasome. The binding of TMC-95 in the proteasome sites is similar to the vinyl sulfone inhibitors, albeit non-covalently to the N-terminal threonine. The further preclinical development of TMC-95 is hampered by its complex total synthesis.^20-27 Novartis has also reported some non-covalent-peptide based 2OS proteasome inhibitors with selectivity towards the chymotrypsin-like active site, but not further cellular or in vivo studies were reported ' ' To the best of our knowledge, non-covalent small molecule inhibitors have not yet been reported in the literature.

NF-kB-MEDIATED DISEASES, Relationship between proteasome inhibition and NF-kB inhibition. Upon the covalent addition of poly-ubiquitin chains (E1-E3 ubiquitin conjugation pathway), proteins are recognize by the multifunctional 26S proteasome, transferred to the 2OS protolytic core particle and degraded by proteolysis. This degradation pathway is critical for the turnover of proteins essential to cell proliferation, cell differentiation and inflammation. The involvement of this proteasomal degradation process is intimately linked to the regulation of NF-kB activation and its control over apoptosis and inflammation.

The mammalian transcription factor NF-kappaB (NF-kB) is an ubiquitous transcription factor responsible for the regulation of more than 150 genes impacting virtually every aspect of cellular adaptation including responses to stress, inflammatory stimuli, activation of immune cell function, cellular proliferation, programmed cell death (apoptosis), and oncogenesis. In normal non-stimulated cells, NF -kB is typically sequestered in the cytoplasm by NF- KB'S inhibitory protein, IkB. Upon stimulation by an appropriate extracellular signal, such as pro-inflammatory cytokines TNF-a and IL-IB, or DNA damaging agents, the NF-kB pathway becomes activated.⁴⁰'^48-51 During this process IkB undergoes a phosphorylated-driven polyubiquination and proteasomal degradation by the 26S proteasoem, resulting in the release of NF- kB.⁴⁷ Liberation of NF-kB allows for its rapid translocation into the nucleus.⁵² Following its nuclear translocation, NF-κB binds to DNA and initiates the transcription of a host of pro-inflammatory signaling genes or survival genes.

Subsequently, deregulation of the NF-kB pathway has been directly implicated in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA), inflammatory bowel disease, helicobacter pylori-associated gastritis, atheroscelerosis, multiple sclerosis, asthma as well as cancer.

REPRESENTATIVE THERAPEUTIC APPLICATIONS:

Multiple Myeloma: In multiple myeloma cells, NF -kB is constitutively activated (Figure 3) and the DNA damaging drags melphalan and doxorubicin (clinically used to treat MM) have been found to induce even higher levels of NF-kB activation.⁶⁵

Current therapeutic treatments of MM: Multiple myeloma (MM) is typically characterized as a type of cancer of plasma cells in the bone marrow. MM is a malignant disorder of differentiated B-cells (plasma cells) and remains incurable,³' ⁵' ^66-72 with a median survival rate of just 33 months.⁷³ Due to the intrinsic resistance of MM cells to classical chemotherapeutics, only few treatment options slow its progression. Traditional MM therapies include the DNA alkylating agents melphalan and doxorubicin in combination with vincristine and/or dexamethasone.⁷² In the last decade, the US Food and Drag Administration has approved three new drags for the treatment of MM:⁷⁴' ⁷⁵ the proteasome inhibitor bortezomib,²' ⁷⁶ the anti-angiogenic and immunomodulator thalidomide and its analogue lenalidomide.⁷⁷' ⁷⁸ Bortezomib- based regimens have since moved to the forefront as front-line therapies.⁵' ⁷⁹' ⁸⁰ The various treatment options/regimes are typically determined a IL-6 based staging method^81-83 and will be indicative of the patient's eligibility for autologous stem cell transplantation. In the last few years, the combination of either thalidomide/dexamethasone, bortezomib-based regimes and/or lenalidomide/dexamethasone combinations have emerged as the most recommended strategies in newly diagnosed patents.⁷² Standard DNA alkylating agent therapies still include melphalan and/or doxorubicin with prednisone, thalidomide or bortezomib and are typically used for patients that have a more advanced stage of the disease and are not transplant candiates.⁷²' ⁸⁴ In addition to the limited survival-times, these treatment options have multiple complications and toxicities related to them.⁸⁵' ⁸⁶

