AU2015249184A1 - Compositions and methods for enhancing proteasome activity - Google Patents

Compositions and methods for enhancing proteasome activity Download PDF

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AU2015249184A1
AU2015249184A1 AU2015249184A AU2015249184A AU2015249184A1 AU 2015249184 A1 AU2015249184 A1 AU 2015249184A1 AU 2015249184 A AU2015249184 A AU 2015249184A AU 2015249184 A AU2015249184 A AU 2015249184A AU 2015249184 A1 AU2015249184 A1 AU 2015249184A1
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compound
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proteasome
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hydrogen
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Daniel Finley
Timothy C. Gahman
Randall W. King
Byung-Hoon Lee
Min Jae Lee
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Harvard College
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Harvard College
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Abstract

Abstract Proteinopathies result from the proteasome not acting efficiently enough to eliminate harmful proteins and prevent the formation of the pathogenic aggregates. As described herein, inhibition of proteasome-associated deubiquitinase Usp14 results in increased proteasone efficiently. The present invention therefore provides novel composition and methods for inhibition of Usp14, enhancement of proteasome activity and treatment of proteinpathies.

Description

Compositions and Methods for Enihancig Proteasome Activity RELATED APPLICATIONS 5 This application claims the benefit of priority to United States Provisional Patent Application serial number 6 1/373,404, filed August 13, 2010, and United States Provisional Patent Application serial number 61/336,959, filed January 28, 2010; the contents of both of which are hereby incorporated by reference, GOVERNMENT SUPPORT 10 This invention was made with U.S. Government support under National Institutes of Health Grant Nos. GM065592, GM66492, and DK082906. The government has certain rights in the invention. BACKGROUND The proteasomne is a large protein complex that contains 33 distinct subunits. I5 Proteasonic complexes function as proteases in part to degrade unneeded or misfolded proteins. Proteasomes regulate many aspects of cell physiology, and protcasome dysfunction has been implicated in a variety of diseases, including cancer and neurodegenerative diseases (Finley D., (2009), Annu. Rev. Biochem., 78, 477-513; Hoeller and Dikic, (2009), Nature, 458, 438-444; Demarto and Gillette, (2007), Cell, 129, 659-662); 20 Dahlmann, B. (2007) BCB Biochem 8, Suppl 1, S3; Schartz AL and Ciechanover A (2009) Ann Rev Pharmacol Toxicol 49, 73-96). Most, but not all. proteasome substrates are targeted for degradation via the covalent attachment of multimeric chains of a small, highly-conserved protein called ubiquitin. Because longer ubiquitin chains interact more strongly with the proteasonme than shorter 25 chains (Thrower et al (2000), EMBO J. 19, 94-102), processes that alter ubiquitin chain length frequently also affect substrate degradation rates. The length of ubiquitin chains attached to substrates tagged for proteasome degradation can be modulated by certain proteasome-associated deubiquitinating enzymes and ubiquitin ligases. These dcubiquitinating enzymes and ligases appear to regulate proteasome activity by 30 disassembling or extending protcasome-bound ubiquitin chains. Mammalian proteasomes contain three major dcubiquitinating enzymes: Rpn 1 1, Uch37, and Usp14 (Finley D., (2009), Annu. Rev. Biochem., 78, 477-513). Rpnl I removes ubiquitin from the tagged substrate by cutting at the junction between the ubiquitin - I chain and the substrate. Because the Rpn I 1 -mediated cleavage occurs following a substrate's commitment to proteolysis. but prior to substrate degradation, Rpn 1 I helps to prevent ubiquitin from being degraded along with the substrate, thus minimizing fluctuations in cellular ubiquitin levels. Additionally, because the proteasome substrate 5 must pass through a narrow translocation channel before encountering the proteasome's sequestered proteolytic sites, removal of a bulky ubiquitin chain may also facilitate substrate translocation. Thus, removal of the ubiquitin chain by Rpnl I promotes substrate degradation through en bloc removal of the ubiquitin chain at a relatively late step in the proteasome pathway (Verma et a/, (2002) Science, 298, 611-615: Yao and Cohen, (2002), 10 Nature, 419, 403-407). In contrast to Rpn 11, Uch37 functions prior to the commitment of a substrate to proteasome degradation. Uch37 disassembles ubiquitin chains at the substrate-distal tip (Lam et i., (1997), Nature, 385, 737-740), and its enzymatic activity shortens chains rather than remove them entirely. It has been proposed that chain trimming by Uch37 increases 15 the ability of the proteasome to discriminate between long and short multiubiquitin chains (Lam et al, (1997), Nature, 385, 737-740). Little is known about how Uch37 may regulate proteasome function in cells. Very little is known about the function of Uspl4. However, the yeast ortholog of Usp 14, Ubp6, has been suggested to disassemble ubiquitin chains at the substrate-distal tip 20 and to function prior to the commitment of a substrate to proteasonie degradation. (Hanna et aL, (2006), Cell, 127(7), 1401-1413). Ubp6 is thought to act as a proteasome inhibitor, and prior work on Ubp6 has indicated a noncatalytic mode of proteasome inhibition (Hanna er al., (2006), Cell, 127(7), 1401-1413). SUMMARY 25 The present invention provides novel compositions and methods for the inhibition of Usp l4, the enhancement of proteasome activity and the treatment of proteinopathies and other diseases for which enhanced protein breakdown may be therapeutic. Aside from proteinopathies, the enhancement of proteasome activity may be therapeutic for any disease characterized by deficient proteasome activity, or deficient activity of other components of 30 the ubiquitin-proteasome pathway, such as in von Hippel-Lindau disease, spinocerebellar ataxia 1, Angelman syndrome, giant axon neuropathy, inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD), and others (Lehman, N. L., (2009), Acta Neuropathologica, 118(3), 329-347;Weihl et al, (2007), Neuromuscular -2 - Disorders, 17, 87-87). Enhancing proteasome activity could also be therapeutic for diseases in which proteasome substrates are involved and contribute to pathology, but which do not satisfy a strict definition of proteopathies. For example, numerous oncoproteins are proteasome substrates and their ability to promote cancer could potentially be attenuated by 5 enhancing proteasome activity. One aspect of the invention relates to a compound represented by formula I, II, III, IV, V, Vi, VII, or VIII. or pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof; wherein the formula are as defined below. 10 Another aspect of the invention relates to a method of inhibiting the dcubiquitination activity of a Usp 14 protein comprising contacting the Usp 14 protein with IUI or a compound of formula I, II, Ill, IV, V, Vi, VI1, or Vill. or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof. 15 Another aspect of the invention relates to a method of enhancing protein degradation by a proteasome in a cell comprising contacting the cell with IUl or a compound of formula I, II, III, IV, V, VI, VII, or VIII, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stereoisomer thereof. 20 Another aspect of the invention relates to a method of treating or preventing a proteinopathy in a subject comprising administering to the subject IUl or a compound of formula I, II, III, IV, V, VI, VII, or VIII, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, or a pharmaceutical composition comprising the same. 25 Another aspect of the invention relates to a method of enhancing proteasome function in a subject comprising administering to the subject IU1 or a compound of formula I, 11, III. IV, V, VI, VII, or VIII, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stereoisomer thereof, or a pharmaceutical composition comprising the same. 30 Another aspect of the invention relates to a method of increasing degradation of Tau, TDP-43 or ataxin-3 in a subject comprising administering to the subject IU1 or a compound of formula 1, II, 111, IV, V, VI, VII, or VIII, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer -3 thereof, or a pharmaceutical composition comprising the same. Another aspect of the invention relates to an isolated proteasome lacking enzymatically active Uch37 and comprising enzymatically active Usp 14. In certain embodiments the proteasome comprises enzymatically inactive Uch37 and/or vinylsulfone 5 Uch37 adducts. In some embodiments the enzymatically active Uspl4 is a recombinant protein. In certain embodiments the proteasome is a human proteasome or a murine proteasome. Another aspect of the invention relates to a method of generating a proteasome comprising enzymatically inactive Uch37 and further comprising enzymatically active 10 Usp14 comprising purifying a proteasome lacking Usp14 but comprising Uch37, treating the purified proteasonc with a deubiquitinase inhibitor, and reconstituting the purified proteasome with enzymatically active Uspl 4. in certain embodiments the proteasone is a human proteasome or a murine proteasome. In some embodiments the proteasome is purified from HEK293 cells. In some embodiments the deubiquitinase inhibitor is 15 ubiquitin-vinylsulfone. In certain embodiments the active Usp 14 is recombinantly produced. Another aspect of the invention relates to a method of screening for an inhibitor of Uspl4 comprising providing a proteasorne comprising enzymatically inactive Uch37 and Further comprising enzymatically active Usp 14, contacting the proteasome with a test 20 compound and a Usp 14 substrate, and determining whether the test compound inhibits the deubiquitination of the substrate. In certain embodiments, the substrate is coupled to a reporter that is detectable after cleavage by a deubiquitinase and/or is an ubiquitin dependant proteasome substrate. In some embodiments the substrate is Ub-AMC or polyubiquitinated cyclin B. In certain embodiments, dcubiquitination of the substrate is 25 demonstrated by inhibition of substrate degradation. In some embodiments the proteasorme comprises vinylsulfone-Uch37 adducts, In certain embodiments the Uspl4 is a recombinant protein, In some embodiments the proteasome is a human proteasome or a murine proteasome. Another aspect of the invention relates to a kit comprising an isolated proteasome 30 lacking enzymatically active Uch37 and comprising enzymatically active Usp14, and instructions of use. In certain embodiments, the kit can comprise a UspI4 substrate. In some embodiments the Usp14 substrate is Ub-AMC and/or polyubiquitinated cyclin B. -4- Additional aspects, embodiments, and advantages of the invention are discussed below in detail. Moreover, the foregoing information and the following detailed description are merely illustrative examples of various aspects and embodiments of the invention, and are intended to provide an overview or framework for understanding the nature and 5 character of the claimed aspects and embodiments. BRIEF DESCRIPTION OF TH E DRAWINGS Figure IA shows an immunoblot that was performed using either recombinant Usp14 protein (Purified Usp 14) or affinity-purified Usp 14 deficient human proteasomcs (Human Proteasome) and anti-Usp14 antibody. The band corresponding to Usp1 4 is 10 indicated. Figure lB shows an immunoblot that was performed using anti-Uch37 antibody and Usp 14-deficient purified human proteasomes (26S) either untreated (-VS) or treated with Ub-VS (+VS). The band corresponding to Uch37 is indicated. Nonspecific bands are indicated with an asterisk. 15 Figure 2A shows a nondenaturing gel analysis that had undergone in-gel suc LLVY-AMC staining (indicating presence of proteasomes) that was performed using commercially available human proteasomes (Biomol), untreated, purified Usp14 deficient human proteasomes (-Ub-VS) or Ub-VS treated purified Usp14 deficient human proteasomes (+Ub-VS). 20 Figure 2B shows a Coomassic Brilliant Blue (CUB) staining of purified, recombinant wild-type Uspl4 (UspI4-wt) or catalytically inactive mutant Usp14 (Uspl4 Cl 14A) either with or without a GST tag, along with a GST control (GST) and a protein size marker (Marker). Figure 2C shows the results of a gel-shift assay of protcasomes alone (-), GST and 25 proteasomes (GST), GST tagged wild-type Usp 14 and proteasomes (GST-Usp 1 4-wt), GST tagged catalytically inactive mutant Usp14 and proteasomes (GST-Usp14-CI 14A), untagged wild-type Usp14 and proteasomes (Uspl4-wt) or untagged catalytically inactive mutant Uspl4 and proteasomes (Uspl4-Cl 14A) that had either been stained with in gel suc-LLVY-AMC staining (top, to show the presence of proteasomes) or Coomassie 30 Brilliant Blue (CBB) staining. Figure 3 shows the results of a Ub-AMC hydrolysis assay for Usp14 activity in the presence or Ub-VS treated human proteasomes. Figure 4A shows a plot of the linear kinetics (R 2 > 0.99) of the initial rates of Ub -5 - AMC hydrolysis by Usp14 and proteasome at I pM Ub-AIMC, I nM proteasome, and the indicated concentration of Usp 14. Figure 4B shows a Michaelis-Menten plot of Usp14-dependent Ub-AMC hydrolysis in the presence of human proteasome for 25 minutes at 1 pM Ub-AMC, 1 nM 5 proteasome, and the indicated concentration of Uspl4. Figure 4C shows a plot of the linear kinetics (R2 > 0.99) of the initial rates of Ub AMC hydrolysis by Usp14 and proteasone at 4 nM Usp 14, 1 nM proteasone and the indicated concentration of Ub-AMC. Figure 4D shows a Michaelis-Menten plot of concentration-dependent Ub-AMC 10 hydrolysis in the presence of Usp 14 and human proteasome for 30 minutes at 4 nM Usp 1 4, I nM proteasome and the indicated concentration of Ub-AMC. Figure 5 shows an immunoblot that was performed using an antibody specific for Cyclin B, which also detects polyubiquitinated Cyclin B (Ubr-ClbB). In this experiment, Ub,,-CIbB was treated with 26S human proteasome alone, human proteasome and wild-type 15 Usp I4 (Usp14-wt) or human proteasome and catalytically inactive Usp14 (Usp14-CA), and subsequently analyzed by irmmunoblotting. Figure 6A shows a diagram of human Usp 14, depicting the ubiquitin-like domain (UBL), the catalytic domain (CAT), the location of exon 4 and the position of Cysl 14. Figure 6B shows immunoblots that were performed on cellular lysates from human 20 293 cells that co-expressed Tau along with either wild-type Usp14 (Usp14-wt), catalytically inactive Usp l4 (Usp 14-CA), short form Uspl4 (Usp 14-SF) or UBL domain deficient Usp 14 (Usp 14-AUBL) and stained using antibodies specific for Tau, Usp 14 or Actin, as indicated. Figure 7 shows immunoblots that were performed on cellular lysates from 293 cells 25 that co-expressed the indicated forms of flag-tagged Usp 14 along with tagged hRpnl I either before (Extract) or after (Purified proteasome) proteasome affinity purification and stained using anti-Flag antibody. Where indicated, Ub-VS was incubated with lysate prior to proteasome purification. Extract samples represent 5% of total. Asterisks, nonspecific signals. Proteasome subunit Rpn 13, load control. Control samples, empty vector. 30 Figure SA shows a statistical plot of the high-throughput large scale compound screening for inhibitors of Usp14 catalytic inhibitors. Figure 8B shows a frequency distribution curve used to determine AMC quenching compounds (control = bottom curve). -6- Figure 9A shows the chemical structure of Usp14 inhibitor IUI. Figure 9B shows a graph depicting (left) the inhibition of Usp14 loaded proteasome (Usp14-Ptsrn) deubiquitinase activity by IUI (left bar = 0 pM, second to left bar = 4 pM, third bar = 8 [LM, right bar = 17 pM) and (right) the lack of quenching of AMC 5 fluorescence by IUl (left bar = 0 pM, right bar = 17 pM). Figure 9C shows a graph depicting (left) the inhibition of Usp 14 loaded proteasome (Usp 14-Ptsm) deubiquitinase activity by ]U 1 (left bar = 0 pM, middle bar = 8 pM, right bar = 17 pM) and (right) the lack of inhibition of Isopeptidase T (IsoT) by IU I (left bar = 0 pM, middle bar = 8 pM, right bar= 17 pM). 10 Figure 9D shows a graph depicting the lack of inhibition of Usp 14 that has not been complexed to the proteasome by either vehicle (DMSO), lU 1 or IlUl IC. Figure 9E shows a graph depicting the lack of inhibition of 26S human proteasomes (Ptsm) that had not been treated with Ub-VS by either vehicle (DMSO) or IUl (i.c, proteasomes which lack Usp14). 15 Figure 1 OA shows the chemical structure of IU I C, an inactive control compound for lUI. Figure lOB shows a plot comparing the deubiquitinase inhibition activity of [U I (bottom circles, bottom triangles) with the deubiquitinase inhibition activity of IUl C (top circles, top triangles, squares). 20 Figure 1 OC shows the ineffectiveness of JUl C in promoting Tau degradation. Immunoblots were performed using lysates of MEF cells that co-expressed Tau and Usp14 and that were treated with 0, 25, 50, 75 or 100 pM IUIC and stained with antibodies specific for either Tau or Actin. Figure 10D shows a graph depicting inhibition of the deubiquitinase activity of the 25 indicated deubiquitinases by IUIC (left bars = 0 pM, right bars = 17 pM). Figure 11A shows a plot depicting the deubiquitinase activity of proteasome bound Usp 14, IsoT or Uch37 that had been treated with the indicated concentration of IUI. Figure 11B shows a graph depicting the inhibition of the deubiquitinase activity of the indicated dcubiquitinases by lUl (left bars = 0 LM, right bars= 17 M). 30 Figure 12A shows immunoblots of purified 26S human proteasomes (~4 nM) that had been incubated with or without Uspl4 (80 nM) and treated either with vehicle (DMSO), IUI C or IUI at the indicated concentrations and stained with antibodies specific for either Usp 14 or Alpha7. The asterisk (*) denotes a nonspecific signal generated by the -7anti-Usp 14 antibody. Figure 12B shows immunoblots as in Figure 12A, except -2-fold molar excess of Usp 14 was incubated with the proteasome in the absence or presence of the indicated compound (30 pM). 5 Figure 13 shows graphs depicting the deubiquitination activity of proteasome bound Usp 14 that had been treated with vehicle (control) or IU 1 and subjected to the indicated number of rounds of ultrafiltration (spins, left panel: no spin = left bar, lx spin = middle bar, 3x spin = right bar), or spin-column gel filtration (right panel; control = left bar, IU 1 = right bar). 