WO2012075393A2

WO2012075393A2 - Activators of proteasomal degradation and uses thereof

Info

Publication number: WO2012075393A2
Application number: PCT/US2011/063061
Authority: WO
Inventors: Alfred L. Goldberg; David Matthew Smith
Original assignee: President And Fellows Of Harvard College
Priority date: 2010-12-02
Filing date: 2011-12-02
Publication date: 2012-06-07
Also published as: WO2012075393A3

Abstract

The present invention provides compounds that induce gate opening of the proteasome, thereby activating the proteasomal degradation of proteins. Such compounds may have beneficial therapeutic effects. Compounds and compositions described herein may be used to treat disorders associated with unfolded, misfolded, or aggregated proteins such as sickle cell anemia, thalassemia, Alzheimer's disease, Parkinson's disease, cystic fibrosis, Huntington's disease, amyotrophic lateral sclerosis (i.e., Lou Gehrig's disease), and Creutzfeld-Jakob disease. The compounds and compositions may also be used to kill or inhibit the growth of a microorganism. The present invention also provides methods for identifying compounds that activate the proteasomal degradation of proteins. In particular, the present invention provides in silico methods for identifying compounds that activate the proteasomal degradation of proteins.

Description

ACTIVATORS OF PROTEASOMAL DEGRADATION AND USES

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of United States

Provisional Patent Application U.S. S.N. 61/419,025, filed December 2, 2010, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Proteasomes are major sites for protein degradation in eukaryotic cells. The

26S proteasome plays a key role in the elimination of misfolded or damaged proteins as well as in the regulation of cell growth. The 26S proteasome is a 2.4 megadalton complex that contains a cylindrical proteolytic core, the 20S proteasome, sandwiched between two 19S regulatory complexes. 20S and 26S proteasomes degrade proteins processively into peptides which vary in length from 2-25 residues (Akopian et al. (1997) J. Biol. Chem. 272: 1791; Kisselev et al. (1998) J. Biol. Chem. 273: 1982; Kisselev et al. (1999), J. Biol. Chem.

274(6):3363-71).

[0003] The active forms of the proteasomes in archaea and eukaryotes are composed of one or two ATPase complexes and the 20S proteasome, a hollow cylinder composed of four stacked rings (Voges et al. (1999) Ann. Rev. Biochem. 68: 1015; DeMartino and Slaughter

(1999) J. Biol. Chem. 274:22123; GroU et al. (1997) Nature 386:463; Lowe et al. (1995) Science 268:533). The alpha rings of the 20S proteasome contain seven a-subunits that surround a narrow channel through which polypeptides pass to enter the central chamber where multiple proteolytic sites are located. The passage of proteins and peptides through this channel is restricted by the N-termini of the a-subunits, which function as a gate (Groll et al.

(2000) Nature Structural Biology 7: 1062; Groll and Huber (2003) Int. J. Biochern. Cell. Biol. 35:606). ATPase ring complexes open this gate and thus regulate proteolysis by the 20S proteasome (Kohler et al. (2001) Mol Cell 7: 1143; Smith et al. (2005) Mol Cell 20:687; Benaroudj et al. (2003) Mol. Cell 11:69).

[0004] In archaea, the proteasome-regulatory complex, PAN, is composed of six identical subunits that are close homologs of the 26S ATPases (Smith et al. (2005) Mol. Cell 20:687; Zwickl, J. Biol. Chem. (1999) 10:274 (37):26008-14; Benaroudj et al. (2003) Mol. Cell 11:69; Ogura and Tanaka (2003) Mol. Cell 11:3; Wilson et al. (2000) J. Bacteriol.

182: 1680; Navon and Goldberg (2001) Mol. Cell 8: 1339). The PAN-20S complex does not require ubiquitin conjugation for proteolysis. It has been a useful experimental system to assess proteasome function and the role of ATP (Smith et al. (2005) Mol. Cell 20:687;

Benaroudj et al. (2003) Mol. Cell 11:69; Ogura and Tanaka (2003) Mol. Cell 11:3; Wilson et al. (2000) J. Bacteriol. 182: 1680; Navon and Goldberg (2001) Mol. Cell 8: 1339).

[0005] Proteasome inhibitors are known which block peptidase sites within the 20S central chamber. These inhibitors have found use as research tools and in the treatment of certain cancers (A.F. Kisselev and A.L. Goldberg, Chem. Biol. (2001) 8:739 and Proteasome Inhibitors in Cancer Therapy, J. Adams, Ed. (Humana Press, Totowa, N.J., 2004)).

Proteasome inhibitors are already widely used for the treatment of hematological cancers (e.g., VELCADE (bortezomib)), and also offer promise as anti-inflammatory treatments and in transplants.

[0006] Conversely, activators of protein degradation would be of particular interest as possible treatments for various age-related neurodegenerative diseases, where there seems to be a failure to efficiently eliminate misfolded proteins. Accumulation of such proteins in inclusions is characteristic of Parkinson's disease, Alzheimer's disease, amytrophic lateral sclerosis, Huntington's disease, and prion disease. Thus an attractive approach for the treatment of these diseases is to enhance the breakdown of the neurotoxic proteins.

[0007] Understanding of the mechanisms that enable proteins to enter the proteasome for degradation may lead to the development of therapies for proteasome-related disorders, including protein folding disorders. Accordingly, there is a need in the art for effective activators of the proteasome for the treatment of protein folding disorders and as research tools.

SUMMARY OF THE INVENTION

[0008] The present invention stems from the discovery that small molecules can activate the proteasomal degradation of proteins. The compounds identified herein enhance the degradation of misfolded proteins by allowing entry of cellular proteins into the proteasome. Entry of proteins into the proteasome is regulated by a gate, which is opened by associated proteasomal regulatory ATPases, whose C-termini function as "keys in a lock" to trigger gate opening. Without wishing to be bound by a prticular theory, compounds described herein may likewise induce gate-opening allowing proteins to enter the proteasome. Accordingly, compounds compositions and methods useful in activating the proteasome and increasing the degradation of cellular proteins are provided herein.

[0009] In one aspect, the present invention provides a compound of the formula (I):

(I)

wherein R 1 , R2",

and X 3^J are described herein; and pharmaceutically acceptable salts thereof. In another aspect, the present invention provides a compound of the formula (Π):

(ID

wherein R⁴, R⁵, R⁶, R⁷, R⁸, and X⁴ are described herein; and pharmaceutically acceptable salts thereof. In yet another aspect, the present invention provides a compound of the formula (III):

(III)

wherein R⁹, R¹⁰, R¹¹, R¹², X⁵, and X⁶ are described herein; and pharmaceutically acceptable salts thereof. In another aspect, the present invention provides a compound of the formula (IV):

wherein R¹³, R¹⁴, R¹⁵, R¹⁶, X⁷, and X⁸ are described herein; and pharmaceutically acceptable salts thereof. In another aspect, the present invention provides a compound of the formula (V):

wherein R¹⁷, R¹⁸, R¹⁹, n, and X⁹ are described herein; and pharmaceutically acceptable salts thereof. In another aspect, the present invention provides a compound of the formula (VI):

(VI)

wherein R 20 , R 21 , R 22 , R 23 , and X 1¹0^U are described herein; and pharmaceutically acceptable salts thereof. In another aspect, the present invention provides a compound of the formula (VII):

(VII)

wherein R 24 , R 25 , R 26 , R 27 , m, k, and q are described herein; and pharmaceutically acceptable salts thereof. In another aspect, the present invention provides a compound of the formula (VIII):

(VIII) wherein R , R , R , R , m, and q are described herein; and pharmaceutically acceptable salts thereof. In another aspect, the present invention provides a compound of the formula (IX):

(IX)

wherein R 24 , R 25 , R 26 , R 27 , m, and q are described herein; and pharmaceutically acceptable salts thereof.

[0100] In another aspect, the present invention provides a compound of the formula

(X):

(X)

wherein R 28 , R 29 , X 11 , and z are described herein; and pharmaceutically acceptable salts thereof.

[0010] The compounds of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X) are thought to open the gate of the proteasome leading to increased proteolysis of cellular proteins. In another aspect, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound described herein for activating proteasomal degradation and, optionally, a pharmaceutically acceptable excipient.

[0011] In another aspect, the present invention provides methods to induce proteasomal degradation of proteins. In another aspect, the present invention provides methods for stimulating proteasomal degradation of proteins comprising contacting a proteasome with a compound or a composition described herein under conditions suitable to increase protein degradation by the proteasome. The step of contacting may be performed in vitro or in vivo, including in a living subject. [0012] In yet another aspect, the present invention provides a method for treating a subject with a disorder associated with unfolded, misfolded, or aggregated proteins comprising administering to the subject an effective amount of a compound or composition of the invention to induce proteasomal degradation of unfolded, misfolded, or aggregated proteins. In certain embodiments, the subject is a human. In certain embodiments, the disorder that the subject is suffering from is sickle cell anemia, thalassemia, Alzheimer's disease, Parkinson's disease, cystic fibrosis, Huntington's disease, amyotrophic lateral sclerosis (i.e., Lou Gehrig's disease), or Creutzfeld-Jakob disease. In certain embodiments, a compound or composition of the invention is useful in enhancing antigen presentation and/or promoting an immune response.

[0013] In one aspect, the present invention provides a method for identifying compounds that activate proteasomal degradation of proteins, the method comprising contacting a fluorogenic peptide with the proteasome in the presence of a test compound; and identifying compounds that cause an increase in the level of fluorescence relative to the level of fluorescence obtained in the absence of the test compound. In certain embodiments, a baseline level of fluorescence is measured from the proteasomal degradation of the fluorogenic peptide by the proteasome in the absence of a test compound. In certain embodiments, compounds that cause an increase in the level of fluorescence are subjected to one or more secondary screens to confrm that the compounds activate the proteasomal degradation of proteins.

[0014] In yet another aspect, the present invention provides a computer-assisted method for identifying compounds that potentially activate the proteasomal degradation of proteins, using a programmed computer comprising a processor, a data storage system, an input device, and an output device, the method comprising a) inputting into the programmed computer through said input device data comprising the atomic coordinates of a subset of the atoms of the proteasome, thereby generating a criteria data set; b) comparing, using said processor, the atomic coordinates to a computer database of chemical structures stored in said computer data storage system; c) selecting from said database, using computer methods, chemical structures that may bind the atomic coordinates; and d) outputting to said output device the selected chemical structures. The present invention also provides a computer system comprising a memory unit comprising x-ray crystallographic structure coordinates defining the 20S proteasome or amino acids of the 20S proteasome; and a processor in electrical communication with the memory unit; wherein the processor generates a molecular model having a three dimensional structure representative of at least a portion of the proteasome. In certain embodients, the three dimensional structure is representative of the intersubunit pockets of the proteasome. In still further embodients, the three dimensional structure is representative of residues on the alpha subunits of the proteasome (e.g., Lys 66, Leu 81 and/or Val 82).

[0015] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control.

DEFINITIONS

[0016] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5^th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern

Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0017] The compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and iraws-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.

[0018] "Labeled": As used herein, the term "labeled" is intended to mean that a compound has at least one element, isotope, or chemical compound attached to enable the detection of the compound. In general, labels typically fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes, including, but not limited to, 2 H, 3 H, 18 F, ³²P, ³⁵S, ⁶⁷Ga, ^99mTc (Tc-99m), ^mIn, ¹²³I, ¹²⁵1, ¹⁶⁹Yb and ¹⁸⁶Re; b) immune labels, which may be antibodies or antigens,which may be bound to enzymes (such as horseradish peroxidase) that produce detectable agents; and c) colored, luminescent, phosphorescent, or fluorescent dyes. It will be appreciated that the labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound that is being detected. In certain embodiments, hydrogen atoms in the compound are replaced with deuterium atoms ( H) to slow the degradation of compound in vivo. Due to isotope effects, enzymatic degradation of the deuterated compounds may be slowed thereby increasing the half-life of the compound in vivo. In certain embodiments of the invention, photoaffinity labeling is utilized for the direct elucidation of intermolecular interactions in biological systems. A variety of known photophores can be employed, most relying on photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (see Bayley, H., Photo generated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam), the entire contents of which are hereby incorporated by reference. In certain embodiments of the invention, the photoaffinity labels employed are o-, m- and p- azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to 4- azido-2,3,5,6-tetrafluorobenzoic acid.

[0019] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90: 10, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.

[0020] Where an isomer/enantiomer is preferred, it may, In certain embodiments, be provided substantially free of the corresponding enantiomer, and may also be referred to as "optically enriched." "Optically enriched," as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound of the present invention is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et ah, Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et ah, Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0021] It will be appreciated that the compounds of the present invention, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein (for example, aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo), and any combination thereof (for example, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,

heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like) that results in the formation of a stable moiety. The present invention contemplates any and all such combinations in order to arrive at a stable substituent/moiety. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples, which are described herein. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

[0022] One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group," as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. "Protecting groups," as used herein, are well known in the art and include those described in detail in Protective Groups in Organic Synthesis, Fourth Ed., Greene, T.W. and Wuts, P.G., Eds., John Wiley & Sons, New York: 2007, the entire contents of which are hereby incorporated by reference.

[0023] A "suitable amino protecting group," as used herein, is well known in the art and include those described in detail in Greene et al. Suitable amino protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-i- butyl-[9-( 10,10-dioxo-l 0, 10,10,10-tetrahydrothioxanthyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), l-(l-adamantyl)-l- methylethyl carbamate (Adpoc), l, l-dimethyl-2-haloethyl carbamate, l, l-dimethyl-2,2- dibromoethyl carbamate (DB-i-BOC), l,l-dimethyl-2,2,2-trichloroethyl carbamate

(TCBOC), l-methyl-l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-i-butylphenyl)-l- methylethyl carbamate (i-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N- dicyclohexylcarboxamido)ethyl carbamate, i-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), /7-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(l,3- dithianyl)] methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4- dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), l,l-dimethyl-2-cyanoethyl carbamate, m- chloro-p-acyloxybenzyl carbamate, /?-(dihydroxyboryl)benzyl carbamate, 5- benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, i-amyl carbamate, S-benzyl

thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2- dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1- dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1, 1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p '- methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, l-methyl-l-(3,5- dimethoxyphenyl)ethyl carbamate, l-methyl-l-(p-phenylazophenyl)ethyl carbamate, 1- methyl-l-phenylethyl carbamate, l-methyl-l-(4-pyridyl)ethyl carbamate, phenyl carbamate, /?-(phenylazo)benzyl carbamate, 2,4,6-tri-i-butylphenyl carbamate, 4- (trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3- phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3- (p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin- 2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5- dimethylpyrrole, N-l,l,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5- substituted l,3-dimethyl-l,3,5-triazacyclohexan-2-one, 5-substituted l,3-dibenzyl-l,3,5- triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N- allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N- (l-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N- benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N- triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl] amine (MMTr), N-9- phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N- ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1- dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N- diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'- dimethylaminomethylene)amine, N,N '-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2- hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5 ,5-dimethyl-3-oxo- l-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N- [phenyl(pentacarbonylchromium- or tungsten)carbonyl] amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl

phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate,

benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,

triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6- trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3 ,5 ,6-tetramethyl-4-methoxybenzenesulfonamide (Mte) , 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4- methylbenzenesulfonamide (iMds), 2,2,5,7, 8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9- anthracenesulfonamide, 4-(4' ,8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0024] A "suitable carboxylic acid protecting group" or "protected carboxylic acid," as used herein, are well known in the art and include those described in detail in Greene et al. Examples of suitably protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p- methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl {e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O- nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

[0025] A "suitable hydroxyl protecting group," as used herein, is well known in the art and include those described in detail in Greene et al. Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), i-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), i-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-l-benzyloxyethyl, 1- methyl-l-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, i-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p '-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, /7-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4', 4' '-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4', 4' '-tris(levulinoyloxyphenyl)methyl, 4,4', 4' '- tris(benzoyloxyphenyl)methyl, 3-(imidazol-l-yl)bis(4' ,4' '-dimethoxyphenyl)methyl, 1 , 1- bis(4-methoxyphenyl)-l '-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, l,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, i-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,

diphenylmethylsilyl (DPMS), i-butylmethoxyphenylsilyl (TBMPS), formate,

benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate

(levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9- fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl /7-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l- napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4- methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-

(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro- 4-( 1 , 1 ,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis( 1 , l-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o- (methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkyl Ν,Ν,Ν',Ν'- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-i-butylethylidene ketal, 1-phenylethylidene ketal, (4- methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide,

cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p- methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, a-methoxybenzylidene ortho ester, l-(N,N- dimethylamino)ethylidene derivative, a-(N,N'-dimethylamino)benzylidene derivative, 2- oxacyclopentylidene ortho ester, di-i-butylsilylene group (DTBS), 1,3— (1, 1,3,3- tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-i-butoxydisiloxane-l,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

[0026] As used herein, substituent names which end in the suffix "-ene" refer to a biradical derived from the removal of two hydrogen atoms from the substitutent. Thus, for example, acyl is acylene; alkyl is alkylene; alkeneyl is alkenylene; alkynyl is alkynylene; heteroalkyl is heteroalkylene, heteroalkenyl is heteroalkenylene, heteroalkynyl is

heteroalkynylene, aryl is arylene, and heteroaryl is heteroarylene.