Rheumatoid Arthritis: Given the critical role of NF-kB-mediated expression of cytokines, this transcription factor has been actively pursued as a therapeutic target for these types of inflammatory disorders.³⁹' ⁸⁷

It is well documented that NF-kB stimulates the expression of multiple genes responsible for many aspects of inflammatory responses and the pathogenesis of inflammatory diseases. ' Cytokines (IL-I , IL-2, IL-6 and TNF-a) expressed by NF- kB induce the amplification of inflammatory signals. Of these cytokines, IL-6 ^{- 2} and TNF-a^93-97 have been identified as key targets in rheumatoid arthritis (RA) and other inflammatory disorders. Pharmacologic intervention in RA was improved drastically with the advent of biologicals that specifically target IL-6⁹¹ or TNF-a^98-100 Unfortunately, these and alternative treatment options for RA are limited, suffer from high costs and involve undesirable methods of administration. Furthermore, these therapies lack data pertaining to their long-term safety, tolerability and sustained efficacy.⁹⁹ hi addition, variability in responses to these anti-inflammatory drugs is found due to the complex network of alternative cytokine-mediated pathways.¹⁰¹ Inhibition of pro-inflammatory transcription factors, such as NF-kB, may therefore represent a better alternative to modulate the complex cytokine network that induces inflammatory response.⁸⁸ In addition, inhibition of NF-kB mediated gene transcription by a small molecule would represent an attractive therapeutic alternative to the current clinical options, which still primarily include anti TNF-a mAbs and anti IL-6 receptor antibodies.⁸⁹' ¹⁰²

Current therapeutic treatment of rheumatoid arthritis (RA): RA is a highly variable and difficult disease to control, can severely deform joints and, in severe cases, shorten a patient's life. No cure for this disease is presently available. The only small molecule therapy for disease modification in rheumatoid arthritis (RA), methotrexate, is effective in only approximately 20% of RA patients and it's use is limited by toxicity issues at higher exposures.^103-105 The most effective disease modifying approaches currently in use for RA rely on protein therapeutic agents that interfere with signaling by the potent proinflammatory cytokines, TNF-a (Infliximab, Adalimumab, Etanercept,) and IL-IB (Anakinra), or an anti-CD20 monoclonal antibody (Rituximab) that depletes B-cells. These biologies are expensive therapeutics, require parenteral administration, and have variable responses in patients.

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EXPERIMENTAL

The following examples are provided in order to demonstrate and further Illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimole); μmol (micromole); nmol (nanomole); gm (gram); mg (milligram); μg (microgram); pg (picogram); L (liter); ml (milliliter); μl (microliter); cm (centimeter); mm (millimeter); μm (micrometer); run (nanometer); °C. (degrees Centigrade or Celsius), s (second), h and hr (hour), and wk (week), and

Example I

This Example demonstrates that trans-substituted imidazolines represented by TCH-Ol 8 show potent inhibition of the 2OS proteasome. TCH-Ol 8 inhibited the chymotrypsin-like Figure 3A and post-glutamyl peptidase -like Figure 3B domains of the 2OS proteasome. No significant activity was seen in the trypsin-like domain (Figure 3C).

Example II

This Example demonstrates that the proteasome inhibitor TCH-018 blocks nuclear translocation of the p65 subunit of NF-kB after activation of the NF -kB pathway. Thus inhibition of the proteasome should inhibit the activation of the pro- survival and pro-inflammatory nuclear transcription factor kappa B (NF-kB). Inhibition of this transcription by a small molecule proteasome inhibitor, blocked the nuclear translocation and subsequent gene transcription of pro-survival genes and pro- inflammatory genes.

