Recent advances in proteasome inhibitor discovery

1. Introduction

2. Currently approved

proteasome inhibitors

3. Compound screening

4. Rational design

5. Expert opinion

Review

Recent advances in proteasomeinhibitor discoveryYuri Pevzner, Rainer Metcalf, Melanie Kantor, Desiree Sagaro &Kenyon Daniel††Moffitt Cancer Center, Screening and Modeling Unit, Chemical Biology Core, Tampa, FL, USA

Introduction: Proteasome inhibition is a quickly advancing subject of research

and has a significant potential to become a potent therapeutic modality for

many diseases and disorders. The aim of this review is to present the reader

with the variety of approaches to the proteasome inhibitor discovery as well

as highlight the diversity of scaffolds being considered for this task.

Areas covered: This review focuses on current developments in proteasome

inhibitor discovery, including an account of research efforts covered in the liter-

ature from the years 2009 -- 2012, although some of the earlier work is also men-

tioned. Specifically, presented are the type of experiments performed, the

compounds and compound families investigated along with their activities and

assessment for potential therapeutic value. In particular, authors highlight differ-

ent paths to discovery of the proteasome inhibitors such as screening of large

libraries, repurposing of existing therapeutics, development of compounds

with known proteasome inhibitory activities as well as utilizing novel scaffolds.

Expert opinion: Discovery of therapeutically successful proteasome inhibitors

depends on a number of factors and demands a multipronged approach.

Screening protocols, choice of assays, desired mode of action, selection of a

binding pocket, targeting and delivery strategy, all require careful consideration

when attempting to target the proteasome.

Keywords: cancer, drug discovery, inhibition, modeling, proteolysis, rational design, screening

Expert Opin. Drug Discov. (2013) 8(5):537-568

1. Introduction

The maintenance of proteins is determined by the rate of expression antagonized byrate of degradation. This turnover process is essential for proper regulation of theamount and function of proteins. The proteasome is vital in this process and is anearly ubiquitous cellular component [1,2].

The pathway for selective proteolysis is a complex process involving discretionaryubiquitination of target proteins by ubiquitin ligases. Ubiqitin is a 76-residue, highlyconserved protein that is first activated by the E1 enzyme, complexed with E2, andfinally conjugated with its target protein by E3 [3]. This results in a polyubiquitinchain marking the desired protein for destruction [4]. These ubiquitin chains canthen bind to the proteasome via specific recognition sites on the regulatory particle(RP), or PA700 cap, of the proteasome [5].

The 26S mammalian proteasome is a massive 2,400 kDa molecule composed of a20S core particle (CP) and one or two 19S 18-subunit RP (Figure 1A). The CP is a700 kDa barrel-shaped particle formed by four axially stacked heptameric rings [6].The b subunits of the inner rings include six proteases whose active sites face an inte-rior lumen where proteolysis occurs (Figure 1B) [7]. These active sites are further anat-omized into a catalytic region (S1) and recognition region (S2) [8]. The S1 pocketsare all very similar and incorporate a catalytic THR1 residue [7]. The S2 pocketsof the active sites display preferential binding to certain residue sequences(Figure 1C) [9]. These sites are known as the b1/PRE3 peptidylglutamyl peptide

10.1517/17460441.2013.780020 © 2013 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X 537All rights reserved: reproduction in whole or in part not permitted

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caspase-like recognition (PGPH), the b2/PUP1 trypsin-like recognition (T-L) and the b5/PRE2 chymotrypsin-like recognition (CT-L) [10]. Other structural variants of theproteasome have been discovered and exhibit slightly alteredfunctions of the beta subunits and regulatory caps. The immu-noproteasome consists of smaller 11S RPs and a modified 20SCP containing ib1, ib2 and ib5 subunits; structural isoformsof the constitutive b1, b2 and b5 subunits, respectively [11].The thymoproteasome is another proteasomal polymorphismmore recently elucidated which incorporates the ib1 andib2 immunoproteasome subunits but integrates a constitu-tively differing tb5 subunit [12].Inhibition of the proteasome has been of recent interest, due

to its effect on cancer cells [13]. Disruption of the ubiquitin-proteasome pathway (UPP) can lead to significant build-up of cytotoxic proteins and activation of apoptotic pathways,particularly in rapidly proliferating cells [14]. Further, termi-nally differentiated cells appear to be protected by proteasomeinhibition, leading to the idea that oncogenic cells are espe-cially susceptible to proteasome inhibition while quiescent cellsmay be preserved to some extent [15]. Proteasome inhibitionmay also be useful in understanding and possibly treating neu-rodegenerative disorders [16], cardiac disease [17,18] and organtransplant rejection [19].Proteasome inhibitors can be classified into many different

groups based on structure and reaction mechanism. Peptidemimics are an emerging class of compounds designed totake advantage of the specificity of proteins as well as theabsorption and stability of small molecules [20]. These typeof compounds and other small molecules contain common

functional groups, such as aldehyde, vinyl sulfone, boronate,epoxyketone, and b-lactone. The typical pharmacophore forthese molecules is the targeting of the CT-L recognition siteand reacting with the catalytic threonine in the activesite [21]. In this review, we survey recent developments inproteasome inhibitor families, screening, and rational design.

2. Currently approved proteasome inhibitors

The peptide boronate, Bortezomib, was the first proteasomeinhibitor approved by the Food and Drug Administration(FDA) for treatment of patients with multiple myeloma [22]

and mantle cell lymphoma [23]. Bortezomib carries a boronicacid “warhead” that forms a covalent bond with the catalyticTHR1 residue of the b5 subunit of the proteasome [24].Although it is considered a reversible inhibitor, Bortezomibhas a slow dissociation rate rendering it virtually irreversible [10].This may contribute to reported side effects which includefatigue, nausea, sensory neuropathy and adverse cardiovasculareffects [25,26]. In clinical use, Bortezomib has been reported tohave up to an 88% response rate [24]. However, despite suc-cesses, about 60% of the patients treated with Bortezomibdevelop resistance to it within an average of 1 year from thebeginning of the treatment [24]. In 2012, the FDA gave anaccelerated approval for an epoxyketone Carfilzomib for treat-ment of patients with multiple myeloma [27]. Carfilzomib, a“second-generation” proteasome inhibitor, was developed toaddress Bortezomib’s toxicity and drug resistance while achiev-ing comparable potency [28]. Carfilzomib is a true irreversibleinhibitor of the proteasome. However, it has an increased selec-tivity for the CT-L activity as compared to Bortezomib and canbe administered on a more aggressive schedule [29]. In clinic,Carfilzomib demonstrated a 22.9% overall response rate andshowed reduced incidence of peripheral neuropathies com-pared to Bortezomib [10,27]. Still, Carfilzomib shows side effectsincluding pneumonia, acute renal failure, pyrexia, congestiveheart failure, cytopenia, fatigue, nausea and dyspnea [27,28].

The fast-tracked approval and successes of Carfilzomibgives validation to the search for follow-on drugs. Intravenousdelivery, the need for aggressive dosing, and side effects, allshow that there is still plenty of room for improvementwhen it comes to proteasome inhibition. An increase in siteselectivity, more effective tissue targeting, improved pharma-codynamic, and pharmacokinetic properties can create betterproteasome inhibitors that are both convenient for patientsand more effective pharmaceutical agents. An expansion ofinhibitor armamentarium would allow for selective targetingof different proteasomal activities through a wide range ofmechanisms, each more fitted to a specific goal.