[0027] The term "acyl," as used herein, refers to a group having the general formula -

C(=0)R^xl, -C(=0)OR^xl, -C(=0)-0-C(=0)R^xl, -C(=0)SR^xl, -C(=0)N(R^xl)₂, -C(=S)R^X1, -C(=S)N(R^X1)₂, and -C(=S)S(R^xl), -C(=NR^X1)R^X1, -C(=NR^xl)OR^xl, -C(=NR^X1)SR^X1, and

XI XI XI

-C(=NR )N(R )₂, wherein R is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched

heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,

heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di- aliphaticamino, mono- or di- heteroaliphaticamino, mono- or di- alkylamino, mono- or di- heteroalkylamino, mono- or

XI

di- arylamino, or mono- or di- heteroarylamino; or two R groups taken together form a 5- to 6- membered heterocyclic ring. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-C0₂H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,

heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0028] The term "aliphatic," as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, and cyclic (i.e. , carbocyclic)

hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl," "alkynyl," and the like. Furthermore, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "aliphatic" is used to indicate those aliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-20 carbon atoms. Aliphatic group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0029] The term "alkyl," as used herein, refers to saturated, straight- or branched- chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom. In certain embodiments, the alkyl group employed in the invention contains 1-20 carbon atoms. In another embodiment, the alkyl group employed contains 1-15 carbon atoms. In another embodiment, the alkyl group employed contains 1-10 carbon atoms. In another embodiment, the alkyl group employed contains 1-8 carbon atoms. In another embodiment, the alkyl group employed contains 1-5 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert- butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which may bear one or more sustitutents. Alkyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0030] The term "alkenyl," as used herein, denotes a monovalent group derived from a straight- or branched-chain hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. In certain embodiments, the alkenyl group employed in the invention contains 2-20 carbon atoms. In certain embodiments, the alkenyl group employed in the invention contains 2-15 carbon atoms. In another embodiment, the alkenyl group employed contains 2-10 carbon atoms. In still other embodiments, the alkenyl group contains 2-8 carbon atoms. In yet other embodiments, the alkenyl group contains 2-5 carbons. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2- buten-l-yl, and the like, which may bear one or more substituents. Alkenyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g. , aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0031] The term "alkynyl," as used herein, refers to a monovalent group derived from a straight- or branched-chain hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. In certain embodiments, the alkynyl group employed in the invention contains 2-20 carbon atoms. In certain embodiments, the alkynyl group employed in the invention contains 2-15 carbon atoms. In another embodiment, the alkynyl group employed contains 2-10 carbon atoms. In still other embodiments, the alkynyl group contains 2-8 carbon atoms. In still other embodiments, the alkynyl group contains 2-5 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2- propynyl (propargyl), 1-propynyl, and the like, which may bear one or more substituents. Alkynyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,

heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0032] The term "amino," as used herein, refers to a group of the formula (-NH₂). A

"substituted amino" refers either to a mono-substituted amine (-NHR^h) of a disubstitued amine (-NR^h ₂), wherein the R^h substituent is any substitutent as described herein that results in the formation of a stable moiety (e.g., a suitable amino protecting group; aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, amino, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy,

heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted). In certain embodiments, the R^h substituents of the di- substituted amino group

(-NR^h ₂) form a 5- to 6- membered hetereocyclic ring.

[0033] The term "alkoxy" refers to a "substituted hydroxyl" of the formula (-OR¹), wherein R¹ is an optionally substituted alkyl group, as defined herein, and the oxygen moiety is directly attached to the parent molecule.

[0034] The term "alkylamino" refers to a "substituted amino' Of the formula (-NR^h ₂), wherein R^h is, independently, a hydrogen or an optionally subsituted alkyl group, as defined herein, and the nitrogen moiety is directly attached to the parent molecule.

[0035] The term "aryl," as used herein, refer to stable aromatic mono- or polycyclic ring system having 3-20 ring atoms, of which all the ring atoms are carbon, and which may be substituted or unsubstituted. In certain embodiments of the present invention, "aryl" refers to a mono, bi, or tricyclic C₄-C₂o aromatic ring system having one, two, or three aromatic rings which include, but not limited to, phenyl, biphenyl, naphthyl, and the like, which may bear one or more substituents. Aryl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,

[0036] The term "azido," as used herein, refers to a group of the formula (-N₃).

[0037] The term "cyano," as used herein, refers to a group of the formula (-CN).

[0038] The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -CI), bromine (bromo, -Br), and iodine (iodo, -I).

[0039] The term "heteroaliphatic," as used herein, refers to an aliphatic moiety, as defined herein, which includes both saturated and unsaturated, nonaromatic, straight chain (i.e., unbranched), branched, acyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, which are optionally substituted with one or more functional groups, and that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more substituents. As will be appreciated by one of ordinary skill in the art, "heteroaliphatic" is intended herein to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,

heterocycloalkenyl, and heterocycloalkynyl moieties. Thus, the term "heteroaliphatic" includes the terms "heteroalkyl," "heteroalkenyl," "heteroalkynyl," and the like.

Furthermore, as used herein, the terms "heteroalkyl," "heteroalkenyl," "heteroalkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "heteroaliphatic" is used to indicate those heteroaliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-20 carbon atoms.

Heteroaliphatic group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g. , aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,

[0040] The term "heteroalkyl," as used herein, refers to an alkyl moiety, as defined herein, which contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g. , in place of carbon atoms.

[0041] The term "heterocyclic," "heterocycles," or "heterocyclyl," as used herein, refers to a cyclic heteroaliphatic group. A heterocyclic group refers to a non-aromatic, partially unsaturated or fully saturated, 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms in size, and bi- and tri-cyclic ring systems which may include aromatic five- or six-membered aryl or heteroaryl groups fused to a non-aromatic ring. These heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms. Heterocyclyl groups include, but are not limited to, a bi- or tri-cyclic group, comprising fused five, six, or seven-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring.

Exemplary heterocycles include azacyclopropanyl, azacyclobutanyl, 1,3-diazatidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl, thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropuranyl, dioxanyl, oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the like, which may bear one or more substituents. Substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g. , aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0042] The term "heteroaryl," as used herein, refer to stable aromatic mono- or polycyclic ring system having 3-20 ring atoms, of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms. Exemplary heteroaryls include, but are not limited to pyrrolyl, pyrazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, pyyrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzoimidazolyl, indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl, quinazolynyl, phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl, thianaphthenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolynyl, isothiazolyl, thiadiazolynyl, oxazolyl, isoxazolyl, oxadiaziolyl, oxadiaziolyl, and the like, which may bear one or more substituents. Heteroaryl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy,

heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0043] The term "hydroxy" or "hydroxyl," as used herein, refers to a group of the formula (-OH). A "substituted hydroxyl" refers to a group of the formula (-OR¹), wherein R¹ can be any substitutent which results in a stable moiety (e.g., a suitable hydroxyl protecting group, aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, nitro, alkylaryl, arylalkyl, and the like, each of which may or may not be further substituted).

[0044] The term "imino," as used herein, refers to a group of the formula (=NR^r), wherein R^r corresponds to hydrogen or any substitutent as described herein, that results in the formation of a stable moiety (for example, a suitable amino protecting group, aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, amino, hydroxyl, alkylaryl, arylalkyl, and the like, each of which may or may not be further substituted). In certain embodiments, imino refers to =NH wherein R^r is hydrogen.

[0045] The term "nitro," as used herein, refers to a group of the formula (-N0₂).

[0046] The term "oxo," as used herein, refers to a group of the formula (=0). [0047] The term "stable moiety," as used herein, preferably refers to a moiety which possess stability sufficient to allow manufacture, and which maintains its integrity for a sufficient period of time to be useful for the purposes detailed herein.

[0048] The term "thio" or "thiol," as used herein, refers to a group of the formula (-

SH). A "substituted thiol" refers to a group of the formula (-SR^r), wherein R^r can be any substituent that results in the formation of a stable moiety (e.g., a suitable thiol protecting group; aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, cyano, nitro, alkylaryl, arylalkyl, and the like, each of which may or may not be further substituted).

[0049] The term "thiooxo," as used herein, refers to a group of the formula (=S).

[0050] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, immunological response, and the like, and are commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. For example, Berge et ah, describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C₁^alkyl)₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and aryl sulfonate.

[0051] The term "subject," as used herein, refers to any animal. In certain

embodiments, the subject is a mammal. In certain embodiments, the term "subject," as used herein, refers to a human (e.g., male, female, adult, or child). The subject may be at any stage of development.

[0052] The term "administer," "administering," or "administration," as used herein, refers to implanting, absorbing, ingesting, injecting, or inhaling the inventive compound.

[0053] The terms "activate" or "stimulate," as used herein, means to increase the protein degradation activity by a proteasome to a level or amount that is statistically significantly more than an initial level, which may be a baseline level of protein degradation activity.

[0054] The term "inhibit," as used herein, means to decrease the protein degradation activity by a proteasome to a level or amount that is statistically significantly less than an initial level, which may be a baseline level of protein degradation activity.

[0055] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In certain embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g. , in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0056] The terms "effective amount" and "therapeutically effective amount," as used herein, refer to the amount or concentration of an inventive compound, that, when

administered to a subject, is effective to at least partially treat a condition from which the subject is suffering.

[0057] The terms "protein," "peptide," and "polypeptide," as used herein, refer to a string of at least three amino acids linked together by peptide bonds. The terms "protein," "peptide," and "polypeptide" may be used interchangeably. Peptide may refer to an individual peptide or a collection of peptides. Peptides typically contain only natural amino acids, although non-natural amino acids (i.e. , compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modifications.

[0058] The terms "polynucleotide" or "oligonucleotide," as used herein, refer to a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides. The polymer may include natural nucleosides (i.e. , adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3- methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine,

C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5 ' -N-phosphoramidite linkages).

[0059] The term "unfolded protein," as used herein, refers to a protein that has yet to adopt the correct three-dimensional conformation that is necessary to achieve its biological function.

[0060] The term "misfolded protein," as used herein, refers to a protein that has adopted an incorrect three-dimensional conformation and thereby fails to achieve its biological function.

[0061] The term "aggregated protein," as used herein, refers to a protein that has aggregated with additional copies of the same protein, with different proteins, or with additional biomolecules and, thus, fails to achieve its biological function.

[0062] The terms "unfolded," "misfolded," and "aggregated," as used interchangeably herein.

[0063] The term "protein folding disorder," as used herein, refers to a disease or disorder whose pathology is related to the presence of an unfolded, misfolded, or aggregated protein. In one embodiment, the disorder is caused when a misfolded protein interferes with the normal biological activity of a cell, tissue, or organ.

[0064] The term "proteasome," as used herein, refers to the 20S proteasome and the

26S proteasome. It is to be understood that the identification of a specific proteasome, such as the 20S proteasome or the 26S proteasome, includes all varieties of the proteasome including singly capped, doubly capped, hybrid, mutants, 20S proteasomes of the present invention include 20S proteasomes from prokaryotes and eukaryotes.

[0065] The term "regulatory complex," as used herein, refers to those that bind to the

20S proteasome. Such regulatory complexes include the regulatory ATPases which further include but are not limited to PAN, the 26S ATPases (e.g., Rptl, Rpt2, Rpt3, Rpt4, Rpt5 and Rpt6 in yeast, and their homologs S7, S4, S6b, SlOb, S6a, and S8 in mammals), the AAA and AAA+ family of ATPases and the like. Other regulatory complexes includes the single protein activator PA200/BLM10 and the 1 IS regulatory complexes such as PA26, PA28a, ΡΑ28β, ΡΑ28γ and the like. It is to be understood that other regulatory complexes can be predicted in their ability to bind to the 20S proteasome based on their C-terminal sequence and homology to regulatory complexes known to bind to the 20S proteasomes, such as PAN or the PA28 family.

[0066] Regulatory ATPases facilitate substrate entry into the 20S proteasome. When

ATP binds to the proteasomal ATPases, the conserved HbYX motif in their C-termini dock into pockets in the 20S a-ring and function like a "key-in-a-lock" to open the gate that limits substrate entry. Specifically, PAN, Rpt2, and Rpt5 exhibit the conserved HbYX motif in their C-termini. Because 7-residue peptides corresponding to the C termini of either PAN, Rpt2 or Rpt5 by themselves bind and trigger gate opening in both archaeal and mammalian proteasomes, this reaction does not require other parts of the ATPase molecule and appears to represent a general mechanism regulating proteasome function (Smith DM et ah, Mol Cell. 2007 September 7; 27(5): 731-744).

[0067] The term "Hb-YX motif," as used herein, refers to the C-terminal Hb-YX motif motif that is required to induce gate opening, wherein Hb is a hydrophobic residue or an aromatic residue; Y is a tyrosine or, optionally, a phenylalanine; and X is any amino acid residue. In some embodiments, a free C-terminal carboxyl group at X is required for binding to Lys₆₆.

[0068] The term "CBZ-YA" refers to the protected dipeptide carboxybenzyl-tyrosine- alanine. CBZ-YA resembles the C-terminus of proteasomal ATPases and thereby activates the proteasomal degradation of proteins.

[0069] The term "fluorogenic peptide," as used herein, refers to peptide derivatives that fluoresce upon hydrolysis such as those disclosed in Kisselev et al., Methods Enzymol. 2005; 398:364-78, the entirety of which is incorporated by reference. Non-limiting examples of fluorogenic peptides include Mca-AKVYPYPME-Dpa(Dnp)-amide (LFP; SEQ ID NO:9); YFP-ssrA; LRR-amc; and nLPnLD-amc (SEQ ID NO: 10); wherein Mca is 4-methyl- coumaryl- amide; Dpa(Dnp) is 3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl; amc is 7- amino-4-methyl-coumarin.

BRIEF DESCRIPTION OF THE DRAWING

[0070] Figure 1 illustrates the 26S proteasome.

[0071] Figure 2 shows a model for the association and gate opening by the

proteasomal ATPases.

[0072] Figure 3 illustrates the proteasomal ATPases C-termini binding to the 20S intersubunit pockets and inducing gate opening.

[0073] Figure 4 illustrates the C-terminal Hb-YX motif as it is required to induce gate opening; wherein Hb is a hydrophobic residue, Y is tyrosine, and X can be most residues with sidechains that are not charged.

[0074] Figure 5 illustrates certain 20S residues that interact with the Hb-YX motif and block gate opening and provides data associated with several 20S proteasome mutants.

[0075] Figure 6 illustrates gate opening of the 20S proteasome with tagged substrates.

[0076] Figure 7 depicts the dose response of carbobenzyloxy-YA (CBZ-YA) for the stimulation of peptide substrate degradation.

[0077] Figure 8 depicts the degradation of Tau by the 20S and 26S proteasomes (3 mM) in the presence of CBZ-YA.

[0078] Figure 9 depicts the stimulation of Tau23 degradation by the 20S proteasome in the presence of several di- or tri-peptides (3 mM).

[0079] Figure 10 depicts the degradation of nLPnLD-amc (SEQ ID NO: 10) by the 20S proteasome in the presence of seven compounds.

[0080] Figure 11 depicts the stimulation of a wild- type yeast 20S proteasome by compound 1892, and the inability of compound 1892 to stimulate the yeast open gate mutant.

[0081] Figure 12 depicts several compounds that stimulate Tau23 degradation by the

20S proteasome.

[0082] Figure 13 depicts the stimulation of Tau degradation by compound 1661 with an EC₅₀ of ~10 μΜ.

[0083] Figure 14 depicts the stimulation of alpha-synuclein degradation by compounds

1661 and 1982.

[0084] Figure 15 depicts a summary of data associated with compounds that have beeen found to activate the proteasomal degradation of proteins. [0085] Figure 16 depicts compounds that have been found to activate the proteasomal degradation of proteins.

[0086] Figure 17 depicts the structure of 20S-PA26^{E102A PAN9} complex solved by X- ray crystallography as reported by Yu, et al., EMBO (2010) 29, 692-702. (A) Stereo views of electron density map (omit map) of a fragment of 20S core particle show the overall quality of the density map. (B) Stereo view of the omit map of PAN's C-terminus in the pocket between neighbouring 20S a-subunits. The numbering of residues follows the sequence of PA26.

[0087] Figure 18 depicts interactions between PAN's C-terminus and the 20S intersubunit pocket as reported by Yu, et al. (2010). (A) Comparison of C-termini' s conformations of PA26 and PAN in the intersubunit pocket. The a-subunits on the right of the same pocket was aligned and superimposed. (B) The position of the tyrosine residue of the C-terminal HbYX motif. (C) Position of the C-terminal HbYX motif in the intersubunit pocket that is represented as space filling.

[0088] Figure 19 depicts the binding of yeast proteasomal ATPases to the yeast 20S proteasome as reported by Yu, et al. (2010). (A) Wild-type T. acidophilum 20S (wt-20S), 20S with al-a2 pocket mutations (20S-ala2 pocket) and with α3-α4 pocket mutations (20S- α3α4 pocket) and the substrate LFP are incubated with the wildtype PAN and PAN mutants that have their C-termini replaced by seven residues of yeast Rptl-6. The stimulation of gate opening was measured by the increase of LFP hydrolysis over the control without any activator. The values are the mean + standard deviation from at least three independent measurements. (B) Averages of 20S with α3-α4 pocket mutations single (left) and double (right) capped with the PANRpt5 mutant. (C) Top view of yeast 20S proteasome a-ring, with each a-subunit marked. The side chains of lysine residues within the pocket, which is required to bind the C-termini of Rpt, are shown.

[0089] Figure 20 depicts the chemical structures of analogs of compound 1892.

[0090] Figure 21 depicts data obtained from the hydrolysis of a substrate by bovine

20S proteasome in the presence of analogs of compound 1892. Analogs 1892- A3, 1892-A5, and 1892-A8 were added to the hydrolysis reaction as acetone solutions whereas the other analogs were added as DMSO solutions. Thus, V_max calculations for 1892- A3, 1892-A5, and 1892-A8 may be underestimated.

[0091] Figure 22 depicts ligand binding data associated with analog 1892-A11.