Inhibition of p65 nuclear translocation by imidazoline 1 (TCH-Ol 3, i.e. the racemic form of TCH-Ol 8): In order to evaluate mechanism for NF-κB mediated transcription of IL-6 production, the effects of imidazoline 1 (TCH-Ol 8) on the nuclear translocation of the p65 subunit of NF-κB was investigated. In order to monitors NF-κB's nuclear translocation, THP-I cells were activated with TNF-α (to induce the nuclear translocation) in the presence and absence of a range of imidazoline 1 concentrations and nuclear extracts were investigated for p65 levels. Nuclear extracts were isolated and analyzed by Western blot using a p65 total (Santa Cruz) antibody. As shown in Figure 4, imidazoline 1 does not induce the activation of NF-κB (lane 3), whereas TNF-α induces the nuclear translocation of p65 (lane 4). The commercial proteasome inhibitor MG-132 blocks TNF-α-induced nuclear translocation (lane 5). Similar to MG-132, imidazoline 1 inhibits the TNF-α-induced nuclear translocation of p65 in a dose response manner (lanes 6-9). This data indicates that imidazoline 1 inhibits the nuclear translocation of NF-κB in THP-I cells after TNF-α activation.

Example III

This Example demonstrates accumulation of IKB by imidazoline 1. The effect of imidazoline TCH-013 on the modification and subsequent degradation of IKB was investigated by Western blot analysis using an IKB polyclonal antibody (Santa Cruz). In control lanes 1-3, IKB remains un-modified and is accumulated in the cytoplasm. Upon treatment with TNF-alpha10 ng/mL for 30 minutes, IKB becomes phosphorylated, ubiquitinylated and degradated by the proteasome (Figure 5). The presence of a weak band in Lane 3 indicates that most of IKB has been degraded. Addition of the proteasome inhibitor MG-132 (lane 5) indicates accumulation of IKB (i.e prevention of its degradation). A second band is visible in lane 5, which is the phosphorylated forms of IDB that is recognized by the antibody. Figure 5 indicates that imidazoline 1 accumulated the unmodified form of IKB (Figure 5, lane 6).

Example IV The following examples demonstrate exemplary embodiments of the present inventions.

This Example demonstrates accumulation of IKB isoforms: The effects of imidazoline 1 (TCH-013) on IKB modification were investigated in more detail using Western blot analysis. HeLa cells were pretreated with test agents for two hours and then challenged with 10 ng/mL TNF-alpha for 30 minutes. Whole cell extracts were prepared in cell lysis buffer containing complete protease inhibitor cocktail. 50 ug of each sample was heated in an equal volume of SDS-PAGE sample buffer containing reducing agent (DTT), resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked in TBS-T (containing 5% nonfat dry milk. IκB-alpha was detected using polyclonal antibody IκB-alpha (SC371). Bound antibodies were detected with enhanced chemiluminescence. Figure 6, indicates that upon treatment with TNF-alpha IKB quickly degrades, resulting in a decrease in the total amount of IKB (lane 2) compared to the untreated control (lane 1). Addition of the kinase inhibitor, parthenolide, prior to TNF-a activation (lane 3) prevents the phosphorylation and thus degradation of IKB. The proteasome inhibitor MG132, also prevented the degradation of IKB following TNF-a activation, resulting in the presence of the phospho-IκB and multiple ubiquitinylated forms of IKB (lane 4). Comparison of lanes 3 and 4 shown that the IKK kinase inhibitor parthinolide prevents the formation of phospo-IκB. However, in the presence of imidazoline 1, IKB appears to be stabilized, and multiple sumoylated and mono-ubiquitinylated forms are predominant (lanes 5- 7), which are not apparent with parthenolide (lane 3). By combining MGl 32 with imidazoline 1, followed by TNF-D activation, a similar profile remains, although the poly-ubiquitinylated IDB and the phosphorylated IKB forms appear to be intensified (lanes 8-10). The absence of a phospho-IκB product in lanes 2 (TNF only) and 5-7 (TNF & imidazoline 1) could indicate that IKB does proceed through the phosphorylation step, but gets blocked in its next post- translational modification by the imidazoline.