3. Compound screening

The practice of screening libraries of compounds for potentiallead molecules is common for a number of targets including,and especially, the proteasome. Following the identification

Article highlights.

. The proteasome is an important cellular componentresponsible for the regulation of protein turnover,therefore its targeting provides an avenue to controlcrucial cellular processes.

. Proteasome inhibition has been considered a promisingstrategy to combating a number of diseasesincluding cancer.

. Compound library screening allows to probe a diversevariety of scaffolds that may yield promising candidates.

. Development of the classes of compounds that havepreviously been shown to inhibit proteasome allows toimprove on existing therapeutics and address concernsthat accompany them.

. Metal complexing is a viable strategy for selectivelytargeting the proteasome, a component essential forboth healthy and diseased cells.

. Drug repurposing or adaptation of existing therapeuticswith no previous proteasome inhibitory indications canprovide a viable strategy for getting promisingcompounds to the clinic.

. Development of novel scaffolds can enrich the pool ofinhibitor candidates and provide new avenues for finetuning the regulation of proteasomal activity.

This box summarizes key points contained in the article.

Y. Pevzner et al.

538 Expert Opin. Drug Discov. (2013) 8(5)

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of potential leads from a screening campaign, compoundscan be further characterized and developed into promisingcandidates. Screening can be performed using large librariescontaining hundreds of thousands of compounds or smaller“diversity” subsets that are representative of the chemicaldiversity of a larger library. A variety of biochemical and cel-lular assays can be used in such screenings as long as they canbe adapted to a high-throughput approach. Alternatively, aninitial computational screening can be performed to discarda substantial portion of compounds as nonbinders prior tobiochemical screening. The advantage of such a techniqueis that a larger library, numbering in millions of com-pounds, can be screened computationally with significantlyless time and cost than a biochemical screen [30]. However,the disadvantage of this is the approximate nature of scoringfunctions that can result in false positives as well as falsenegatives [31,32].

Daniel and colleagues analyzed 1,990 drug-like compoundsof the National Cancer Institute (NCI) Diversity Set Library [33]

in a combination of biochemical and cellular assays and foundthat compound NCI-109268 (Table 1, 1a) inhibited the CT-Lactivity of the proteasome in Jurcat T cells and 20S rabbitproteasome isolates with an IC50 » 6 µM [34]. Similarly, theNCI Diversity Set [33] was screened by Lavelin and colleaguesusing a novel live cell image screening method based on thetranslocation of a fluorescent reporter, the nuclear localizationsignal-deficient p53 derivative, from the cytoplasm to thenucleus [35]. The most active of the several resulting hits inhib-ited the 26S proteasome at EC50 = 100 µM (Table 1, 1b,bromocopper; (dipyridin-2-ylmethylideneamino)-[methylsulfanyl

(sulfoniumylidene)methyl]milacicazanide). Lawrence et al. alsoscreened the NCI Diversity Set [33] specifically targeting theCT-L activity of the 20S proteasome. Following the initialscreen, a smaller focused library of naphthoquinone analogs ofthe lead compound (Table 1, 1c, 4-[(3-chloro-1,4-dioxonaph-thalen-2-yl)amino]-N-pyridin-2-ylbenzenesulfonamide) wassynthesized and evaluated in subsequent assays in order todetermine the structure--activity relationship (SAR) of itsCT-L inhibition [36]. Ge and colleagues performed a libraryscreening of 20,000 compounds in a biochemical assay thatidentified a hydronaphthoquinone containing compound tosynthesize and evaluate a focused library. This approach haslead to an identification of a compound (Table 1, 1d, N-(3-(1H-Tetrazol-5-ylthio)-4-hydroxynaphthalen-1-yl)-4-(tri-fluoromethyl)biphenyl-4-sulfonamide) with an inhibition ofthe CT-L activity in the range of IC50 » 0.44 to 1.01 µM inintact breast cancer cells [37]. Another set of naphthoquinonederivatives based on the hit identified by Lawrence et al. wasdesigned by Xu and colleagues [38]. Biochemical assay andtumor cell line evaluation of these compounds identified acompound (Table 1, 1e) with activity improved or comparableto that of the original compound found by Lawrence et al. [38].

A secondary metabolite secreted by the fungus Aspergillusterreus, terrein (Table 1, 1f) displayed proteasome inhibitionwhen Demasi et al. performed a screening of fungi extractsagainst a purified 20S horse proteasome [39]. The initial screenwas followed by assays to determine the site specificity ofterrein and its effect on proteolytic activity of the 20S isolatesas well as its performance in cells [39]. Terrein inhibitedCT-L and T-L activities at IC50 = 300 µM and promoted

β Ring subunits

β1

β2

β5

20S CP

Caspase-like

(PGPH)

Trypsin-like

Chymotrypsin-like

(T-L)

(CT-L)

A. B. C.

19SCap

19SCap

Lid

Base

α Subunits

α Subunits

β Subunits

Proteasome

Figure 1. Structural diagram of the constitutive proteasome. A. 19S RPs presented in grey (lid) and red (base) and 20S CP in

blue (a) and turquoise (b). B. 20S CP shown with b1, b2 and b5 subunits localized in central lumen. C. b1, b2 and b5 subunits

displayed as an electrostatic surface potential map with red representing negative charge, white representing neutral and

blue representing positive. The proteolytic threonine 1 (green) is highlighted in each.

Recent advances in proteasome inhibitor discovery

Expert Opin. Drug Discov. (2013) 8(5) 539

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Table 1. Survey of screening compounds.

Compound reference number 2-D Compound structure Description Inhibition activity* Refs.

Compound screening1a A,B,C 20S (CT-L) 6 µM [34]

1b A 26S (CT-L) 100 µM EC50 [35]

1c A,C,F (CT-L) 1.0 ± 0.63 µM [36]

1d A,C (CT-L) 0.44 -- 1.01 µM [37]

1e A,C (CT-L) 3.65 µM [38]

1f A,C,D (CT-L) (T-L) 300 µM [39]

*Inhibition activity reported as IC50 unless otherwise noted.

A: Biochemical assay; B: Cellular assay; C: CT-L activity assayed; D: T-L activity assayed; E: PGPH activity assayed; F: Covalent inhibitor; G: Noncovalent inhibitor;

H: Peptide; I: Peptidomimetic; J: Structure determined, no data reported.

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80% cell death in fibroblasts at 150 and 250 µM after only4 h [39]. Blackburn et al. employed a multistep strategy con-sisting of: library screening, X-ray crystallography, andstructure-based design to develop a series of highly potentand selective inhibitors of the CT-L activity of the 20S consti-tutive and immunoproteasome [40]. The inhibitors were devel-oped using structure-guided design based on the crystalstructure of a hit compound that was identified by a high-

throughput cell-based screening of a library of 350,000 com-pounds [40]. After a series of focused library screens, the mostpotent compound (Table 1, 1g, N-(2,2-dimethylpropyl)-N˜2˜-[1H-indol-3-yl(oxo)acetyl]-L-asparaginyl-N-(2-methyl-benzyl)-3-pyridin-4-yl-L-alaninamide) was identified withinhibitory activity IC50 = 0.0012 µM with a proposed nonco-valent mode of inhibition [40]. Long and colleagues developedan assay utilizing human colon cell lines 4Ub-Luc DLD-1 [41]

Table 1. Survey of screening compounds (continued).