[0092] Figure 23 depicts ligand binding data associated with analog 1892-A4. [0093] Figures 24A-C depict peptide sequence alignments of the archaeal 20S alpha ring with each of seven different human 20S alpha rings. The sequence identifications of the peptides are as follows: gi\10640633-t20s\emb\CAC12411.1 \/l-233 (SEQ ID NO: l), gi\48146005-al \emb\CAG33225.1 \/l-246 (SEQ ID NO:2), gi\47496581- a2\emb\CAG29313.1 \/ 1-234 (SEQ ID NO:3), gi\34783332-a3\gb\AAH22817.2\/l-260 (SEQ ID NO:4), gi\12314028-a4\emb\CAC04017.1 \/l-248 (SEQ ID NO:5), gi\48145811- a5\emb\CAG33128.1 \/l-241 (SEQ ID NO:6), gi 118490859-a6\gb \AAH22372.11/ -263 (SEQ ID NO:7), and gi\48145983-a7\emb\CAG33214.1 \/l-255 (SEQ ID NO:8).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0094] The present invention stems, in part, from the discovery that small molecules have been identified that can activate the proteasomal degradation of proteins. The compounds identified herein enhance the degradation of misfolded proteins by regulating the entry of cellular proteins into the proteasome.

[0095] The 26S proteasome is a large ATP-dependent proteolytic complex that catalyzes the degradation of most cellular proteins (Glickman MH and Ciechanover A, Physiol. Rev. 2002; 82:373-428.; Voges D et al., Annu. Rev. Biochem. 1999; 68: 1015-1068), generally after linkage to a ubiquitin chain (Glickman MH and Ciechanover A, 2002;

Goldberg AL, Neuron 2005; 45:339-344; Goldberg AL, Nature 2003; Dec

18;426(6968):895-9). Proteasomes are also found in archaea and certain eubacteria, where ubiquitin is not present, but proteolysis is still ATP-dependent (Goldberg AL, 2005). The active forms of proteasomes in archaea and eukaryotes are composed of an ATPase complex bound to one or both ends of the 20S core proteasome (DeMartino GN and Slaughter CA, J Biol Chem 1999; 274:22123-22126; Voges D et al., Annu Rev Biochem 1999; 68: 1015- 1068). This hollow cylinder is composed of four stacked rings (Groll et al., Nature 1997; 386:463-471; Lowe et al., Science 1995; 268: 533-539). Its alpha rings contain seven a- subunits that surround a narrow channel, through which polypeptides enter the 20S's central chamber, where its multiple proteolytic sites are located. The passage of proteins and peptides through this channel is restricted by the N-termini of the a-subunits, which function as a gate (Groll et al., Nat Struct Biol 2000; 7: 1062-1067; Groll and Huber, Int J Biochem Cell Biol 2003; 35:606-616). The ATPase ring complexes open this gate and thus regulate proteolysis by the 20S particle (Benaroudj et al., Mol Cell 2003; 11:69-78; Kohler et al., Mol Cell 2001; 7: 1143-115; and Smith et al., 2005). In eukaryotes, the base of the 19S regulatory complex contains six different ATPases (Rptl-6) (Fu et al., Embo J 2001; 20:7096-7107). [0096] In archaea, the proteasome-regulatory complex, PAN, is composed of six identical subunits that are close homologs of the 26S ATPases (Benaroudj et al., 2003; Navon and Goldberg, Mol Cell 2001; 8: 1339-1349; Ogura and Tanaka, Mol Cell 2003; 11:3-5; Zwickl et al, J Biol Chem 1999; 274:26008-26014). In electron micrographs, PAN resembles a top-hat structure capping the 20S cylinder and appears similar to densities in the base of the 19S complex (Smith et al., 2005). These ATPase rings associate with the outer a- ring of the 20S where in the presence of ATP, they unfold globular proteins and promote their translocation through the gated entry channel into the 20S particle (Benaroudj et al., The Ubiquitin-Proteasome System; Wiley- VCH:2005; Kohler et al., 2001; Smith et al., 2005; Voges et al, 1999).

[0097] The simpler PAN-20S complex offers major advantages for exploring proteasome function (Benaroudj et al., 2003; Navon and Goldberg, 2001; Ogura and Tanaka, 2003; Smith et al., 2005). For example, it does not require ubiquitin conjugation for proteolysis, the archaeal 20S particle contains only one type of a and β subunits, and these complexes assemble when expressed in E. coli. Our recent studies have established that upon binding of ATP or nonhydrolyzable ATP analogs, PAN associates with the 20S particle and triggers gate opening in the a-ring. This gate-opening by PAN, even without ATP hydrolysis, facilitates the diffusion of peptides and unfolded proteins into the 20S particle. Similarly, ATP binding in the eukaryotic 26S complex also causes gate opening and entry of unfolded proteins into the 20S particle (Smith et al., 2005).

[0098] As discussed herein, there remains a need for the development of small molecules that can activate the proteasomal degradation of proteins. Compounds described herein advantageously bind to the 20S proteasome at one or more binding sites that otherwise would be available for binding with a regulatory complex that is required to allow the entry of a protein into the 20S proteasome where degradation takes place. According to further embodiments, the present invention provides compounds that bind to the intersubunit pockets in the a-ring of the 20S proteasome and inhibit complex formation with the regulatory complex {i.e., often containing an ATPase ring) which is necessary to allow proteins to enter the 20S proteasome for protein degradation.

[0099] Further embodiments of the present invention are based on the discovery that certain compounds can bind to the 20S proteasome and induce gate opening while other compounds can bind to the 20S proteasome and not induce gate opening, leaving the gate in its default closed state. While both classes of compounds bind to the 20S proteasome and thereby prevent binding of regulatory complexes thereto, the different capability of each class in inducing or not inducing gate opening provides distinct advantages in therapeutic applications where the stimulation of protein degradation is desirable. For example, compounds that bind to the 20S proteasome and induce gate opening could stimulate the degradation of proteins in an ATP-independent and/or ubiquitin-independent manner.

[00100] The present invention provides compounds of the general formulae (I) through (X), and pharmaceutical compositions thereof. These compounds are useful as activators of proteasomal degradation, and thus are useful for the treatment of diseases or disorders associated with unfolded, misfolded, or aggregated proteins in a subject. In further embodiments, compounds and compositions of the invention are used to treat disorders associated with unfolded, misfolded, or aggregated proteins such as sickle cell anemia, thalassemia, Alzheimer's disease, Parkinson's disease, cystic fibrosis, Huntington's disease, amyotrophic lateral sclerosis (i.e. , Lou Gehrig' s disease), and Creutzfeld-Jakob disease.

[00101] The inventive activators of proteasomal degradation of proteins, as described herein, can exhibit antimicrobial activity (e.g. , anti-bacterial, anti-fungal, anti-malarial activity). In certain aspects, the compounds of the invention sufficiently activate the proteasomal degradation of proteins in a microorganism to kill or inhibit the growth of the microorganism. In certain embodiments, the inventive compounds are useful as antibiotics.

[00102] In certain aspects, the compounds and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies. In further embodiments, an inventive compound may be administered concurrently with another agent to treat disorders associated with unfolded, misfolded, or aggregated proteins such as sickle cell anemia, thalassemia, Alzheimer' s disease, Parkinson' s disease, cystic fibrosis,

Huntington' s disease, amyotrophic lateral sclerosis (i.e. , Lou Gehrig's disease), and

Creutzfeld-Jakob disease. In other embodiments, an inventive compound may be

administered concurrently with another agent to treat a neurodegenerative disease. In still further embodiments, an inventive compound may be administered concurrently with another inhibitor of a deubiquitinating enzyme or an inhibitor of ubiquitin- specific proteases. In still further embodiments, an inventive compound may be administered concurrently with an antibiotic agent. In further embodiments, an inventive compound may be administered concurrently with another antifungal agent. In further embodiments, an inventive compound may be administered concurrently with another antiparasitic agent.

[00103] In certain aspects, inventive methods are provided to screen and identify compounds that activate the proteasomal degradation of proteins. In certain embodiments, the invention provides a method for identifying compounds that activate the proteasomal degradation of proteins, by contacting a fhiorogenic peptide with the proteasome in the presence of a test compound; and identifying compounds that cause an increase in the level of fluorescence relative to the level of fluorescence obtained in the absence of the test compound. In certain embodiments, a baseline level of fluorescence is measured from the proteasomal degradation of the f iorogenic peptide by the proteasome in the absence of a test compound.

[00104] In certain aspects, inventive methods are provided to screen compounds, in silico, that induce gate opening of the proteasome and activate the proteasomal degradation of proteins. It is a further embodiment of the present invention to use the atomic coordinates of a proteasome to design, identify, and screen potential binding compounds that bind to the proteasome or a subunit of the proteasome, such as the 20S proteasome, and alter the proteasome' s physical, chemical, and/or physiological properties. Novel compounds obtained from this screen may further be identified as being able to induce gate opening of the 20S subunit and activate the proteasomal degradation of proteins.

Compounds

[0101] Compounds of the present invention include activators of the proteasomal degradation of proteins. Compounds of the invention and salts thereof include those, as set forth above and described herein, and are illustrated in part by the various classes, subclasses, subgenera, and species disclosed herein. The inventive compounds may be useful in the treatment of a variety of diseases. In certain embodiments, the compounds are useful in the treatment of a disorder associated with unfolded, misfolded, or aggregated proteins (e.g., Alzheimer's disease, Huntington's disease, Parkinson's disease, sickle cell anemia, and thalassemia) in a subject and complications thereof.

[0102] In certain embodiments, an inventive compound is of formula (I):

or a pharmaceutically acceptable salt thereof,

wherein

R¹ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; -CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; -OC(=0)OR^A; -OC(=0)R^A; - OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais

independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or hetero arylthio moiety;

each of R 2 and R 3 is, independently, hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

X is O, S, or NR^Y1;

X² is O, S, or NR^Y2;

X³ is O, S, or NR^Y3; and

each of R , R , and R is, independently, hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

provided that the compound is not

₃

[0103] In certain embodiments, X is S. In further embodiments, X is O. In still further embodiments, X 1 is NR Yl . In certain embodiments, X 1 is NH. In certain

embodiments, X 1 is S; X2 is O; and X 3 is O.

[0104] In certain embodiments, X² is S. In further embodiments, X² is O. In still further embodiments, X 2 is NR Y2. In certain embodiments, X 2 is NH.

[0105] In certain embodiments, X³ is S. In further embodiments, X³ is O. In still further embodiments, X 3 is NR Y3. In certain embodiments, X 3 is NH.

[0106] In certain embodiments, R¹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In certain embodiments, R¹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci-ioheteroaliphatic. In certain embodiments, R¹ is a substituted or unsubstituted aryl. R¹ is substituted or unsubstituted phenyl. In still furt or unsubstituted naphthyl. In certain

embodiments, R is

or wherein each R is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroalkyl; and g¹ is 0 or an integer between 1 and 7, inclusive. In certain embodiments, R¹ is a substituted

naphthyl. In further embodiments, R¹ is

[0107] In certain embodiments, R² is a substituted or unsubstituted aryl. In further embodiments, R 2 is a substituted or unsubstituted phenyl. In still further embodiments, R 2 is

2

Z2

a substituted phenyl. In certain embodiments, R is wherein each R is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroalkyl; and g is 0 or an integer between 1 and 5, inclusive.

[[00110088]] In certain embodiments, R³ is C_1-6-alkyl. In certain embodiments, R³ is hydrogen.

[0109] In certain embodiments, R^¹ is hydrogen. In certain embodiments R^¹ is C_1-6- alkoxy. In certain embodiments R Zl is methoxy. In certain embodiments R Zl is ethoxy. In certain embodiments R Zl is propoxy. In certain embodiments R Zl is butoxy.

[0110] In certain embodiments, R^Z2 is hydrogen. In certain embodiments R^Z2 is C_1-6- alkyl. In certain embodiments R Z2 is methyl. In certain embodiments R Z2 is ethyl. In certain embodiments R Z2 is propyl. In certain embodiments R Z2 is butyl. In certain embodiments fluoro. In certain embodiments R is chloro. In certain embo is bromo. In

certain embodiments R^Z2 is iodo. In still further embodiments, R² is

[0111] In certain embodiments, g¹ is 0. In further embodiments, g¹ is 1. In still further embodiments, g¹ is 2. In other embodiments, g¹ is 3. In certain embodiments, g¹ is 4. In further embodiments, g¹ is 5. In still further embodiments, g¹ is 6. In other embodiments, g¹ is 7.

[0112] In certain embodiments, g² is 0. In further embodiments, g² is 1. In still further embodiments, g 2 is 2. In other embodiments, g 2 is 3. In certain embodiments, g 2 is 4. In further embodiments, g is 5.

[0113] In other embodiments, an inventive compound is of formula (II):

(Π)

or a pharmaceutically acceptable salt thereof;

wherein

each of R⁴, R⁵, and R⁶ is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; - CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; - OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; each R 7 and R 8 is, independently, hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

X⁴ is O, S, or NR^Y4; and

R Y4 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

provided that the compound is not

H₃

[0114] In certain embodiments, X⁴ is S. In further embodiments, X⁴ is O. In still further embodiments, X⁴ is NR^Y4. In further embodiments, X⁴ is NH.

[0115] In certain embodiments, R⁴ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R⁴ is Ci_6 alkyl. In further embodiments, R⁴ is -CN. In further embodiments, R⁴ is hydrogen.

[0116] In certain embodiments, R⁵ is a substituted or unsubstituted aryl. In further embodiments, R⁵ is a substituted or unsubstituted phenyl. In certain embodiments, R⁵ is 9 wherein each R is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroalkyl; and g is 0 or an integer between 1 and 5, inclusive. In still further embodiments, R⁵ is a substituted phenyl. In certain embodiments,

[0117] In certain embodiments, R⁶ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R⁶ is C_1-6 alkyl. In certain embodiments, R⁶ is methyl. In further embodiments, R⁶ is hydrogen.

[0118] In certain embodiments, R⁷ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In certain embodiments, R is Q-Cealkyl. In further embodiments, R is hydrogen.

[0119] In certain embodiments, R⁸ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In certain embodiments, R is hydrogen. In certain embodiments R 8 is Ci 8

-6-alkyl. In certain embodiments R is methyl. In certain embodiments R 8 is ethyl. In certain embodiments R 8 is propyl. In certain embodiments R 8 is butyl.

[0120] In certain embodiments, R^Z3 is hydrogen. In certain embodiments R^Z3 is C_1-6- alkyl. In certain embodiments R Z3 is methyl. In certain embodiments R Z3 is ethyl. In certain embodiments R Z3 is propyl. In certain embodiments R Z3 is butyl. In certain embodiments

R Z3 is fluoro. In certain embodiments R Z3 is chloro. In certain embodiments R Z3 is bromo. In certain embodiments R Z3 is iodo.

[0121] In certain embodiments, g³ is 0. In further embodiments, g³ is 1. In still further embodiments, g 3 is 2. In other embodiments, g 3 is 3. In certain embodiments, g 3 is 4. In further embodiments, g is 5.

[0122] In certain embodiments, an inventive compound is of formula (III):

(III)

or a pharmaceutically acceptable salt thereof;

wherein

each of R⁹ and R¹⁰ is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; -CN; -SCN; - SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; -OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio;

arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

each of R 11 and R 12 is, independently, hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

each R is independently hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

X⁵ is O, S, or NR^Y5;

X⁶ is N or CR^Y6;

R Y5 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and

R Y6 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(=0)R^B; -C0₂R^B; -C(=0)N(R^B)₂; -

SOR B ; -S0₂R B ; -C(R B )₃; aryloxy; alkylthio; arylthio; alkylamino, dialkylamino,

heteroaryloxy; or hetero arylthio moiety;

provided that the compound is not

[0123] In certain embodiments, X⁵ is O. In further embodiments, X⁵ is S. In other embodiments, X 5 is NR Y5. In other embodiments, X 5 is NH.

[0124] In certain embodiments, X⁶ is N. In further embodiments, X⁶ is CR^Y6.

[0125] In certain embodiments, X⁵ is O; and X⁶ is N. In certain embodiments, X⁵ is S; and X⁶ is N.

[0126] In certain embodiments, R⁹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R⁹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In certain embodiments, R⁹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkenyl. In certain embodiments, R⁹ is substituted or unsubstituted styrene. In further embodiments, R⁹ is a substituted or unsubstituted aryl. In still further embodiments, R⁹ is a substituted or

unsubstituted phenyl. In certain embodiments, R is each

Z4

R is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroalkyl; and g⁴ is 0 or an integer between 1 and 5, inclusive.

[0127] In certain embodiments, R¹⁰ is hydrogen. In further embodiments, R¹⁰ is -CN.

[0128] In certain embodiments, R¹¹ is a substituted or unsubstituted aryl. In certain embodiments, R¹¹ is a substituted or unsubstituted phenyl. In further embodiments, R¹¹ is a

substituted phenyl. In still further embodiments, R is wherein R is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; a substituted or unsubstituted, branched or unbranched heteroaryl; or

B3 substituted or unsubstituted, branched or unbranched aryl. In certain embodiments R is C .

B3 B3

6-alkyl. In certain embodiments R is methyl. In certain embodiments R is ethyl. In

B3 B3

certain embodiments R is propyl. In certain embodiments R is butyl. In certain

embodiments, R¹¹ is .

12

[0129] In certain embodiments, R is substituted or unsubstituted, branched or

12

unbranched, cyclic or acyclic C^o aliphatic. In further embodiments, R is hydrogen. In

12 12

certain embodiments R is Ci-6-alkyl. In certain embodiments R is methyl. In certain

12 12 12 embodiments R is ethyl. In certain embodiments R is propyl. In certain embodiments R is butyl.