10 Figure 14A shows a plot depicting an IC5o curve of proteasome bound Usp14 treated with the indicated concentration of IU I for 45 minutes. Figure 14B shows a plot depicting an IC 5 O curve of proteasome bound Usp14 treated with the indicated concentration of IU I for 30 minutes. Figure 15 shows an immunoblot that was performed using an antibody specific for 15 Cyclin B and polyubiquitinated Cyclin B (Ub,-CIbB) that had been treated with 4 nM of 26S human proteasome, either alone, with wild-type Usp 14 (Usp I 4-wt), with I U I, and/or with protcasome inhibitor as indicated. The immunoblot was either subject to a long exposure (Long exp) or a short exposure (Short exp). Figure 16A shows an immunoblot that was performed using an antibody specific 20 for Cyclin B and polyubiquitinated Cyclin B (Ub, 1 -CIbB) that had been treated with 4 nM of 26S human proteasome, either alone or in combination with wild-type Usp 14 (60 nM) along with either vehicle or IU l (34 pM). Figure 16B shows an immunoblot that was performed using an antibody specific for T7-tagged Sic I ' and polyubiquitinated Sic I " (Ub 1 -Sic11'*) that had been treated with 25 5 nM of 26S human proteasome, either alone or in combination with wild-type Usp 14 (75 nM) along with either vehicle or IU 1 (75 pM), Figure 17 shows plots depicting the ion counts of Liquid Chromatography/Mass Spectrometry (LC/MS) traces of lysates from M EF cells that had not been treated with IU I (No IUl), or been treated with Ifl for I or 24 hours. The bottom panel depicts the ion 30 count for an IUI standard solution at I pg/mL. Figure 18 shows plots depicting the ion counts of LC/MS traces of various concentrations of IU l standard. Figure 19 shows the UV spectrum of IU I, depicting absorption maxima at 255 nm - 8and 305 nm (left) and HPLC chromatograms showing the time-dependence of IU l internalization into cells, followed at 300 nm (right). Figure 20A shows a graph depicting the JUl concentration in MEF cells after normalization by cell number as detected by UV absorption assay. 5 Figure 20B shows a graph depicting the IU 1 concentration in 293 cells after normalization by cell number as detected by UV absorption assay. Figure 21A shows immunoblots that were performed using lysates of MEF cells that co-expressed Tau and Usp 14 and that were treated with 0, 25, 50, 75 or 100 [IM IU I and stained with antibodies specific for Tau, LacZ or Actin. 10 Figure 211B shows the result of quantitative RT-PCR analysis of Tau RNA levels in MEF cells that co-expressed Tau and Usp 1 4 and that were treated with 0, 25, 50, 75 or 100 pM IUl. Figure 22A shows immunoblots that were performed using tysates of MEF cels that co-expressed TDP43" and UspI4 and that were treated with 75 pM IUl for the 15 indicated number of hours and stained with antibodies specific for TDP431"- LacZ or Actin. Figure 22B shows immunoblots that were performed using lysates of MEF cells that co-expressed either Atx3-Q80 or Atx3-Q22 along with Uspl4 and that wcre treated with 0, 50 or 100 pM IU I and stained with antibodies specific for Atx3 or Actin. 20 Figure 22C shows immunoblots that were performed using lysates of MEF cells that co-expressed either wild-type GFAP (GFAP-wt), K63Q mutant GFAP (GFAP-K63Q) or E2 10K GFAP (GFAP-E2OK) along with Usp14 and that were treated with 0, 25, 50 or 100 pM IUl and stained with antibodies specific for GFAP or Actin. Figure 22D shows the result of quantitative RT-PCR analysis of TDP-43 RNA 25 levels in MEF cells that co-expressed TDP-43 and Usp-14 and that was treated with 75 pM IUI for each indicated time. Figure 23A shows immunoblots stained with anti-DNPH or anti-Actin antibodies that were performed on DNPH-treated lysates of MEF cells that were preincubated with vehicle or 75 pM IU I and treated with 63 LaM menadione, as indicated. 30 Figure 23B shows immunoblots stained with anti-DNPH or anti-Actin antibodies that were performed on DNPH-treated lysates of HEK293 cells that were preincubated with IUl (75 pM) or protcasome inhibitors (20 pM MG132, 10 pM PS-341) for 4 h, then treated with menadione (300 pM) for 60 min. -9- Figure 24 shows a plot depicting MEF cell viability as assessed by MTT assay upon treatment with the indicated concentration of IU I for 48 hours. Figure 25A shows a plot depicting MEF cell viability as assessed by MT assay upon treatment with the indicated concentration of I U 1 for 6 hours. 5 Figure 25B shows a plot depicting MEF cell viability as assessed by MTT assay upon treatment with the indicated concentration of IU I for 12 hours. Figure 25C shows a plot depicting MEF cell viability as assessed by MTT assay upon treatment with the indicated concentration of IU I for 24 hours. Figure 26A shows a plot depicting 293 and HcLa cell viability as assessed by MTT 10 assay upon treatment with the indicated concentration of IUI for 6 hours. Figure 26B shows a plot depicting 293 and HeLa cell viability as assessed by MTT assay upon treatment with the indicated concentration of IUI for 12 hours. Figure 26C shows a plot depicting 293 and HeLa cell viability as assessed by MTT assay upon treatment with the indicated concentration of IUl for 24 hours. 15 Figure 27A shows fluorescent microscopy images of TUNEL stained MEF cells that had been treated with 100 gM I U I or control for 6 hr. Figure 27B shows a graph depicting the quantification of the TUNEL staining analysis depicted in Figure 3 IA. Figure 28 shows a plot depicting the percent confluence of MEF cells that had been 20 treated with the indicated concentration of vehicle or IUI for the indicated period of time. Figure 29 depicts one approach to compounds of the invention. Figure 30 depicts a graph showing MTT assay for cell viability: specifically, IU 1 effects on cell survival upon oxidative stress. Experiment performed in HEK293 cells with Menadione (dose-dependent, 4hr) and IUl (50 uM, 6 hr). MEF cells show this effect as 25 well. The effect on the IC 5 for menadione is almost 4-fold. Figure 31 depicts a table of selected compounds of the invention, including some percent inhibition and IC 5 values. Percent inhibition was measured at 8 ptM from IU 1-I to IU-46, at 4 LM from IU 1-47 to IU 1-96, and at 17 ItM for C'l to C9. Figure 31A shows immunoblots that were performed using lysates of Uvpl14-/ 30 MEF cells that co-expressed tau and LacZ and that were treated with 0, 25, 50, 75 or 100 uM IU 1 and stained with antibodies specific for tau, LacZ or Actin. Figure 31B shows immunoblots that were performed using lysates of UVp14-/ MEF cells that co-expressed TDP-43lt! and LacZ and that were treated with 75 pM JUl for - 10the indicated number of hours and stained with antibodies specific for TDP-431", LacZ or Acting. Figure 32 shows irnmunoblots that were performed using lysates of wild-type MEF cells that co-expressed tau and Ub-independent proteasome substrate cODC-EGFP and that 5 were incubated with 50 pLM JU I for 6 h and stained with antibodies specific for tau, cODC EGFP or Actin. Proteasone inhibitors (30 PM MG 132, 10 pIM PS-341) were treated 4 hr before lysis. Figure 33 shows immunoblots that were performed using lysates of wild-type MEF cells that co-expressed HA-tagged Ub and/or Flag-tagged TDP-43 and that were incubated 10 with 50 pM 1IJ For 6 hr and stained with antibodies specific for Flag-TDP-43, HA-Ub, or Actin. Proteasome inhibitors (20 PM MG132, 10 LM PS-341) were added 4 hr before lysis. Arrows indicate likely ubiquitinated TDP-43 species. HC, heavy chain of antibody. Figure 34A shows immunoblots that were performed using lysates of wild-type or UspI14 ' MEF cells that were treated with 1Ul (0, 25, 50, 75, or 100 pM) for 6 hr and 15 stained with antibodies specific for Ub, Actin, CP subunit a7, or RP subunit mRPT5. Figure 34B shows quantification of ubiquitin levels in Figure 34A. Polyubiquitin and monoubiquitin levels from wild-type and Usjp4~' MEF cells were quantified after treatment of various concentration of IUl. Ub signals were normalized to that of endogenous actin, Quantification was achieved by densitometry of a film image (left bar = 20 poly UB (+/+ MEF); second bar to the left = poly Ub (Usp14 MEF); third bar = mono Ub (+/+ MEF); right bar = mono Ub (Usp/i4 MEF)). Figure 35A shows the specificity of 1Ul for USP14 which is observed independently of Ub-AMC concentration. Assays of Ub-AMC hydrolysis were done as in Figure 11B, except lower concentrations of Ub-AMC were used (left bar = 0 pM; right bar 25 = 17 pM). Figure 35B shows the summary of K\ values for Ub-AMC of deubiquitinating enzymes in this study. KI values of DUBs used in the selectivity assays were obtained from the literature. Unknown K,, values were determined in this study, as indicated. These values are significant because the DUB assays should be most sensitive to inhibition when 30 substrate is at a low concentration as compared to the Km of the enzyme in question. CD, catalytic domain. Figure 36 shows that IU I inhibits proteasome-associated USP 14 activity without a - 11 detectable lag period. 2.5 n.M of human proteasome was mixed with 30 nM of recombinant USP14 protein. The reaction was then initiated by adding I M Ub-AMC. After 30 min, I U I (100 pM) or vehicle (DMSO) was added to the sample. Figure 37 confirms Figure 13, except prolonged incubation (5 and 8 hr) was tested. 5 The percent USP14 activity was normalized to 26S peptidase activity (i.e. LLVY-AMC hydrolysis). IUI was added to 100 PM (left bar = DMSO; right bar = IU1). Figure 38 shows in vitro chain trimming assays that were performed with human proteasome purified in the presence of ADP (ADP prep) and assayed in the presence of ADP. Immunoblot was performed using an antibody specific for human cyclin B (CCNB). 10 IUl is effective at inhibition of chain trimming at approximately 5 aM, as expected from Ub-AMC hydrolysis data. Figure 39 shows that IUl does not affect cyclin B degradation in the presence of USP 14-CA. Assays were done as in Figure 5. Figure 40A shows 'H-NMR spectroscopic data of U I. 15 Figure 40B shows LC/MS analysis of JUl. TIC, total ion count. SPC, shared peak count extracted from the peak with the indicated retention time. Figure 41A shows 'H-NMR spectroscopic data of IU I C. Figure 41B shows LC/MS analysis of IUIC. TIC, total ion count. SPC, shared peak count extracted fi-on the peak with the indicated retention time. 20 Figure 42A shows a graph depicting rapid release of the internalized IUl from wild-type MEF cells. After wild-type MEFs were incubated with 50 IM of lUl for one hour, the culture media were replaced with fresh media without IUl. Internalized IUI was monitored at the indicated times and its concentration was normalization by cell number as detected by UV absorption assay. 25 Figure 42B shows the comparable concentration of IU I from I hr to 48 hr in the scrum-containing media of H EK293 cells. Figure 42C shows the comparable concentration of IU I from I hr to 48 hr in the serum-containing media of Usp4- MEF cells. Figure 43A shows the quantitative analysis of tau levels after IUl treatment in 30 wild-type MEFs. Quantification was performed with infrared dye-conjugated secondary antibodies using Odyssey imaging system. Tau signal intensities were normalized to that of endogenous actin and relative amounts are shown. - 12- Figure 43B shows the quantitative analysis of tau levels after IU I treatment in Uspt4 MEFs. Quantification was performed as in Figure 43A. Figure 44A shows that the immunofluorescence signal of transiently expressed mCherry-NBRI (top row) was significantly increased after treatment with 200 LIM of 5 bafilomycin A, (BafAi), an autolysosome formation inhibitor, for 6 hr in wild-type MEFs. Figure 44B shows that the stimulation of tau degradation by IUI is not mediated by autophagy. Wild-type MEF cells were transfected with a plasmid expressing tau and then treated with 200 UM of BafA and/or 75 IM of JUl for 6 hrs, and analyzed by SDS PAGE/immunoblot using the Odyssey infrared imaging system. 10 Figure 44C shows the quantification of normalized tau protein level from three independent experiments (mean ± SD) as performed in Figure 44B using Odyssey software, Figure 45A shows that IU I treatment does not affect the integrity of proteasome. Total cell extracts (50 ag/lanc) before and after a 6-hr IU I treatment (100 LOM) were 15 resolved by native PAGE, and the proteasorne was visualized using either an in-gel activity stain with a fluorogenic peptide substrate (LLVY-AMC), or immunoblotting with antibodies specific to subunit a6. RP 2 -CP and RP-CP indicate distinct forms of the 26S proteasome. Figure 45B shows that IUl treatment does not induce the transcription of Psmb5 20 gene, a proteasome subunit. A luciferase reporter gene containing the murine Psmb5 promoter (-1 kb to 0 kb) was transiently expressed in wild-type and Usp/i4 MEFs and promoter activity was assessed following incubation of 25 or 50 IM of IUl for 8 hr. For normalization of luciferase activity, a control experiment using the promoter-less pGL3 plasmid was performed. Values are mean ± SD from three independent experiments. RLU, 25 relative light un its. Figure 45C shows that JUl treatment does not induce the transcription of UbB, a ubiquitin gene. Quantitative RT-PCR was performed using total mRNA from +/+ (left panels) and Usp4t MEFs (right) after incubation with a graded doses of IU 1 for 6 hr. Figure 45D shows that JUl treatment does not induce the transcription of a6, a 30 proteasome subunit. Quantitative RT-PCR was performed using total mRNA from +/+ (left panels) and Usp14* MEFs (right) after incubation with a graded doses of IU 1 for 6 hr. Figure 45E shows that IUl treatment does not induce the transcription of a7, a - 13proteasome subunit. Quantitative RT-PCR was performed using total mRNA from +/+ (left panels) and UspI4i' MEFs (right) after incubation with a graded doses of IU I for 6 hr. Figure 46A depicts a graph showing MTT assay for cell viability: specifically, IUIC effects on cell survival upon oxidative stress. Experiment performed in HEK293 5 cells with Menadione (dose-dependent, 4hr) and IU I C (50 uM, 6 hr). Figure 46B shows a graph depicting the internalized IUlC concentration in wild type MEF cells. After the indicated time-course treatment of 50 uM of IUI C, IUlC levels were measured by LC/MS. The concentration shown was normalized by cell number as detected by UV absorption assay. 10 Figure 46C shows a graph depicting the internalized IU I C concentration in 293 cells, After the indicated time-course treatment of 50 uM of IU I C, IU IC levels were measured by LC/MS. The concentration shown was normalized by cell number as detected by UV absorption assay. Figure 46D shows a graph depicting rapid release of the internalized IU I C from 15 wild-type MEF cells. The experiment was performed as in Figure 42A. Figure 46E shows a graph depicting rapid release of the internalized I U I C from 293 cells. The experiment was performed as in Figure 42A. Figure 47 shows an immunoblot that were performed using lysates of wild-type MEF cells that transiently expressed Tau and that were treated with 0, 20, 40, 60 or 80 tM 20 of lU1-47, a more potent IUI derivative, and stained with an antibody specific for Tau. DETAILED DESCRIPTION Proteinopathies are a class of diseases and disorders that result from the aggregation of abnormal or misfolded proteins. Often, and perhaps typically, such proteins are eliminated from cells through protcasome-mediated degradation. However, in the case of 25 proteinopathics, the proteasome does not act efficiently enough to eliminate all of the harmful proteins and prevent the formation of the pathogenic aggregates. As is demonstrated herein, under normal growth conditions, the protcasome is subject to tonic inhibition brought about by the trimming of substrate-bound ubiquitin chains by Usp14. Ubiquitin chain trimming inhibits the proteasome because it removes 30 from proteasome substrates the signal (a ubiquitin chain) that allows recognition by the proteasome; the proteasome-bound substrate can therefore escape without being degraded. Consequently, an inhibitor of chain trimming by Usp]4 promotes protein degradation by the proteasone. Thus, as a result of this inhibitory mechanism, the mammalian proteasome -14pathway does not ordinarily operate at full efficiency because the pathway is partially inhibited by Usp 14. The methods and compositions of the present invention enhance proteasome activity by inhibiting the deubiquitinase activity of Usp14. As demonstrated herein, this enhanced 5 proteasone activity increases the ability of a cell to eliminate abnormal or misfolded proteins, including those associated with human disease. The methods and compositions of the present invention are therefore useful for the enhancement of proteasome function and the treatment of proteinopathics. Definitions 10 In order for the present invention to be more readily understood, certain terms and phrases are defined below and throughout the specification. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. 15 The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the lccments so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the 20 elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another 25 embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, 30 additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of' or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the - 15other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase "at least one," in 5 reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of clements. This definition also allows that elements may optionally be present other than the elements 10 specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including 15 elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any 20 methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," 25 "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of' shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. The definition of each expression, e.g., alkyl, m, n, and the like, when it occurs 30 more than once in any structure, is intended to be independent of its definition elsewhere in the same structure. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted - 16atom and the substituent, and that the substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. The term "substituted" is also contemplated to include all permissible substituents of 5 organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein below. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the 10 heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds. The term "lower" when appended to any of the groups listed below indicates that 15 the group contains less than seven carbons (i.e. six carbons or less). For example "lower alkyl" refers to an alkyl group containing 1-6 carbons, and "lower alkenyl" refers to an alkyenyl group containing 2-6 carbons, The term "saturated," as used herein, pertains to compounds and/or groups which do not have any carbon-carbon double bonds or carbon-carbon triple bonds. 20 The term "unsaturated," as used herein, pertains to compounds and/or groups which have at least one carbon-carbon double bond or carbon-carbon triple bond. The term "aliphatic," as used herein, pertains to compounds and/or groups which are linear or branched, but not cyclic (also known as "acyclic" or "open-chain" groups). The term "cyclic," as used herein, pertains to compounds and/or groups which have 25 one ring, or two or more rings (e.g., spiro, fused, bridged). The term "aromatic" refers to a planar or polycyclic structure characterized by a cyclically conjugated molecular moiety containing 4n+2 electrons, wherein n is the absolute value of an integer. Aromatic molecules containing fused, or joined, rings also are referred to as bicylic aromatic rings. For example, bicyclic aromatic rings containing heteroatoms 30 in a hydrocarbon ring structure are referred to as bicyclic heteroaryl rings. The term "hydrocarbon" as used herein refers to an organic compound consisting entirely of hydrogen and carbon. For purposes of this invention, the chemical elements are identified in accordance - 17with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. The term "heteroatom" as used herein is art-recognized and refers to an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, 5 oxygen, phosphorus, sulfur and selenium. The term "alkyl" means an aliphatic or cyclic hydrocarbon radical containing from I to 12 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-buty L, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-methylcyclopentyl, and I -cyclohexylethyl. 