Compound reference number 2-D Compound structure Description Inhibition activity* Refs.

1g A,C,D,E,G (CT-L) 1.2 � 10-3 µM [40]

1h J - [42]

1i J - [42]

1j A,C,D,E,G (CT-L) 1.9 µM [43]

1k A,C,G (CT-L) 0.34 ± 0.04 µM [44]






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that express 4-ubiquitin luciferase reporter protein to screen12,000 plant extracts and 62,000 pure compounds [42]. Thisassay led to identification and characterization of severalcycloartane derivatives from the leaves of Neobutonia melleri,some of which (Table 1, 1h -- Neoboutomellerone and1i -- 22-de-O-acetylneoboutomellerone) showed activity ina cellular proteasome assay [42]. A combination of compu-tational and biochemical screening was employed byBasse et al. [43]. Following the initial docking of a 300,000compound library, 200 were evaluated experimentally andseveral compounds were validated showing low micromolarinhibition of one or more proteolytic activities of 20S protea-some. The most active compound (Table 1, 1j) inhibited theCT-L activity at IC50 = 1.9 µM [43]. Similar to the approachtaken by Blackburn et al. [40], an investigation combininglibrary screening, crystallographic structure determinationand structure-guided SAR was conducted by Gallastegui andcolleagues [44]. The SAR study was performed on a series ofnoncovalent hydroxyurea derivatives which emerged from aninitial fluorogenic tetrapeptide substrate library screen [44].The most active compound (Table 1, 1k, 1-hydroxy-1-[(2R)-4-3-[(3S,5S,7S)-tricyclo[3.3.1.1˜3,7˜]dec-1-yloxy]phenyl-but-3-yn-2-yl]urea) exhibited inhibition of the CT-L activity ofthe proteasome at IC50 = 0.34 µM [44].Compound library screening is an important and widely

used approach that helps to search for novel inhibitors byallowing exploration of broad and diverse body of com-pounds. As this review demonstrates, the diversity of com-pounds exhibiting proteasome inhibitory activity hints at theimportance of venturing into the vastness of chemical space,a task that compound screening helps to achieve.

4. Rational design

In addition to screening compound libraries, there are a num-ber of compound families that have received significant atten-tion for targeting the proteasome in recent years. These haveemerged from earlier studies, current clinically approved drugs,and/or promising leads. Important structural features that thesecompounds possess are often used in the development of novelscaffolds. Substitution of these moieties onto a lead compoundmay increase its inhibitory activity or specificity toward one ormore of the proteasome’s catalytic sites. These scaffolds ofteninclude peptides, peptidomimetics, and natural products.

4.1 Boron-containing compoundsThe development of Bortezomib as a proteasome inhibitorapproved by the FDA for the treatment of multiple myelomahas accelerated active efforts to improve on its capability anddiminish some of its drawbacks for the next generation ofboron-containing proteasome inhibitors. Although Bortezo-mib is a potent and effective proteasome inhibitor, it hasbeen found to have multiple and often serious side effectsincluding: fatigue, nausea, sensory neuropathy and adversecardiovascular effects [25,26]. As a part of the effort to develop

second-generation proteasome inhibitors with high potencyand reduced side effects, the ability of Bortezomib to selec-tively bind to the b5 site of the 20S proteasome via its boronicacid moiety is a frequent design strategy.

Zhu et al. reported a SAR study on a series of tripeptidealdehyde and boronate-based compounds that demonstratelow nanomolar inhibitory activity [45], the most potent(Table 2, 2a, N-(2-pyrazinecarbonyl)-L-leucine-L-(2-naph-thyl)-alanine-L-leucine boronic acid) exhibiting inhibition ofthe CT-L activity of the isolated mouse 20S proteasome atIC50 = 0.00079 µM [45]. This study was followed by a com-prehensive SAR of a large number of dipeptidyl boronateinhibitors [26]. The best performing compounds (Table 2, 2b,[(1R)-1-[[(2S)-3-phenyl-2-[((S)-(-)-1,2,3,4-tetrahedro-naph-thoic-1-carbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic acid and 2c, [(1R)-1-[[(2S)-3-phenyl-2-[(5,6,7,8-tet-rahedro-naphthoic-1-carbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl] boronic acid) in this study were assayed in10 tumor cell lines that included multiple myeloma, large celllung cancer, human non-small cell lung cancer, colon carcinoma,and other cell lines, and showed an improvement over Bortezo-mib in seven out of ten cell lines with IC50 values ranging from0.0098 to 0.07 µM [26]. To further develop dipeptidyl boronicacids as proteasome inhibitors, in 2010, Zhu and colleagues per-formed another SAR study on this class of compounds [46].However, this time, in an effort to increase the activity and sta-bility, the peptides were composed of b-amino acids [46]. Thestudy also included toxicity assessments and docking simulationsfor binding mode characterization [47]. The best performingcompound (Table 2, 2d, [(1R)-1-[[(3R)-3-(4-methoxyphenyl)-3-[(phenylcarbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic acid), although reduced in potency compared to Borte-zomib (IC50 = 0.0096 µM vs 0.00248 µM), was less toxic inzebrafish embryo and Sprague--Dawley rats and possessed a bet-ter pharmacokinetic profile [46]. Following this study, a morecomprehensive SAR evaluation of b-amino acid dipeptidylboronic-acid inhibitors was performed using a similar approachthat included a biochemical 20S proteasome screen followedby cell line cytotoxicity studies as well as pharmacokinetic andtoxicity evaluation in rodent models [47]. The most potent com-pound (Table 2, 2e, (R)-1-[3-(3-fluorophenyl)-3-((S)-1,2,3,4-tet-rahydronaphthalene-4-carboxamido)propanamido]-3-methyl-butyl boronic acid) performed comparably to Bortezomib in a20S proteasome inhibitory assay (IC50 = 0.00102 µM vs0.00154 µM) and showed potent cytotoxicity while maintainingimproved toxicity and pharmacokinetic profiles [47].

The recently discovered proteasome inhibitor tyropep-tin [48,49] was chosen by Watanabe et al. as the scaffoldfor development of tyropeptin boronic acid derivatives [50].A resulting lead compound (Table 2, 2f, (1R)-1-[N-(1-naph-thylacetyl)-L-O-methyltyrosyl-L-valyl]amino-3-methyl-1-butylboronic acid) inhibited the CT-L activity of the purifiedhuman 20S proteasome with IC50 = 0.0026 µM [50]. To fur-ther characterize tyropeptin boronic acid derivatives, a SARstudy was expanded to a total of 53 derivatives and tested for

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Table 2. Survey of boron-containing compounds.

Compound reference

number

2-D Compound structure Description Inhibition activity* Refs.

Boron-containing compounds2a A,C,D,E,H (CT-L) (7.9 ± 1.1) � 10-5 µM [45]

2b A,C,F,H (CT-L) (1.2 ± 0.1) � 10-3 µM [26]

2c A,C,F,H (CT-L) (1.6 ± 0.2) � 10-3 µM [26]

2d A,C,F,H (CT-L) (9.6 ± 1.3) � 10-3 µM [46]

2e A,C,H (CT-L) 1.02 � 103 µM [47]

2f A,C,D,E,F,H (CT-L) 2.6 � 10-3 µM [50]






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Table 2. Survey of boron-containing compounds (continued).