This Example demonstrates accumulation of ubiquitinylated IkB. Ubiquitinylation of I D B Q was confirmed by performing a complementary experiment in which IκBa was immunoprecipitated and the ubiquitinylated species was detected by western blot analysis. HeLa cells were transfected with pMT123, a plasmid encoding an epitope tagged (HA) ubiquitin. pMT123 was received as a kind gift from Professor Dirk Bohamnn, University of Rochester. Cells were transfected with ρMT123 using Lipofectamine 2000. Twenty-four hours later, transfected cells were pretreated with test agents for two hours and then challenged with 10 ng/mL TNF-a . (Sigma) for 90 minutes. The extended time (90 minutes vs 30 minutes (see preliminary data section 2.4 and 2.5) was used to investigate the fate of IkB, following ample IkBactivation. Whole cell extracts were prepared in cell lysis buffer (20 mM Tris-HCL [pH 7.5], 150 mM NaCl, 1 niM Na₂EDTA, 1 πiM EGTA, 1% Triton) containing complete protease inhibitor cocktail. For immunoprecipitation, the whole cell extract was incubated overnight at 4°C with a specific antibody for IkBa (sc-371) covalently bound to protein G agarose. Samples were heated in an equal volume of SDS-PAGE sample buffer containing reducing agent (DTT), resolved by SDS-PAGE, and transferred to a PVDF membrane. The membranes were blocked in TBS-T (50 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk. Ubiquitinylated species of IDB Q were detected using polyclonal antibody to HA conjugated to HRP (sc-8017). Bound antibodies were detected with enhanced chemiluminescence. Figure 7 indicates that imidazoline 1 clearly induces accumulation of polyubiquitinylated IDB's (lanes 5-7) as compared to the TNF-a control (lane 2), consistent with the profile of proteasome inhibitors such as MG-132.

This Example demonstrates inhibition of NF-κB mediated gene transcription. In order to test the ability of the imidazolines to inhibit NF-κB -mediated gene transcription, we evaluated the compounds using a luciferase reporter assay. Human cervical epithelial HeLa cells with stably transfected NF-κB -luc gene were purchased from Panomics (Freemont, CA.). These cells maintain, through hygromycin selection, a chromosomal integration of a luciferase reporter construct regulated by multiple copies of the NF-DB response element. The cells were activated with TNF-a (25 ng/mL) in the presence or absence of the imidazolines. DMSO and TNF-a were used as negative and positive controls respectively and luciferase production was evaluated after 8h using the Steady-Glo luciferase assay system. All samples were normalized to the TNF-a activated control. Treatment of HeLa/ NF-κB -luc cells with the imidazolines without any TNF-a activation did not induce a significant amount of luciferase activity, suggesting that the compounds had no effect on NF-κB activation. Pre-treatment of the cells with the imidazolines followed by TNF-a stimulation resulted in a dose dependent decrease in luciferase production (Figure 8). A representative dose-response of imidazoline 1 is shown in Figure 8. Compounds 1-9 were evaluated and their respective IC50 values are listed in Table 1. (error margins shown in Table 2). Limited structure activity relationship of the carboxylic acid moiety identified imidazoline ester 1 as a potential viable alternative to 2a.

Table 1. IC₅₀ values for inhibition of luciferase production in pNF-κB -luc HeIa cells following TNF-a( alpha) activation. Data are an average of 2 independent experiments.

This Example demonstrates inhibition of NF-kB mediated IL-6 production in human blood. In order to evaluate the potential of these compounds to inhibit NF-kB mediated gene transcription beyond cell culture, human blood samples were pretreated with imidazoline 1 followed by IL- lβ to stimulate NF-kB mediated gene transcription of IL-6. IL-I β was chosen as stimulus, because second to IL-6, this mediator has been implicated as part of the primary mechanism for bone destruction in patients with multiple myeloma. In addition, IL- lβ is know to be a potent inducer of IL-6 in multiple myeloma IL-6 was chosen as an end-point read-out, because this NF-kB-regulated cytokine plays a key role in the pathogenesis of multiple myeloma.

After obtaining the appropriate approval for de-identified human cell lines, human whole blood was obtained through the Jasper Research Clinic, Kalamazoo, MI, from a single healthy, fasted human volunteer and was collected in glass citrated tubes by venipuncture. Only samples with a white blood count falling within the normal range (4,800-10,800 white blood cells per liter) were used. To support the viability of white blood cells, blood was diluted 1 :10 in RPMI- 1640 media. Aliquots of diluted blood (1 mL) were pre-incubated with vehicle (0.1% DMSO, final concentration) or imidazoline (at various concentrations) for 2 hours at 37° C, 5% CO₂. IL-I β was added to a final concentration of 200 LVmL and the samples were further incubated for 22 hours at 37° C, 5% CO₂. At the end of the incubation period, the blood samples were centrifuged at 3000 RPM for 5 minutes. The plasma was removed, snap frozen and stored at -80° C. IL-6 levels were determined by ELISA. As clearly indicated in Figure 11 the circulating cytokine levels in IL-I β stimulated samples were significantly higher (>1000 fold) than in unstimulated or the vehicle treated blood. IL-6 levels significantly reduced in the presence of the imidazoline in a clear dose response (Figure 9). The IC₅₀ of the racemic compound 1 was determined to be 0.8 M for IL-6 inhibition. In addition to imidazoline 1, compounds 6-9 were also evaluated for activity (Table 2).⁶⁸