10 The term "substituted alkyl" means an aliphatic or cyclic hydrocarbon radical containing from I to 12 carbon atoms, substituted with 1, 2, 3, 4. or 5 substiuents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio, 15 alkyenylthio, alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl, fluroralkylsul fonyl, alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl, fluroralkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfony, aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl, fluroralkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl, fluroralkoxysulfinyl, alkenyloxysulfinyl, 20 alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylearbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalky Icarbonyloxy, fluoroalkylcarbonyloxy, alkenylearbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluroralkylsulfonyloxy, 25 alkenylsulfonyloxy, alkynylsulfonyloxy, haloalkoxysul fonyloxy, fluroralkoxysulfonyloxy, alkenyloxysulfonyloxy, alkynyloxysul fonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy, fluroralkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy, fluroralkoxysulfinyloxy, alkenyloxysufinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, 30 cyano, nitro, azido, phosphinyl, phosphoryl, silyl and silyloxy. The term "alkylene" is art-recognized, and as used herein pertains to a bidentate moiety obtained by removing two hydrogen atoms of an alkyl group, as defined above. - 18- The term "alkenyl" as used herein means a straight or branched chain hydrocarbon containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5 5 hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl. The term "alkynyl" as used herein means a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but arc not limited, to acetylenyl, I -propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and I -butynyl. 10 The term "carbocycly" as used herein means monocyclic or multicyclic (e.g., bicyclic, tricyclic, etc.) hydrocarbons containing from 3 to 12 carbon atoms that is completely saturated or has one or more unsaturated bonds, and for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system (e.g. phenyl). Examples of carbocyclyl groups include 1-cyclopropyl, 1 -cyclobutyl, 2-cyclopentyl, I 15 cyclopentenyl, 3-cyclohexyl, I -cyclohexenyl and 2-cyclopentenylnethyl. The term "heterocyclyl", as used herein include non-aromatic, ring systems, including, but not limited to, monocyclic, bicyclic (e.g. fused and spirocyclic) and tricyclic rings, which can be completely saturated or which can contain one or more units of unsaturation, for the avoidance of doubt, the degree of unsaturation does not result in an 20 aromatic ring system, and have 3 to I 2 atoms including at least one heteroatoi, such as nitrogen, oxygen, or sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention, the following are examples of heterocyclic rings: azepines, azetidinyl, morpholinyl, oxopiperidinyl, oxopyrrolidinyl, piperazinyl, piperidinyl, pyrrolidinyl, quinicludinyl, thiomorpholinyl, tetrahydropyranyl and 25 tetrahydrofiranyl. The heterocyclyl groups of the invention are substituted with 0, 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio, alkycnylthio, alkynylthio, sulfonic acid, alkylsulfonyl, 30 haloalkylsulfonyl, fluroralkylsulfonyl, alkenylsulfonyl, alkynylsuilfonyl, alkoxysulfonyl, haloalkoxysulfonyl, fluroralkoxysulfonyl, alkenyloxysulfonyl, a lkynyloxysu [fony, aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl, fluroralkylsulfinyl, alkenylsul finy], alkynylsul finyl, alkoxysul tfinyl, haloalkoxysulfinyl, fluroralkoxysulfinyl, -19alkenyloxysulfinyl, alkynyloxysulfiny, aminosulfinyl, forrnyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyL, alkylcarbonyloxy, haloaLkylcarbonyloxy, fluoroalkylcarbonyloxy. 5 alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluroralkylsul fonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy. haloalkoxysulfonyloxy, fluroralkoxysulfonyloxy, alkenyloxysulfonyloxy, alkynyloxysuifonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy, fluroralkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy, alkoxysul finyloxy, haloalkoxysulfinyloxy, fluroralkoxysulfinyloxy, alkenyloxysulfinyloxy, 10 alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of said substiucnts bound to the heterocyclyI group through an alkylene moiety (e.g. methylene). The term "N-heterocyclyl" as used herein is a subset of heterocyclyl, as defined herein, which have at least one nitrogen atom through which the N-heterocyclyl moiety is 15 bound to the parent moiety. Representative examples include pyrrolidin-I-yl, pipeildin- 1 yl, pipcrazin-l-yl, hexahydropyrimidin-l-yl, morpholin- I-y!, I,3-oxazinan-3-yl and 6 azaspiro[2.5]oct-6-yl. As with the hcterocyclyl groups, the N -hcterocyclyl groups of the invention are substituted with 0, 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, 20 alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sulfhydiyl, alkylthio, haloalkylthio, fluoroalkylthio, alkyenylthio, alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl, fluroralkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl, fluroralkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfony, aminosulfonyl, sul finic acid, alkylsulfinyl, haloalkylsulfinyl, 25 fluroralkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl, fluroralkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, 30 alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluroralkylsulfonyloxy, alkenylsulfonyloxy, alkynyisulfonyloxy, haloalkoxysulfonyloxy, fluroralkoxysulfonyloxy, alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy, fluroralkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy, - 20 alkoxysulfinyloxy, haloalkoxysulfinyloxy, fluroralkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy. and any of said substituents bound to the N-heterocyclyl group through an alkylene moiety (e.g. methylene). 5 The term "aryl," as used herein means a phenyl group, naphthyl or anthracenyl group. The aryl groups of the present invention can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sul fhydryl, alkylthio, 10 haloalkylthio, fluoroalkylthio, alkyenylthio, alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl, fluroralkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl, fluroralkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfony, aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl, fluroralkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl, fluroralkoxysulfinyl, 15 alkenyloxysulfinyl, alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylearbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, 20 fluroralkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluroralkoxysulfonyloxy, alkenyloxysulfonyloxy, alkyny loxysulfonyloxy, alkylsuIlfinyloxy, haloalkylsulfinyloxy, fluroralkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy, fluroralkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino, arnido, aminosulfortyl, aminosulfinyl. 25 cyano, nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of said substiuents bound to the heterocyclyl group through an alkylene moiety (e.g. methylene). The term "arylene," is art-recognized, and as used herein pertains to a bidentate moiety obtained by removing two hydrogen atoms of an aryl ring, as defined above. The term "arylalkyl" or "aralkyl" as used herein means an aryl group, as defined 30 herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of aralkyl include, but are not limited to, benzyl, 2-phenylethyl, 3 phenylpropyl, and 2-naphth-2-ylethyl. The term "biaryl," as used herein means an aryl-substituted aryl, an aryl-substituted -21 heteroaryl, a heteroaryl-substituted aryl or a heteroaryl-substituted heteroaryl, wherein aryl and heteroaryl are as defined herein. Representative examples include 4-(phenyl)phenyl and 4-(4-fluorophenyl)pyridinyl. The term "heteroaryl" as used herein include aromatic ring systems, including, but 5 not limited to, monocyclic, bicyclic and tricyclic rings. and have 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention: azaindolyl, benzo(b)thienyl, benzirnidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, bcnzotriazolyl, benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, 10 indolyl, indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl. pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrim.idinyl, pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl, thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl, thienyl, thiomorpholinyl, triazolyl or tropanyl, The heteroaryl groups of the invention are 15 substituted with 0, 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, sulfhydiyl, alkylthio, haloalkylthio, fluoroalkylthio, alkyenylthio, alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl, fluroralkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, 20 alkoxysulfonyl, haloalkoxysulfonyl, fluroralkoxysuIfonyl, alkenyloxysulfonyl, alkynyloxysulfony, aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfiyl, fluroralkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl, furoralkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, 25 carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluroralkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluroralkoxysul fonyloxy, alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulIfinyloxy, 30 haloalkylsulfinyloxy, fluroralkylsu Ifinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysu lfinyloxy, fluroralkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, - 22 cyano, nitro, azido. phosphinyl, phosphoryl, silyl, silyloxy, and any of said subsituents bound to the heteroaryl group through an alkylene moiety (e.g. methylene). The term "heteroarylene," is art-recognized, and as used herein pertains to a bidentate moiety obtained by removing two hydrogen atoms of a heteroaryl ring, as defined 5 above. The terrn "heteroarylalkyl" or "heteroaralkyl" as used herein means a heteroaryl, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of heteroarylalkyl include, but are not limited to, pyridin 3-yl methyl and 2-(thien-2-yl)ethyl. 10 The term "halo" or "halogen" means -Cl, -Br, -l or -F. The term "haloalkyl" means an alkyl group, as defined herein, wherein at least one hydrogen is replaced with a halogen, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl. 15 The term "fluoroalkyl" means an alkyl group, as defined herein, wherein all the hydrogens are replaced with fluorines. The term "hydroxy" as used herein means an -OH group. The term "alkoxy" as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of 20 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert butoxy, pentyloxy, and hexyloxy. The terms "alkyenyloxy", "alkynyloxy" "carboeyelyloxy", and "heterocyclyloxy" are likewise defined. The term "haloalkoxy" as used herein means an alkoxy group, as defined herein, wherein at least one hydrogen is replaced with a halogen, as defined herein. Representative 25 examples of haloalkoxy include, but are not limited to, chloromethoxy, 2-fluorocthoxy, trifluoromethoxy, and pentafluoroethoxy. The term "fluoroalkyloxy" is likewise defined. The term "aryloxy" as used herein means an aryl group, as defined herein, appended to the parent molecular moiety through an oxygen. The term "heteroaryloxy" as used herein means a heteroaryl group, as defined herein, appended to the parent molecular 30 moiety through an oxygen. The terms "heteroaryloxy" is likewise defined. The term "arylalkoxy" or "arylalkyloxy" as used herein means an arylalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen. The term "heteroarylalkoxy" is likewise defined. Representative examples of aryloxy and -23hoteroarylalkoxy include, but are not limited to, 2-chlorophenylmethoxy, 3-trifluoromethyl phenylethoxy, and 2,3-dimethylpyridinylimethoxy. The term "sulfhydryl" or "thio" as used herein means a -SH group. The term "alkylthio" as used herein means an alkyl group, as defined herein, 5 appended to the parent molecular moiety through a sulfur. Representative examples of alkylthio include, but are not limited, methylthio. ethylthio, tert-butylthio, and hexylthio. The terms "haloalkylthio", "fluoroalkylthio", "alkyenylthio", "alkynylthio", "carbocyclylthio", and "heterocyclylthio" are likewise defined. The term "arylthio" as used herein means an aryl group, as defined herein, appended 10 to the parent molecular moiety through an sulfur. The term "heteroarylthio" is likewise defined. The term "arylalkylthio" or "aralkylthio" as used herein means an arylalkyl group, as defined herein, appended to the parent molecular moiety through an sulfur, The term "heteroarylalkylthio" is likewise defined. 15 The term "sulfonyl" as used herein refers to -S(=0)2- group. The term "sulfonic acid" as used herein refers to -S(=0)20H. The term "alkylsulfonyl" as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein. Representative examples of alkylsulfonyl include, but are not limited to, methylsulfonyl 20 and ethylsulfonyl. The terms "haloalkylsulfonyl", "fluroralkylsulfonyl", "alkenylsulfonyl", "alkynylsulfonyl", "carbocyclylsulfonyl", "heterocyclylsulfonyl", "arylsulfonyl", "aralkylsulfonyl", "heteroarylsulfonyl" and "heteroaralkylsulfonyl" are likewise defined. The term "alkoxysulfonyl" as used herein means an alkoxy group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein. 25 Representative examples of alkoxysulfonyl include, but are not limited to, methoxysulfonyl, ethoxysulfonyl and propoxysulfonyl. The terms "haloalkoxysulfonyl", "fluroralkoxysulfonyl", "alkenyloxysulfonyl", "alkynyloxysulfonyl", "carbocyclyloxysulfonyl", "heterocyclyloxysulfonyl", "aryloxysulfonyl", "aralkyloxysulfonyl", "heteroaryloxysulfonyl" and "heteroaralkyloxysuIlfonyl" are likewise 30 defined. The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanosuIlfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfony] groups, respectively. The terms triflate, tosylate, mosylate, and - 24 nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-to luenesu I fonate ester, methanesulfonate ester, and nonafluorobutanesul fonate ester functional groups and molecules that contain said groups, respectively. The term "aminosulfonyl" as used herein means an amino group, as defined herein, 5 appended to the parent molecular moiety through a sulfonyl group. The term "sulfinyl" as used herein refers to -S(=0)- group. Sulfinyl groups are as defined above for sulfonyl groups. The term "sulfinic acid" as used herein refers to S(=O)OH. The term "oxy" refers to a -0- group. 10 The term "carbonyl" as used herein means a -C(=0)- group. The term "thiocarbonyl" as used herein means a -C(=S)- group. The term "formyl" as used herein means a -C(=0)H group. The term "alkylcarbonyl" as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. 15 Representative examples of alkylcarbonyl include, but are not limited to, acetyl, I oxopropyl, 2,2-dimethyl- I -oxopropyl, I -oxobutyl, and I -oxopentyl. The terms "haloalkylcarbonyl", "fluoroalkylcarbonyl", "alkenylcarbonyl", "alkynylcarbony]", "carbocyclylcarbonyl", "heterocyclylcarbonyl", "arylcarbonyl", "aralkylcarbonyl", "heteroarylearbonyl", and "heteroaralkylcarbonyl" are likewise defined. 20 The term "carboxy" as used herein means a -CO2H group. The term "alkoxycarbonyl" as used herein means an alkoxy group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, and tert-butoxycarbonyl. The terms 25 "haloalkoxycarbonyl", "fluoroalkoxycarbonyl", "alkenyloxycarbonyl", "alkynyloxycarbonyl", "carbocyclyloxycarbonyl", "heterocyclyloxycarbonyl", "aryloxycarbonyl", "aralkyloxycarbonyl", "heteroaryfoxycarbonyl", and "heteroaralkyloxycarbonyl" are likewise defined. The term "al.kylcarbonyloxy" as used herein means an alkylcarbonyl group, as 30 defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of a[kylcarbonyloxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, and tert-butyIcarbonyloxy. The terms "haloalkylcarbonyloxy", "fluoroalkylcarbonyloxy", "alkenylcarbonyloxy", "alkynylcarbonyloxy", - 25 - "carbocyclylcarbonyloxy", "hetcrocyclylcarbonyloxy", "arylcarbonyloxy", "aralkylearbonyloxy", "heteroarylcarbonyloxy", and "heteroaralkylcarbonyloxy" are likewise defined. The term "alkylsulfonyloxy" as used herein means an alkylsulfonyl group, as 5 defined herein, appended to the parent molecular moiety through an oxygen atom. The terms "haloalkylsul fonyloxy", "fluroralkylsulfonyloxy", "alkenylsulfonyloxy", "alkynylsulfonyloxy", "carbocyclylsulfonyloxy", "heterocyclyl su Ifonyloxy", "arylsulfonyloxy", "aralkylsulfonyloxy", "heteroarylsulfonyloxy". "heteroaralkylsulfonyloxy", "haloalkoxysulfonyloxy", "fluroralkoxysulfonyloxy", 10 "alkenyloxysulfonyloxy", "alkynyloxysulfonyloxy", "carbocyclyloxysul fonyloxy", "heterocyclyloxysulfonyloxy", "aryloxysulfonyloxy", "aralkyloxysulfonyloxy", "heteroaryloxysulfonyloxy" and "heteroaralkyloxysulfonyloxy" The term "amiino" as used herein refers to -NH 2 and substituted derivatives thereof wherein one or both of the hydrogens are independently replaced with substituents selected 15 from the group consisting of alkyl. haloalkyl, fluoroalkyl, alkenyl, alkynyl, carbocyclyl, hcterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carbocyclylcarbonyl, ieterocyclylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarnbonyl, heteroaralkylearbonyl and the sufonyl and sulfinyl groups defined above: or when both 20 hydrogens together are replaced with an alkylene group (to form a ring which contains the nitrogen). Representative examples include, but are not limited to methylamino, acetylamino, and dimethylamino. The term "amido" as used herein means an amino group, as defined herein, appendcd to the parent molecular moiety through a carbonyl. 