Compound reference

number


2g A,C,D,E,F,H (CT-L) 4.1 � 10-3 µM [51]

2h A,C,D,E,F,H (Cytotoxicity) 4.9 � 10-3 µM [51]

2i A,C,F,H (CT-L) 0.28 ± 0.04 µM [54]

2j A,C,F,H (CT-L) 0.54 ± 0.14 µM [54]

2k A,C,F,H (CT-L) 2.5 � 10-4 µM [55]

2l A,C,I (CT-L) (3.5 ± 0.5) � 10-3 µM [56]




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both purified 20S proteasome inhibition and cytotoxicity inmultiple myeloma-derived cells [51]. The compound exhibitingthe most potency in a biochemical assay (Table 2, 2g, 3-phe-noxyphenylacetamide) showed inhibition of the CT-L activitywith an IC50 = 0.0041 µM, while the most cytotoxic com-pound (Table 2, 2h, 3-fluoro picolinamide) possessed an IC50

of 0.0049 µM [51]. Nakamura et al., in addition to exploitingthe boron peptide moiety, have turned to structural featuresfrom the natural product Belactosin C -- a compound isolatedfrom Streptomyces sp. and shown to inhibit rabbit 20S protea-some [52,53]. The result was a series of low micromolar com-pounds with both cell growth inhibition and 20S proteasomeinhibition capacity, representatives of which are shownin Table 2 (2i and 2j) [54]. A computationally generated three-dimensional (3D) pharmacophore model was used as a startingpoint to elucidate a series of boron-containing dipeptide inhib-itors by Lei et al. [55]. The most active compound (Table 2, 2k,[(1R)-1-[[(2S)-3-(1-Naphthyl)-2-[(1-naphthyl-2-carbonyl)-amino]-1-oxopropyl]amino]-3-methylbutyl]boronic acid)inhibited the CT-L activity of the 20S proteasome with anIC50 of 0.00025 µM [55]. The assays were followed by adocking study of the most active compound to elucidate itsbinding mode within the b5 subunit of the 20S particle [55].As part of their search for novel proteasome inhibitors,Iqbal et al. have explored chiral boronate-derived peptidomi-metic compounds [56]. The most active of those (Table 2, 2l,2-[[(1R)-1-[[(2S-5-[[Imino(nitroamino)methyl]-2-[((R,S)-10-cyano-2-cyclopentyldecanoyl)amino-1-oxopentyl]amino]-3-methylbutyl]]-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborole and 2m, 2-[[(1R)-1-[[(2S-5-[[Imino(nitroamino)methyl]-2-[((R,S)-10-N-phtha-limido-2-cyclopentyldecanoyl)amino-1-oxopentyl]amino]-

3-methylbutyl]]-(3aS,4S,6S,7aR)-hexahydro3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborole) inhibited CT-L activityof 20S proteasome at IC50 = 0.0035 µM and showed activ-ity in a number of human and rodent cancer cell lines [56].Furthermore, these compounds served as a starting pointfor a study of a series of (2S,3R)-2-amino-3-hydroxybutyricacid derivatives that led to discovery of a potent inhibitor(Table 2, 2n, [(1R)-1-[[(2S,3R)-3-hydroxy-2-[(6-phenylpy-ridine-2-carbonyl)amino]-1-oxobutyl]amino]-3-methylbu-tyl]boronic acid) that demonstrated efficacy in a cellular assayalong with a favorable toxicity profile and was selected for pre-clinical profiling for multiple myeloma therapy [57].

Boron-containing compounds have proven themselves aspotent proteasome inhibitors, albeit not without caveats.Thus, it is important to build on this progress and improveon past efforts to take boronates from “the lesser of two evils”to an optimal choice of treatment.

4.2 Fluorine-containing compoundsFluorine-containing compounds have been playing anincreasingly important role in drug design over the past sev-eral years [58-61]. The popularity of fluorine is largely due tothe characteristics of carbon-bonded fluorine such as smallsize, ability to withdraw electrons, and hydrophobicity of flu-orocarbons that facilitates tighter binding to their targets.Additionally, it has been proposed that addition of fluoro sub-stituents may help enhance metabolic stability of anticancercompounds [62,63]. In particular, the trifluoromethyl groupwith its unique properties and suitable geometry is oftenused to improve the stability and activity of compounds [64].Due to this, many investigators have turned to this class ofcompounds in the search for potent proteasome inhibitors.

Table 2. Survey of boron-containing compounds (continued).

Compound reference

number


2m A,C,I (CT-L) (3.5 ± 1.3) � 10-3 µM [56]

2n A,C,I (CT-L) (3.8 ± 1) � 10-3 µM [57]






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Table 3. Survey of fluorine- and aldehyde-containing compounds.

Compound reference

number


Flourine-containing compounds3a A,C,D,E,G,I (CT-L) 1.6 ± 0.1 µM

(PGPH) 2.7 ± 0.1 µM(T-L) 8.4 ± 1.3 µM

[65]

3b A,C,D,E,G,I (CT-L) 32 ± 2 µM(PGPH) 6 ± 0.5 µM(T-L) 30% inhibition

[65]

3c A,C,D,E,G,I (CT-L) 5.9 ± 0.5 µM(PGPH) 2.7 ± 0.1 µM(T-L) 4.4 ± 1.2 µM

[65]

3d A,C (CT-L) 4.44 µM [62]

3e A,C,D,E,G,H 26S (CT-L) 2.0 � 10-3 µM [70]

Aldehyde-containing compounds4a A,C,F,H (CT-L) 0.028 ± 0.006 µM [81]




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Formicola et al. developed a series of pseudopeptides basedon the tryfluoromethyl-b-hydrazino acid scaffold the mostactive of which (Table 3, 3a, 2-[3-(N-6-Amino-2-[2-(3-phe-noxyphenyl)acetylamino]hexanoylhyd)hydrazino)-4,4,4-tri-fluoro-butyrylamino]-3-phenyl-propionic acid methyl ester,trifluoro-acetic acid salt, 3b, 2-3-[N-(6-Amino-2-tert-butoxy-carbonylaminohexanoyl)hydrazino]-4,4,4-trifluorobutyryla-mino-3-phenylpropionic acid methyl ester, citric acid saltand 3c, 2-3-[N-(2-Amino-6-benzyloxycarbonylaminohexa-noyl)hydrazino]-4,4,4-trifluoro-butyrylamino-3-phenylpro-pionic acid methyl ester) have shown differential inhibitorycapabilities toward the CT-L, T-L and PGPH activities of20S proteasome at micromolar concentrations [65]. It haspreviously been shown that an active phenolic natural productextracted from rhizome of the tropical Southeast Asian plantCurcuma longai curcumin [66-68], inhibited the proteasomeactivity in human colon cancer cells [69]. Padhye et al. devel-oped curcumin’s fluoro Knoevenagel condensates, Schiff bases,and copper complexes [62]. The study resulted in a potentinhibitor (Table 3, 3d, 4-Salicylidene-1,7-bis(4-hydroxy,3-methoxyphenyl)-1,6-heptadiene-3,5-dione) of both 20S pro-teasome CT-L activity and colon cancer cell growth [62]. Further-more, over the course of developing a series of fluorinatedinhibitors, the most potent one of which (Table 3, 3e (S)-2-azido-N-((S)-1-((S)-1-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-ylamino)-1-oxo-3-(perfluorophenyl)propan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)-3-phenylpropanamide,N3PhePhePhe(F5)Leu-EK) inhibited the CT-L activity ofpurified 26S proteasome at IC50 = 0.002 µM Geurink et al.demonstrated that the presence of fluorinated phenylalaninederivatives in peptide-based compounds improved their CT-Linhibitory potency and selectivity [70].