The inventors observed that the imidazoline 1, when added post-treatment (add imidazoline 1 hour after IL-IB activation of NF-kB pathway), instead of pre- treatment (2 hours prior to IL-IB treatment), was still able of significantly reducing IL-6 production (IC₅₀ 2.5 M).

This Example inhibition of NF-DB mediated TNF-a production in human blood: NF-DB regulates multiple cytokines, including IL-2, IL-6, IL-8 and TNF-a. In order to exclude the potential of imidazoline 1 to inhibit IL-6 production via an NF- kB independent mechanism, the same human blood samples were evaluated inhibition of another NF-kB regulated cytokine, TNF-a. It should be noted that TNF-a is also a cytokine directly related to the pathogenesis of MM and strong stimulus for IL-6 production in MM. The levels of TNF-a production were measured using the same blood samples as described above and similarly a strong dose-response inhibition of TNF-a was found following IL-IB stimulation. Plasma was harvested 22 hours after stimulation and IL-IB induced TNF-a production was measured using a human TNF- a ELISA (R&D Systems) assay. The circulating TNF-a levels in IL-IB stimulated blood samples were significantly higher (-400 fold) than in unstimulated or the vehicle treated blood (Figure 10). Pretreatment of the blood for 2 hours with the imidazoline 1, followed by IL-IB stimulation resulted in a strong dose-dependent inhibition of TNF-a production, as compared to the vehicle control (Figure 10). The IC₅₀ of compound 1 (TCH-013, racemate) was determined to be 1.2 DM for TNF-a inhibition. In addition, 6-9 were also evaluated for inhibition of TNF-a production (Table 2). This Example Cell shows cytotoxicity effects. In order to exclude the possibility of decreased cytokine production due to an increase of imidazoline mediated cell death, both the HeLa cells and the white blood cells were evaluated for cell death at various concentrations of imidazoline 1. An LDH release assay showed that incubation of HeLa/NF-kB-luc cells with up to 10 micro M imidazoline 1 for 8 hours did not induce any significant amount of cell death (data not shown). FACS analysis of these samples confirmed the cells were healthy (data not shown).⁶⁸ In addition, to evaluate the toxicity of imidazoline 1 in human blood, de-identified human whole blood was obtained from the Jasper Clinic (Kalamazoo, Michigan) and diluted 1 : 1 in PBS. Lymphocytes were isolated from whole blood by layering over Lymphoprep solution. The samples were centrifuged at 800 X g for 30 minutes in a swinging bucket rotor at room temperature with the break turned off. The lymphocyte band was transferred to a fresh tube and diluted with RPMI- 1640 containing 10% fetal bovine serum until there were I X 10⁶ cells/mL. The lymphocytes were ali quoted into culture dishes and treated with 20, 10, 3, and 1 DM compound 1 in quadruplicate. The cells were incubated at 37°C, 5% CO₂ for twenty-four hours. Cells were diluted 1 : 1 in trypan blue solution and live cells were counted. No significant cell cytotoxicity could be detected at 10 DM or lower (Figure 11, white blood cell count after 24 hours at various concentration of imidazoline 1). Therefore, the inhibition of luciferase production and inhibition of IL-6 and TNF-a production was not due to a decrease in cell number of the HeLa cells or white blood cells, respectively.

Whole blood cytokine inhibition versus cellular reporter assay. The correlation between the activity seen in HeLa cells (NF-kB-luc reporter assay) and in whole blood (IL-6 and TNF-a, ELISA assay) was highly consistent and reproducible (Table 2). As mentioned above, compounds 1 (and enantiomers Ia and Ib), 3-9 were evaluated for inhibition of IL-6 and TNF-a production and the data is summarized in Table 2. Comparison of the IC₅₀ values from all assays indicates that the R,R enantiomers of 1 (Ib) is the most potent compound thus far (IL-6: IC₅₀0.2 microM). To illustrate the error margin from experiment to experiment most accurately, error margins are listed as the standard error of the log EC₅₀'s.