25 The term "cyano" as used herein means a -C=N group, The term "nitro" as used herein means a -NO 2 group. The term "azido" as used herein means a -N 3 group. The term "phosphinyl" as used herein includes -PH 3 and substituted derivatives thereof wherein one, two or three of the hydrogens are independently replaced with 30 substituents selected from the group consisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkoxy, haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, aryloxy, aralkyloxy, beteroaiyloxy, heteroaralkyloxy, and amino. -26- The term "phosphoryl" as used herein refers to -P(=O)OH 2 and substituted derivatives thereof wherein one or both of the hydroxyls are independently replaced with substituents selected from the group consisting of alkyl, haloalkyl, fluoroalkyl, alkenylt alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkoxy, 5 haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, aryloxy, aralkyloxy, heteroaryloxy, heteroaralkyloxy, and amino. The term "silyl" as used herein includes HSi- and substituted derivatives thereof wherein one, two or three of the hydrogens are independently replaced with subsitutuents selected from alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, 10 aralkyl, heteroaryl, and heteroaralkyl. Representitive examples include trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsi lyl )ethoxy]methyi (SEM). The term "silyloxy" as used herein means a silyl group, as defined herein, is appended to the parent molecule through an oxygen atom. 15 The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesul fonyl, p-toluenesul fonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chenistry; this list is typically presented in a table entitled Standard 20 List of Abbreviations. As used herein, the term "administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. As used herein, the phrases "neurodegenerative disorder" and "neurodegenerative 25 disease" refers to a wide range of diseases and/or disorders of the central and peripheral nervous system, such as neuropathologies, and includes but is not limited to, Parkinson's disease, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), dncrvation atrophy, otoselerosis, stroke, dementia, multiple sclerosis, Huntington's disease, encephalopathy associated with acquired immunodeficiency disease (AIDS), and other 30 diseases associated with neuronal cell toxicity and cell death. As used herein, the phrase "pharmaceutically acceptable" refers to those agents, compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and - 27 animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, the phrase "pharmaceutically-acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid 5 filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Sonic examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as 10 lactose, glucose and sucrose: (2) starches, such as corn starch and potato starch: (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate: (4) powdered tragacanth; (5) malt; (6) gelatin; (7) tale; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene 15 glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as cthyl oleate and ethyl laurate; (I 3) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible 20 substances employed in pharmaceutical formulations. As used herein, the phrase "pharmaceutically-acceptable salts" refers to the relatively non-toxic, inorganic and organic salts of compounds. As used herein, the phrase "proteinopathy" refers to any disease associated with the accumulation and/or aggregation of abnormal or misfolded proteins. Though 25 proteinopathics are frequently neurodegenerative diseases, proteinopathics also include diseases of other tissues, including the liver, muscle and heart, and include some cancers. As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy. As used herein, the phrase "subject suspected of having" means a subject exhibiting 30 one or more clinical indicators of a disease or condition. In certain embodiments, the disease or condition is cancer, a neurodegencrative disorder or pancreatitis. As used herein, the phrase "subject in need thereof' means a subject identified as in need of a therapy or treatment of the invention. -28- As used herein, the phrase "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans. caused by an agent. The phrases "therapeutically-effective amount" and "effective amount" mean the amount of an agent that produces some desired effect in at least a sub-population of cells. A 5 therapeutically effective amount includes an amount of an agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. For example, certain agents used in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment. 10 As used herein, the term "treating" a disease in a subject or "treating" a subject having or suspected of having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of an agent, such that at least one symptom of the disease is decreased or prevented from worsening. As used herein, "any of the aforementioned compounds" is any compound of 15 formula 1, 11, 11, IV, V, VI, Vill, and VIII, Inhibitors of Usp14 One aspect of the invention relates to a compound represented by formula I: R1 A-N 20 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stereoisomer thereof: wherein, independently for each occurrence, A is aryl, heteroaryl, carbocyclyl, heterocyclyl, or biaryl; R is hydrogen, alkyl, haloalkyl, fluoroalkyl, lower alkyoxy, halo or trifluoromethyl; G is -N= or -C(R2 25 Z is =C(R')-, =C(R 2 )- or =N-; -29-
R
2 is hydrogen, alkyl, haloalkyl, fluoroalkyl, lower alkyoxy, halo or trifluorornethyl;
R
10 R or, when G is -C(R2= and Z is =C(R 2 )-, the two R 2 taken together are R' 0
R
0 , N \ 10 ~Rl-j / R 1 N / Rl R
/
10 1 NN N Rio N R 10 R O N\O RI NI N fr
R
1
R
10 , R , R01 0o HR9 0 N'R N 'N'R 9 N'N'R9 N'OH N' 0R 9 XisNlKYi Y, I R9 d 0-R 9 5 or heteroaryl; Y is -CH 2 NR'R-', -CH 2 (N-heterocyclyl), -CH 2
NH(CH
2 )riNNH(alkyl), -CH2NH(CH2)nN(alkyl) 2 , -CH 2
NH(CH
2 ),(N-heterocyclyl), -CH 2 N(alky])(CH2)nN H(alkyl),
-CH
2 N(alkyl)(CH2) 1 N(alkyl)2, -CH 2 N(alkyl)(CH 2 )n(N-heterocyclyl),
-CH
2
NH(CH
2 )n0(alkyl), -CH 2 N(alky)(CH2)nO(alkyl), -NRR, -NR 5 NRR, -NR(N 10 heterocyclyl), or -N-heterocyclyl; n is 1, 2, 3 or 4; R' is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; R 4 is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, 15 aralkyl, heteroaryl, or heteroaralkyl; R' is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; R' is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; 20 R 7 is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; R8 is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl;
R
9 is alkyl; or two R 9 taken together with the nitrogen to which they are bound are 25 an N-hcterocyclyl group; and -30- R"' is hydrogen, alkyl, haloalkyl, fluoroalkyl, alkyoxy, alkoxyalkyl, halo. trifluoromethyl, sulfoxymethyl. sulfonamido, amino, amido, N-heterocyclyl, aminoalkyl, amidoalkyl, or N-hetrocyclylalkyl. In certain embodiments, the present invention relates to any of the aforementioned 5 compounds, provided that when A is 4-fluorophenyl, R' is methyl, G is -C(R 2)=, R 2 is 0 methyl, X is and Y is -CH 2 (piperidin-1-yl), Z is not =C(H)-. In certain embodiments, the present invention relates to any of the aforementioned compounds, provided that when A is 4-methylphenyl, R' is methyl, G is -C(R 2 )=, R 2 is 0 methyl, X is and Y is -CH 2 (4-methylpiperidin-I-yl), Z is not =C(H)- (i.e., C 100). 10 In certain embodiments, the present invention relates to any of the aforementioned 0 compounds, provided that when A is 4-chlorophenyl, R' is methyl, G is -N=, X is and Y is -NH 2 , Z is not =N- (i.e., CI 21). In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein G is -N=. 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein G is -C(R2=. In certain embodiments, the present invention relates to any of the aforementioned com)ounds, wherein the compound is represented by formula 11: Rx A-N z R2 20 II or is a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof. - 31 - Another aspect of the invention relates to a compound represented by formula I11: 0 x A-N I 7fZ 0 III or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chernically-protected form, 5 enantiorner or stercoisoner thereof: wherein, independently for each occurrence, A is aryl, heteroaryl, carbocyclyl, heterocyclyl, or biaryl; Z is =C(R)- or =N
R
2 is hydrogen, alkyl, haloalkyl, fluoroalkyl, lower alkyoxy, halo or trifluoromethyl: RIO R 0 N R1O R / RIO or, R 2 and X taken together are R R R 10 R, RI 0 N R 10 N / R10 N 3~N R1 0
R
10 10 R , RIO or N-N H R 9 0 N'R9 N'NS N'N'R9 NOH N'OR9 Xis Nfry It'Y Y Y
R
9 ' O-R 9 or heteroaryl; Y is -CH 2
NR
3
R
4 , -CH 2 (N-heterocyclyl), -CH 2 NA(CH2)nNH(alkyl),
-CH
2
NH(CH
2 )nN(a1kyl)2, -CH 2 N H(CH2),(N-heterocyclyl), -CH 2 N(alkyl)(CH2),NH(alkyl), 1 5 -CH 2 N(alkyl)(CH 2 )nN(alkyl) 2 , -CH 2 N(alkyl)(CH2)(N-heterocyclyl),
-CH
2 NH(CH2)n4O(alkyl), -CH 2 N(alky)(CH 2 ),O(alkyl), -NR 3
R
4 , -NR 5
NRR
7 , -NR'(N heterocyclyl), or -N-heterocyclyl; n is 1, 2, 3 or 4;
R
3 is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, 20 aralkyl, heteroaryl, or heteroaralkyl; R4 is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; -32- R' is hydrogen, alkyl, substituted alkyl, alkoxyalkyl. haloalkyl, fluoroalkyl, aryl. aralkyl, heteroaryl, or heteroaralkyl;
R
6 is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl; 5 R 7 is hydrogen, alkyl, substituted alkyl, alkoxyalkyl, haloalkyl, fluoroalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl;
R
9 is alkyl; or two R 9 taken together with the nitrogen to which they are bound are an N-heterocyclyl group: and R1 0 is hydrogen, alkyl, haloalkyl, fluoroalkyl, alkyoxy, alkoxyalkyl, halo, 10 trifluoromethyl, sulfoxymethyl, sulfonamido, amino, amido, N-heterocyclyl, arninoalkyl, amidoalkyl, or N-hetrocyclylalkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, provided that the compound is not C12. In certain embodiments, the present invention relates to any of the aforementioned 15 compounds, provided that the compound is not C31 1. Another aspect of the invention relates to a compound represented by formula IV: OR" A N'N N S N IV or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, 20 enantiomer or stercoisomer thereof; wherein, independently for each occurrence, A is aryl, heteroaryl, carbocyclyl, heterocyclyl, or biaryl; and R"1 is hydrogen, alkyl, alkylcarbonyl, aralkyl, haloalkyl, fluoroalkyl, alkoxyalkyl, trifluorornethyl, or silyl. In certain embodiments, the present invention relates to any of the aforementioned 25 compounds, provided that the compound is not C73. - 33 - Another aspect of the invention relates to a compound represented by Formula V:
R
12 N-A
R
1 3 %J/ R 1,
R
13 S
R
13 0 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, 5 enantiomer or stercoisom er thereof; wherein, independently for each occurrence, A is aryl, heteroaryl, carbocyclyl, heterocyclyl, or biaryl,
R
2 is hydrogen or alkyl; and R13 is hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, 10 fluoroalkyloxy, formyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, sulfoxymethyl, sulfonamido, amino, amido, azido, aninosulfonyl, aminosulIfinyl, cyano, 15 nitro, phosphinyl, phosphoryl, silyl, silyloxy, and any of said substiucnts bound through a methylene or ethylene moiety: or one or two instances of R", and the carbon to which it is bound, taken together are -N=. In certain embodiments, the present invention relates to any of the aforementioned compounds, provided that the compound is not C106. 20 Another aspect of the invention relates to a compound represented by formula VI:
R
1
R
14 A-N
R
1 5
R
15 VI or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chen i ca lly -protected form, enantiomer or stercoisomer thereof; wherein, independently for each occurrence, 25 A is aryl, heteroaryl, carbocyclyl, heterocyclyl, or biaryl; R' is hydrogen, alkyl, haloalkyl, fluoroalkyl, lower alkyoxy, halo or trifluoromethyl; R 1 is hydrogen or X; - 34 - RN R 3 R3 R 13 both R i, taken together, are R 13
R
13 or R 1 3
RN
3 and R'3 is hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, formyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, 5 alkenylcarbonyl, alkynylearbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylearbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy, alkynylearbonyloxy, sulfoxymethyl, sulfonamido, amino, arnido, azido, aminosulfonyl, aminosulfinyl, cyano, nitro, phosphinyl, phosphoryl, silyl, silyloxy, and any of said substiuents bound through a 10 methylene or ethylene moiety; or one or two instances of R' 3 , and the carbon to which it is bound, taken together are N. In certain embodiments, the present invention relates to any of the aforementioned compounds, provided that the compound is not Cl1 8. Another aspect of the invention relates to a compound represented by formula VII: 0 A-N I eS 15 S VIU or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof; wherein, independently for each occurrence, A is aryl, heteroaryl, carbocyclyl, heterocyclyl, or biaryl; and 20 Y is -CH 2
NR
3
R
1 , -CH 2 (N-hetcrocyclyl), -CH 2 NH(CH2)nNH(alkyl),
-CH
2
NH(CH
2 )nN(alkyl) 2 , -CH 2
NH(CH
2 )n(N-heterocyclyl), -CH 2 N(alkyl)(CH 2 )nNH(alkyl), -CH2N(alkyl)(CH 2 )nN(alkyl) 2 , -CH 2 N(alkyl)(CHI 2 )(N-heterocyclyl),
-CH
2
NH(CH
2 )nO(alkyl), -CH 2 N(alkyl)(CH2)nO(alkyl), -NRR, -NRNR R' or heterocyclyl), 25 In certain embodiments, the present invention relates to any of the aforementioned compounds, provided that the compound is not C133. Another aspect of the invention relates to a compound represented by formula VII: -35- CN A N S'R1 0 VIll or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof; wherein, independently for each occurrence, 5 A is aryl, heteroaryl, carbocyclyl, heterocyclyl, or biaryl; and R 2 is hydogen or alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, provided that the compound is not C 139. In certain embodiments, the present invention relates to any of the aforementioned 10 compounds, wherein A is aryl or hcteroaryl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is phenyl, pyridin-2-yl, pyridin-3-yl or pyrimidin-2-yl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, 15 alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, formyl, alkylearbonyl, haloalkylearbonyl, fluoroalkylcarbonyl, alkenylearbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fl uoroalky Icarbony loxy, alkenylcarbonyloxy, alkynylearbonyloxy, suI foxymethyl, sulfonamido, amino, amido, 20 azido, aminosulfonyl, aminosulfinyl, cyano, nitro, phosphinyl, phosphoryl, silyl, silyboxy, and any of said substiuents bound to the phenyl, pyridin-2yl, pyridin-3-y or pyrimidin-2-y through a methylene or ethylene moiety. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is phenyl, optionally substituted with 1, 2, 3, 4 or 5 substituents 25 independently selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is In certain embodiments, the present invention relates to any of the aforementioned -36compounds, wherein A is phenyl substituted in the two position (ortho substituted) with a substitutent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. In certain embodiments, the present invention relates to any of the aforementioned CI 0 F 5 compounds, wherein A is , , or , In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is phenyl substituted in the three position (meta substituted) with a substitutent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido cyano, and nitro. 10 In certain embodiments, the present invention relates to any of the aforementioned CI 0- F compounds, wherein A is -or 1 6 . In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is phenyl substituted in the four position (para substituted) with a substitutent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, 15 hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. In certain embodiments, the present invention relates to any of the aforementioned CI \\O F compounds, wherein A is N CF 3 / 1 NO 2 Br / \ CF 2
CF
3 00
-NH
2 H Na 0 - - 4 2o 20 Tn certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is phenyl substituted in the two and four positions with substitutents independently selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. -37- In certain embodiments. the present invention relates to any of the aforementioned CI F .F F compounds, wherein A is - or . In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is pyridin-2-yl, optionally substituted in the four position with a 5 substituent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. In certain embodiments, the present invention relates to any of the aforementioned N compounds, wherein A is . In certain embodiments, the present invention relates to any of the aforementioned 10 compounds, wherein A is pyrimidin-2-yI, optionally substituted in the four position with a substituent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is N . 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is biaryl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is 4-(phenyl)phen-1-yl or 4-(2-pyridinyl)phen- 1-yl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting 20 of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro, In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein A is - or N In certain embodiments, the present invention relates to any of the aforementioned 25 compounds, wherein R' is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is alkyl. In certain embodiments, the present invention relates to any of the aforementioned -38compounds, wherein R' is haloalkyl, In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R t is fluoroalkyL In certain embodiments, the present invention relates to any of the aforementioned 5 compounds, wherein R' is methyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is halomethyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 1 is fluoromethy L 10 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is ethyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is haloethyl. In certain embodiments, the present invention relates to any of the aforementioned 15 compounds, wherein R' is fluoroethyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 2 is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 2 is alkyl. 20 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 2 is methyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 2 is ethyl. In certain embodiments, the present invention relates to any of the aforementioned 25 compounds, wherein R' is hydrogen; and R 2 is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is alkyl; and R 2 is alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is methyl; and R 2 is methyl. 30 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is ethyl; and R 2 is ethyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Z is =C(R 8 )-; and R is hydrogen. -39- In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Z is =C(R )-; and Rs is alicyL In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Z is =N-. 5 In certain embodiments, the present invention relates to any of the aforementioned 0 compounds, wherein X is . In certain embodiments, the present invention relates to any of the aforementioned N'R 9 compounds, wherein X is . In certain embodiments, the present invention relates to any of the aforementioned H I N'N,R9 N'N'R9 N Y YLt 10 compounds, wherein X is or . In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein X is heteroaryL. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein X is pyrrolof1,2-a]pyrazin-3-yl. 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 9 is alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 9 is methyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH2NR 3 R. 20 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH2NR*R 4 : and R3 is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH 2
NRIR
4 ; and R 3 is alkyl, In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH2NR3R 4 ; and 25 R 4 is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH 2 NR R 4 ; and R 4 is alkyl -40 - In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH 2
NR
3
R
4 ; and R 4 is alkoxyalkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH2NR 3 R4, R 3 is hydrogen; and R4 is alkyl. 5 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH 2 NR"R : R is alkyl; and R 4 is alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH 2 NR3R 4 , R' is hydrogen: and R 4 is alkoxyalkyl. In certain embodiments, the present invention relates to any of the aforementioned 10 compounds, wherein Y is -CH 2
NR
3
R
4 ; R 3 is alkyl; and R4 is alkoxyalkyl. In certain embodiments, the present invention relates to any of the aforementioned HN N HN N N HN compounds, wheremi Y is V N pN N N [SN / N or 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH 2 (N-heterocyclyl), which is optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of alkyl, haloalkyl, fluoroalkyl, halo, hydroxyl, alkoxy, haloalkoxy, fluoroalkoxy, amino and nitro. 20 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH2(piperidin- I -yl), -CH2(piperazin-I-yl), -CH2(hexahydropyrimidin- I -yl), -CH2(morpholin- I -yl) or -CH2(1,3-oxazinan-3-yl), which is optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of alkyl, haloalkyl, fluoroalkyl, halo, hydroxyl, alkoxy, 25 haloalkoxy, fluoroalkoxy, amino and nitro. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH2(piperidin-1-yl) or -CH 2 (piperazin-I -yl), which is optionally -41 substituted with one, two, three, four or five substituents independently selected from the group consisting of alkyl, haloalkyl, fluoroalky], halo, hydroxyl, alkoxy, haloalkoxy, fluoroalIkoxy, amino and nitro. In certain embodiments, the present invention relates to any of the aforementioned O 5 compounds, wherein Y is