With a good track record, fluorine-containing pharmaceu-tical agents have proven themselves both within and outsideof the proteasome inhibition arena. Further inquiry into andthe development of these compounds have the potential toresult in effective clinical candidates.

4.3 Aldehyde-containing compoundsAldehydes, and specifically peptide aldehydes, have proventhemselves to be promising compounds when it comes to pro-teasome inhibition [7,71-77]. In addition, a wealth of experi-mental data has been accumulated over the years, which hasallowed investigators to draw important conclusions andbuild on the existing body of work. In a recent study Maand colleagues, guided by a crystal structure of a peptide alde-hyde inhibitor MG101 [78] in complex with 20S proteasome,synthesized and evaluated the activities of a new series ofpeptide aldehyde derivatives in a combination of computa-tional and biochemical experiments. The best performingcompound (Table 3, 4a, Cbz-Glu(OtBu)-Phe-Leucinal) inan isolated 20S proteasome assay exhibited activity atIC50 = 0.028 µM, outperforming a known potent MG101derivative peptide aldehyde MG132 [79,80] by an order ofmagnitude [81]. These results suggest that aldehyde-containing

compounds are a promising avenue to explore in the searchfor effective proteasome inhibitors.

4.4 Vinyl sulfone and vinyl ester-containing

compoundsVinyl sulfones are another example of a pharmacophoric groupoften used in developing proteasome inhibitors [82-86]. Theybelong to a class of inhibitors that contain Michael acceptorsand have been shown to inhibit the proteasome via formationof a covalent bond with the catalytic THR1 residues of theproteasomal active sites [21,87,88].

Ettari and coworkers developed a series of conformationallyconstrained tripeptidyl vinyl sulfones, the best of which(Table 4, 5a, Benzyl-1-((S)-1-((S,E)-1-(ethylsulfonyl)-5-meth-ylhex-1-en-3-ylamino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-ylcarbamate, 5b, Allyl-1-((S)-1-((S,E)-1-(ethylsulfonyl)-5-methylhex-1-en-3-ylamino)-4-methyl-1-oxo-pentan-2-yl)-2-oxo-1,2-dihydropyridin-3-ylcarbamate and 5c,Allyl-1-((S)-1-((S,E)-1-(ethylsulfonyl)-5-methylhex-1-en-3-ylamino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-ylcarbamate) showed good selectivity and inhibition of CT-L activity of 20S proteasome in the submicromolar range [82].As a strategy to enhance proteasome inhibition and cellularuptake, Vivier et al. incorporated vinyl sulfone groups into aseries of inhibitors [83]. The most potent of these compounds(Table 4, 5d, N1-(2-(Dipropylamino)ethyl)-N4-((S)-4-methyl-1-((S)-4-methyl-1-((S)-4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2-ylamino)-1-oxopentan-2-yl)terephthalimide,hydrochloride salt and 5e, N1-((Diethylamino)ethyl)-N4-(4-methyl-1-oxo-1-(1-oxo-3-phenylpropan-2-ylamino)pen-tan-2-yl)terephthalamide), while possessing decreasedcytotoxicity in comparison with their aldehyde-containinganalogs, provided potential low micromolar candidatesfor further assessment [83]. Similarly, Screen and colleaguesinvestigated the effects of replacing epoxyketone withvinyl sulfone moieties in proteasome inhibitors [84]. Thesubstitution resulted in an increased specificity toward pro-teasomal CT-L activity, which was proposed to be the rea-son for decreased overall cytotoxicity of vinyl sulfoneinhibitors [84].

Vinyl ester groups were proposed to inhibit the proteasomeby interacting with the catalytic threonine residue in a mannersimilar to that of vinyl sulfones [85]. Baldisserotto et al. exam-ined a series of cyclic peptides containing the moiety andshowed selective and potent inhibition of the PGPH activityin a biochemical assay at IC50 = 0.065 µM in 20S purifiedproteasome and in a cellular assay at IC50 = 0.091 µM bythe most active compound (Table 4, 5f c[Ser-Leu-Leu-Glu(Leu-VE)]) [85]. In a later study, however Baldisserotto andcolleagues saw a marked reduction in the inhibitory activityof a series of N-allyl vinyl ester-based peptides [89]. The mostpotent N-allylic vinyl ester derivative (Table 4, 5g HMB-Leu-Leu-N-AllylLeu-VE) exhibited its strongest inhibition atIC50 = 0.29 µM comparing to IC50 = 0.059 µM of the refer-ence vinyl ester peptide toward the T-L activity of the 20S



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Table 4. Survey of vinyl sulfone, vinyl ester, and epoxyketone-containing compounds.

Compound reference

number

2-D Compound structure Description Inhibition

activity*

Refs.

Vinyl sulfone-containing compounds5a A,C,D,E,I (CT-L) 0.33 µM Ki [82]

5b A,C,D,E,I (CT-L) 0.61 µM Ki [82]

5c A,C,D,E,I (CT-L) 0.37 µM Ki [82]

5d B,G,H 0.71 ± 0.34 µM [83]

5e B,G,H 0.64 µM [83]

5f A,B,C,D,E,F,H (PGPH)0.065 µM (A)(PGPH)0.091 µM (B)

[85]

5g A,C,D,E,H (T-L)0.29 ± 0.33 µM

[89]




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proteasome [89]. In another study, Chen and colleagues evalu-ated a set of vinyl ester-containing peptides that had alsoshown mixed results [86]. Finally, based on the 26S protea-some and cell extract assays, Screen et al. concluded that con-trary to the earlier studies vinyl ester-containing peptidesshow no inhibition of any of the proteolytic activities [84].This result shows the importance of assaying the inhibitoryactivities of compounds in 26S proteasome assays in additionto 20S and cell-based assays in order to confirm the inhibitionof the entire proteasome particle and rule out other waysthrough which an inhibitor may be exhibiting activity.

High specificity toward the CT-L activity of the protea-some, tight covalent binding to catalytic threonine and rela-tively low overall reactivity [83] make vinyl sulfones anattractive alternative to some of the other families of protea-some inhibitors. In addition to these hallmarks of a potentproteasome inhibitor, vinyl sulfones exhibit a potential foralleviating some of the side effects that other alternatives suf-fer. Therefore, further development of these compounds isneeded to ensure that this potential is realized in the clinic.

4.5 Epoxyketone-containing compoundsAs the success of Bortezomib has been fueling efforts todevelop boron-containing compounds, Carfilzomib [90,91]

has paved the way for the development for epoxyketone-containing compounds. Although a highly selective andpotent inhibitor of the CT-L activity of the proteasome [90-96],Carfilzomib shares some of the patient inconveniences of Bor-tezomib such as frequent intravenous dosing that often has tobe carried out for months [91]. As a result, efforts have beenundertaken to develop inhibitors that provide the samepotency and selectivity, yet are more patient friendly.