[0130] In certain embodiments, R⁷⁴ is hydrogen. In certain embodiments R⁷⁴ is C_\-e-

Z4 Z4

alkyl. In certain embodiments R is methyl. In certain embodiments R is ethyl. In certain embodiments R is propyl. In certain embodiments R is butyl. In certain embodiments

R Z4 is fluoro. In certain embodiments R Z4 is chloro. In certain embodiments R Z4 is bromo. In certain embodiments R is iodo.

[0131] In certain embodiments, g⁴ is 0. In further embodiments, g⁴ is 1. In still further embodiments, g⁴ is 2. In other embodiments, g⁴ is 3. In certain embodiments, g⁴ is 4. In further embodiments, g⁴ is 5.

[0132] In certain embodiments, an inventive compound is of formula (IV):

(IV)

or a pharmaceutically acceptable salt thereof;

wherein

each of R¹³ and R¹⁴ is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; - CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; - OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; each of R¹⁵ and R¹⁶ is, independently, hydrogen; a nitrogen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

X⁷ is O, S, or NR^Y7,

X⁸ is N or CR^Y8; and

R Y7 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and

R Y8 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(=0)R^A; -C0₂R^A; -C(=0)N(R^A)₂; - SOR^A; -S0₂R^A; -C(R^A)₃; aryloxy; alkylthio; arylthio; alkylamino, dialkylamino,

heteroaryloxy; or hetero arylthio moiety;

provided that the compound is not

[0133] In certain embodiments, X⁷ is O. In further embodiments, X⁷ is S. In other embodiments, X 7 is NR Y7. In other embodiments, X 7 is NH.

[0134] In certain embodiments, X⁸ is N. In further embodiments, X⁸ is CR^Y8.

[0135] In certain embodiments, X⁷ is O; and X⁸ is N. In certain embodiments, X⁷ is S; and X⁸ is N.

[0136] In certain embodiments, R 13 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In still further embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In certain

embodiments, R 13 is hydrogen. In certain embodiments R 13 is Ci-6-alkyl. In certain embodiments R 13 is methyl. In certain embodiments R 13 is ethyl. In certain embodiments

R 13 is propyl. In certain embodiments R 13 is butyl. In certain embodiments R 13 is fluoro. In certain embodiments R 13 is chloro. In certain embodiments R 13 is bromo. In certain embodiments R 13 is iodo.

[0137] In certain embodiments, R¹⁴ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In still further embodiments, R¹⁴ is a substituted or unsubstituted aryl. In certain embodiments, R¹⁴ is a substituted or unsubstituted heteroaryl. In further embodiments, R ⁴ is

. In still further embodiments,

R¹⁴ is a substituted or unsubstituted pyranone or chromeneone.

[0138] In certain embodiments, R¹⁵ is a substituted or unsubstituted aryl. In further embodiments, R¹⁵ is a substituted or unsubstituted phenyl. In still further embodiments, R¹⁵

is a substituted phenyl. In certain embodiments, R¹⁵ is

wherein R^B4 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; or substituted or unsubstituted, branched or unbranched aryl. In further

embodiments, R is wherein R is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroalkyl; -OR^D; -C(=0)R^D; -C0₂R^D; -CN; -SR^D; -SOR^D; -S0₂R^D; -N0₂; -N₃; -N(R^D)₂; -NHC(=0)R^D; -NR^DC(=0)N(R^D)₂; - OC(=0)OR^D; -OC(=0)R^D; -OC(=0)N(R^D)₂; -NR^DC(=0)OR^D; or -C(R^D)₃; wherein each occurrence of R^D is independently a hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl; aryl; heteroaryl; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or hetero arylthio; and r is 0 or an integer between 1 and 5, inclusive.

[0139] In certain embodiments, R^C4 is hydrogen. In certain embodiments R^C4 is C_1-6- alkyl. In certain embodiments R C4 is methyl. In certain embodiments R C4 is ethyl. In certain embodiments R C4 is propyl. In certain embodiments R C4 is butyl. In certain embodiments

R C4 is fluoro. In certain embodiments R C4 is chloro. In certain embodi R C4 is bromo.

In certain embodiments R is iodo. In still further embodiments, R is

[0140] In certain embodiments, R¹⁶ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In certain embodiments, R¹⁶ is Ci-6-alkyl. In certain embodiments, R¹⁶ is a nitrogen protecting group. In certain embodiments, R¹⁶ is acyl. In further embodiments, R¹⁶ is hydrogen. [0141] In certain embodiments, r is 0. In further embodiments, r is 1. In still further embodiments, r is 2. In other embodiments, r is 3. In certain embodiments, r is 4. In further embodiments, r is 5.

[0142] In certain embodiments, an inventive compound is of formula (V):

(V)

or a pharmaceutically acceptable salt thereof;

wherein

each of R 17 and R 18 is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; - CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; - OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R¹⁹ is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

X⁹ is O, S, or NR^Y9;

R is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

n is 0 or an integer between 1 and 4, inclusive; provided that the compound is not

[0143] In certain embodiments, X⁹ is O. In further embodiments, X⁹ is S. In other embodiments, X⁹ is NR^Y9. In other embodiments, X⁹ is NH.

[0144] In certain embodiments, R 17 is a substituted or unsubstituted aryl. In certain embodiments, R 17 is a substituted or unsubstituted phenyl. In certain embodiments, R 17 is

unsubstituted phenyl. In certain embodiments, R is

wherein each R is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroalkyl; and g⁵ is 0 or an integer between 1 and 5, inclusive.

[0145] In certain embodiments, R 18 is hydrogen. In certain embodiments R 18 is C_1-6- alkyl. In certain embodiments R 18 is methyl. In certain embodiments R 18 is ethyl. In certain embodiments R 18 is propyl. In certain embodiments R 18 is butyl. In certain embodiments R 18 is fluoro. In certain embodiments R 18 is chloro. In certain embodiments R 18 is bromo. In certain embodiments R 18 is iodo.

[0146] In certain embodiments, R¹⁹ is a substituted or unsubstituted aryl. In further embodiments, R¹⁹ is a substituted or unsubstituted phenyl. In still further embodiments, R¹⁹ substituted heteroaryl. In certain embodiments, R¹⁹

, where independently hydrogen or C_1-6 alkyl. In

certain embodiments, R¹⁹ is

[0147] In certain embodiments, R Z5 is hydrogen. In certain embodiments R Z5 is C_1-6- alkyl. In certain embodiments R Z5 is methyl. In certain embodiments R Z5 is ethyl. In certain embodiments R is propyl. In certain embodiments R is butyl. In certain embodiments

R Z5 is fluoro. In certain embodiments R Z5 is chloro. In certain embodiments R Z5 is bromo. In certain embodiments R Z5 is iodo.

[0148] In certain embodiments, g⁵ is 0. In further embodiments, g⁵ is 1. In still further embodiments, g⁵ is 2. In other embodiments, g⁵ is 3. In certain embodiments, g⁵ is 4. In further embodiments, g⁵ is 5.

[0149] In certain embodiments, R^wl is hydrogen. In certain embodiments R^wl is C_1-6- alkyl. In certain embodiments R^wl is methyl. In certain embodiments R^wl is ethyl. In certain embodiments R^wl is propyl. In certain embodiments R^wl is butyl. In certain embodiments, R ^V2 is hydrogen. In certain embodiments R ^V2 is Ci-6-alkyl. In certain embodiments R ^V2 is methyl. In certain embodiments R ^V2 is ethyl. In certain embodiments

R ^V2 is propyl. In certain embodiments R ^V2 is butyl.

[0150] In still certain embodiments, n is 0. In further embodiments, n is 1. In still further embodiments, n is 2. In other embodiments, n is 3. In certain embodiments, n is 4.

[0151] In certain embodiments, an inventive compound is of formula (VI):

(VI)

or a pharmaceutically acceptable salt thereof;

wherein

each of R 20 , R 21 , and R 22 is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; - CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; - OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R 21 and R 2"2 may optionally join to form a fused cyclic structure; R is hydrogen; a nitrogen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or

unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

X¹⁰ is O, S, or NR^Y10; and

R YIO is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

provided that the compound is not

[0152] In certain embodiments, X is O. In further embodiments, X is S. In other embodiments, X¹⁰ is NR^Y1°. In other embodiments, X¹⁰ is NH.

[0153] In certain embodiments, R²⁰ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 20 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o heteroaliphatic. In still further embodiments, R 20 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic

20 B B

Ci-20 alkyl. In certain embodiments, R is C Cioalkylene-COiR , wherein R is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched

heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; or substituted or unsubstituted, branched or unbranched heteroaryl. In further embodiments, R B is H. In still further embodiments, R 20 is

[0154] In certain embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In certain embodiments, R 21 is Ci-6-alkyl. In further embodiments, R 21 is hydrogen. [0155] In certain embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In certain embodiments, R 22 is Ci-6-alkyl. In further embodiments, R 22 is hydrogen.

[0156] In certain embodiments, R²¹ and R²² join to form a fused cyclic structure. In still further embodiments, R 21 and R 22 join to form a cyclopentene ring.

[0157] In certain embodiments, R²³ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 23 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In certain embodiments,

R 23 is a nitrogen protecting group. In certain embodiments, R 23 is acyl. In further

embodiments, R 23 is hydrogen.

[0158] In certain embodiments, an inventive compound is of formula (VII):

(VII)

or a pharmaceutically acceptable salt thereof;

wherein

each of R 24 , R 25 , and R 26 is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; - CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; - OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R 27 is hydrogen; a nitrogen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or

m is 0 or an integer between 1 and 5, inclusive;

k is 0 or an integer between 1 and 3, inclusive; and

q is 0 or an integer between 1 and 4, inclusive;

nsisting of

[0159] In certain embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 24 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci-io heteroaliphatic. In still further embodiments, R 24 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic

Ci alkyl. In certain embodiments, R 24 is hydrogen. In certain embodiments, R 24

-20 is C_1-6- alkyl. In certain embodiments, R 24 is methyl. In certain embodiments, R 24 is ethyl. In certain embodiments, R 24 is propyl. In certain embodimen R 24 is butyl. In further embodiments, two R²⁴ sub stituents join to form

.

[0160] In certain embodiments, R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C^oheteroaliphatic. In certain embodiments, R 25 is a substituted or unsubstituted heteroaryl. In further embodiments, R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In certain embodiments, R 25 is selected from the group consisting of hydrogen, -CH₃, -CH₂CH₃, - CH₂CH₂CH₃, -CH(CH₃)₂, -CH₂CH₂CH₂CH₃, -CH₂CH(CH₃)₂, -C(CH₃)₃, -OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₃)₂, -OCH₂CH₂CH₂CH₃, -OCH₂CH(CH₃)₂, -OC(CH₃)₃, -N0₂, -CN,

. In certain embodiments, R²⁵ is one of the followin heteroc clic moieties:

[0161] In certain embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In further embodiments, R²⁶ is hydrogen. In certain embodiments, R²⁶ is Ci-6-alkyl. In certain embodiments, R²⁶ is methyl. In certain embodiments, R²⁶ is ethyl. In certain embodiments, R²⁶ is propyl. In certain embodiments, R²⁶ is butyl. In certain embodiments, R²⁶ is fluoro. In certain embodiments, R²⁶ is chloro. In certain embodiments, R²⁶ is bromo. In certain embodiments, R²⁶ is iodo.

[0162] In certain embodiments, R²⁷ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In further embodiments, R 27 is hydrogen. In certain embodiments, R 27 is Ci 27

-6-alkyl. In certain embodiments, R is methyl. In certain embodiments, R 27 is ethyl. In certain embodiments, R 27 is propyl. In certain embodiments, R²⁷ is butyl.

[0163] In certain embodiments, m is 0. In further embodiments, m is 1. In still further embodiments, m is 2. In other embodiments, m is 3. In certain embodiments, m is 4. In further embodiments, m is 5.

[0164] In certain embodiments, k is 0. In further embodiments, k is 1. In still further embodiments, k is 2. In other embodiments, k is 3. [0165] In certain embodiments, q is 0. In further embodiments, q is 1. In still further embodiments, q is 2. In other embodiments, q is 3. In certain embodiments, q is 4.

[0166] In certain embodiments an inventive compound is of formula (VIII):

(VIII)

or a pharmaceutically acceptable salt thereof;

wherein

R 27 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

m is 0 or an integer between 1 and 5, inclusive; and

q is 0 or an integer between 1 and 4, inclusive.

[0167] In certain embodiments, R²⁴ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 24 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o heteroaliphatic. In still further embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic

Ci In certain embodiments, R 24 is hydrogen. In certain embodiments, R 24

-20 alkyl. is C_1-6- alkyl. In certain embodiments, R 24 is methyl. In certain embodiments, R 24 is ethyl. In certain embodiments, R 24 is propyl. In certain embodimen 24 is butyl. In further embodiments, two R substituents join to form

[0168] In certain embodiments, R ^J is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci^o heteroaliphatic. In certain embodiments, R 25 is a substituted or unsubstituted heteroaryl. In further embodiments, R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In certain embodiments, R 25 is selected from the group consisting of hydrogen, -CH₃, -CH₂CH , - CH₂CH₂CH₃, -CH(CH₃)₂, -CH₂CH₂CH₂CH₃, -CH₂CH(CH₃)₂, -C(CH₃)₃, -OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₃)₂, -OCH₂CH₂CH₂CH₃, -OCH₂CH(CH₃)₂, -OC(CH₃)₃, -N0₂, -CN,

, . In certain embodiments, R²⁵ is one of the following heterocyclic moieties:

[0169] In certain embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In further embodiments, R is hydrogen. In certain embodiments, R²⁶ is Ci-6-alkyl. In certain embodiments, R²⁶ is methyl. In certain embodiments, R²⁶ is ethyl. In certain embodiments, R²⁶ is propyl. In certain embodiments, R²⁶ is butyl. In certain embodiments, R²⁶ is fluoro. In certain embodiments, R²⁶ is chloro. In certain embodiments, R²⁶ is bromo. In certain embodiments, R²⁶ is iodo.

[0170] In certain embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In further embodiments, R 27 is hydrogen. In certain embodiments, R is C^-allcyl. In certain embodiments, R is methyl. In certain embodiments, R 27 is ethyl. In certain embodiments, R 27 is propyl. In certain embodiments, R²⁷ is butyl.

[0171] In certain embodiments, m is 0. In further embodiments, m is 1. In still further embodiments, m is 2. In other embodiments, m is 3. In certain embodiments, m is 4. In further embodiments, m is 5.

[0172] In certain embodiments, q is 0. In further embodiments, q is 1. In still further embodiments, q is 2. In other embodiments, q is 3. In certain embodiments, q is 4.

[0173] In certain embodiments an inventive compound is of formula (IX):

(IX)

or a pharmaceutically acceptable salt thereof;

wherein

unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; m is 0 or an integer between 1 and 5, inclusive; and

q is 0 or an integer between 1 and 4, inclusive.

[0174] In certain embodiments, R²⁴ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 24 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci^oheteroaliphatic. In still further embodiments, R 24 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic

Ci ments, R 24 is hydrogen. In certain embodiments, R 24

-20 alkyl. In certain embodi is C_1-6- alkyl. In certain embodiments, R 24 is methyl. In certain embodiments, R 24 is ethyl. In certain embodiments, R 24 is propyl. In certain embodimen R 24 is butyl. In further embodiments, two R²⁴ sub stituents join to form

or

[0175] In certain embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic. In further embodiments, R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci^o heteroaliphatic. In certain embodiments, R 25 is a substituted or unsubstituted heteroaryl. In further embodiments, R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci-20 alkyl. In certain embodiments, R 25 is selected from the group consisting of hydrogen, -CH₃, -CH₂CH₃, - CH₂CH₂CH₃, -CH(CH₃)₂, -CH₂CH₂CH₂CH₃, -CH₂CH(CH₃)₂, -C(CH₃)₃, -OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₃)₂, -OCH₂CH₂CH₂CH₃, -OCH₂CH(CH₃)₂, -OC(CH₃)₃, -N0₂, -CN,

. In certain embodiments, R²⁵ is one of the following heteroc clic moieties:

[0176] In certain embodiments, R²⁶ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In further embodiments, R²⁶ is hydrogen. In certain embodiments, R²⁶ is C^-alky!. In certain embodiments, R²⁶ is methyl. In certain embodiments, R is ethyl. In certain embodiments, R is propyl. In certain embodiments, R²⁶ is butyl. In certain embodiments, R²⁶ is fluoro. In certain embodiments, R²⁶ is chloro. In certain embodiments, R²⁶ is bromo. In certain embodiments, R²⁶ is iodo.

[0177] In certain embodiments, R²⁷ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl. In further embodiments, R 27 is hydrogen. In certain embodiments, R 27 is C^-allcyl. In certain embodiments, R 27 is methyl. In certain

embodiments, R 27 is ethyl. In certain embodiments, R 27 is propyl. In certain embodiments,

R 27 is butyl. In certain embodiments, R 27 is a nitrogen protecting group.

[0178] In certain embodiments, m is 0. In further embodiments, m is 1. In still further embodiments, m is 2. In other embodiments, m is 3. In certain embodiments, m is 4. In further embodiments, m is 5.

[0179] In certain embodiments, q is 0. In further embodiments, q is 1. In still further embodiments, q is 2. In other embodiments, q is 3. In certain embodiments, q is 4.

[0180] In certain embodiments an inventive compound is of formula (X):

(X)

or a pharmaceutically acceptable salt thereof;

wherein

each of R 28 and R 29 is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; - CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; - OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

X^u is O, S, or NR^Y11 ;

R Yl 1 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

z is 0 or an integer between 1 and 3, inclusive;

provided that the compound is not

[0181] In certain embodiments, X¹¹ is O. In further embodiments, X¹¹ is S. In other embodiments, X¹¹ is NR^YU. In other embodiments, X¹¹ is NH.