Table 2. ^aLog EC₅₀ values for inhibition of luciferase production in pNF-κB-luc HeLa cells following TNF-a activation. ^b Standard error of log EC₅₀ values for inhibition of luciferase production in pNF-κB-luc HeLa cells following TNF-D activation. ^cEC₅₀ values calculated from the log EC₅₀ values for inhibition of luciferase production in pNF-κB-luc HeLa cells following TNF-aactivation. ^dLog IC₅₀ values for inhibition of TNF-a production in human blood following IL- lβ stimulation. ^eStandard error of log IC₅₀ values for inhibition of TNF-a production in human blood following IL- lβ stimulation. ^fICso values calculated from the log IC₅₀ values for inhibition of TNF Q production in human blood following IL- lβ stimulation. ^gLog IC₅₀ values for inhibition of IL-6 production in human blood following IL- lβ stimulation. ''Standard error of log IC₅₀ values for inhibition of IL-6 production in human blood following IL- lβ stimulation. ¹IC₅₀ values calculated from the log IC₅₀ values for inhibition of IL- 6 production in human blood following IL- lβ stimulation.

This Example demonstrates Inhibition of the chymotrypsin-like activity of the

2OS proteasome is consistent with the inhibition of NF-kB mediated gene

transcription. The in vitro activity of the chymotrypsin-like activity of the 2OS proteasome was evaluated for several active and inactive imidazolines. The activity correlated well with the inhibition of NF-kB mediated gene transcription of (Hela-luc reporter assay) as well as with the activity of the compounds in reducing cytokine production in human blood (Figure 12).

This Example demonstrates effeciacy for treatment of Multiple Myeloma: Given the critical role NF-kB-mediated IL-6 production plays in the growth and parthenogenesis of multiple myeloma, we investigated the use of imidazoline 1 in somewhat more detail in a MM cell line (RPMI-8226). Multiple myeloma is a key target disease for NF-kBas well as IL-6 inhibitors, since nearly all multiple myeloma cells (including RPMI-8226 cells) contains constitutively active NF-kB. Similar to bortezomib (or velcade), imidazoline 1 was found to be effective as a single agent in MM cells. The CC₅₀ of imidazoline 1 in the RPMI-8226 MM cell lines was 4.0 microM (Figure 13), which is comparable to the clinically significant doxorubicin (CC50 1.2 microM) but better than melphalan (CC50 30.6 microM) in this particular cell line. Importantly, unlike other MM drugs no significant cytotoxicity towards white blood cells was observed at effective concentrations (<20 microM).

This Example demonstrates Structure activity relationship. A series of imidazolines were prepared and investigated for their ability to reduce NF-kB activity (in cell culture, HeLa-luc reporter assay) and cytokine production (in human blood, ELISA assay). The IC50 values of the compounds are listed in Table 3 below:

Table 3. ^aLog EC₅₀ values for inhibition of luciferase production in pNF-κB-luc HeLa cells following TNF- D activation. ^bStandard error of log EC₅₀ values for inhibition of luciferase production in pNF-κB-luc HeLa cells following TNF-D activation. ⁰EC₅₀ values calculated from the log EC₅₀ values for inhibition of luciferase production in pNF-κB-luc HeLa cells following TNF-D activation. ⁸Log IC₅₀ values for inhibition of IL-6 production in human whole blood following IL- lβ stimulation. ^hStandard error of log IC₅₀ values for inhibition of IL-6 production in human whole blood following IL- lβ stimulation. ¹IC₅₀ values calculated from the log IC₅₀ values for inhibition of IL-6 production in human whole blood following IL- lβ stimulation.