Zhou et al. performed a systematic SAR analysis in orderto develop orally bioavailable Carfilzomib analogs [97]. Thestudy resulted in a orally bioavailable and selective candidate(Table 4, 6a, (2S)-3-Methoxy-2-[(2S)-3-methoxy-2-[(2-methyl-1,3-thiazol-5-yl)formamido]propanamido]-N-[(2S)-1-[(2R)-

2-methyloxiran-2-yl]-1-oxo-3-phenylpropan-2-yl]propanamide)with favorable pharmacological properties [97].

In a proteasome inhibitor pharmacophore study, Screenand colleagues concluded that epoxyketones, althoughexhibiting preference toward the CT-L activity, also inhibitboth T-L and PGPH activities and result in overallincreased cytotoxicity in comparison with the vinyl sulfoneanalogs [84].

Approval of Carfilzomib, undoubtedly, gave epoxyketonesa necessary credibility in the arena of proteasome inhibition.However, the success of epoxyketones in the clinic is still lim-ited by many common patient inconveniences and sideeffects. A continued development of this family of com-pounds should, over time, address many of these concernsand improve the success rate of epoxyketones making thema more safe and effective therapeutic.

4.6 SyrbactinsTwo representatives of this class of natural products: syringo-lins, isolated from Pseudomonas syringae pv syringae [98] andstructurally related glidobactins isolated from a species of Bur-kholderiales [99] had received increased attention as candidatesfor proteasome inhibition [100-103]. This is largely because arepresentative of this class Syringolin A (SylA) has beenobserved to induce apoptosis in human neuroblastoma andovarian cancer cells [104] and subsequent evidence that itinhibited eukaryotic proteasomes via a novel mechanism inwhich the catalytic threonine of the proteasome performsMichael-type addition to the double bond of C4 on the12-membered ring system of the inhibitor [105].

Building on these initial findings Clerc et al., guided by crystalstructure, rationally designed a successful SylA-based lipophilicderivative (Table 5, 7a) that showed a 100-fold improvement inpotency over the original SylA toward the CT-L activity [100].In a later study, the same group developed a series of pegylatedSylA derivatives, the most potent of which (Table 5, 7b, SylA-L-L) inhibited CT-L activity of the purified human 20S

Table 4. Survey of vinyl sulfone, vinyl ester, and epoxyketone-containing compounds (continued).

Compound reference

number


activity*

Refs.

Epoxyketone-containing compounds6a A,C,H (CT-L)

0.055 ± 0.019 µM[97]






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proteasome at Ki = 0.401 µM [101]. In order to get further insightinto the activities and modes of action of syrbactins, Clerc et al.developed a Syringolin A--Glidobactin A hybrid (Table 5, 7c) inaddition to GlbA. Both turned out to be potent proteasomeinhibitors, but have displayed different specificity toward consti-tutive proteasome, immunoproteasome, and thymoproteasomecatalytic subunits [106]. Archer and colleagues followed up onthe study of the SylA--GlbA hybrid with its crystal structureand additional assays including biochemical, cellular, and mul-tiple tumor cell lines. The study revealed that the SylA--GlbAhybrid is a promising proteasome inhibitor with a biochemically

assayed CT-L activity inhibition at Ki = 0.0125 µM andcell culture proteasome inhibition at IC50 = 2.87 µM(Table 5, 7c) [102]. Van der Linden and associates employed astrategy of combining structural features of syringolins andother moieties known to improve proteasome inhibition suchas epoxyketones and vinyl sulfones. The study yielded selectiveCT-L inhibitors (Table 5, 7d, tBuO-Val-urea-Val-Leu-EK2and 7e, Benzylamide-Val-urea-Val-Leu2-EK) with activityexceeding that of syringolin A (IC50 = 0.016 µM and 0.008 µMvs 1.3 µM) as well as a potent (IC50 = 0.46 µM) PGPH activityinhibitor (Table 5, 7f, Benzylamide-Val-urea-Val-Leu2-EK2) [103].

Table 5. Survey of syrbactins.

Compound

reference number


Syrbactins7a A,C,D,E,F,H (CT-L)

(8.65 ± 1.33) � 10-3µM Ki

[100]

7b A,C,F,H (CT-L) 0.401 ± 0.037 µM Ki [101]

7c A,C,D,E,F,H (CT-L) 0.0125 µM KI (A)(CT-L) 2.87 ± 1.32 µM Ki (B)

[102]

7d A,C,D,E,H 26S (CT-L) 0.016 µM [103]

7e A,C,D,E,H 26S (CT-L) 0.008 µM [103]

7f A,C,D,E,H 26S (PGPH) 0.46 µM [103]




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A relative newcomers to the field of proteasomeinhibition, Syrbactins have established themselves as apowerful tool in the hands of medicinal chemists. Contin-ued development of these compounds will result in potentand specific proteasome inhibitors that may see success inthe clinic.

4.7 FlavonoidsThis large class of compounds, frequently found in edibleplants, has received considerable attention due to the evidencesuggesting it has marked antitumor, anti-inflammatory andantioxidant activities [107]. Moreover, flavonoid activities havebeen linked with proteasome inhibition [108,109]. In recent yearsinquiry has continued into the inhibitory effects this widevariety of compounds has on the proteasome.

Chang investigated 26 flavonoids for their inhibitorypotential of 26S isolates across all proteolytic activities [110].The most potent compound (Table 6, 8a, 5,6,3,4-tetrahy-droxy-7-methoxyflavone) exhibited broad micromolar inhibi-tory activity with a slight selectivity toward CT-L [110]. Shimperformed biochemical screening of several flavonoids isolatedfrom the stem of Spatholobus suberectus against the CT-L activity of the 20S proteasome and identified severalcompounds (Table 6, 8b Isoliquiritigenin, 8c, Genisteinand 8d, a.7-Hydroxyflavanone) with inhibitory activity atlow micromolar concentrations [111].

One representative of this class of compounds, epigallocate-chin 3-gallate or EGCG, has been extensively studied as a pro-teasome inhibitor due to evidence linking this compound withthe reduction or growth inhibition of several types ofcancer [112-119] as well as evidence of EGCG’s targeting of theproteasome specifically [120]. In the effort to improve bioavail-ability of EGCG analogs, Huo and colleagues screened severalcompounds for their activity against both 20S and 26S protea-somes using biochemical and cellular assays in addition to per-forming an assay measuring the resistance of each compoundto methylation, which decreases bioavailability, in additionto elucidating the binding mode of EGCG and some of itsanalogs [121-124]. The experiment revealed the most cytotoxiccompound (Table 6, 8e) that is a prodrug of the mostmethylation-resistant inhibitor whose binding mode was thenmodeled computationally to elucidate its mode of actionwithin the b5 site of the proteasome [125]. A complimentarystudy by Bonfili et al. employed a combination of computa-tional studies and experimental assays to evaluate the stabilityof EGCG under neutral-alkaline and cell physiological condi-tions as well as to identify biologically active EGCG oxidativering-fission product (Table 6, 8g) [126]. The metabolite showedboth comparable inhibition of isolated 20S (IC50 = 173 µMfor CT-L activity) across all proteolytic activities as well as similarto EGCG activity in human cervical carcinoma cells [126].

Flavonoids do not exhibit the same level of potency seen inother compounds, which largely renders them unfit for treat-ment purposes. Their low toxicity, however, in combinationwith observed proteasome inhibition and antioxidant

activities make flavonoids a low risk agent that can beincorporated in a daily diet as part of a preventative strategy.