CF
3 N- 0- 0-CF 3 N N pSpN NN pN 0 / / 0 O N N NJ N0 p' H' V -N or In certain embodiments, the present invention relates to any of the aforementioned 10 compounds, wherein Y is -CH2NH(CH 2 )NH(aLkyl), -CH 2
NH(CH
2 )nN(alkyl)2,
-CH
2 N H(CH 2 %)N(alkylenc), -CH 2 N(alkyI)(CH 2 )nNH(alkyl), -CH 2 N(alkyl)(CH 2 )nN(alkyl) 2 or -CH 2 N(alkyl)(CH 2 )nN(alkylene). In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -CH2NH(CHl2)nO(alky[) or -CH 2 N(alkyl)(CH 2 ),0(alkyl). 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein n is 1. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein n is 2. In certain embodiments, the present invention relates to any of the aforementioned 20 compounds, wherein n is 3. -42 - In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein n is 4. In certain embodiments, the present invention relates to any of the aforementioned / / N N 0 compounds. wherein Y is [ .N o 5 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR 3
R
4 . In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR'R 4 ; and R3 is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned 10 compounds, wherein Y is -NRR; and R is alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NRR 4 ; and R4 is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NRtR 4 ; and R 4 is alkyl. 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR 3 R4: R3 is hydrogen: and R 4 is alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR'R 4 ; R3 is hydrogen: and R 4 is hydrogen. In certain embodiments, the present invention i-elates to any of the aforementioned compounds, 20 wherein Y is -NRR 4 ; R 3 is alkyl; and RI is alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR 5 NRr R 7 or -NlR(N-heterocyclyl). In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR 5
NR
6
R
7 ; and R5 is hydrogen. 25 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR 5
NR
6
R
7 ; and R5 is alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR 5 NRR'; and R5, RI and R' are, independently, hydrogen or alkyl. 30 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR5(N-heterocyclyl); and R 5 is hydrogen. -43 - In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Y is -NR(N-heterocyclyl): and R 5 is alkyl. In certain embodiments, the present invention relates to any of the aforementioned Q 0 N NH N NN compounds, wherein Y is H H or H 5 In certain embodiments, the present invention relates to any of the aforementioned
R
10 R / compounds, wherein Z is =C(If)-; and the two R taken together are R 10
R
0 , N R 10 RO4 10 N R N 10 N\/N N %-N RO I
R
10 R , 0 or N-N In certain embodiments, the present invention relates to any of the aforementioned R R 10 compounds, wherein Z is =C(R 2 )-; and the two R2 taken together are RI 0
RI
0 N R10 R Ri l( R 10 RIO or R 10 In certain embodiments, the present invention relates to any of the aforementioned
R
10 R P 0 compounds, wherein Z is =C(R 2 )-; and the two R2 taken together are R 10
R
0 . In certain embodiments, the present invention relates to any of the aforementioned N ' R' 0 compounds, wherein Z is =C(R2)-; and the two R2 taken together are RIO RIO 15 In certain embodiments, the present invention relates to any of the aforementioned R10i4 P' 0 compounds, wherein Z is =C(R 2 )-; and the two R2 taken together are R'O. -44- In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R"' is hydrogen, alkyl, haloalkyl, fluoroalkyl, alkyoxy, alkoxyalkyl, halo or trifluoromethyl. In certain embodiments, the present invention relates to any of the aforementioned 5 compounds, wherein R' is hydrogen, amino, amido, N-heterocyclyl, aminoalkyl, arnidoalkyl, or N-hetrocyclylalkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 1 "' is hydrogen, halo or N-heterocycly. In certain embodiments, the present invention relates to any of the aforementioned 10 compounds, wherein R"' is hydrogen, chloro or piperidin-l-y. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R' is hydrogen or N-heterocyclylalkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R") is hydrogen or piperidin-1-ylmethyl. 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R'( 0 is hydrogen or alkyl, In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R"' is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned 20 compounds, wherein R" is hydrogen or alkyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R" is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R ' is methyl. 25 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R " is hydrogen. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 2 is methyl. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R" is hydrogen. 30 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein exactly one R", and the carbon to which it is bound, is -N=. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 4 is hydrogen. -45- In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein R 1 is X. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein Z is =C(R-2); the two R 2 taken together are R"0 R10 Rio Rio ; and R'" is hydrogen, halo or N-heterocyclyl. 5 In certain embodiments, the present invention relates to any of the aforementioned N / Rio compounds, wherein Z is =C(R 2 )-; the two R 2 taken together are R 10 Rio and R' 0 is hydrogen or N-heterocyclylalkyl. Another aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or N 7 F /&N 10 stercoisomer thereof, selected from the group consisting of O F \ N N ~ F N F N O 0 H N FoN 1 yF"N O 0 H NN F N F N -46 - FNNNF N F N F N o H H F N N F NN 0 o H N N F N F N F HO CN N N Q N F N N N N N O N /\ N N 6-4N Fb NO' C NJ OKN > N / ' N N N OC- N D -47- N N
F
3 C N NN C N 0 2 N N B r N N N and F N 5 Another aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or F N O stercoisomer thereof, selected from the group consisting of C N 0 2 N N Br NN F " N and -48- Another aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiorner or ON F / N stercoisomer thereof, of the following formula Another aspect of the invention relates to a compound, or a pharmaceutically 5 acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or 0 F N N stercoisomer thereof, of the following formula Another aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or 0 N stereoisomer thereof, selected from the group consisting of 0 N N CW N W N 10 o 0 N N O o 00 WNN W_ N N-e 49 -49 o 0 H NW N W -a'N OW -NO W N W _ N o H 0 O O N W \N W ) N 5 N N \ o 0 N N-, W \N W-N and /;wherein W is methyl, fluoro, chloro, nitro, methoxy, ethIoxy -SO2NH2 or -C(=0)NH2. Another aspect of the invention relates to a compound, or a pharmaceutically 10 acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomner or -50- 0 N WN stercoisorner thereof, selected from the group consisting of wherein W is alkyl, fluoro, chloro, nitro, methoxy, ethoxy, -SO 2
NH
2 or -C(=0)NH- 2 . In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is methyl. 5 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is fluoro. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is chloro. In certain embodiments, the present invention relates to any of the aforementioned 10 compounds, wherein wherein W is nitro. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is methoxy. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is ethoxy. 15 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is -SO 2 NH2 In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is -C(=0)NH 2 Another aspect of the invention relates to a compound, or a pharmaceutically 20 acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or 0 W ~
NH
2 stercoisomer thereof, selected from the group consisting of / NN wherein W is alkyl, fluoro, chloro, nitro, methoxy, ethoxy, -SO 2
NH-
2 or -C(=0)NH2. In certain embodiments, the present invention relates to any of the aforementioned compounds, wherein wherein W is chloro. 25 Another aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, selected from the group consisting of -51 - 0 o 0 N NN I N CFaCF2 -0 N NIN CN N CFN CC N 5NN3 N NCF N and 0
N
3 / &N 5 Another aspect of the invention i-elates to a compound, or a pharmaceutically acceptable salt, solIvate, hydrate, prodrug, chemical ly-lproected. form, enantiomer or -.. N C0 N H stercoisomer thereof, selected from the group consisting of o 0 N'N N : CI N H c; O N H and 0 Ci ' N H 10 -52 - Another aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or Me2N N N stereoisomer thereof, selected from the group consisting of and 0 ( N -(N 5 Another aspect of the invention relates to a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or CI N N stercoisomer thereof, selected from the group consisting of N N N JN C1 N ' NCI N and Many of the compounds of the invention may be provided as salts with 10 pharmaceutically compatible counterions (i.e., pharmaceutically acceptable salts). A pharmaceuticallyy acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a prodrug of a compound of this invention. A pharmaceuticallyy acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt upon administration to a 15 recipient. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. Acids commonly employed to form pharmaceutically acceptable salts include 20 inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, salicylic, - 53 tartaic. bitartaric, ascorbic, rnalcic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para bromophenylsulfonic, carbonic, succinic. citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, 5 pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, nalcate, butyne-1,4 dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, 10 hydroxybenzoate, methoxybenzoatc, phthalate, terephatha late, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, ph1enylbutyrate, citrate, lactate, .beta. -hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-I -sulfonate, naphthalene-2-sulfonate, mandelate and the like salts. Pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and 15 hydrobromic acid, and those formed with organic acids such as malcic acid. Suitable bases for forming pharmaceutically acceptable salts with acidic functional groups include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such 20 as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine; diethylamine: triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2-hydroxycthyl)am ine, 2-hydroxy-tert-butylamine, or tris-( hydroxymethyl)methyl amine, N,N-di-lower alkyl-N (hydroxy lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxycthyl)amine, or tri-(2 25 hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like. Certain compounds of the invention and their salts may exist in more than one crystal form and the present invention includes each crystal form and mixtures thereof. Certain compounds of the invention and their salts may also exist in the form of 30 solvates, for example hydrates, and the present invention includes each solvate and mixtures thereof. Certain compounds of the invention may contain one or more chiral centers, and exist in different optically active forms. When compounds of the invention contain one -54chiral center, the compounds exist in two enantiorneric forms and the present invention includes both enantiomers and mixtures of enantiomers, such as racemic mixtures. The enantioners may be resolved by methods known to those skilled in the art, for example by formation of diastercoisomeric salts which may be separated, for example, by 5 crystallization; formation of diastercoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a 10 chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step may be used to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric 15 transformation. When a compound of the invention contains more than one chiral center, it may exist in diastercoisomeric forms. The diastercoisomeric compounds may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers may be separated as described above. The present invention 20 includes each diastercoisomer of compounds of the invention and mixtures thereof. Certain compounds of the invention may exist in different tautomeric forms or as different geometric isomers, and the present invention includes each tautomer and/or geometric isomer of compounds of the invention and mixtures thereof. Certain compounds of the invention may exist in different stable conformational 25 forms which may be separable. Torsional asymmetry due to restricted rotation about an asymmetric single bond, for example because of steric hindrance or ring strain, may permit separation of different conformers. The present invention includes each conformational isomer of compounds of the invention and mixtures thereof. Certain compounds of the invention may exist in zwitterionic form and the present 30 invention includes each zwitterionic form of compounds of the invention and mixtures thereof The present invention also includes pro-drugs. As used herein the term "pro-drug" refers to an agent which is converted into the parent drug in vivo by some physiological - 55 chemical process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form). Pro-drugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved 5 solubility in pharmacological compositions over the parent drug. An example, without limitation, of a pro-drug would be a compound of the present invention wherein it is administered as an ester (the "pro-drug") to facilitate transmittal across a cell membrane where water solubility is not beneficial, but then it is metabolically hydrolyzed to the carboxylic acid once inside the cell where water solubility is beneficial. Pro-drugs have 10 many useful properties. For example, a pro-drug may be more water soluble than the ultimate drug, thereby facilitating intravenous administration of the drug. A pro-drug may also have a higher level of oral bioavailability than the ultimate drug. After administration, the prodrug is enzymatically or chemically cleaved to deliver the ultimate drug in the blood or tissue. 15 Exemplary pro-drugs upon cleavage release the corresponding free acid, and such hydrolyzable ester-forming residues of the compounds of this invention include but are not limited to carboxylic acid substituents (e.g., -C(0) 2 H or a moiety that contains a carboxylic acid) wherein the free hydrogen is replaced by (C-CA)alkyl, (C2-Cl)alkanoyloxymethyl,
(C
4
-C
9 )l-(alkanoyloxy)ethyl, 1-methyl-1-(alkanoyloxy)-ethyl having fi-om 5 to 10 carbon 20 atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, I (alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-I (alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N (alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N (alkoxycarbonyl)amino)ethyl having fr-om 4 to 10 carbon atoms, 3-phthalidyl, 4 25 crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(C -C 2 )alkylamino(C 2 -Cf)alkyl (such as P-dimethylaminocthyl), carbamoyl-(C-C 2 )alkyl, N,N-di(Cj-C 2 )-alkylcarbamoyl-(C
C
2 )alkyl and piperidino-, pyrrolidino- or morpholino(C2-C 3 )alkyl. Other exemplary pro-drugs release an alcohol or amine of a compound of the invention wherein the free hydrogen of a hydroxyl or amine substituent is replaced by 30 (C-C)alkanoyloxymethyl, I-((C -Co)alkanoyloxy)ethyl, 1-methyl-1-((C C6)alkanoyloxy)ethyl, (C -C 6 )alkoxycarbonyl-oxymethyl, N-(C -C 6 )alkoxycarbonylamino methyl, succinoyl, (C-C)alkanoy IL a-am ino(C-C 4 )alkanoyl, arylactyl and a-aminoacyl, or a-aminoacyl-a-aminoacyl wherein said a-aminoacyl moieties are independently any of the - 56 naturally occurring L-amino acids found in proteins, -P(O)(OH)2, -P(0)(O(C 1
-C
6 )alkyl)2 or glycosyl (the radical resulting from detachment of the hydroxyl of the hemiacetal of a carbohydrate). The phrase "protecting group" as used herein means temporary substituents which 5 protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2 "d cd.; Wiley: New Yoik, 1991). Protected forms of the 10 inventive compounds are included within the scope of this invention. The term "chemically protected form," as used herein, pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as a masked or masking group). It may be convenient or desirable to prepare, purify, and/or 15 handle the active compound in a chemically protected form. By protecting a reactive flinctional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic 20 Synthesis (T. Green and P. Wuts, Wiley, 1991), and Protective Groups in Organic Synthesis (T. Green and P. Wuts: 3rd Edition; John Wiley and Sons, 1999). For example, a hydroxy group may be protected as an ether (-OR) or an ester (-OC(=O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenyliethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl 25 ester (-OC(=O)CH 3 ,-OAc). For example, an aldchyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (C(=0)) is converted to a diether (C(OR) 2 ), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid. 30 For example, an amine group may be protected, for example, as an aide ( NRC(=O)R) or a urethane (-NRC(=O)OR), for example, as: a methyl amide ( NHC(=O)CH3); a benzyloxy aide (-NIHC(=0)0CH 2
C
6 1HsNHCbz); as a t-butoxy aide ( NHC(=0)OC(CH 3 ),-NHBoc); a 2-biphenyl-2-propoxy amid ( - 57 -
NHC(=O)OC(CH
3
)