This Example demonstrates In vivo activity. The activity of the imidazolines was evaluated in BALB/C mice using a standard LPS challenge. In the control group, mice were injected ip with 1.0 mg/Kg or 0.1 mg/Kg LPS in saline solution to initiate a massive inflammatory response. In the treatment groups, mice were given 200 mg/Kg TCH-018 in vegetable oil by oral lavage, 1 hour prior to LPS injection. After the LPS injection, blood was analyzed for TNF-a levels. Figure 14 indicates that LPS induced high levels of TNF-a after 2 hours (2,000 pg/mL TNF-a) in female mice treated with 1.0 mg/Kg LPS. In the mice pretreated with TCH-018 this level was significantly reduced (400 pg/mL). Similar results were obtained with male mice treated with 0.1 mg/Kg LPS. See, Figure 14. LPS challenge in BALB/C mice with and without TCH-018. This Example shows TCH-018 inhibits the 2OS proteasome: The ability of TCH- 018 to inhibit the 2OS proteasome was determined in vitro using purified human 2OS proteasome and the following fluorogenic peptides as substrates: Suc-LLVY-AMC (substrate for CT-L activity), Boc-LRR-AMC (substrate for T-L activity) and Z-LLE- AMC (substrate for PGPH activity). The rates of hydrolysis were monitored by fluorescence increase and the initial linear portion of the curves were used to calculate the IC₅₀ values, resulting in inhibition of the CT-L, (IC₅₀ 0.95 uM) and PGPH (also referred to a caspase-like) (IC₅₀ 1.6 uM) activities of the 2OS catalytic core. The T-L activity was not inhibited (IC₅₀ >10 uM, Figure 16). An apparent slight activation of the trypsin-activity (~ 10%) was seen at the highest concentration of TCH-018 (10 uM), not uncommon to proteasome inhibition.

This Example shows TCH-018 inhibits via non-competitive binding: A classical non-competitive inhibitor has no effect on substrate binding (i.e. the inhibitor and substrate bind independently at different sites). A non-competitive inhibitor decreases the V_max, but has no effect on the K_m value. Kinetic analysis of CT-L activity indicate that when substrate (Suc-LLVY-AMC) concentration were increased and measurements were taken at five different concentrations of TCH-018 or vehicle, the V_Max of the CT-L activity diminished with the increasing concentration of substrate and the K_M of the substrate remained constant (Figure 17a). The collected data followed the model of non-competitive inhibition: calculated maximum velocities, V_max, were 0.018, 0.243, 0.722, 0.855, 0.822 and 0.991 FU/sec released by 1 nM protein over thirty minutes at 37° C. Over a concentration range from 10 uM - 0.6 uM of TCH-018 respectively, the Michaelis constant, K_M, remained constant at 54 uM for all concentrations. This data indicates that the substrate concentration has no affect on the activity of the inhibitor. The mechanism for inhibition of PGPH-like (or caspase- like) activity was found to be un-competitive. This is a classic representation of a noncompetitive type of inhibition. However, it should be noted that non-competitive kinetics could be mis-interpreted if the agent binds irreversibly in the catalytic domains

This Example shows TCH-018 does not bind in the catalytic domains. To eliminate (or confirm) irreversible binding of TCH-018 in the catalytic domains, we incubated the 2OS proteasome with the biotinylated sulfone (Figure 17b, Ada- Lys(biotinyl)-(Ahx)3-(Leu)3 -vinyl sulfone) to label the three core catalytic sites following the work of Overkleeft and Ploegh. The affinity label (1 uM) is incubated for 1 h with purified human 2OS proteasome (200 ng) where it binds covalently and irreversibly to the three catalytic sites (Figure 1, βl, β2 and β5). The complex was denatured, exposed to gel electrophoresis (12%), transferred to a PVDF membrane, where after blocking with 3% gelatin in TBS, the covalent biotin-protein adducts are visualized using avidin-HRP. Panel b illustrates that the biotin-labeled sulfone bind irreversibly to the three domains, βl, β2 and β5 (lane 1). Pretreatment of the 2OS proteasome of 1 h with different concentrations of TCH-018, prior to the biotinylated sulfone, does not interfere with the ability of the probe to bind (lanes 3-7), whereas the competitive inhibitor MG- 132 or Velcade (i.e. bortezomib) blocks access to the βl and β5 sites as reported previously.⁷⁰ This data eliminates the possibility that TCH- 018 binds irreversibly to any of the three catalytic domains. Thus, indicating that TCH-018 inhibits the proteolytic activity at the βl (PGP-like) and β5 (CT-like) sites via a non-competitive mechanism at a site other that the catalytic sites.