4.8 SalinosporamidesSalinosporamides have received considerable attention dueto recent success of a marine natural product-derived com-pound Salinosporamide A (salino A) that has been showingpromising signs and may become an option for cancertreatment [127-129].

Nguyen et al. have synthesized and enzymatically assayeda series of (-)-salinosporamide A (salino A) derivatives andidentified compounds that exhibit activities comparableto those of the original salino A and better than the activi-ties of Bortezomib with the most potent compound(Table 6, 9a (-)-homosalino) inhibiting CT-L activity atIC50 = 0.0007 µM and 0.0023 µM in 20S and 26S isolatesas compared to salino A at IC50 = 0.0008 µM and0.0025 µM and Bortezomib at IC50 = 0.0026 µM and0.0074 µM [130]. In a different approach, Rachid and col-leagues identified the genes of and utilized the enzymes ofStreptomyces sp. responsible for the biosynthesis of cinna-baramides, compounds closely related to salinosporamides,in order to generate novel compounds with inhibitorypotential toward the proteasome [131]. The activity of theresulting compounds was then tested in 20S isolates andin several tumor cell lines. The most potent (Table 6, 9b,17-chlorocinnabaramide A) of the compounds showedactivity (IC50 = 0.0088 µM against CT-L activity), butfailed to outperform salino A (IC50 = 0.0031 µM) [131].

Marine natural product-derived compounds have servedas a source for promising pharmaceutical agents in thepast [132,133], therefore it is not surprising to see a representa-tive of this class of compounds showing potential when itcomes to inhibition of the proteasome. However, it is a longway from the ocean to the clinic, and in order to surmountthe obstacles faced by a promising lead, an ongoing researcheffort is essential. Salinosporamide has shown the potentialand will, undoubtedly, continue to be of interest to researcherslooking for an effective proteasome inhibitor.

4.9 TMC-95ATMC-95A is a cyclic tripeptide from ascomycete fungusApiospora montagnei. It is one of the few potent noncovalentinhibitors of the proteasome that inhibits all three proteolyticactivities [134]. Its synthesis, however, is a difficult task [135-138]

and in the past few years there have been numerous attemptsto develop its mimics and derivatives [139-141].

Groll and colleagues developed a series of linear TMC-95Amimics, the most potent of which (Table 6, 10a) exhibited anincreased specificity toward CT-L activity of the proteasomein a biochemical assay superior to TMC-95A [142]. A seriesof pegylated monomers and dimers of a known linearTMC-95A mimic [143] were synthesized and evaluated in abiochemical assay by Marechal et al. and found that the pegy-lated dimers are much more potent toward the CT-L activity



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Table 6. Survey of flavonoids, salinosporamides, and TMC-95A compounds.

Compound

reference number

2-D compound structure Description Inhibition activity* Refs.

Flavonoids8a A,C,D,E,H 26S (CT-L) 14.0 µM [110]

8b A,C (CT-L) 4.88 ± 1.5 µM [111]

8c A,C (CT-L) 9.26 ± 1.2 µM [111]

8d A,C (CT-L) 5.21 ± 1.5 µM [111]

8e A,C (CT-L) 29 µM [123,125]

8f A,C,D,E (CT-L) 173 ± 26 µM Ki [126]




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Table 6. Survey of flavonoids, salinosporamides, and TMC-95A compounds (continued).

Compound

reference number

2-D compound structure Description Inhibition activity* Refs.

9a A,C,D,E 20S (CT-L)(7.0 ± 0.4) � 10-3 µM26S (CT-L)(2.3 ± 1.1) � 10-3 µM

[130]

9b A,C,D,E (CT-L)(8.8 ± 5.5) � 10-3 µM

[131]

TMC-95A10a A,C,G,I (CT-L) 1.5 ± 0.1 µM [142]

10b A,C,G,I (CT-L) 0.18 ± 0.01 µM [144]






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of the proteasome 20S proteasome with the most active(Table 6, 10b) exhibiting activity at IC50 = 0.18 µM as comparedto IC50 = 141 µM of the original linear mimic [144].The noncovalent binding mode of TMC-95A makes this

compound an attractive alternative to its more potent covalentcounterparts. If developed further this compound can exhibitselective, potent and yet reversible, thus potentially less toxic,inhibition of the proteasome.

4.10 Metal complexesOver the years, a number of metal complexes have been inves-tigated for their potential to inhibit activity of the proteasome.Among those receiving attention are gold [145,146], nickel [147,148],zinc [147,148] and to a greater degree copper [34,149-152].Gold complexes initially showed promise when bis-

chelated gold(I) phosphine complexes had induced apoptosisin breast cancer cells [153]. Specific targeting of the proteasomeby gold complexes was performed by Milacic and colleagueswho showed that gold(III) complex had inhibited all threeactivities of 20S proteasome with a preference for CT-Lactivity [145]. More recently, Frezza et al. have tested Nickel(II) and Zinc(II) complexes for their inhibitory potentialagainst 26S and 20S proteasome [148]. While Nickel(II)-con-taining complex did not exhibit any marked activity againstthe target, Zinc(II) complex (Table 7, 11a, [Zn(LIA)2]) hadshown promise by inhibiting CT-L activity in both particlesat low micromolar concentrations [148].Special attention has been given to copper complexes as

potential proteasome inhibitors, in particular as a promisingstrategy in cancer therapy [34,149-152]. It has been shown thatmany types of cancers accumulate copper in their cells at levelsgreater than those of healthy cells, providing avenue for tar-geted treatment of tumors [154-157]. In one of the first studiesthat exploited the copper accumulation in cancer cells, itwas reported that certain copper organic compounds werepotent and selective inhibitors of the CT-L and speculatedthat these otherwise innocuous compounds can becomepotent in the copper-rich environment of cancer cells [34].This hypothesis was supported when the 8-hydroxyquinolineanalog clioquinol and a known copper binding compoundpyrrolidine dithiocarbamate inhibited the CT-L activity ofthe proteasome, blocked proliferation, and induced apoptosisin breast cancer cells while remaining nontoxic to noncancer-ous breast cells [149].In 2009, Hindo et al. characterized and evaluated a series of

Copper (II) complexes for 20S proteasome inhibition. Two ofthe complexes showed inhibition of CT-L activity of the pro-teasome and induction of prostate cancer cell death with themost potent (Table 7, 11b, [Cu(LI)Cl]) inducing cell deathat IC50 = 3.82 µM [151]. Milacic and colleagues performedextract-based, whole-cell, and proliferation assays to screen aseries of 8-hydroxyquinoline analogs [150]. The result showedthat otherwise nontoxic compounds (see Table 7, 11c) exhibittumor-suppressing and proteasome-inhibitory activity whenmixed with copper [150]. Zuo et al. have used a combination

of biological assays and computational methods to profileactivities and modes of action of several amino acid Schiffbase--copper (II) complexes. The investigation resulted in twocomplexes (Table 7, 11d [Cu(L1)(Phen)]·9H2O and 11e, [Cu(L2)(Phen)]·3H2O) that inhibit CT-L activity of both purified20S and cellular 26S proteasomes as well as induce growth inhi-bition and apoptosis in human breast and prostate cancercells [152].