2 C6HC 6 HsNHBoc), as a 9-fluorenylmethoxy amid (~NHFmoc), as a 6 nitrovcratryloxy amide (-NHNvoc), as a 2-trimethylsilylethyloxy amide (-NHTeoc), as a 2,2,2-trichloroethyloxy aide (-NHTroc), as an allyloxy aide (-NHAIloc), as a 2 (phenylsulfonyl)ethyloxy amide (-NHPscc); or, in suitable cases (e.g., cyclic amines), as a 5 nitroxide radical. For example, a carboxylic acid group may be protected as an ester or an aide, for example, as: a benzyl ester; a t-butyl ester; a methyl ester: or a methyl amide. For example, a thiol group may be protected as a thiocther (-SR), for example, as; a benzyl thiocther; or an acetamidomethyl ether (-SCH 2 NHfC(=O)CH 3 ). 10 Pharmaceutical Compositions The invention provides pharmaceutical compositions comprising inhibitors of Usp14. In one aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically 15 acceptable carriers (additives) and/or diluents. In another aspect, the agents of the invention can be administered as such, or administered in mixtures with pharmaccutically acceptable carriers and can also be administered in conjunction with other agents. Conjunctive therapy thus includes sequential, simultaneous and separate, or co administration of one or more compound of the invention, wherein the therapeutic effects of 20 the first administered has not entirely disappeared when the subsequent compound is administered. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or 25 non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled 30 release patch or spray applied to the skin: (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. As set out above, in certain embodiments, agents of the invention may be compounds containing a basic functional group, such as amino or alkylamino, and are, thus, -58capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or through a separate reaction of a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the 5 salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate., valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylatc, mcsylatc, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, for example, Berge ct al. (1977) "Pharmaceutical Salts", J. Phann. 10 Sci. 66:1-19). The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, 15 sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stcaric, lactic, malic, tartaric, citric, ascorbic, palmitic, malcic, hydroxymalcic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2 acetoxybcnzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. 20 In other cases, the compounds of the present invention may be compounds containing one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with phariaceutically-acceptable bases. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a 25 suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include 30 ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra). Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, -59flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium 5 metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tctraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. 10 The formulations of the compounds of the invention may be presented in unit dosage form and may bc prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier 15 material to produce a single dosage form will generally be that amount of the agent which produces a therapeutic effect. In certain embodiments, a formulation of the present invention comprises an excipient, including, but not limited to, cyclodextrins, liposomes, micelle forming agents, e.g.. bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and an agent of 20 the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a agent of the present invention. Methods of preparing these formulations or compositions may include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. 25 Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrtips and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl 30 carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. - 60 - Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents 5 as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia 10 or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present 15 invention may also be administered as a bolus, electuary or paste. In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (I) fillers or extenders, such as starches, lactose, sucrose, 20 glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary 25 ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearatc, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering 30 agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more -61 accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert dilucnt, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a 5 suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in 10 the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. Compositions of the invention may also be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, 15 Filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, 20 optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro encapsulated form, if appropriate, with one or more of the above-described excipients. Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by 25 mixing one or more compounds of the invention with one or more suitable nonirritating recipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. 30 Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. - 62 - Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants 5 which may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. 10 Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc. silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. 15 Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound 20 in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more 25 pharmaceutically-acceptable sterile isotonic aqueous or nonaqucous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. 30 Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl olcate. Proper - 63 fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some cases, in order to prolong the effect of a drug, it is desirable to slow the 5 absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug forn is accomplished 10 by dissolving or suspending the drug in an oil vehicle. Injectable depot forms arc made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycol ide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include 15 poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. Exemplary formulations comprising agents of the invention are determined based on various properties including, but not limited to, chemical stability at body temperature, functional efficiency time of release, toxicity and optimal dose. 20 The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. 25 Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. Therapeutic Methods of the Invention 30 The present invention further provides novel therapeutic methods of treating proteinopathies and other diseases for which enhanced protein breakdown may be therapeutic, including neurodegenerative diseases, comprising administering to a subject, (e.g., a subject in need thereof), an effective amount of a compound of the invention. - 64 - A subject in need thereof may include, for example, a subject who has been diagnosed with a proteinopathy or a subject who has been treated for a proteinopathy, including subjects that have been refractory to the previous treatment. The methods of the present invention may be used to treat any proteinopathy. 5 Examples of such proteinophaties include, but are not limited to, Alzheimer's disease, cerebral p-amyloid angiopathy, retinal ganglion cell degeneration, prion diseases (e.g. bovine spongiform encephalopathy, kuru, Creu tzfeldt-Jakob disease. variant Creutzfeldt Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia) tauopathics (e.g. frontotemporal dementia, Alzheimer's disease, progressive supranuclear 10 palsy, corticobasal degeration, frontotemporal lobar degeneration), frontemporal lobar degeneration, amyotrophic lateral sclerosis, Huntington's disease, familial British dementia, Familial Danish dementia, hereditary cerebral hemorrhage with amyloidosis (lclandic), CADASIL, Alexander disease, Seipinopathies, familial amyloidotic neuropothy, senile systemic amyloidosis, serpinopathies, AL amyloidosis, AA amyloidosis, type Il diabetes, 15 aortic medial amyloidosis, ApoAl amyloidosis, Apoll amyloidosis. ApoAIV amyloidosis, familial amyloidosis of the Finish type, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis/myopathy, cataracts, medullary thyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma, hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis, corneal lactofcrin amyloidosis, corneal 20 lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic tumor amylois, seminal vesical amyloid, cystric fibrosis, sickle cell disease, critical illness myopathy, von Hippel-Lindau disease, spinocerebellar ataxia 1, Angelman syndrome, giant axon neuropathy, inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD), 25 In some embodiments, the subject pharmaceutical compositions of the present invention will incorporate the substance or substances to be delivered in an amount sufficient to deliver to a patient a therapeutically effective amount of an incorporated therapeutic agent or other material as part of a prophylactic or therapeutic treatment. The desired concentration of the active agent will depend on absorption, inactivation, and 30 excretion rates of the drug as well as the delivery rate of the compound. It is to be noted that dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the - 65 person administering or supervising the administration of the compositions. Typically, dosing will be determined using techniques known to one skilled in the art. The dosage of the subject agent may be determined by reference to the plasma concentrations of the agent. For example, the maximum plasma concentration (Cmax) and 5 the area under the plasma concentration-time curve from time 0 to infinity (AUC (0-4)) may be used. Dosages for the present invention include those that produce the above values for Cnax and AUC (0-4) and other dosages resulting in larger or smaller values for those parameters. Actual dosage levels of the active ingredients in the pharmaceutical compositions of 10 this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of 15 administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. 20 A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the agents of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage 25 until the desired effect is achieved. In general, a suitable daily dose of an agent of the invention will be that amount of the agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the agent may be administered as two, three, 30 four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. The precise time of administration and amount of any particular agent that will yield the most effective treatment in a given patient will depend upon the activity, - 66 pharmacokinctics, and bioavailability of a particular agent, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like. The guidelines presented herein may be used to optimize the treatment, e.g., 5 determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing. While the subject is being treated, the health of the subject may be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour 10 period. All aspects of the treatment, including supplements, amounts, times of administration and formulation, may be optimized according to the results of such monitoring. The patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters, the first such reevaluation typically occurring at the end of four weeks from the onset of therapy, and subsequent reevaluations 15 occurring every four to eight weeks during therapy and then every three months thereafter. Therapy may continue for several months or even years, with a minimum of one month being a typical length of therapy for humans. Adjustments, for example, to the amount(s) of agent administered and to the time of administration may be made based on these reevaluations. 20 Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is attained. In addition, the combined use an agent that modulates an autotrophy-associated gene product and a second agent, e.g. another agent usefuli for the treatment of the autophagy-related disease, may reduce the required dosage 25 for any individual agent because the onset and duration of effect of the different compounds and/or agents may be complimentary. One aspect of the invention relates method of inhibiting the dcubiquitination activity of a Usp14 protein comprising contacting the Usp14 protein with any one of the aforementioned compounds (including IU1), or a pharmaceutically acceptable salt, solvate, 30 hydrate, prodrug, chemically-protected form, enantiomer or stereoisomer thereof. Another aspect of the invention relates to a method of enhancing protein degradation by a proteasome in a cell comprising contacting the cell with any one of the aforementioned compounds (including IUl), or a pharmaceutically acceptable salt, solvate. -67 hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, Another aspect of the invention relates to a method of treating or preventing a proteinopathy in a subject comprising administering to the subject any one of the aforementioned compounds (including IUI), or a pharmaceutically acceptable salt, solvate, 5 hydrate, prodrug, chemically-protected form, enantiomer or stereoisomer thereof. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the proteinopathy is selected from the group consisting of Alzheimer's disease, cerebral p-amyloid angiopathy, retinal ganglion cell degeneration,, bovine spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, variant Creutzfcldt-Jakob 10 disease, Gerstma nn-Straussler-Schein ker syndrome, fatal familial insomnia, frontotemporal dementia, Alzheimer's disease, progressive supranuclear palsy, corticobasal degcration, frontotemporal lobar degeneration, frontemporal lobar degeneration, amyotrophic lateral sclerosis, Huntington's disease, familial British dementia, Familial Danish dementia, hereditary cerebral hemorrhage with amyloidosis (lclandic), CADASIL, Alexander disease, 15 familial amyloidotic neuropothy, senile systemic amyloidosis, serpinopathies, AL amyloidosis, AA amyloidosis, type 11 diabetes, aortic medial amyloidosis, ApoAl amyloidosis, Apoll amyloidosis, ApoAIV amyloidosis, familial amyloidosis of the Finish type, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis/myopathy, cataracts, medullary thyroid carcinoma, cardiac atrial amyloidosis, 20 pituitary prolactinoia, hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis, corneal lactoferrin amyloidosis, corneal lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic tumor amylois, seminal vesical amyloid, cystric fibrosis, sickle cell disease and critical illness myopathy. In certain embodinents, the present invention relates to any of the aforementioned 25 methods, wherein the proteinopathy is Alzheimer's disease, frontotemporal lobar degeneration, amyotrophic lateral sclerosis or Machado-Joseph disease. Another aspect of the invention relates to a method of treating or preventing a disease, for which enhanced protein breakdown may be therapeutic, in a subject comprising administering to the subject any one of the aforementioned compounds (including IU1), or 30 a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, or a pharmaceutical composition thereof In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the disease is selected from the group consisting of von Hippel-Lindau -68disease, spinocerebellar ataxia I, Angelman syndrome, giant axon neuropathy, inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD). Another aspect of the invention relates to a method of enhancing proteasome function in a subject comprising administering to the subject any one of the aformentioned 5 compounds(including IUI), or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, or a pharmaceutical composition thereof. Another aspect of the invention relates to a method of increasing degradation of Tau, TDP-43 or ataxin-3 in a subject comprising administering to the subject any one of the 10 aformentioned compounds(including 1Ul), or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, or a pharmaceutical composition thereof. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein said subject is human. Isolated Reconstituted Proteasomes 15 Certain aspects of the invention relate to isolated proteasomes that lack enzymatically active Uch37 but comprise cnzymatically active Usp 14. Sch proteasomes can be from any suitable organism. In certain embodiments the proteasomes of the invention are mammalian proteasomes, such as human or murine proteasomes. Such proteasomes may contain enzymatically inactive Uch37 or may lack Uch37 altogether. The proteasomes 20 of the invention are useful, for example, in methods of screening for specific inhibitors of Usp14. See, for example, International Patent Application Publication WO 2008/147536 Al, hereby incorporated by reference in its entirety. In certain embodiments, the proteasomes of the invention include enzymatically inactive Uch37. Uch37 can be rendered inactive through any method known in the art, 25 including, for example, through mutation of its enzymatic site, through treatment with a Uch37 specific inhibitor, or through treatment with a non-specific deubiquitinase inhibitor (e.g, through treatment with ubiquitin-vinylsulfone). Treatment of Uch37 with ubiquitin vinylsulfone results in the generation of vinylsulfone-Uch37 adducts, which are inactive for deubiquitinase activity. 30 Another aspect of the invention relates to methods of generating proteasomes of the invention. Such methods may include steps of purifying a proteasome lacking Uspl4 but comprising Uch37, treating the purified proteasome with a dcubiquitinase inhibitor, and/or reconstituting the purified proteasome with enzymatically active Usp14. -69- Purification of the proteasome lacking Usp14 but comprising Uch37 can be done using any suitable method known in the art. For example, human proteasornes can be affinity-purified from a HEK293 cell line that expresses HTBI-l-tagged hRpn 11. The cells can be lysed and the proteasomes affinity purified with NeutrAvidin agarose resin to 5 produce proteasomes lacking Uspl4 but containing Uch37. Any suitable Uch37 inhibitor can be used in the methods of the invention, including Uch3 7 specific inhibitors and non-specific deubiquitinase inhibitor (e.g., ubiquitin vinylsulfone). The active Usp14 that is used to reconstitute the proteasomes of the invention can be 10 from any suitable source, including, for example, Uspl 4 purified from a mammalian cell or recombinantly produced Usp 14. Another aspect of the invention relates to methods of screening for inhibitors of Usp14 comprising providing a proteasome of the invention, contacting the proteasome with a test compound and a Usp 14 substrate, and determining whether the test compound 15 inhibits the deubiquitination of the substrate. Deubiquitination of the substrate can be detected either directly or indirectly using any suitable method. For example, in certain embodiments, the substrate is coupled to a reporter that is detectable after cleavage by a dcubiquitinase and/or is an ubiquitin dependant proteasome substrate (e.g, Ub-AMC). In other certain embodiments, 20 deubuquitination of the substrate is demonstrated by inhibition of substrate degradation. Another aspect of the invention relates to a kit comprising the isolated proteasome of the invention, instructions of use, and/or a Uspl4 substrate. In some embodiments the Usp14 substrate is Ub-AMC and/or polyubiquitinated cyclin B. EXAM PLES 25 The invention now being generally described, it will be more readily understood by reference to the following, which is included merely for purposes of illustration of certain aspects and embodiments of the present invention, and is not intended to limit the invention. Example I -- Synthesis of Inhibitors 30 Figure 29 depicts one approach to the preparation of pyrroles of the invention. By forming a 1,3-diazole, instead of a pyrrole, similar diazole componds may be prepared. By varying the ring substitution on aryl amine la, or substituting an alkyl amine, heteroaryl amine, aralkyl amine, etc., a wide variety of compounds may be prepared. Likewise, -70compouind Ie may be reacted with a number of nucleophiles to provide a wide variety of compounds. Experimental procedures corresponding to the compounds shown in Figure 29 are provided below. Synthesis of 1-(4-chliorophenyl)-2,5-dimethylpyrrole (1c). A mixture of la 5 (7.65g, 60.0mmol) and lb (34.2 g, 300.0 mmol) in acetic acid (40 mL) was heated to 100 T for I hour, the solvent was then evaporated and the residue was purified by silica column chromatography to give Ic (11.07 g, yield: 89.8%). Synthesis of 2-chlioro-1-[1-(4-chlorophenvl)-2,5-dimethylpyrrol-3-yllethan- 1 one (le). To a solution of AIC13 (7.98 g, 60.0 mmol) in 1, 2-dichlorocthane (50 mL) was 10 added Id (6.78 g, 60.0 mmol) at 0 *C. The resulting mixture was stirred for 30 min and added to a solution of le (6.17 g, 30.0 mmoL) in 1,2-dichlorocthane (50 mL) at 0 C. The reaction mixture was theii warmed to room temperature for 2 hour and poured into ice water (20 mL). The mixture was extracted with dichloromethane (15 mL x 3), dried over MgSO 4 and purified by silica column chromatography to give le, 3.37 g, yield: 39.9% 15 Synthesis of 1-1 1-(4-chlorophenyl)-2,5-dimethylpyrrol-3-ylI-2-piperidylethan- I one (1). To a solution of le (85 mg, 0.3 mmol) and triethylamine (61 mg. 0.6 mmol) in acetonitrile(10 mL) was added I f (28 mg, 0.33 mmol). After being heated to reflux for I hour, the mixture was concentrated and the residue was dissolved in dichloromethane (30 rnL), washed with sat. NaHC0 3 (10 mL), dried over MgSO 4 and purified by silica column 20 chromatography to give 1 (83 mng, yield: 83.8%). LC/MS: 331.1 (M+1) l H NMR (CDCb, 300 MHz): 7.45-7.49 (2H, dd), 7.10-7.13 (2H, dd), 6.39 (2H, ds), 3.56(2H, s), 2.53-2.56 (4H, m), 2.30 (3H, s), 1.98 (3H, s), 162-1.70 (4H, m), 1.44-1.49 (21H, in). Example 2 -- Uspl4 Mediates Substrate Deubiquitination To test whether Usp 14 is a potent inhibitor of human protcasoies, a purification 25 procedure was developed to generate proteasomes that lack detectible levels of dcubiquitinase Usp 14 (modified from Wang et a/., (2007), Biochemistry, 46, 3553-3565). Briefly, human proteasomes were affinity-purified on a large scale from a stable HEK293 cell line harboring HTBH-tagged hRpnl 1. The cells were Dounce-homogenized in lysis buffer (50 mM NaH 2