Consistent with this hypothesis, the addition of TCH-018 to the sulfone, resulted in additive activities for CT-inhibition (Figure 18). This follows the same trend as the combination studies with the competitive binding agent, bortezomib, as described in C3.2. Therefore, (1) Figure 16 illustrates the inhibition of proteolytic activity of the 2OS proteasome by TCH-018. (2) The kinetics of binding in Figure 17a, indicates a non-competitive mechanism, and (3) Figure 17b indicates that binding of TCH-018 to the proteasome does not occur at any of the three catalytic sites. We recognize that individually these data do not fully constitute the case for allosteric inhibition. However, taken together these data strongly suggest that TCH- 018 inhibits the 2OS proteasome by binding a site other than the catalytic sites resulting in inhibition of its catalytic activity (typically/often referred to as negative allosteric inhibition).

This Example shows TCH-018 does not inhibit other proteases: Inhibition was not limited to human proteasome tested, but TCH-018 was equally active with the human 2OS immunoproteasome and was also effective toward 20S proteasome isolated from S. cerevisiae (IC₅₀ 2.8 uM). However, when TCH-018 was evaluated for its modulation of other proteases including calpain, caspase and cathepsin B, the compound did not exhibit any detectable inhibition (data not shown). This data indicates that TCH-018 is not a general protease inhibitor, but has primarily (or selectively) targets the 2OS proteasome.

This Example shows TCH-018 binds reversibly. In order to investigate whether the TCH-018 binding is reversible or irreversible, we evaluated plot of V_max versus amount of enzyme added to distinguish between a reversible and irreversible noncompetitive inhibitor. Irreversible inhibition can be distinguished from reversible binding by plotting V_max vs. [E]. If the compound binding is reversible, the curve will have a smaller slope than the control curve and go through the origin. An irreversible inhibitor's curve will have the same slope but intersect on the horizontal axis at the position equivalent to the amount of enzyme that is irreversibly inactivated by the inhibitor. As illustrate in Figure 19, TCH-018 (2.5 uM) and control both intersect the plot origin, indicative of classical non-competitive reversible binding. This data indicates that TCH-018 behaves as a classical non-competitive reversible inhibitor.

This Example shows that TCH-062 binds irreversibly. TCH-062 was found to irreversibly bind to the 2OS proteasome and as evaluated by the plot of V_max versus amount of enzyme added (Figure 20).

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS:

1. A pharmaceutical composition comprising a small molecule non-covalent inhibitor of the 26S proteasome.

2. The composition of Claim 1, wherein said inhibitor inhibits the 2OS catalytic core of the 26S proteasome.

3. The composition of claim 1, wherein the small molecule inhibitor is selected from the group consisting of imidizolines 1-9 as set forth in Figure 8.

4. The composition of claim 1 , wherein the compound is a therapeutically effective amount for reducing a symptom of a disease selected from the group consisting of cancer and inflammatory disease.

5. The composition of claim 1, wherein the cancer is multiple myeloma.

6. The composition of claim 1 , wherein the inflammatory disease is rheumatoid arthritis.

7. A method, comprising, a) providing, i) a pharmaceutical composition comprising a small molecule non- covalent inhibitor of the catalytic region of a 26S proteosome and ii) a patient, wherein said patient is in need of treatment and b) administering said pharmaceutical composition to said patient as a therapeutically effective amount

8.The method of Claim 7, wherein said inhibitor inhibits the 2OS catalytic core of the 26S proteasome.

9. A method, comprising, a) providing, iii) a pharmaceutical composition comprising a small molecule noncompetitive inhibitor of the 26S proteosome and iv) a patient, wherein said patient is in need of treatment; and b) administering said pharmaceutical composition to said patient.

10. The method of claim 9, wherein the small molecule is selected from the group consisting of imidizolines 1-9 as set forth in Figure 8.

11. The method of claim 9, wherein the patient shows a symptom selected from the group consisting of a cancer and an inflammatory disease.

12. The method of claim 11 , wherein the cancer is multiple myeloma.

13. The method of claim 11 , wherein the inflammatory disease is rheumatoid arthritis.