[0182] In certain embodiments, z is 0. In further embodiments, z is 1. In still further embodiments, z is 2. In other embodiments, z is 3.

[0183] In certain embodiments, R²⁸ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C 28

1_-2o aliphatic. In still further embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_{1 -2}o alkyl. In certain

embodiments, R 28 is hydrogen. In certain embodiments, R 28 is Ci-6-alkyl. In certain embodiments, R 28 is methyl. In certain embodiments, R 28 is ethyl. In certain embodiments,

R 28 is propyl. In certain embodiments, R 28 is butyl. In certain embodiments, R 28 is fluoro. In certain embodiments, R 28 is chloro. In certain embodiments, R 28 is bromo. In certain embodiments, R 28 is iodo. In certain embodiments, R 28 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_{1 -2}o heteroaliphatic. In still further embodiments,

R 28 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o

heteroalkyl. In still further embodiments, R 28 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci-io haloalkyl. In certain embodiments, R 28 is -CH₂F. In certain embodiments, R 28 is -CHF₂. In certain embodiments, R 28 is -CH₂F. In certain embodiments, R 28 is -CF₃. In certain embodiments, R 28 is a substituted or unsubstituted aryl.

In further embodiments, R 28 is a substituted or unsubstituted phenyl. In still further

embodiments, R is a substituted phenyl. In certain embodiments, R is

wherein g⁶ is 0 or an integer between 1 and 5, inclusive; and R²⁶ is hydrogen; an oxygen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; or substituted or unsubstituted, branched or unbranched aryl. In certain embodiments, R is methyl. In certain embodiments, R is ethyl. In certain embodiments, R^z6 is propyl. In certain embodiments, R^z6 is butyl.

[0184] In certain embodiments, g⁶ is 0. In further embodiments, g⁶ is 1. In still further embodiments, g⁶ is 2. In other embodiments, g⁶ is 3. In certain embodiments, g⁶ is 4. In

6 ·

further embodiments, g⁶ is 5.

[0185] tain embodiments, R ^s is

In certain embodiments,

[0186] In certain embodiments, R is substituted or unsubstituted, branched or

9Q

unbranched, cyclic or acyclic C^o aliphatic. In still further embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C^o alkyl. In certain

9Q 9Q

embodiments, R is hydrogen. In certain embodiments, R is substituted or unsubstituted,

9Q

branched or unbranched, CrCs-alkyl. In certain embodiments, R is hydrogen. In certain

9Q 9Q embodiments, R is a branched CrCg-alkyl. In certain embodiments, R is

_{In certain embodimentS ; R}29 _is

certain embodiments R²⁹ is

_certam embodiments, R²⁹

is substituted or unsubstituted, branched or

9Q unbranched, cyclic or acyclic Ci-io heteroaliphatic. In still further embodiments, R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci-ioheteroalkyl. In certain embodiments, R is a substituted or unsubstituted aryl. In certain embodiments, R is a substituted or unsubstituted heteroaryl.

Pharmaceutical Compositions

[0187] The compounds described herein may be combined with one or more pharmaceutical excipients to form a pharmaceutical composition that is suitable for administration to an animal including a human. As would be appreciated by one of this art, the excipients may be chosen based on the route of administration as described below, the agent being delivered, or the time course of delivery of the agent. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

[0188] In one aspect, the present invention provides a pharmaceutical composition comprising a compound for activating proteasomal degradation as described herein and, optionally, a pharmaceutically acceptable excipient.

[0189] In one aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective ound of formula (I):

wherein R 1 , R2", R 3^J, X 1¹, X2", and X 3^J are as described herein; and, optionally, a

pharmaceutically acceptable excipient.

[0190] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (II):

(Π)

wherein R⁴, R⁵, R⁶, R⁷, R⁸, and X⁴ are described herein; and, optionally, a pharmaceutically acceptable excipient.

[0191] In yet another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (III):

(III)

wherein R⁹, R¹⁰, R¹¹, R¹², X⁵, and X⁶ ar ree ddeescribed herein; and, optionally, a

pharmaceutically acceptable excipient. [0192] In still another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (IV):

(IV)

wherein R¹³, R¹⁴, R¹⁵, R¹⁶, X⁷, and X⁸ are described herein; and, optionally, a

pharmaceutically acceptable excipient.

[0193] In one aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (V):

(V)

wherein R¹⁷, R¹⁸, R¹⁹, n, and X⁹ are described herein; and, optionally, a pharmaceutically acceptable excipient.

[0194] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (VI):

(VI)

wherein R ^u, R ¹, R , R ^J, and X^1U are described herein; and, optionally, a pharmaceutically acceptable excipient.

[0195] In yet another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (VII):

(VII) wherein R , R , R , R , m, k, and q are described herein; and, optionally, a pharmaceutically acceptable excipient.

[0196] In still another aspect, the present invention provides a therapeutically effective amount of a compound of formula (VIII :

wherein R , R , R , R , m, and q are described herein; and, optionally, a pharmaceutically acceptable excipient.

[0197] In one aspect, the present invention provides a therapeutically effective amount of a compound of formula (IX):

(IX)

wherein R 24 , R 25 , R 26 , R 27 , m, and q are described herein; and, optionally, a pharmaceutically acceptable excipient.

[0198] In one aspect, the present invention provides a therapeutically effective amount of a compound of formula (X):

(X) wherein R , R , X , and z are described herein; and, optionally, a pharmaceutically acceptable excipient.

[0199] In another aspect, the present invention provides a pharmaceutical composition comprising a compound selected from the group consisting of:

[0200] Pharmaceutical compositions of the present invention and for use in accordance with the present invention may include a pharmaceutically acceptable excipient or carrier. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi- solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack

Publishing Co., Easton, PA, 1980) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;

Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical compositions of this invention can be administered to humans and/or to animals, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, or as an oral or nasal spray.

[0201] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredients {i.e., microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipid complexes), 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 carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0202] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80.

[0203] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0204] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension or crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide- polyglycolide. Depending upon 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 poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0205] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the particles with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the particles.

[0206] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.

[0207] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.

[0208] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[0209] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0210] Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The particles are admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. [0211] In certain embodiments, the pharmaceutically acceptable topical formulations of the invention comprise at least a compound of the invention and a penetration enhancing agent. The choice of topical formulation will depend or several factors, including the condition to be treated, the physicochemical characteristics of the inventive compound and other excipients present, their stability in the formulation, available manufacturing equipment, and costs constraints. As used herein the term "penetration enhancing agent" means an agent capable of transporting a pharmacologically active compound through the stratum coreum and into the epidermis or dermis, preferably, with little or no systemic absorption. A wide variety of compounds have been evaluated as to their effectiveness in enhancing the rate of penetration of drugs through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveys the use and testing of various skin penetration enhancers, and Buyuktimkin et al. , Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh T. K., Pfister W. R., Yum S. I.

(Eds.), Interpharm Press Inc., Buffalo Grove, 111. (1997). In certain exemplary embodiments, penetration agents for use with the invention include, but are not limited to, triglycerides {e.g., soybean oil), aloe compositions {e.g., aloe- vera gel), ethyl alcohol, isopropyl alcohol, octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N- decylmethylsulfoxide, fatty acid esters {e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate), and N-methyl pyrrolidone.

[0212] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the microparticles or nanoparticles in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.

[0213] In certain embodiments, the compositions may be in the form of ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. In certain exemplary embodiments, formulations of the compositions according to the invention are creams, which may further contain saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleyl alcohols, stearic acid being particularly preferred. Creams of the invention may also contain a non-ionic surfactant, for example, polyoxy-40-stearate. In certain embodiments, the active component is admixed under sterile conditions with a pharmaceutically acceptable excipient and any needed preservatives or buffers as may be required. Ophthalmic formulations, eardrops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms are made by dissolving or dispensing the compound in the proper medium. As discussed above, penetration enhancing agents can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix (e.g., PLGA) or gel.

[0214] The ointments, pastes, creams, and gels may contain 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.

[0215] Powders and sprays can contain 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 .

[0216] It will also be appreciated that the compounds and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with an agent effective against a neurodegenerative disease or an antibiotic), or they may achieve different effects (e.g. , control of any adverse effects).

[0217] It will also be appreciated that certain of the compounds of present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof. According to the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or a prodrug or other adduct or derivative of a compound of this invention which upon administration to a patient in need is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof. Methods of Treatment

[0218] The present invention provides compounds that mimic the C-termini of the proteasome-regulatory ATPases, induce gate opening of the proteasome, activate the proteasomal degradation of proteins, and can produce beneficial therapeutic effects. In further embodiments, compounds and compositions described herein are used to treat disorders associated with unfolded, misfolded, or aggregated proteins such as sickle cell anemia, thalassemia, Alzheimer's disease, Parkinson's disease, cystic fibrosis, Huntington's disease, amyotrophic lateral sclerosis {i.e., Lou Gehrig's disease), and Creutzfeld-Jakob disease. In further embodiments, compounds and compositions of the invention are used to kill or inhibit the growth of a microorganism.

[0219] The inventive methods stimulate gate opening of the proteasome to induce proteasomal degradation of proteins. In one aspect, the present invention provides methods comprising the compounds of the invention for stimulating proteasomal degradation of proteins comprising: contacting a subject with a compound or a composition of the invention under conditions suitable to induce the proteasomal degradation of proteins in the subject.

[0220] Further aspects of the invention include a method for treating a disorder associated with unfolded, misfolded, or aggregated proteins in a subject comprising:

administering to the mammal an effective amount of a compound or composition of the invention to stimulate gate opening of the proteasome to induce proteasomal degradation of unfolded, misfolded, or aggregated proteins. In certain embodiments, the subject is human. In further embodiments, the proteasome is a 20S proteasome.

[0221] In certain embodiments, the disorder is selected from the group consisting of a neurodegenerative disease; a bacterial infection; a fungal infection; sickle cell anemia;

thalassemia; Alzheimer's disease; Parkinson's disease; cystic fibrosis; amyotrophic lateral sclerosis {i.e., Lou Gehrig's disease); Creutzfeld-Jakob disease; kuru; GSS disease;

Huntington's disease; polyglutamine diseases; prion-related disorders; fatal familialinsomnia; scrapie; transmissible spongiform encephalopathy (TSE); bovine spongiform

encephalopathy; Alexander's disease; primary systemic amyloidosis; secondary systemic amyloidosis; senile systemic amyloidosis; amyloidosis in senescence; ocular disease:

cataract; retinitis pigmentosa; macular degeneration; islet amyloid; medullar carcinoma of the thyroid; hereditary renal amyloidosis; hemodialysis-related amyloidosis; Desmin-related cardiomyopathy; Charcot-Marie tooth disease; diabetes insipidis; diabetes insipidis; protein- destabilization, such as Gaucher disease and Sanfilippo syndrome type B

(mucopolysaccharidosis III B); and congophilic angiopathy. In further embodiments, the disorder is a neurodegenerative disease.

[0222] In still further embodiments, the unfolded, misfolded, or aggregated protein is selected from the group consisting of beta-casein; tau; tau 23; amyloid beta; alpha-synuclein; amyloid protein; huntingtin; atrophin-1, ataxins; prion protein; BSE; superoxide dismutase; glial fibrillary acidic protein; immunoglobulin light chain or fragments thereof; serum amyloid-A or fragments thereof; transthyretin or fragments thereof; apolipoprotein A-II; crystallins or crystallin filaments; rhodopsin; calcitonin; fibrinogen; beta 2-microglobulin; desmin; peripheral myelin protein 22 (PMP-22); aquaporin; vasopressin receptor; connexin 32; and cystic fibrosis transmembrane conductance regulator (CFTR).

[0223] In one aspect, the inventive activators of proteasomal gate opening can enhance antigen presentation and promote an immune response (Rock and Goldberg, Annu. Rev.

Immunol. 1999 17:739-779; Rock et al, Nat. Immunol. 5, 670-7 (2004)). The proteasome degrades host proteins derived from host cells, bacterial or viral proteins derived from infected cells, or cancerous proteins derived from cancerous cells. Further, the proteasomal degradation of proteins derived from infected or cancerous cells yields antigenic peptide fragments that are presented on major histocompatibility complex (MHC) class I molecules. Presentation of the antigenic peptide fragments enables circulating cytotoxic lymphocytes to recognize and eliminate infected or cancerous cells. Thus, the inventive activators of proteasomal gate opening can be administered to a subject to increase the presentation of the antigenic peptide fragments on MHC class I molecules, stimulate a cytotoxic immune response, and accelerate the elimination infected or cancerous cells.

[0224] In certain embodiments, the administration to a subject of the inventive activators of proteasomal gate opening causes a reduction or elimination of cells that are infected with bacteria. In certain embodiments, the cells are infected with Gram-negative bacteria. Exemplary Gram-negative bacteria include, but are not limited to, Escherichia coli, Salmonella (e.g., Salmonella enteritidis, and Salmonella typhi), Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Pseudomonas, Moraxella (e.g., Moraxella catarrhalis), Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella, and Neisseria (e.g., Neisseria gonorrhoeae, Neisseria meningitidis). In certain embodiments, the cells are infected with Gram-positive bacteria. Exemplary Gram-positive bacteria include, but are not limited to, Streptococci bacteria such as Streptococcus Group A, Streptococcus Group B, Streptococcus Group G (e.g.,

Streptococcus anginosus, Streptococcus pneumoniae), Streptococcus viridans, and Streptococcus pyogenes; Staphylococci bacteria such as Staphylococcus aureaus (e.g., Staphylococcus aureus, Staphylococcus aureus (MSA), and Staphylococcus saprophytics; and other bacteria such as Micrococcus luteus, Enterococcus faecalis, and Enterococcus faecalis.

[0225] In certain embodiments, the inventive activators of proteasomal gate opening may be used to treat any infection including, but not limited to, anthrax, bacterial meningitis, botulism, brucellosis, campylobacteriosis, cholera, diphtheria, gonorrhea, impetigo, legionellosis, leprosy (Hansen's disease), leptospirosis, listeriosis, lyme disease, melioidosis, methicillin-resistant Staphylococcus aureus (MRSA) infection, nocardiosis, pertussis (whooping cough), plague, pneumococcal pneumonia, psittacosis, Q fever, rocky mountain spotted fever (RMSF), scarlet fever, shigellosis, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhoid fever, typhus, urinary tract infection (UTI), skin infections, GI infections, genito-urinary infections, and systemic infections.

[0226] In further embodiments, the administration to a subject of the inventive activators of proteasomal gate opening causes a reduction or elimination of cells that are infected with a virus. Exemplary viruses include, but not limited to, adenovirus, rhinovirus, human papilloma virus, human immunodeficiency virus, hepatitis virus, Newcastle disease virus, cardiovirus, corticoviridae, cystoviridae, epstein-barr virus, filoviridae, hepadnviridae, hepatitis virus, herpes virus, influenza virus, inoviridae, iridoviridae, metapneumovirus, orthomyxoviridae, papovavirus, parainfluenza virus, paramyxoviridae, parvoviridae, polydnaviridae, poxyviridae, reoviridae, respiratory syncytial virus, rhabdoviridae, semliki forest virus, tetraviridae, toroviridae, vaccinia virus, vesicular stomatitis virus, hantavirus, and cytomegalovirus.

[0227] In still further embodiments, the administration to a subject of the inventive activators of proteasomal gate opening causes a reduction or elimination of cells that are cancerous. Exemplary cancers include, but are not limited to, carcinomas, sarcomas, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, central nervous system (CNS) cancer, breast cancer, cervical cancer, choriocarcinoma, colon and rectum cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, intra-epithelial neoplasm, kidney cancer, larynx cancer, leukemia, liver cancer, lung cancer (e.g., small cell and non- small cell), lymphoma including Hodgkin's and non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cavity cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, renal cancer, cancer of the respiratory system, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system.

[0228] The inventive activators of proteasomal degradation of proteins, as described herein, can exhibit antimicrobial activity (e.g. , anti-bacterial, anti-fungal, anti-malarial activity). In certain aspects, the compounds of the invention sufficiently activate the proteasomal degradation of proteins in a microorganism to kill or inhibit the growth of the microorganism.

[0229] In certain embodiments, the invention provides a method for killing or inhibiting the growth of a microorganism, the method comprising contacting a

microorganism with an effective amount of a compound or composition of the invention to kill or inhibit the growth of the microorganism. In further embodiments, the compound or composition of the invention sufficiently activates the proteasomal degradation of proteins within the microorganism to kill or inhibit the growth of the microorganism.

[0230] In certain embodiments, the compound or composition of the invention inhibits an infection. In certain embodiments, the infection is a bacterial infection. In certain embodiments, the infection is a fungal infection. In certain embodiments, the infection is a parasitic infection.

[0231] In certain embodiments, the bacterial infection is caused by caused Gram- negative bacteria. In certain embodiments, the infection is caused by caused Gram-positive bacteria.

[0232] In general, methods of using the compounds of the present invention comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention. As discussed above, the compounds of the invention are typically activators of the proteasomal degradation of proteins and, as such, are useful in the treatment of disorders associated with unfolded, misfolded, or aggregated proteins.