PO
4 [pH 7.5], 100 mM NaCI, 10% glycerol, 5 mMi MgCl 2 , 0.5% NP 30 40, 5 mM ATP, and I mM DTT) containing protease inhibitors. Lysates were cleared, then incubated with NeutrAvidin agarose resin (Thermo Scientific) overnight at 4 'C. The beads were then washed with excess lysis buffer followed by the wash buffer (50 mM Tris-HCI [pH 7.5], 1 mM MgCl 2 and I mM ATP). For VS-proteasomes, I to 1.5 pLM of Ub-VS -71 - (Boston Biochem) was added to the resin and incubated at 30 'C for 2 i. Residual Ub-VS was removed by washing the beads with at least 20 bed vol of wash buffer. 26S proteasones were eluted from the beads by cleavage, using TEV protease (Invitrogen). Using this proteasome purification procedure, Human proteasornes were affinity 5 purified from a hRpnl 1-tagged line of HEK293 cells. Purification of proteasomes lacking Usp 14 but containing related deubiquitinase Uch37 was confirmed by western blot using an anti-Usp14 and anti-Uch37 antibodies (Figure IA and IB, respectively). The purified Usp 14-free proteasome (also described as 26S proteasormes) retained high levels of deubiquitinating activity that could be irreversibly inhibited by treating the proteasome with 10 ubiquitin-vinylsulfone (Ub-VS, Yao et al., (2006) Nat. Cell Biol., 8, 994-1002). Ub-VS inhibits dcubiquitination of substrates by forming adducts with the Cys amino acid located in the active site of thiol protease class deubiquitinating enzymes. As demonstrated in Figure I B, addition of Ub-VS to 26S proteasomes resulted in enzymatically inactive VS Uch37 adducts forming with all detectable Uch37. 15 In order to generate pure, recombinant Usp 14 enzyme, GST-Usp 14 (WT and CI 14A variants) was expressed in E. coli strain Rosetta 2 (DE3) cclls (Novagen). Cultures were grown at 37 'C until OD 00 reached 0.6 to 0.8, and expression was induced overnight with I mM IPTG at room temperature. Cells were then harvested in PBS containing protease inhibitors and lysed by French press. The cleared lysates were incubated with 20 GST Sepharose 4B resin (GE Healthcare) at 4C for 1 h, and subsequently washed with excess PBS, followed by PBS containing 100 mM NaCl. The GST moiety was removed by thrombin in the cleavage buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2.5 mlM CaC 2 , and 0. 1 % 2-mercaptoethanol) for 3 h at room temperature. GST-tagged Usp 14 proteins for proteasome binding assays were elutcd before thrombin cleavage using clution buffer (10 25 mM reduced glutathione in 50 mM Tris-HCl [pH 8.0]), The inhibited "VS-proteasomes" described above, which lack endogenous dcubiquitination activity due to Ub-VS treatment, were successfully reconstituted with recombinant Usp14 (Figure 2). An Ub-AIC hydrolysis assay was performed with 1 nM of Ub-VS treated human proteasome (VS-Proteasome) alone, 400nM ofUspl14 alone, or VS 30 proteasome that had been reconstituted with 4 or 40 nM of recombinant Usp 14 protein. As has been described above, the deubiquitination activity of the VS-proteasome was almost completely inhibited (Figure 3). In contrast, the reconstituted Usp 14/VS proteasome demonstrated substantial deubiquitination activity (Figure 3). In fact, the Usp14/VS - 72 proteasome demonstrated an 800-fold increase in Ub-AMC hydrolyzing activity over that of isolated Usp14 alone (Figure 3). Thus, the enzymatic activity of Usp14 is increased by its complexing with the proteasome. Therefore, the Ub-AMC assay allows the success of reconstitution to be followed. 5 The Ub-AMC assay was also used to examine the kinetics of Ub-AVIC hydrolysis by the reconstituted Usp 14-proteasome complexes. Ub-AMC hydrolysis by Usp 14/VS proteasomes that had been reconstituted with various amounts of UspI14 was monitored over a period of 30 minutes (Figure 4). Analysis of the results of this assay demonstrated the affinity of Uspl4 for the proteasome is approximately 4 nM. 10 Example 3 -- Usp14 Inhibits Proteasomal Degradation The effect of Usp14 on the degradation ofubiquitinated substrates was examined using an in vito degradation assay using the ubiquitin-dependant proteasome substrate polyubiquitinated cyclin B (Ubr-ClnB). In these experiments, Ubr-ClnB was incubated with human protcasomes (4 nM), containing either wild-type or catalytically inactive Usp 14 15 (60 nM). The catalytically inactive Usp used in these assays was Usp14-Cl 14A, which contains a mutation in Usp 4's active site for dcubiquitination. Notably, both wild-type Usp14 and Uspl4-Cl 14A are able to bind to 26S mammalian proteasomes (Figure 2). As demonstrated in Figure 5, Usp 14 strongly inhibits the degradation of cyclin B, while the active site mutant of Usp14 showed little inhibitory effect. The lack of inhibition of Ub, 20 CInB degradation by the active site mutant indicates that the ubiquitin chain trimming activity of wild-type Usp14 is required for Usp14's inhibition of proteasome degradation. Indeed, extensive trimming of the ubiquitin groups from cyclin B was evident by immunoblot analysis in the samples containing wild-type Usp 14, but was nearly eliminated when catalytically inactive Usp 14 was used (Figure 5). 25 An effect of Usp14 on Tau degradation in human cells was observed in the human cell line, HEK293. Tau was coexpressed with exogenous wild-type or catalytically inactive Uspl4 and Tau protein levels were determined by western blot. Expression of wild-type Usp 14, but not enzymatically inactive Usp14, stabilized Tau in the human cell line (Figure 6). In fact, expression of enzymatically inactive Usp14 in H1EK293 cells resulted in 30 accelerated Tau degradation (Figure 613). This dominant negative effect likely reflects the displacement of endogenous, wild-type Usp 14 from the proteasome. This hypothesis was confirmed using a mutant form of Usp 14 that lacks the N-terminal UBL domain (Usp 14 AUBL). The N-terminal UBL domain (Figure 6A) is the principal proteasome-binding site - 73 on Usp 14. Deletion of the UBL attenuated the dominant negative effect (Figure 7), indicating that proteasome binding is required for the mediation of this effect. The short form (SF) of Usp 14 is an endogenous Usp 14 splice variant that is expressed from mnRNA that lacks a junctional exon (exon 4) between the N-terminal 5 ubiquitin-like domain of Usp14 and its catalytic domain (Wilson et a/., (2002). Nat. Genet., 32, 420-425; Figure 6A). Like the catalytically inactive mutant of Usp14, Usp14-SF exhibited a dominant negative effect on Tau stability in HEK293 cells (Figure 6A). This suggests that Usp 14-SF may be an endogenous inhibitor of Usp 14. Consistent with this possibility, Usp14-SF is able to bind proteasome, but unlike the wild-type enzyme, it is not 10 activated enzymatically by proteasome binding (Figure 7). Example 4 -- Specific Inhibitors of Usp14 As demonstrated above, chain trimming at the proteasome by Usp14 is a key regulatory step in the ubiquitin-dependent protcolytic pathway. Therefore, in order to identify enhancers of proteasome function, a high-throughput screen for small molecule 15 Usp 14 inhibitors was performed using VS-proteasomes reconstituted with recombinant Usp14 and assayed with Ub-AMC (Figure 8). Compounds were screened for Usp 14/26S inhibition in 384-well low-volume plates in duplicate. Data processing was done by a robust Z-score method and each compound was plotted using Spotfire software. Compounds over the cut-off of Z > 5 were mostly 20 autofluorescent and were therefore not counted. To exclude quenching compounds that only affect AMC fluorescence, 312 primary hits were tested for quenching of AMC amine, and pure quenchers were scored as false-positives and excluded from further analysis (Figure 813). Of the 63,052 compounds analyzed in the high-throughput screen, 215 were identified as true inhibitors of Usp 14. 25 In order to identify compounds that specifically inhibited Usp14 but were not general dcubiquitinase inhibitors, the 215 hit compounds were counterscreened against a panel of deubiquitinating enzymes. Among the hit compounds that inhibited the activity of Usp14 but not any other tested deubiquitinase, I-[1-(4-fluorophenyl)-2,5-dimethlypyiol-3 yl]-2-pyrrolidin-1-ylethanone (IU I, Figure 9) was selected for further analysis. 30 Example 5 -- Specific Inhibition of Usp14 by JU1 Additional studies were performed on the specific Usp14 inhibitor IUI (Figure 10). To serve as a negative control, a compound that is structurally similar to ID], termed - 74 - "IUIC" (Figure 9B) which does not inhibit Usp14 dcubiquitinase (Figure lOB) or enhance proteasome function (Figure IOC) was also identified. The specificity of JUl for Usp14 was determined by testing its ability to inhibit the activity of eight deubiquitinating enzymes of human origin. As seen in Figures I 1 and 9C, 5 despite being a potent inhibitor of proteasome-bound Uspl4, IUI failed to significantly inhibit the other tested deubiquitinating enzymes, including Uch37. Note that Figure 9E shows that IUI does not inhibit the proteasome-bound form of Uch37. Furthermore, IUI also failed to inhibit the activity of Usp14 that had not been loaded onto a proteasome (Figure 9D), indicating that IUl specifically inhibits the proteasome-bound, activated form 10 of Usp14. As the binding of Usp14 to the proteasome enhances Usp14 activity, it was possible that IUl inhibited UspI4 activity by interfering with the Uspl4/proteasome interaction. Therefore, ability of IU 1 to interfere with the ability of Usp 14 to bind to the proteasome was examined. Purified human proteasomes were incubated with recombinant Usp 14 either 15 in the presence or the absence of various concentrations of IUL. As seen in Figure 12, JU l did not antagonize Usp 14 complexing with the proteasome, indicating that the inhibitory activity of lU l is not the result of an inhibition of the formation of UspI4/protcasome complexes. The reversibility of IUI inhihibition of Uspi4 was next assayed. Uspl4/protcasomc 20 complexes were treated with IU, followed by centrifugation with a Micron-YM3 filter up to three times. After each spin, the protein complex was tested for deubiquitinase activity. As demonstrated in Figure 13, the activity of Uspl4 returned following centrifugation, thereby indicating that inhibition of Uspl4 by IU I is rapidly reversible. Consistent with this observation, mass spectrometry analysis of IUI inhibited Uspl4 failed to detect any 25 covalent I U] -Usp 14 adducts. The Uspl4 inhibitory activity of lUl was further quantified by generating two independent IC5e curves for Usp 14/26S proteasome complexes treated with various concentrations of JUl for either 45 minutes (Figure 14A) or 30 minutes (Figure 14B). The data plot of each experiment was fit into a four parameter logistic model (the Hill-slope 30 model) based on guidelines from the NIH Chemical Genonics Center. The results of these experiments indicated that the IC5rj value of IUI is 2-5 paM (Figure 14). Using methods similar to those described in Example 3, Cyclin B was used as a substrate to test whether IU I influenced the trimming of ubiquitin chains by proteasome - 75 complexes. To separate chain trimming from substrate degradation, these assays were done in the presence of proteasome inhibitors. The effectiveness of the proteasome inhibitors is evidenced by the accumulation of unmodified cyclin B in the assay (Figure 15). When proteasomes that lack Usp 14 were tested, JUl had little or no effect on the release of 5 ubiquitin chains from cyclin B (Figure 15), which is likely mediated by another deubiquitinating enzyme on the proteasome, Rpn 11. Upon the addition of Usp 14, however, chain trimming by the proteasome complexes was strongly enhanced, as apparent from the increased electrophoretic mobility of the ubiquitinated forms of cyclin B. The further addition of IUI to the Usp14/protcasome complexes reversed this effect, and reduced the 10 chain trimming to a level similar to that of the protcasome complexes that lacked Usp 14 (Figure 15). It was next tested whether IU I could serve as an enhancer of substrate degradation by the proteasome. Using the methods described in Example 3, an in vitro Ub-ClinB degradation assay was performed, but this time in the absence or presence of 34 pM IU . 15 The addition of IUI to proteasomes that lack Usp14 had no effect on substrate degradation or chain trimming. Confirming the results described above, addition of Usp 14 to the proteasome complex enhanced chain trimming and dramatically inhibited substrate degradation. The addition of IU1 stimulated the activity of Usp14-containing proteasomes in degrading Ub-cyclin B and inhibited ubiquitin chain trimming (Figure 16). 20 Example 6 -- Cellular Entry of IU I The IUl experiments described above were performed in vitro. In order for IUl to enhance proteasome degradation in vivo, it is necessary the IUl be able to enter cells. In order to examine this, entry of IU 1 into cells was assayed by electrospray mass spectrometry using an Agilent series 1200 LC/6130 system with a reversed-phase Cs 25 column. IUI was added to MEFs at 50 pM for various periods of time. Cell lysates were colleted and ethyl acetate extraction was used to prepare mass spectrometer samples. Ion count of LC/MS traces (m/z at 30 1) at 0 hr, I hr and 24 hr are shown (Figure 17). This assay revealed that, when added to the medium at 50 pM, IU 1 reached a steady-state concentration of-19 pM within cells by I hour, and maintained approximately the same 30 level over the time course of the experiment (Figures 17 and 18). Similar results, extending through two days, were obtained using a separate UV absorption assay (Figure 19). Additionally, IUl concentrations were maintained in the medium as for at least two days. These results indicate that IUl is stable compound within both cells and standard media. - 76 - Example 7 -- Enhancement of in vivo Proteasomal Degradation by IUI To determine whether IU could enhance proteasome function in living cells, Tau was expressed in MEF cells, which were then treated with IUl at concentrations from 25 to 100 pM. Specifically, after 36 hours of Tau and LacZvS expression, MEF cells were 5 incubated with 0, 25, 50, 75 or 100 pM of IUl for 6 hours. As seen in Figure 21 A, IU I reduced Tau levels at all concentrations tested. No effect was seen on Tau rnRNA levels (Figure 21B). Other proteins that have been implicated in protcotoxic mechanisms were also tested. Using similar methods to those described above, it was demonstrated that TDP-43 10 (implicated in frontotemporal lobar degeneration and amyotrophic lateral sclerosis), ataxin 3 (implicated in Machado-Joseph disease) and glial fibrillary acidic protein (GFAP, implicated in Alexander disease) were similarly depleted from cells upon IUI treatment (Figure 22A-C). On the other hand, IUI had little or no effect upon the in vivo degradation of the ubiquitin-independent proteasome substrate, GFP-ODC. Together, these results 15 indicate that IUI is a general enhancer of the ubiquitin-mediated proteasome degradation. Oxidized proteins form another class of proteasome substrates that play an important role in human health. Harmful oxidized proteins accumulate upon aging and are implicated in a variety of age-related diseases and disorders (Stadtman (2006) Protein oxidation and aging. Free Radic. Res. 40, 1250-1258; Ahmed et al (2007) Protein 20 oxidative modifications and replicative senescence of WI-38 human embryonic fibroblasts. Ann. NY Acad. Sci, 1119, 88-96; Moskovitz et al (200 1). Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc. Nat. Acad. Sci. USA, 1981, 12920-12925.). Protein oxidation was induced by treating cells with menadionc. and oxidized species were visualized using an antibody specific for protein 25 carbonyls. Specifically, MEFs were preincubated with vehicle or 75 pM IU I for 4 hours and then treated with 63 pM menadione for 45 minutes. The cells were lysed and lysates were incubated with DNPH and immunoblotted with anti-DNPH antibody to assay for oxidized proteins. Accumulation of oxidized proteins was reduced in cells treated with IUl than in untreated cells (Figure 23). When proteasome inhibitor PS-341 was added together 30 with IU, the effect of IUl was eliminated, indicating that IUI does not prevent oxidation reaction, but rather it enhances the proteasomal degradation of the oxidized proteins. These data indicate that there is a Usp 14-inhibited ubiquitin-dependent mechanism for the degradation of proteins damaged by reactive oxygen species. Menadione is toxic to cells, -77and IUl treatment reduced this toxicity substantially in HEK293 cells (Fig. 30), strongly supporting the hypothesis that proteins are critical targets of oxidative damage in cells. IU I also reduced the toxicity of an unrelated oxidizing agent, hydrogen peroxide (data not shown). IUI C, the JUl variant that is inactive against Usp14, failed to reduce menadione 5 cytotoxicity (data not shown). Importantly, these experiments indicate that IUI can promote cell survival during proteotoxic stress. Example 8 -- The Effects of 1Ul on Cellular Proliferation and Viability The effect of IU I on cell viability was next examined by MTT assay. IU 1 was added to MEF, HEK293 and HeLa cells at various concentrations, followed by addition of 10 M4TT solution after 6, 12, 24 or 48 hours of IU I incubation. Effects on cell viability became apparent at concentrations over 100 pM, well above the doses required to enhance the degradation of Tau, TDP-43, ataxin-3, and oxidized proteins (Figures 21-23). Moreover, IUI did not noticeably induce apoptosis in MEF cells, as assessed by TUNEL assay (Figure 3 1). 15 Cell proliferation of MEFs (Figure 28) and 293 cells that had been exposed to various concentrations of lUI was measured by microscopy in real time. The results of this assay revealed only a slight inhibition in cellular proliferation at 120 pM, but no apparent inhibition at lower concentrations (Figure 28). Taken together with the results of the cell viability assays presented above, this indicates that lU I's inhibition of ubiquitin chain 20 trimming by Uspl4 does not grossly compromise cell function. EQUIVALENTS The present invention provides, in part, methods for the enhancement of protein turnover by the proteasome and the treatment of diseases involving either proteasome substrates, upstream components of the ubiquitin-proteasome pathway, or the proteasome 25 itself. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The appended claims are not intended to claim all such embodiments and variations, and the full scope of the invention should be determined by reference to the claims, along with their full scope of 30 equivalents, and the specification, along with such variations. All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, In case of conflict, the present application, -78including any definitions herein, will control. -79-

Claims (105)

  1. 2. The compound of claim I, wherein A is aryl or heteroaiyl.
  2. 3. The compound of claim I, wherein A is phenyl, pyridin-2-yl, pyridin-3-yl or pyrimidin-2-yI, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy, carbocyclyloxy, heterocyclyloxy, haloalkoxy, fluoroalkyloxy, formyl, alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, amino, amido, azido, aminosuIfonyl, aminosulfiny], cyano, nitro, phosphinyl, phosphoryl, silyl, -81 - silyloxy, and any of said substiuents bound to the heterocyclyl group through a methylene or ethylene moiety.
  3. 4. The compound of claim 1. wherein A is phenyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro.
  4. 5. The compound of claim 1, wherein A is 6. The compound of claim 1, wherein A is phenyl substituted in the two position (ortho substituted) with a substitutent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro.
  5. 7. The compound of claim 1, wherein A is or F
  6. 8. The compound of claim 1, wherein A is phenyl substituted in the three position (meta substituted) with a substitutent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido cyano, and nitro. CI 0
  7. 9. The compound of claim 1, wherein A is or F
  8. 10. The compound of claim 1, wherein A is phenyl substituted in the four position (para substituted) with a substitutent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fl uoroalkyloxy, amino, azido, cyano, and nitro. - 82 -
  9. 11. The compound of claim 1, wherein A is F N CF 3 NO 2 [-nj Br CF 2 CF 3 0 / N~k \H - 1ttN1 / \= N 3 NH NH2 or N
  10. 12. The compound of claim 1, wherein A is phenyl substituted in the two and four positions with substitutents independently selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fuoroalkyloxy, amino, azido, cyano, and nitro. CI F F F
  11. 13. The compound of claim 1, wherein A is or
  12. 14. The compound of claim 1, wherein A is pyridin-2-yl, optionally substituted in the four position with a substituent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. N
  13. 15. The compound of claim 1, wherein A is .
  14. 16. The compound of claim 1, wherein A is pyrimidin-2-yl, optionally substituted in the four position with a substituent selected from the group consisting of alkyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro. N
  15. 17. The compound of claim 1, wherein A is N Cl. 17. The compound of claim 1, wherein A is biaryL
  16. 19. The compound of claim 1, wherein A is 4-(phenyl)phen-l-yI or 4-(2 pyridinyl)phen- I -yl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, halo, haloalkyl, - 83 - fluoroalkyl, hydroxy, alkoxy. haloalkoxy, fluoroalkyloxy, amino, azido, cyano, and nitro.
  17. 20. The compound of claim 1, wherein A is -or N .
  18. 21. The compound of any one of claims 1-20, wherein R' is hydrogen.
  19. 22. The compound of any one of claims 1-20, R' is alkyl, haloalkyl or fluoroalkyl.
  20. 23. The compound of any one of claims 1-20, R' is methyl, halomethyl or fluoromethyl.
  21. 24. The compound of any one of claims 1-20, R' is ethyl, haloethyl or fluoroethyl.
  22. 25. The compound of any one of claims 1-20, R 2 is hydrogen.
  23. 26. The compound of any one of claims 1-20, R 2 is alkyl, haloalkyl or fluoroalkyl.
  24. 27. The compound of any one of claims 1-20, R 2 is methyl, halomethyl or fluoromethyl.
  25. 28. The compound of any one of claims 1-20, R 2 is ethyl, haloethyl or fluoroethyl.
  26. 29. The compound of any one of claims 1-20, R' is hydrogen; and R 2 is hydrogen.
  27. 30. The compound of any one of claims 1-20, R' is alkyl; and R 2 is alkyl.
  28. 31. The compound of any one of claims 1-20, R' is methyl: and R 2 is methyl.
  29. 32. The compound of any one of claims 1-20, R' is ethyl; and R 2 is ethyl.
  30. 33. The compound of any one of claims 1-32, Z is =C(R 8 )-; and R 5 is hydrogen.
  31. 34. The compound of any one of claims 1-32, Z is =C(Rs)-; and R 8 is alkyl.
  32. 35. The compound of any one of claims 1-32, Z is =N
  33. 36. The compound of any one of claims 1-24, wherein Z is =C(R2)-; and the two R2 R 10 ~ / R 1 0 N\ R 10 R 1 O 0 R'N taken together are R1 0 R 0 , R 0 R 1 0 , R 0 , R N R 10 N R R 10 RIO or N-N 0
  34. 37. The compound of any one of claims 1-32, X is . N'R9
  35. 38. The compound of any one of claims 1-32, X is . - 84 - H I N'N, R N'NR9
  36. 39. The compound of any one of claims 1-32, X is or
  37. 40. The compound of any one of claims 1-39, Y is -CH 2 NR'R 4 .
  38. 41. The compound of any one of claims 1-39, Y is -CH 2 N RR 4 ; and R 3 is hydrogen.
  39. 42. The compound of any one of claims 1-39, Y is -CH 2 NR 3 R 4 ; and R3 is alkyl.
  40. 43. The compound of any one of claims 1-39, Y is -CH 2 NR'R; and R 4 is hydrogen.
  41. 44. The compound of any one of claims 1-39, Y is -CH 2 NR 3 R and R 4 is alkyl.
  42. 45. The compound of any one of claims 1-39, Y is -CH 2 NR 3 R, R 3 is hydrogen; and R 4 is alkyl.
  43. 46. The compound of any one of claims 1-39, Y is -CH 2 NR3R'; R 3 is alkyl; and R 4 is alkyl. HN- \N- HN
  44. 47. The compound of any one of claims 1-38, Y is N N-/ HN pN N N '-'P Nr 0
  45. 48. The compound of any one of claims 1-39, Y is -CH 2 (N-heterocyclyl), which is optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of alkyl, haloalkyl, fluoroalkyl, halo, hydroxyl, alkoxy, haloalkoxy, fluoroalkoxy, amino and nitro.
  46. 49. The compound of any one of claims 1-39, Y is -CH2(piperidin-l-yl), -CH2(pipcrazin-] -yl), -CH 2 (hexahydropyrimidin- 1 -yl), -CHI- 2 (morphol in- I -yl) or -CH2(1,3-oxazinan-3-yl), which is optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of alkyl, haloalkyl, fluoroalkyl, halo, hydroxyl, alkoxy, haloalkoxy, fluoroalkoxy, amino and nitro. -85-
  47. 50. The compound of any one of claims 1-39, Y is -CH2(piperidin-1-yl) or -CH2(piperazin-1-yl), which is optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of alkyl, haloalkyl, fluoroalkyl, halo, hydroxyl, alkoxy, haloalkoxy, fluoroalkoxy, amino and nitro. O
  48. 51. The compound of any one of claims 1-39, Y is F' V N CF 3 N 0- O-CF 3 0 0 N N N N NJ or N
  49. 52. The compound of any one of claims 1-39, Y is -CH 2 NH(CH 2 )nNH(alkyl), -CH 2 NH(CH 2 )nN(alkyl) 2 , -CH2NH(CH 2 )N(alkylene), -CH 2 N(alkyl)(CH 2 )n 1 NH(alkyl), -CH- 2 N(alkyl)(CH 2 ),N(alkyl) 2 or -CH 2 N(alkyl)(CH2)N(alkylene).
  50. 53. The compound of any one of claims 1-38, Y is -CH 2 NH(CH 2 )nO(alkyl) or -CH 2 N(alkyl)(CH 2 )A0(alkyl).
  51. 54. The compound of claim 52 or 53, wherein n is 1,
  52. 55. The compound of claim 52 or 53, wherein n is 2. - 86 -
  53. 56. The compound of claim 52 or 53, wherein n is 3.
  54. 57. The compound of claim 52 or 53, wherein n is 4. / / N N N O
  55. 58. The compound of any one of claims 1-39, Y is Fr
  56. 59. The compound of any one of claims 1-39, Y is -NR2R].
  57. 60. The compound of any one of claims 1-39, Y is -NR'R 4 ; and R is hydrogen.
  58. 61. The compound of any one of claims 1-39, Y is -NR R ; and R' is alkyl.
  59. 62. The compound of any one of claims 1-39, Y is -NR 3 R 4 ; and R 4 is hydrogen.
  60. 63. The compound of any one of claims 1-39, Y is -NR 3 R 4 ; and R 4 is alkyl.
  61. 64. The compound of any one of claims 1-39, Y is -NR 3 R 4 R 3 is hydrogen; and R 4 is alkyl.
  62. 65. The compound of any one of claims 1-39, Y is -NR"R 4 ; R" is alkyl; and R 4 is alkyl,
  63. 66. The compound of any one of claims 1-39, Y is -NR 5 NR 6 R 7 or -NR 5 (N heterocyclyl).
  64. 67. The compound of any one of claims 1-39, Y is -NR 5 NR 6 R 7 ; and R is hydrogen.
  65. 68. The compound of any one of claims 1-39, Y is -NR5NR 6 R 7 ; and R5 is alkyl.
  66. 69. The compound of any one of claims 1-39, Y is -NR 5 NRR'; and R5, R 6 and R 7 are, independently, hydrogen or alkyl.
  67. 70. The compound of any one of claims 1-39, Y is -NR-(N-heterocyclyl); and R 5 is hydrogen.
  68. 71. The compound of any one of claims 1-39, Y is -NR(N-hoterocyclyl); and R' is alkyl.
  69. 72. The compound of any one of claims 1-39, Y is H , H , H or N PH,
  70. 73. A compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, -87- chemically-protected form, enantiomer or stercoisomer thereof, selected from the 0 ( N group consisting of NN O 0 FNN F N F N FN 0 H FNN F N H O F N F N N N -_ F-N -. 0 o HN F N F N HO- 8 FN N~NF ' F-- N - 88 F oNNN 4N N N 0D 0 D N Cl- N oN ND IIN N N N > _ N 3 NN KN CN 0 N 0 3 N \ N N CI NO> 0 N> N 0 2 N N - 89 - Br &N N N F N F N CI N 0 2 N N 0(9Q- FQNKJ> Br N F N W - N W 6N N N W N W N o 0 W'aN WaN o 0 0 NJ o 0 - 90 - 00 H W N N W N N W' N W' NO 0 Hl 0 N N -rN W N Oo O ON O N WON W' N N N \ W-ON W N co o 0 W N WN N C N o-%- 0 d W N N N N "C - N CI \-9N N o 0 CF 3 GF 2 'a N i'"'N -91 - N N N CI NC N 0 F 0 NM N C) Na N CI N NN o 0 N N o 0 N' N \/ 0 0eN, N NN .l N CIHN 0 0 N N - 92 - N CN N Cl N CI N and Cl a N wherein W is methyl, fluoro, chloro, nitro, methoxy, cthoxy, -S0 2 NH 2 or -C(=O)NH 2 .
  71. 74. A pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, of any one of claims 1-73, or IUI, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, and a pharmaceutically acceptable excipient.
  72. 75. A method of inhibiting the dcubiquitination activity of a Usp 14 protein comprising contacting the Usp14 protein with a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stereoisomer thereof, of any one of claims 1-73, or IUI, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof.
  73. 76. A method of enhancing protein degradation by a proteasome in a cell comprising contacting the cell with a compound, or a phannaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stereoisomer thereof, of any one of claims 1-73, or IUl, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof.
  74. 77. A method of treating or preventing a proteinopathy in a subject comprising administering to the subject a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, of any one of claims 1-73, or IUI, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantioner or stercoisomer thereof, or a pharmaceutical composition of claim 74.
  75. 78. The method of claim 77, wherein the proteinopathy is selected from the group consisting of Alzhcimer's disease, cerebral p-amyloid angiopathy, retinal ganglion - 93 - cell degeneration,. bovine spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, frontotemporal dementia, Alzheimier's disease, progressive supranuclear palsy, corticobasal degeration. frontotemporal lobar degeneration, frontemporal lobar degeneration, amyotrophic lateral sclerosis, Huntington's disease, familial British dementia, Familial Danish dementia, hereditary cerebral hemorrhage with amyloidosis (1clandic), CADASI L. Alexander disease, familial amyloidotic neuropothy, senile systemic amyloidosis, serpinopathics, AL amyloidosis, AA amyloidosis, type I1 diabetes, aortic medial amyloidosis, ApoAl amyloidosis, Apoll amyloidosis, ApoAIV amyloidosis, familial amyloidosis of the Finish type, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis/myopathy, cataracts, medullary thyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma, hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis, corneal lactoferrin amyloidosis, corneal lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic tumor amylois, seminal vesical amyloid, cystric fibrosis, sickle cell disease and critical illness myopathy.
  76. 79. The method of claim 77, wherein the proteinopathy is Alzhcimer's disease, frontotemporal lobar degeneration, amyotrophic lateral sclerosis or Machado-Joseph disease.
  77. 80. A method of treating or preventing a disease, for which enhanced protein breakdown may be therapeutic, in a subject comprising administering to the subject a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, cnantiomer or stercoisomer thereof, of any one of claims 1-73, or IU, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, or a pharmaceutical composition of claim 74.
  78. 81. The method of claim 80, wherein the disease is selected from the group consisting of von Hippel-Lindau disease, spinocerebellar ataxia 1, Angel man syndrome, giant axon neuropathy, inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBNIPFD).
  79. 82. A method of enhancing proteasome function in a subject comprising administering to the subject a compound, or a pharmaceutically acceptable salt, solvate, hydrate, -94- prodrug, chemically-protected form, enantiomer or stercoisorner thereof, of any one of claims 1-73, or IUI, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, or a pharmaceutical composition of claim 74.
  80. 83. A method of increasing degradation of Tau, TDP-43 or ataxin-3 in a subject comprising administering to the subject a compound, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, of any one of claims 1-73, or IUI, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, chemically-protected form, enantiomer or stercoisomer thereof, or a pharmaceutical composition of claim 74.
  81. 84. The method of any one of claims 77-83, wherein said subject is human.
  82. 85. An isolated proteasome comprising enzymatically inactive Uch37 and further comprising enzymatically active Usp 14.
  83. 86. The proteasome of claim 85, wherein said proteasome comprises vinylsulfone Uch37 adducts.
  84. 87. The proteasome of claim 85, wherein said Usp14 is a recombinant protein.
  85. 88. The proteasome of claim 85, wherein said proteasome is a human proteasome or a murine proteasome.
  86. 89. An isolated proteasome comprising enzymatically active Usp14 and lacking enzymatically active Uch37.
  87. 90. The proteasome of claim 89, wherein said Uspl4 is a recombinant protein.
  88. 91. The proteasome of claim 89, wherein said proteasome is a human proteasome or a murine proteasome.
  89. 92. A method of generating a proteasome comprising cnzymatically inactive Uch37 and further comprising enzymatically active Usp14 comprising: (a) purifying a proteasome lacking Uspl4 but comprising Uch37; (b) treating said purified proteasomne with a deubiquitinasc inhibitor; and (c) reconstituting said purified proteasome with enzymatically active Usp 14.
  90. 93. The method of claim 92, wherein said proteasome is a human proteasome or a murine proteasome.
  91. 94. The method of claim 92, wherein said proteasome lacking UspI4 but comprising Uch37 is purified from HEK293 cells. -95- Ally Docket No.: HMV-194.25
  92. 95. The method of claim 92, wherein said dcubiquitinase inhibitor is ubiquitin vinylsulfone.
  93. 96. The method of claim 92, wherein said active Usp14 is recombinantly produced.
  94. 97. A method of screening for an inhibitor of Uspl4 comprising: (a) providing a proteasome comprising enzymatically inactive Uch37 and further comprising enzymatically active Usp14 (b) contacting said proteasome with a test compound and a Usp14 substrate; and (c) determining whether said test compound inhibits the doubiquitination of said substrate.
  95. 98. The method of claim 97, wherein said substrate is coupled to a reporter that is detectable after cleavage by a dcubiquitinase.
  96. 99. The method of claim 98, wherein said substrate is Ub-AMC.
  97. 100. The method of claim 97, wherein said substrate is an ubiquitin-dependant proteasome substrate.
  98. 101. The method of claim 100, wherein deubuquitination of said substrate is demonstrated by inhibition of substrate degradation.
  99. 102. The method of claim 101, wherein said substrate is polyubiquitinated cycling B.
  100. 103. The method of claim 97, wherein said proteasome comprises vinylsulfone-Uch37 add ucts.
  101. 104. The method of claim 97, wherein said Usp14 is a recombinant protein.
  102. 105. The method of claim 97, wherein said proteasome is a human proteasome or a urine proteasome.
  103. 106. A kit comprising an isolated proteasome of any one of claim 85-91, and instructions of use.
  104. 107. The kit of claim 106 further comprising a Usp 14 substrate
  105. 108. The kit of claim 107, wherein said Usp14 substrate is Ub-AMC or polyubiquitinated cyclin B. -96-
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