[0233] It will also be appreciated that the compounds and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder. In certain embodiments, an inventive compound may be administered concurrently with another agent to treat disorders associated with unfolded, misfolded, or aggregated proteins such as sickle cell anemia, thalassemia, Alzheimer's disease, Parkinson's disease, cystic fibrosis, Huntington's disease, amyotrophic lateral sclerosis (i.e., Lou Gehrig's disease), and Creutzfeld-Jakob disease. In other embodiments, an inventive compound may be administered concurrently with another agent to treat a neurodegenerative disease. In further embodiments, an inventive compound may be administered concurrently with an inhibitor of a deubiquitinating enzyme or an inhibitor of ubiquitin- specific proteases. In further embodiments, an inventive compound may be administered concurrently with an antiparasitic agent. In still further embodiments, an inventive compound may be

administered concurrently with an antibiotic agent. In further embodiments, an inventive compound may be administered concurrently with an antifungal agent.

[0234] The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of therapeutic agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, mute of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see, for example, Goodman and Oilman 's The Pharmacological Basis of Therapeutics, Tenth Edition, A. Gilman, J.Hardman and L. Limbird, eds., McGraw-Bill Press, 155-173, 2001, which is incorporated herein by reference in its entirety).

Screening methods

[0235] In one aspect, the present invention provides a method for identifying compounds that activate the proteasomal degradation of proteins. In certain embodiments, proteasomes are isolated using standard biochemical approaches such as those described in the international PCT Patent Application Publication WO 2008/048373. In one aspect, a bacterial culture can be grown in order to produce proteasomes native to that organism (see Knipfer and Shrader (1997) Mol. Microbiol. 25:375). In another aspect, genes encoding the alpha and beta subunits of a 20S proteasome may be cloned, and large amounts of the proteasomes may be generated in an organism such as E. coli (Kim et al. (1995) J. Biol. Chem. 270:29799). The isolation of proteasomes from a variety of mammalian tissues such as rabbit, bovine, or human tissues and/or from eukaryotic organisms such as yeast, are also known in the art.

[0236] In certain embodiments, bovine 20S proteasome from the liver is prepared as described by Kisselev, A. F., Kaganovich, D., and Goldberg, A. L. 2002 J. Biol. Chem. 277, 22260-22270. Yeast 20S proteasomes are affinity -purified as described by Leggett et al, Mol. Cell. 2002 Sep;10(3):495-507. The 26S proteasomes from any mammalian tissue or eukaryotic cell can be affinity purified by the method of Besche et al. 2009 Biochemistry, Mar 24;48(11):2538-49. Protein expression and cell lysis are performed as described by Kisselev et al. and Leggett et al., using a PA26 pET-15b construct that was transformed into BL21(DE3) competent cells. Proteins are purified with Ni-NTA affinity chromatography.

[0237] The ability of one or more of the compounds described herein to inhibit or activate protein degradation can be assayed using standard fluorogenic substrates (Kisselev and Goldberg (2001) Chem. Biol. 8:739; Kisselev et al, (2002) J. Biol. Chem. 25:22260; and Kisselev et al., (2003) J. Biol. Chem. 278:35869. The substrate succinyl Leu-Leu- Val-Tyr- amc can be used, for example, to establish efficacy for the proteasomes (Knipfer and Shrader (1997) Mol. Microbiol. 25:375; and Akopian et al, (1997) J. Biol. Chem. 272: 1791; the entirety of each of which is incorporated herein by reference. Standard methods using fluorogenic peptides are also available to confirm a lack of potency against the mammalian proteasome, as would be desirable for an antimicrobial drug (Kisselev et al, (2003) supra).

[0238] In addition to the standard substrates of the proteasome' s active sites, we have shown that gate opening can be sensitively monitored with longer fluorescent substrates, such as Suc-Arg-Pro-Phe-His-Leu-Val-Tyr-MCA (SEQ ID NO: 11) or MOCAc-Ala-Pro-Ala-Lys- Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂ (SEQ ID NO: 12). These substrates are degraded rapidly by the proteasome and this process is stimulated 4-10 fold by Z-Tyr-Ala, which mimics the C-terminal HbYX motif in inducing gate-opening. Moreover, these longer peptide substrates mimic the behavior of protein substrates {e.g., Tau) better than the standard proteasome tetrapeptide substrates, such as Suc-LLVY-MCA (SEQ ID NO: 13) or Z-GGL-MCA, and thus are valuable as screening agents or in counterscreens.

[0239] In another embodiment, the invention provides a cell-based assay comprising contacting a cell expressing a proteasome with a test compound {e.g., protein, polypeptide, peptide and/or small molecule) and determining the ability of the test compound to inhibit or activate protein degradation. Determining the ability of the test compound to inhibit or activate protein degradation can be accomplished, for example, by measuring the

accumulation of a short-lived model protein that is normally degraded rapidly by the proteasome (e.g., ΙκΒ or ubiquitin-pro-P-galactosidase) (Kisselev and Goldberg (2001) supra).

[0240] In another embodiment, in vivo validation is accomplished using

neurodegenerative cell culture models. One experimental model for the validation of a test compound is to determine if the test compound can prevent and/or reduce aggregate formation from mutant proteins associated with one or more disorders and/or diseases described herein (e.g., neurodegenerative diseases). Aggregates of fluorescently labeled proteins are easily observed under the microscope or can be isolated by filtration, and therefore give an easy readout of effectiveness of each compound. Thus, it is possible to test if a test compound can prevent and/or reduce aggregate formation and/or induce the clearance and/or reduction of aggregates once formed. Many suitable cell culture models of

neurodegenerative diseases are available, such as, a cell culture model of ALS whereby a neuronal cell line expresses mutant SOD1 which forms visible intercellular aggregates a neuroblastoma cell line (SY5Y) that upon transient transfection with a-synuclein (a model for Parkinson's disease) forms intercellular aggregates that appear to be degraded by the 20S and not the 26S proteasome (Tofaris et al. (2001) FEBS Let.); a PC 12 cell line that constitutively expresses α-synuclein; and a model of a tauopathy (Alzheimer's disease model) whereby tau is transiently transfected into a neuroblastoma cells line and causes cellular toxicity. One or more of these cell models can be used to assay whether a test compound has the capacity to reduce aggregate formation, clear pre-formed aggregates, and/or prevent tau induced toxicity. Additional techniques to monitor aggregates in cellular models of Parkinson's, Alzheimer's, and Huntington's disease are described in Menzies et al., Hum Mol Genet. 2010 Dec l;19(23):4573-86.

[0241] In yet another embodiment, an assay of the present invention is a cell-free assay in which a proteasome or a biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 20S proteasome or a

biologically active portion of the proteasome is determined. Binding of the test compound to a proteasome can be determined either directly or indirectly as described further herein. [0242] In further embodiments, screening methods were adapted from those reported by Yu Y., et al, EMBO J. 2010 Feb 3;29(3):692-702; Rabl J., et al, Mol Cell. 2008 May 9;30(3):360-8; and Smith DM, et al, Mol Cell. 2007 Sep 7;27(5):731-44.

[0243] A further aspect of the invention provides a method for identifying compounds that activate the proteasomal degradation of proteins, the method comprising contacting a fluorogenic peptide with the proteasome in the presence of a test compound; and identifying compounds that cause an increase in the level of fluorescence relative to the level of fluorescence obtained in the absence of the test compound. In further embodiments, a baseline level of fluorescence is measured from the proteasomal degradation of the fluorogenic peptide by the proteasome.

[0244] In certain embodiments, the activators are small molecules. In further embodiments, the activators bind a 20S proteasome. In further embodiments, the proteasome is a eukaryotic 20S proteasome. In further embodiments, the proteasome is a mammalian 20S proteasome. In further embodiments, the proteasome is a rabbit 20S proteasome. In further embodiments, the proteasome is a human 20S proteasome. In certain embodiments, the activators bind a site other than an a-ring of a 20S proteasome. In certain embodiments, the activators bind an a-ring of a 20S proteasome. In still further embodiments, the activators bind one or more pockets in the a-ring of an a-ring of a 20S proteasome. In further embodiments, the activators bind the intersubunit pockets of a 20S proteasome.

[0245] In certain embodiments, each of the contacting steps is incubated with ATP or a nonhydrolyzable ATP analog. In further embodiments, the nonhydrolyzable ATP analog is ATP7S. In still further embodiments, each of the contacting steps is heated. In certain embodiments, each of the contacting steps is heated to 37 °C. In further embodiments, each of the contacting steps is heated to 45 °C. In still further embodiments, the fluorogenic peptide is nLPnLD-amc (SEQ ID NO: 10). In certain embodiments, the fluorogenic peptide is LRR-amc. In further embodiments, the fluorogenic peptide is YFP-ssrA. In still further embodiments, the fluorogenic peptide is Mca-AKVYPYPME-Dpa(Dnp)-amide (LFP; SEQ ID NO:9). In certain embodiments, the fluorogenic peptide is Suc-Arg-Pro-Phe-His-Leu- Val-Tyr-MCA (SEQ ID NO: 11). In certain embodiments, the fluorogenic peptide is

MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂ (SEQ ID NO: 12). In certain embodiments, the test compound is a small molecule. In further embodiments, the test compound is a peptide. In further embodiments, the test compound is a peptide derivative. In further embodiments, the proteasome is a 20S proteasome. In still further embodiments, the proteasome was expressed in Escherichia coli. In certain embodiments, the methods further comprise one or more tertiary assays to determine if the compound that increases fluorescence can reproducibly activate the proteasomal degradation of proteins. In further embodiments, one or more of the tertiary assays measures the degradation of proteins by a proteasome. In still further embodiments, the protein is Tau. In certain embodiments, Tau is Tau23. In certain embodiments, the protein is alpha-synuclein. In certain embodiments, the proteasome is a 20S proteasome. In certain embodiments, the proteasome is an archaeal 20S proteasome. In further embodiments, the proteasome is a eukaryotic 20S proteasome. In certain embodiments, the proteasome is a mammalian 20S proteasome. In further embodiments, the proteasome is a primate 20S proteasome. In further embodiments, the proteasome is a human 20S proteasome. In further embodiments, the proteasome is a bovine 20S proteasome. In further embodiments, the proteasome is a bovine 20S proteasome from bovine liver. In further embodiments, the proteasome is a rabbit 20S proteasome. In further embodiments, the proteasome is a rat 20S proteasome. In further embodiments, the proteasome is a mouse 20S proteasome. In further embodiments, the proteasome is a dog 20S proteasome. In still further embodiments, the proteasome is a wild-type yeast 20S proteasome. In certain embodiments, the proteasome is a 26S proteasome. In certain embodiments, the invention provides a method for identifying compounds that activate the wild-type proteasomal degradation of proteins to a greater extent than they activate the proteasomal degradation of proteins by an open gate mutant of the proteasome. In certain embodiments, the open gate mutant is a yeast open gate mutant. In certain embodiments, the open gate mutant is a bovine open gate mutant. In certain embodiments, the open gate mutant is a human open gate mutant. In certain embodiments, the open gate mutant is a eukaryotic open gate mutant. In certain embodiments, the open gate mutant is a prokaryotic open gate mutant.

[0246] In certain embodiments, assays are provided to determine the ability of compounds to activate the proteasomal degradation of proteins. Representative data that was used to calculate EC₅₀ values is provided in Figures 22 and 23. In further embodiments, inventive compounds exhibit EC₅₀ values < 100 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values < 50 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values < 40 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values < 30 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values < 20 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values <10 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values < 7.5 μΜ. In certain embodiments, inventive compounds exhibit EC₅₀ values < 5 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values < 2.5 μΜ. In certain embodiments, inventive compounds exhibit EC₅₀ values < 1 μΜ. In certain embodiments, inventive compounds exhibit EC₅₀ values < 0.75 μΜ. In certain embodiments, inventive compounds exhibit EC₅₀ values < 0.5 μΜ. In certain embodiments, inventive compounds exhibit EC₅₀ values < 0.25 μΜ. In certain embodiments, inventive compounds exhibit EC₅₀ values < 0.1 μΜ. In further embodiments, inventive compounds exhibit EC₅₀ values < 75 nM. In further embodiments, inventive compounds exhibit EC₅₀ values < 50 nM. In further embodiments, inventive compounds exhibit EC₅₀ values < 25 nM. In further embodiments, inventive compounds exhibit EC₅₀ values < 10 nM. In other embodiments, exemplary compounds exhibit EC₅₀ values < 5 nM. In other embodiments, exemplary compounds exhibit EC₅₀ values < 1 nM.

[0247] Uses according to the present invention, the inventive compounds may be assayed in any of the available assays known in the art for identifying activators of proteasomal degradation. For example, the assay may be cellular or non-cellular, in vivo or in vitro, high- or low-throughput format.

In silico screening

[0248] Proteasomal gate opening is induced by the association of activator compounds with key regulatory residues in the intersubunit pockets in the alpha ring of the 20S proteasome. Thus, it is a further object of the present invention to use atomic coordinates corresponding to key regulatory residues along the intersubunit pockets in the alpha ring of the 20S proteasome to design, identify, and/or screen potential binding compounds that induce gate opening of the proteasome and activate the proteasomal degradation of proteins. In certain embodiments, the alpha ring derives from an archaeal 20S proteasome. In further embodiments, the the alpha ring derives from a eukaryotic 20S proteasome. In still further embodiments, the the alpha ring derives from a mammalian 20S proteasome. In further embodiments, the the alpha ring derives from a bovine 20S proteasome. In further embodiments, the the alpha ring derives from a human 20S proteasome.

[0249] In one embodiment, the invention provides in silico screening methods based upon the atomic coordinates of intersubunit pockets in the alpha ring of an archaeal 20S proteasome in complex with the C-terminus of the archaeal proteasome ATPase, PAN (the "20S-PA26^E102A-^PAN9 complex"), the structure of which was solved by X-ray crystallography as reported by Yu, et ah, EMBO (2010) 29, 692-702, which is incorporated herein by reference in its entirety. The archaeal 20S proteasome comprises a single type of alpha subunit whereas the mammalian 20S proteasome (e.g., bovine or human) comprises seven types of alpha subunits (alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, and alpha 7). Some residues are conserved between the alpha subunit of the archaeal 20S proteasome and each of the seven alpha subunits of the mammalian 20S proteasome, although the residue numbering may differ among alpha subunits. Thus, in certain embodiments, the key regulatory residues in each of the mammalian alpha subunits may be similar or identical with the key regulatory residues of the alpha subunit of the archaeal 20S proteasome (e.g., Example 3 and Figure 24A-C).

[0250] As discussed, the atomic coordinates of the intersubunit pockets in the alpha ring of the proteasome can also be used to computationally screen for small molecule compounds that bind to the 20S proteasome in order to select, design, and develop potential binding compounds of the proteasome. In certain embodiments, one or more amino acid mutations of the 20S proteasome are computationally represented. It should be understood that a potential binding compound may bind to an intersubunit pockets in the alpha ring or any other site which is not identified as such. In some preferred embodiments, compound may bind to an intersubunit pockets in the alpha ring. In some embodiments, binding of a compound may perturb the alpha subunit (e.g., cause the alpha subunit to rotate) and thereby cause gate opening. In certain embodiments, the binding compound may bind specifically to one or more sites on the proteasome. It should be appreciated that peptides or small molecules that mimic the C-termini of proteasomal regulatory ATPases could be used.

[0251] In one embodiment, the present invention provides a method for the design and identification of a potential binding compound for a proteasome comprising the steps of (a) using a three-dimensional structure of the intersubunit pockets in the alpha ring of a 20S proteasome; (b) employing the three-dimensional structure to design and/or select the potential binding compound; and (c) synthesizing and/or choosing the potential binding compound.

[0252] Suitable computer programs which may be used in the design and selection of potential binding compounds (e.g., by selecting suitable chemical fragments) include, but are not limited to, GRID (Goodford; J. Med. Chem. (1985) 28:849 857); MCSS (Miranker, A. and M. Karplus, Proteins: Structure. Function and Genetics, (1991) 11:29-34); AUTODOCK (Goodsell, D. S, and A. J. Olsen, Proteins: Structure. Function, and Genetics (1990) 8: 195 202); and DOCK (Kuntz, I. D. et al, J. Mol. Biol. (1982) 161:269-288).

[0253] Suitable computer programs which may be used in connecting the individual chemical entities or fragments include, but are not limited to, CAVEAT (Bartlett, Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. (1989) 78: 182-196); and 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.), HOOK (Molecular Simulations, Burlington, Mass.) and as reviewed in Martin, Y. C, J. Med. Chem., (1992) 35:2145 2154).

[0254] In addition to the method of building or identifying a potential binding compound in a step-wise fashion (e.g., one fragment or chemical entity at a time as described above), potential binding compounds may be designed as a whole or "de novo" using either an empty binding site or, optionally, including some portion(s) of a known inhibitor(s).

Suitable computer programs include, but are not limited to, LUDI (Bohm, Comp. Aid. Molec. Design (1992) 6:61-78); LEGEND (Nishibata, Y. and Itai, A., Tetrahedron, 47, p. 8985

(1991) ); and LEAPFROG (Tripos Associates, St. Louis, Mo.). Other molecular modeling techniques may also be employed in accordance with this invention; see, for example, Cohen, et al., J. Med. Chem. (1990) 33: 883-894, and Navia, Current Opinions in Structural Biology

(1992) 2:202-210, the entirety of each of which is hereby incorporated herein by reference.

[0255] Once a potential binding compound has been designed and/or selected and/or synthesized and/or chosen by the above methods, the efficiency with which that compound binds to the 20S proteasome may be tested and optimized by computational evaluation. A compound designed and/or selected and/or synthesized and/or chosen as potential inhibitor compound may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target site. Such non- complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole- dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the potential inhibitory compound and the site at which it is bound to the 20S proteasome may make a neutral or favorable contribution to the enthalpy of binding. Suitable computer software which may be used to evaluate compound deformation energy and electrostatic interactions, includes, but is not limited to, Gaussian 92, revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa., 1992); AMBER, version 4.0 (P. A. Kollman, University of California at San Francisco, 1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass. 1994); and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif., 1994). These programs may be implemented, for example, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art of which the speed and capacity are continually modified.

[0256] In certain embodiments, binding compounds may be specifically designed and/or selected and/or synthesized and/or chosen by the above methods to induce non- complementary (e.g., electrostatic) interactions, such as repulsive charge-charge, dipole- dipole and charge-dipole interactions. In certain embodiments, the sum of all electrostatic interactions between the potential binding compound and the site at which it is bound to the 20S proteasome will make a contribution to the enthalpy of binding that is not neutral.

[0257] Thus, in certain embodiments, the above method comprises using a suitable computer program in designing and/or selecting a potential binding compound.

Additionally, in certain embodiments, step (c) of the above method comprises using a suitable computer program in conjunction with synthesizing and/or choosing the potential binding compound.

[0258] Thus, in certain aspects, the invention provides a computer-assisted method for identifying compounds that potentially activate the proteasomal degradation of proteins, using a programmed computer comprising a processor, a data storage system, an input device, and an output device, the method comprising: a) inputting into the programmed computer through said input device data comprising the atomic coordinates of a subset of the atoms generated from a complex of the 20S proteasome, thereby generating a criteria data set; b) comparing, using said processor, the atomic coordinates to a computer database of chemical structures stored in said computer data storage system; c) selecting from said database, using computer methods, chemical structures that may bind the atomic coordinates; and d) outputting to said output device the selected chemical structures.

[0259] Furthermore, in certain embodiments, the above method further comprises the steps of using a suitable assay, as described herein, to characterize the potential binding compound's influence on the proteasome' s activity, stability, folding, and/or intracellular localization. In certain embodiments, the above method further comprises: (d) contacting the potential binding compound with mutated 20S proteasome, in the presence of a substrate; and (e) determining the amount of substrate conversion of the mutated from compared to a wild- type (non-mutated) 20S proteasome, to determine the effect of the potential binding compound on proteasomal activity.

[0260] Alternatively, in certain embodiments, the above method further comprises the steps of: (d) contacting the potential binding compound with a cell that expresses mutated 20S proteasome; and (e) determining the effect of the binding compound on proteasomal activity in the cell.

[0261] Additionally, the present invention provides a method of evaluating the binding properties of a potential binding compound comprising the steps of: (a) soaking a potential binding compound with crystalline 20S proteasome to provide the crystalline 20S proteasome complexed to a binding compound; (b) determining the three-dimensional structure of the crystalline 20S proteasome, or the mutant thereof, complexed to the binding compound by molecular replacement using the three-dimensional structure of the crystalline 20S proteasome, or the mutant thereof, as defined by atomic coordinates; and (c) analyzing the three-dimensional structure of the crystalline 20S proteasome, or the mutant thereof, complexed to the binding compound, to evaluate the binding characteristics of the potential binding compound. To evaluate binding properties of binding compounds, assays may be used, such as, calorimetric techniques (e.g. isothermal titration calometry, differential scanning calometry), or Biacore™ can be used for initial screening. Other assays are known in the art. For further optimization, co-crystallization may be useful to determine the structure of the binding compound complexed with the 20S proteasome, or a mutant thereof.

[0262] The term "molecular replacement" refers to a method that involves generating a preliminary model of the three-dimensional structure of a 20S proteasome or 20S

proteasome-inhibitor complex whose structure coordinates are not known, by orienting and positioning a 20S proteasome structure whose atomic structure coordinates are known.

Phases can then be calculated from this model and combined with the observed amplitudes of the unknown crystal structure to give an approximate structure. This, in turn, can be subject to any of several forms of refinement to provide a final, accurate structure. Any program known to the skilled artisan may be employed to determine the structure by molecular replacement. Suitable molecular replacement programs include, but are not limited to, AMORE (the CCP4 suite: Acta Crystallogr. D. (1994) 50:760-763; Navaza, Acta Cryst. (1994) A50, 157- 163) and CNS (Acta Crystallogr. D (1998) 54:905 921).

[0263] In certain embodiments of the above-indicated methods, the atomic coordinates correspond to those of the intersubunit pockets in the alpha ring of an archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise amino acids selected from amino acids Arg₂o through Tyr₁₂₆ of the archaeal 20S proteasome. In further embodiments, the s atomic coordinates comprise amino acids selected from amino acids Phe₉₁ through Tyr₁₂₆ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise amino acids selected from amino acids Arg₂₀ through Val₈₂ of the archaeal 20S proteasome. In other embodiments, the atomic coordinates comprise amino acids selected from amino acids Arg₂o through Lys₃₃ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise amino acids selected from amino acids Leu₈₁ and Val₈₂ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise Arg₂₀ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise Leu₂₁ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise Val₂₄ of the archaeal 20S proteasome. In certain

embodiments, the atomic coordinates comprise Lys₃₃ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise K₆6 of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise Leu₈₁ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise Valg₂ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise Phe^ of the archaeal 20S proteasome. In certain embodiments, the atomic coordinates comprise Tyr₁₂₆ of the archaeal 20S proteasome.

[0264] In certain embodiments of the above-indicated methods, the atomic coordinates correspond to those of the intersubunit pockets in the alpha ring of a mammalian 20S proteasome. In certain embodiments, the subset of the atoms comprises amino acids of the al-a2 binding pocket of a mammalian 20S proteasome. In certain embodiments, the subset of the atoms comprises amino acids of the α2-α3 binding pocket of a mammalian 20S proteasome. In certain embodiments, the subset of the atoms comprises amino acids of the α3-α4 binding pocket of a mammalian 20S proteasome. In certain embodiments, the subset of the atoms comprises amino acids of the α4-α5 binding pocket of a mammalian 20S proteasome. In certain embodiments, the subset of the atoms comprises amino acids of the α5-α6 binding pocket of a mammalian 20S proteasome. In certain embodiments, the subset of the atoms comprises amino acids of the α6-α7 binding pocket of a mammalian 20S proteasome. In preferred embodiments, the subset of the atoms comprises amino acids of the α3-α4 binding pocket of a mammalian 20S proteasome.

[0265] Another aspect of the invention relates to a computer system comprising a memory unit comprising x-ray crystallographic structure coordinates defining amino acids of an intersubunit pocket in the alpha ring of the 20S proteasome; and a processor in electrical communication with the memory unit; wherein the processor generates a molecular model having a three dimensional structure representative of at least a portion of said intersubunit pocket in the alpha ring of the 20S proteasome.

[0266] In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids selected from the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids selected from amino acids Arg₂₀ through Tyr₁₂₆ of the archaeal 20S proteasome. In further embodiments, the x- ray crystallographic structure coordinates comprise amino acids selected from amino acids Phe₉₁ through Tyr₁₂₆ of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids selected from amino acids Arg₂₀ through Valg₂ of the archaeal 20S proteasome. In other embodiments, the x-ray

crystallographic structure coordinates comprise amino acids selected from amino acids Arg₂o through Lys₃₃ of the archaeal 20S proteasome. In certain embodiments, the x-ray

crystallographic structure coordinates comprise amino acids selected from amino acids Leu₈₁ and Valg₂ of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise Arg₂o of the archaeal 20S proteasome. In certain

embodiments, the x-ray crystallographic structure coordinates comprise Leu₂₁ of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise Val₂₄ of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise Lys₃₃ of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise K₆6 of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise Leugi of the archaeal 20S proteasome. In certain embodiments, the x- ray crystallographic structure coordinates comprise Valg₂ of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise Phe₉₁ of the archaeal 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise Tyr₁₂₆ of the archaeal 20S proteasome.

[0267] In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids selected from a mammalian 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids of the l-a2 binding pocket of a mammalian 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids of the α2-α3 binding pocket of a mammalian 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids of the α3-α4 binding pocket of a mammalian 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids of the α4-α5 binding pocket of a mammalian 20S proteasome. In certain embodiments, the x- ray crystallographic structure coordinates comprise amino acids of the α5-α6 binding pocket of a mammalian 20S proteasome. In certain embodiments, the x-ray crystallographic structure coordinates comprise amino acids of the α6-α7 binding pocket of a mammalian 20S proteasome. In preferred embodiments, the x-ray crystallographic structure coordinates comprise amino acids of the α3-α4 binding pocket of a mammalian 20S proteasome.

[0268] In still further embodiments, the crystallographic structure coordinates defining the 20S proteasome are altered in that one or more of the locations of known 20S proteasome amino acid mutations are computationally represented by the crystallographic structure coordinates.

[0269] These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

EXAMPLES

[0270] Screening methods were adapted from those reported by Yu et al., Interactions of PAN's C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome-ATPase interactions, EMBO J. 2010 Feb 3;29(3):692-702; Rabl J., et al, Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases, Mol Cell. 2008 May 9;30(3):360-8; and Smith, et al., Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry, Mol Cell. 2007 Sep 7;27(5):731-44.

[0271] Bovine 20S proteasome from the liver was prepared as described by Kisselev,

A. F., Kaganovich, D., and Goldberg, A. L. 2002 /. Biol. Chem. 277, 22260-22270. Yeast 20S proteasome was affinity-purified as described by Leggett et al., Mol. Cell. 2002

Sep;10(3):495-507. Protein expression and cell lysis were performed as described by Kisselev et al. and Leggett et al., using a PA26 pET-15b construct that was transformed into BL21(DE3) competent cells. Proteins were purified with Ni-NTA affinity chromatography.

EXAMPLE 1

High throughput screening

[0272] A high throughput screen was developed at the Institute of Chemical and Cell

Biology (ICCB) at Harvard Medical School to identify agents that affect gate opening.

Agents were used that enhance gate-opening and stimulate the entry and hydrolysis of a fluorogenic substrate. One such agent that was used was the seven-residue HbYX-containing peptide, corresponding to the C-terminal residues of ATPases in mammalian 26S

proteasomes. The terminal HbYX sequence interacts with specific residues in these pockets to cause gate-opening. This screen utilized bovine 20S proteasomes (B20S) isolated from the liver and fluorometric substrates that could only enter the proteasome rapidly when the gate was open. [0273] Gate opening was measured by the addition of B20S (0.001 μg to 0.1 μg) to a reaction buffer (30 μί) of 50 mM Tris at pH 7.5, 5% glycerol, and 1 mM DTT that contained nLPnLD-AMC (SEQ ID NO: 10; 25 μΜ). The B20S showed a low basal rate of substrate hydrolysis (i.e., hydrolytic cleavage of the AMC substituent). Addition of the peptide containing the HbYX motif (i.e., KANLQYYA-RPT5 peptide; SEQ ID NO: 14) to the B20S resulted in increased levels of substrate hydrolysis. These 30 μL-scale reactions were conducted in two identical 384-well plates. A third series of control reactions, to monitor the fluorescence of the added compounds, was also conducted in a 384-well plate that contained the reaction buffer without a substrate or B20S.

[0274] Screening compounds were added via 300 nL pinning to the three series of reactions in the three 384-well plates and reaction mixtures were incubated at 25 °C for 30 minutes. After 30 minutes, the fluorescence (380/460 nM) of each well was measured by a plate reader and Rpt5 peptide (KANLQYYA (SEQ ID NO: 14); 25 to 100 μΜ) was added to stimulate gate opening. Following incubation for 30 minutes, the fluorescence of the plates was measured as before to identify compounds that inhibited activation of the Rpt5 peptide or that acted synergistically with the Rpt5 peptide activation. The Rpt5 peptide could be replaced with any number of peptides that contain the HbYX motif (e.g., GTPEGLYL (SEQ ID NO: 15), AHLDVLYR (SEQ ID NO: 16), QYYA (SEQ ID NO: 17), YYA, CBZ-YA, CBZ- FA, YFA, and FFA). Compounds that activated nlPnLD-AMC hydrolysis at 3X the rate of the basal rate were selected as hit compounds. Binding affinities and related characteristics of the hit compounds and analogs thereof (e.g., ECso's, Kd's, Vmax, and Hill coefficients) were calculated by fitting the data to a standard ligand binding curve using SigmaPlot^® from Systat Software Inc.

EXAMPLE 2

Secondary and tertiary screens

[0275] The hit compounds were then selected for further secondary and tertiary screens to investigate the mechanism of action of the hit compounds. The secondary screens were conducted according to the above high throughput screening protocol on a series of plates containing only the hit compounds. The tertiary screens were likewise conducted according to the above high throughput screening protocol, but with different substrates of the active sites, or with activators other than the Rpt5 peptide. For example, Tau, alpha-synuclein, and Casein degradations were performed in reaction buffers as described above and with similar concentrations of B20S. Tau, alpha- synuclein, and Casein were used at 10- to 20-fold higher concentrations than the B20S and reaction mixtures were incubated as described above. Tau, alpha-synuclein, and Casein were detected by running the reaction on an SDS gel and staining with coomasie. The following chart depicts combinations of agents used in the above-described screening assays.

EXAMPLE 3

[0276] Peptide sequence alignments of the alpha rings of the bovine 20S to the archaeal 20S proteasomes.

[0277] Peptide sequence alignments of the seven alpha rings of bovine 20S (PDB: llRU) to the single alpha ring of the archaeal 20S from 3IPM were performed to determine which residues of the bovine 20S are likely to interact with the C-termini of the proteasome ATPases. The archaeal 20S from 3IPM contains the bound C-termini of PAN. PAN's C- terminus was docked into the bovine 20S intersubunit pockets. All residues from the bovine 20S that were within 4 angstroms of PAN's C-termini are noted below. The representative residues indicated below likely contribute to HbYX binding. Thus, the residues indicated below can be used when computationally docking small molecules to any of the seven bovine alpha rings. Further, the residues indicated below can be used to computationally dock small molecules to any of the seven mammalian alpha rings because there is is substantial homology among some of the mammalian alpha rings (e.g., between bovine and human alpha rings).

[0278] The following residues from the bovine 20S alpha rings that were within 4 angstroms of PAN's C-termini are noted below.

Alpha 1: G20, R21, L22, V25, E26, A31, Q34, G35, L37, H68, T81, G82, M83, T84, A157, Y158 Alpha 2: G16, K17, L18, 121, E22, L25, A27, G31, A32, K50, T56, K63, Y74, G77, S151, F155

Alpha 3: G16, R17, A27, H30, A31, G32, R50, N51, E63, K64, G78, N155

Alpha 4: G14, H15, Q23, A25, K28, G29, S30, K47, V59, G75, D148, S150, H154

Alpha 5: E18, G19, R20, E25, A30, L33, G34, S35, K52, R53, T55, M59, 164, K66, S79, G80, L81, I82, S159

Alpha 6: G17, R18, Q31, G32, S33, R51, A52, Q53, Q60, K62, S150, N152, F154

Alpha 7: G18, R19, M27, A29, S33, S34, L52, L54, Y58, N63, R65, A78, G79, L80, S154,

Y158

Claims

What is claimed is:

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof;

wherein

R¹ is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; -CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; -OC(=0)OR^A; -OC(=0)R^A; - OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^A is

2 3

each of R and R is, independently, hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

X^l O, S, or NR^Y1;

X² is O, S, or NR^Y2;

X³ is O, S, or NR^Y3; and

Y 1 Y2 Y3

provided that the compound is not

2. The compound of claim 1, wherein X 1 is S; X2 is O; and X 3 is O.

3. The compound of claim 1, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C^o aliphatic.

4. The compound of claim 1, wherein R¹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci-io heteroaliphatic.

5. The compound of claim 1, wherein R¹ is a substituted or unsubstituted aryl.

6. The compound of claim 1, wherein R¹ is a substituted or unsubstituted naphthyl.

7. The compound of claim 1, wherein R¹ is naphthyl.

8. The compound of claim 1, wherein R¹ is

The compound of claim 1, wherein R is a substituted or unsubstituted

10. The compound of claim 1, wherein R is a substituted or unsubstituted phenyl.

11. The compound of claim 1, wherein R is a substituted phenyl.

12. The compound of claim 1, wherein R is CI CI

13. The compound of claim 1, wherein R is hydrogen.

14. A compound of formula (II):

(Π)

or a pharmaceutically acceptable salt thereof;

wherein

each of R⁴, R⁵, and R⁶ is, independently, hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR^A; -C(=0)R^A; -C0₂R^A; - CN; -SCN; -SR^A; -SOR^A; -S0₂R^A; -N0₂; -N₃; -N(R^A)₂; -NHC(=0)R^A; -NR^AC(=0)N(R^A)₂; - OC(=0)OR^A; -OC(=0)R^A; -OC(=0)N(R^A)₂; -NR^AC(=0)OR^A; or -C(R^A)₃; wherein each occurrence of R^Ais independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; each of R 7 and R 8 is, independently, hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

X⁴ is O, S, or NR^Y4; and

R Y4 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl;

provided that the compound is not

15. The compound of claim 14, wherein X⁴ is S.

16. The compound of claim 14, wherein R⁴ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

17. The compound of claim 14, wherein R⁴ is -CN.

The compound of claim 14, wherein R⁵ is a substituted or unsubstituted

19. The compound of claim 14, wherein R⁵ is a substituted or unsubstituted phenyl.

The compound of claim 14, wherein R⁵ is a substituted phe

The compound of claim 14, wherein R i s

22. The compound of claim 14, wherein R⁶ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

23. The compound of claim 14, wherein R⁶ is C_1-6 alkyl.

24. The compound of claim 14, wherein R⁶ is methyl.

25. The compound of claim 14, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

26. The compound of claim 14, wherein R is hydrogen.

27. The compound of claim 14, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

28. The compound of claim 14, wherein R is C_1-6 alkyl.

29. The compound of claim 14, wherein R is methyl.

30. A compound of formula (III):

(III)

or a pharmaceutically acceptable salt thereof;

wherein

X⁵ is O, S, or NR^Y5;

X⁶ is N or CR^Y6;

R Y5 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and

heteroaryloxy; or hetero arylthio moiety;

provided that the compound is not

31. The compound of claim 30, wherein X⁵ is O; and X⁶ is N.

32. The compound of claim 30, wherein R⁹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

33. The compound of claim 30, wherein R⁹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

34. The compound of claim 30, wherein R⁹ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkenyl.

35. The compound of claim 30, wherein R⁹ is a substituted or unsubstituted aryl.

36. The compound of claim 30, wherein R⁹ is a substituted or unsubstituted phenyl.

37. The compound of claim 30, wherein R¹⁰ is hydrogen.

38. The compound of claim 30, wherein R¹⁰ is -CN.

39. The compound of claim 30, wherein R¹¹ is a substituted or unsubstituted aryl.

40. The compound of claim 30, wherein R¹¹ is a substituted or unsubstituted phenyl.

41. The compound of claim 30, wherein R¹¹ is a substituted phenyl.

42. The compound of claim 30, wherein R¹¹ is

wherein

R is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; or substituted or unsubstituted, branched or unbranched aryl.

43. The compound of claim 30, wherein R¹¹ is

44. The compound of claim 30, wherein R 12 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

45. The compound of claim 30, wherein R 12 is hydrogen.

46. A compound of formula (IV):

(IV)

or a pharmaceutically acceptable salt thereof;

wherein

X⁷ is O, S, or NR^Y7,

X⁸ is N or CR^Y8; and

R Y7 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

R Y8 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(=0)R^A; -C0₂R^A; -C(=0)N(R^A)₂; - SOR ; -S0₂R ; -C(R )₃; aryloxy; alkylthio; arylthio; alkylamino, dialkylamino,

heteroaryloxy; or hetero arylthio moiety;

provided that the compound is not

47. The compound of claim 46, wherein X 7 is S; and X 8 is N.

48. The compound of claim 46, wherein R 13 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

49. The compound of claim 46, wherein R 13 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

50. The compound of claim 46, wherein R 13 is hydrogen.

51. The compound of claim 46, wherein R¹⁴ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

52. The compound of claim 46, wherein R¹⁴ is a substituted or unsubstituted aryl.

53. The compound of claim 46, wherein R¹⁴ is a substituted or unsubstituted heteroaryl.

54. The compound of claim 46, wherein R¹⁴ is a substituted or unsubstituted pyranone or chromeneone.

55. The compound of claim 46, wherein R is

56. The compound of claim 46, wherein R¹⁵ is a substituted or unsubstituted aryl.

57. The compound of claim 46, wherein R is a substituted or unsubstituted phenyl.

The compound of claim 46, wherein R is a substituted phi

59. The compound of claim 46, wherein R¹⁵ is

wherein

60. The compound of claim 46, wherein R is

wherein R is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroalkyl; - OR^D; -C(=0)R^D; -C0₂R^D; -CN; -SR^D; -SOR^D; -S0₂R^D; -N0₂; -N₃; -N(R^D)₂; -NHC(=0)R^D; - NR^DC(=0)N(R^D)₂; -OC(=0)OR^D; -OC(=0)R^D; -OC(=0)N(R^D)₂; -NR^DC(=0)OR^D; or - C(R^D)₃; wherein each occurrence of R^D is independently a hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl; aryl; heteroaryl; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio; and r is 0 or an integer between 1 and 5, inclusive.

The compound of claim 46, wherein R

62. The compound of claim 46, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

63. The compound of claim 46, wherein R is hydrogen.

64. A compound of formula (V):

or a pharmaceutically acceptable salt thereof;

wherein

X⁹ is O, S, or NR^Y9;

n is 0 or an integer between 1 and 4, inclusive;

provided that the compound is not

65. The compound of claim 64, wherein X⁹ is NR

66. The compound of claim 65, wherein R is hydrogen.

The compound of claim 64, wherein R is a substituted or unsubstituted

68. The compound of claim 64, wherein R is a substituted or unsubstituted phenyl.

The compound of claim 64, wherein R is a substituted phi

The compound of claim 64, wherein

71. The compound of claim 64, wherein R is a substituted or unsubstituted aryl

72. The compound of claim 64, wherein R is a substituted or unsubstituted phenyl.

The compound of claim 64, wherein R is a substituted or unsubstituted heteroaryl.

The compound of claim 64, wherein R

A compound of formula (VI):

(VI)

or a pharmaceutically acceptable salt thereof;

wherein

R 21 and R 2"2 may optionally join to form a fused cyclic structure;

R 23 is hydrogen; a nitrogen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or

X¹⁰ is O, S, or NR^Y10; and

R Y10 is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl;

provided that the compound is not

76. The compound of claim 75, wherein X is S.

77. The compound of claim 75, wherein R 21 and R 22 join to form a fused cyclic structure.

78. The compound of claim 75, wherein R 21 and R 22 join to form a cyclopentene ring.

79. The compound of claim 75, wherein R 20 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

80. The compound of claim 75, wherein R 20 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o heteroaliphatic.

81. The compound of claim 75, wherein R 20 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

20 B B

82. The compound of claim 75, wherein R is Q-Cealkylene-COiR , wherein R is hydrogen; a protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl.

83. The compound of claim 82, wherein R is H.

84. The compound of claim 83, wherein R is

.

85. The compound of claim 75, wherein R 21 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

86. The compound of claim 75, wherein R 22 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

87. The compound of claim 75, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

88. The compound of claim 75, wherein R 23 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

89. The compound of claim 75, wherein R 23 is hydrogen.

90. A compound of formula (VI

(VII)

or a pharmaceutically acceptable salt thereof;

wherein

m is 0 or an integer between 1 and 5, inclusive; k is 0 or an integer between 1 and 3, inclusive; and

q is 0 or an integer between 1 and 4, inclusive;

provided that the compound is not selected from the group consisting of

91. The compound of claim 90, wherein m is 0.

92. The compound of claim 90, wherein m is 1.

93. The compound of claim 90, wherein m is 2.

94. The compound of claim 90, wherein k is 1.

95. The compound of claim 90, wherein q is 0. he compound of claim 90, wherein two R substituents join to form

97. The compound of claim 90, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci_2o aliphatic.

98. The compound of claim 90, wherein R 24 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic Ci^o heteroaliphatic.

99. The compound of claim 90, wherein R 24 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

100. The compound of claim 90, wherein R 24 is selected from the group consisting of hydrogen, -CH₃, -CH₂CH₃, -CH₂CH₂CH₃, -CH(CH₃)₂, -OCH₃, -OCH₂CH₃, -OCH₂CH₂CH₃, -OCH(CH₃)₂, -N0₂, and -CN.

101. The compound of claim 90, wherein R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

102. The compound of claim 90, wherein R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C₁_₂o heteroaliphatic.

103. The compound of claim 90, wherein R 25 is a substituted or unsubstituted heteroaryl.

104. The compound of claim 90, wherein R²⁶ is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

105. The compound of claim 90, wherein R 27 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

106. The compound of claim 90, wherein R is hydrogen.

107. The compound of claim 90 wherein the compound is of formula (VIII):

(VIII)

or a pharmaceutically acceptable salt thereof;

wherein

m is 0 or an integer between 1 and 5, inclusive; and

q is 0 or an integer between 1 and 4, inclusive.

108. The compound of claim 107, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o aliphatic.

109. The compound of claim 107, wherein R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o heteroaliphatic.

110. The compound of claim 107, wherein R is a substituted or unsubstituted heteroaryl.

111. The compound of claim 107, wherein R is selected from the group consisting of hydrogen, -CH₃, -CH₂CH₃, -CH₂CH₂CH₃, -CH(CH₃)₂, -CH₂CH₂CH₂CH₃, -CH₂CH(CH₃)₂, -C(CH₃)₃, -OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₃)₂, -OCH₂CH₂CH₂CH₃, -

112. The compound of claim 107, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

113. The compound of claim 90 wherein the compound is of formula (IX):

(IX)

or a pharmaceutically acceptable salt thereof;

wherein

m is 0 or an integer between 1 and 5, inclusive; and

q is 0 or an integer between 1 and 4, inclusive.

114. The compound of claim 113, wherein R 25 is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o heteroaliphatic.

115. The compound of claim 113, wherein R 25 is selected from the group consisting of hydrogen, -CH₃, -CH₂CH₃, -CH₂CH₂CH₃, -CH(CH₃)₂, -CH₂CH₂CH₂CH₃, -CH₂CH(CH₃)₂, -C(CH₃)₃, -OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₃)₂, -OCH₂CH₂CH₂CH₃,

116. The compound of claim 113, wherein R is substituted or unsubstituted, branched or unbranched, cyclic or acyclic C_1-2o alkyl.

117. The compound of claim 113, wherein R 25 is hydrogen.

118. A compound of formula (X :

(X)

or a pharmaceutically acceptable salt thereof;

wherein

X^u is O, S, or NR^Y11 ;

z is 0 or an integer between 1 and 3, inclusive;

provided that the compound is not

119. A pharmaceutical composition for modulating proteasomal degradation comprising any of the compound of any one of claims 1-118 and, optionally, a pharmaceutically acceptable excipient.

120. The pharmaceutical composition of claim 119 for stimulating proteasomal

degradation.

121. A pharmaceutical composition for modulating proteasomal degradation comprising a therapeutically effective amount of a compound selected from the group consisting of:

108

109

and, optionally, a pharmaceutically acceptable excipient.

122. The pharmaceutical composition of claim 121 for use in stimulating proteasomal degradation.

123. A method for stimulating proteasomal degradation of proteins comprising:

contacting a proteasome with a compound of any of claims 1-118 or a composition of claim 121 under conditions suitable to increase proteasomal degradation of proteins.

124. A method for treating a subject with a disorder associated with unfolded, misfolded, or aggregated proteins comprising:

administering to the mammal an effective amount of a compound of any of claims 1- 118 or a composition of claim 121 to increase proteasomal degradation of unfolded, misfolded, or aggregated proteins.

125. The method of claim 124, wherein the subject is human.

126. The method of claim 124, wherein the proteasome is a 20S proteasome.

127. The method of claim 124, wherein the compound of any of claims 1-118 or a composition of claim 121 is administered in combination with an additional therapy for a disorder associated with unfolded, misfolded, or aggregated proteins in a subject.

128. The method of claim 127, wherein the disorder is selected from the group consisting of a neurodegenerative disease; a bacterial infection; a fungal infection; Alzheimer's disease; Parkinson's disease; cystic fibrosis; amyotrophic lateral sclerosis (i.e., Lou Gehrig's disease); Creutzfeld-Jakob disease; kuru; GSS disease; Huntington's disease; polyglutamine diseases; prion-related disorders; fatal familialinsomnia; scrapie; transmissible spongiform

encephalopathy (TSE); bovine spongiform encephalopathy; Alexander's disease; primary systemic amyloidosis; secondary systemic amyloidosis; senile systemic amyloidosis;

amyloidosis in senescence; ocular disease: cataract; retinitis pigmentosa; macular

degeneration; islet amyloid; medullar carcinoma of the thyroid; hereditary renal amyloidosis; hemodialysis-related amyloidosis; Desmin-related cardiomyopathy; Charcot-Marie tooth disease; diabetes insipidis; diabetes insipidis; sickle cell anemia; thalassemia; protein- destabilization, such as Gaucher disease and Sanfilippo syndrome type B

(mucopolysaccharidosis III B); and congophilic angiopathy.

129. The method of claim 128, wherein the disorder is a neurodegenerative disease.

130. The method of claim 127, wherein the unfolded, misfolded, or aggregated protein is selected from the group consisting of beta-casein; tau; tau 23; amyloid beta; alpha-synuclein; amyloid protein; huntingtin; atrophin-1, ataxins; prion protein; BSE; superoxide dismutase; glial fibrillary acidic protein; immunoglobulin light chain or fragments thereof; serum amyloid-A or fragments thereof; transthyretin or fragments thereof; apolipoprotein A-II; crystallins or crystallin filaments; rhodopsin; calcitonin; fibrinogen; beta 2-microglobulin; desmin; peripheral myelin protein 22 (PMP-22); aquaporin; vasopressin receptor; connexin 32; and cystic fibrosis transmembrane conductance regulator (CFTR).

131. A method for killing or inhibiting the growth of a microorganism, the method comprising: contacting a microorganism with an effective amount of a compound of any of claims 1-118 or a composition of claim 121 to kill or inhibit the growth of the

microorganism.

132. The method of claim 131, wherein the microorganism is a Gram-positive

Actinomyces.

Ill

133. The method of claim 131, wherein the microorganism is a Gram-positive Mycobacteria.

134. The method of claim 131, wherein the microorganism is a Gram-negative bacterium.

135. A method for identifying compounds that activate the proteasomal degradation of proteins, the method comprising:

a) contacting a fluorogenic peptide with a proteasome and measuring the baseline level of fluorescence that results from proteasomal degradation of the fluorogenic peptide by the proteasome;

b) contacting the fluorogenic peptide with the proteasome in the presence of one or more test compounds; and

c) identifying compounds that cause an increase in the level of fluorescence relative to the baseline level of fluorescence obtained in the absence of the activator compounds.

136. The method of claim 135, wherein each of the contacting steps is incubated with ATP or a nonhydrolyzable ATP analog.

137. The method of claim 136, wherein the nonhydrolyzable ATP analog is ADP, ATP7S, or AMPPNP.

138. The method of claim 135, wherein each of the contacting steps is heated.

139. The method of claim 135, wherein each of the contacting steps is heated to 37 °C.

140. The method of claim 135, wherein the fluorogenic peptide is GFP-ssrA.

141. The method of claim 135, wherein the fluorogenic peptide is nLPnLD-amc (SEQ ID NO: 10).

142. The method of claim 135, wherein the fluorogenic peptide is LRR-amc.

143. The method of claim 135, wherein the fluorogenic peptide is YFP-ssrA.

144. The method of claim 135, wherein the fhiorogenic peptide is Mca-AKVYPYPME- Dpa(Dnp)-amide (LFP; SEQ ID NO:9).

145. The method of claim 135, wherein the test compound is a small molecule.

146. The method of claim 135, wherein the proteasome is a 20S proteasome.

147. The method of claim 135, wherein the proteasome was expressed in Escherichia coli.

148. The method of claim 135, further comprising one or more tertiary assays to determine if the compound that increases fluorescence can reproducibly activate the proteasomal degradation of proteins.

149. The method of claim 141, wherein one or more of the tertiary assays measures the degradation of proteins by a proteasome.

150. The method of claim 149, wherein the protein is Tau.

151. The method of claim 149, wherein Tau is Tau23.

152. The method of claim 149, wherein the protein is alpha- synuclein.

153. The method of claim 149, wherein the proteasome is a 20S proteasome.

154. The method of claim 149, wherein the proteasome is an archaeal 20S proteasome.

155. The method of claim 149, wherein the proteasome is a eukaryotic 20S proteasome.

156. The method of claim 149, wherein the proteasome is a mammalian 20S proteasome.

157. The method of claim 149, wherein the proteasome is a rabbit 20S proteasome.

158. The method of claim 149, wherein the proteasome is a wild-type yeast 20S proteasome.

159. The method of claim 149, wherein the proteasome is a 26S proteasome.

160. The method of claim 149, wherein the proteasome is an open gate mutant.

161. A computer-assisted method for identifying compounds that potentially activate the proteasomal degradation of proteins, using a programmed computer comprising a processor, a data storage system, an input device, and an output device, the method comprising:

a) inputting into the programmed computer through said input device data comprising the atomic coordinates of a subset of the atoms generated from a complex of the proteasome, thereby generating a criteria data set;

b) comparing, using said processor, the atomic coordinates to a computer database of chemical structures stored in said computer data storage system;

c) selecting from said database, using computer methods, chemical structures that may bind the atomic coordinates; and

d) outputting to said output device the selected chemical structures.

162. The computer-assisted method of claim 161, wherein the subset of the atoms comprises residues in the intersubunit pockets in the alpha ring of the 20S proteasome that contribute to binding to the C-terminal region of a regulatory complex.

163. The computer-assisted method of claim 161, wherein the regulatory complex is a proteasomal ATPase.

164. The computer-assisted method of claim 161, wherein the regulatory complex is selected from the group consisting of archaeal PA28, PA26, PA200/BLM10, and PI31.

165. The computer-assisted method of claim 161, wherein the the subset of the atoms comprises amino acids selected from amino acids Arg₂o through Tyr₁₂₆ of the alpha subunit of the archaeal 20S proteasome.

166. The computer-assisted method of claim 161, wherein the subset of the atoms comprises amino acids selected from amino acids Arg₂o through Valg₂ of the alpha subunit of the archaeal 20S proteasome.

167. The computer-assisted method of claim 161, wherein the subset of the atoms comprises K₆6 of the alpha subunit of the archaeal 20S proteasome.

168. The computer-assisted method of claim 161, wherein the proteasome is an archaeal 20S proteasome.

169. The computer-assisted method of claim 161, wherein the proteasome is a eukaryotic 20S proteasome.

170. The computer-assisted method of claim 161, wherein one or more amino acid mutations of the 20S proteasome are computationally represented.

171. A computer system comprising a memory unit comprising x-ray crystallographic structure coordinates defining an intersubunit pocket in the alpha ring of the 20S proteasome; and a processor in electrical communication with the memory unit; wherein the processor generates a molecular model having a three dimensional structure representative of at least a portion of said intersubunit pocket.

172. The computer system of claim 171, comprising amino acids selected from amino acid residues Arg₂o through Tyr₁₂₆ of the intersubunit pocket.

173. The computer system of claim 171, comprising the coordinates of the amino acid K₆6 of the alpha subunit of the intersubunit pocket.