Metal complexing compounds described above provideresearchers with an important avenue of specifically target-ing diseased cells. In particular, the characteristic accumula-tion of copper in cancerous cells provides an additional levelof precision to the inhibitors targeting the proteasome, aprotein equally important to both cancerous and healthycells. Moreover, a wide range of copper binding moietiesmeans additional flexibility in inhibitor design. Theseunique characteristics, in combination with the deeperinsight into the mechanisms of proteasome inhibition willundoubtedly lead up to some intriguing developments inthe field.

4.11 Known therapeutics as proteasome inhibitorsIn addition to discovering novel proteasome inhibitors and thedevelopment of existing compounds to improve their perfor-mance, another approach is evaluating known therapeutic com-pounds for their potential to inhibit the proteasome [158,158-161].The mechanism of action of such compounds in terms of pro-teasome inhibition is usually not known. However, their widerange of activities which often include antiproliferative andanticancer effects makes them good candidates for furtherinquiry regarding their potential as proteasome inhibitors. Inaddition, having been clinically evaluated, such compoundshave well understood toxicity profiles.

Schoof and colleagues synthesized and profiled a series ofderivatives of thiostrepton, a potent antibiotic and an anti-malarial agent [162-164]. Among other goals, the study wasaimed at finding a link between the antiplasmodial activityand the inhibition of the proteasome [158]. As a result, a seriesof new compounds (see Table 7, 13a for a representative com-pound) showed inhibition of CT-L and PGPH activities ofproteasome that correlated with their antiplasmodialactivity [158].

Over the years, a number of anti-HIV drugs including rito-navir [165] and bevirimat [159,166] have been shown to inhibitproteasome. Bevirimat, a known anti-HIV-1 agent is derivedfrom betulinic acid (BA), a compound with a number ofknown biological activities including antibacterial, antimalar-ial, anti-inflammatory, anticancer, antioxidant and anthelmicactivities [167]. Qian and colleagues biochemically assayedinhibition of CT-L activity of the proteasome by a series ofBA derivatives and identified several low micromolar inhibi-tors, the most potent of which (Table 7, 13b) exhibited activityat IC50 = 1.42 µM [167].

Fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-ben-zimidazol-2-yl) carbamate) is a known anthelmintic drug

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Table 7. Survey of metal complexes and known therapeutic classes of compounds.

Compound reference

number


activity*

Refs.

Metal complexes11a A,C,F 26S (CT-L) 5.7 µM

20S (CT-L) 16.6 µM[148]

11b A,C (CT-L) 3.82 ± 0.01 µM [151]

11c A,C (CT-L) 26% inhib.at 5 µM(CT-L) 52% inhib.at 10 µM

[150]

11d A,C (CT-L) 0.0133 µM [152]






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Table 7. Survey of metal complexes and known therapeutic classes of compounds (continued).

Compound reference

number


activity*

Refs.

11e A,C (CT-L) 0.0113 µM [152]

Known therapeutics classes of compounds13a See reference for structure A,C,D,E (CT-L) 0.32 µM

(PGPH) 0.1 µM[158]

13b A,C (CT-L) 1.42 µM [167]

13c A,C (CT-L) 1.2 -- 1.6 µM [160]

13d A,C,D,E (CT-L) 0.75 µM(T-L) 0.53 µM(PGPH) 0.46 µM

[161]




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used to treat pinworm in animals [160]. Its two benzimidazolederivatives mebendazole(methyl 5-benzoyl-2-benzimidazole-carbamate) and albendazole (methyl (5-propylthio)-1H-ben-zimidazol-2-yl carbamate methylester) have also been usedto treat pulmonary helminitic infection and alveolar echino-coccosis in humans [168] with low incidence of severe sideeffects despite long-term high dosage treatment [169,170]. Dograand Mukhopadhyay performed a comprehensive inquiry intothe effects that FZ had on tumor cells and proteasomalactivity specifically. The study demonstrated, among otherfindings, that FZ inhibits growth and induces apoptosisof human non-small cell lung cancer cells and inhibitsproteasome function at low micromolar concentrations(Table 7, 13c) [160].

Santoro and colleagues assessed the ability of porphyrins,molecules that among a multitude of other uses serve as pho-tosensitizers in photodynamic cancer therapy [171,172], to actas proteasome inhibitors. Inhibitory capacity of several vari-ous types of porphyrins toward 20S and 26S proteasomeswere assessed using cell lysates and protein extracts [161].The experiment demonstrated that tetracationic porphyrinsand some of their metallo derivatives may inhibit all threeactivities of the proteasome, with the most active compound(Table 7, 13d, meso-tetrakis(4-N-methylpyridyl)-porphyrin)exhibiting inhibition in the submicromolar range [161].

Lastly, the anti-alcoholism drug disulfiram, that has alsobeen shown to bind copper [173,174] has been closely investi-gated as a potential anticancer agent and a proteasome inhibitorin particular [174,175]. Chen et al. demonstrated an induction ofapoptosis in breast cancer cells and xenografts treated withdisulfiram and showed an inhibition of CT-L activity of puri-fied 20S proteasome by disulfiram (Table 7, 13e) in presenceof copper at IC50 » 7.5 µM [174]. Conticello and colleaguessimilarly saw cytotoxic effect that disulfiram--copper complexexhibited in hematological malignant cells, however an inquiryinto the effects on the 20S proteasome showed no direct evi-dence of activity of either disulfiram--copper or disulfiramalone [175].

Drug repurposing has, of late, been considered a viablestrategy for getting drugs through the regulatory hurdles andinto clinic [176]. Existing therapeutics have known toxicity

profiles and although their primary clinical purpose is differ-ent from the one sought, their effects may provide importantinitial clues toward their potential off-target activity. A closerlook at the multitude of known therapeutics is bound to resultin a number good candidates for development of proteasomeinhibitors.

4.12 Newly emerging classes of compoundsIn addition to some of the classes of compounds that havealready established themselves as proteasome inhibitors,others are just emerging onto the scene. As investigatorsexplore alternatives to some of the existing inhibition strate-gies and continue to look for potent compounds with goodbioavailability and fewer side effects, new families of com-pounds have been emerging and receiving more attention inrecent years. One of such compound is a cyclic heptapeptideisolated from a myxobacterium argyrin. Initially, it was shownthat inhibition of proteasome by argyrin A has led to the sta-bilization of the tumor suppressor protein p27 [177]. In a seriesof subsequent studies, argyrin A was characterized and theproteasome inhibitory capacities of its derivatives wereelucidated (Table 8, 12a, argyrin F) [178,179].

Building on their previous work with vinyl esters [85],Baldisserotto and colleagues synthesized and evaluated a seriesof novel N-acylpyrrole-containing peptide-based compoundswith the hypothesis that the new pharmacophore will beable to mimic the functionality of vinyl ester moiety [180].These were evaluated in purified proteasome and cellularassays where the most potent compounds (Table 8, 12b,Z-NH-(CH2)7-Val-Ser-Leu-VAP) exhibited activities atIC50 = 0.036 µM and IC50 = 0.058 µM toward PGPHactivity in biochemical and cellular assays respectively [180].

The previously mentioned curcumin, a natural product,whose fluorine-containing derivatives were developed into pro-teasome inhibitors [62], itself is a promising anticanceragent [181-183]. Curcumin has been the subject of a recent studywhere its amino acid conjugates (Table 8, 12c, (1E,6E)-1,7-Bis(4-glycinoyl-3-methoxyphenyl)hepta-1,6-diene-3,5-dionehydrochloride, 12d, (1E,6E)-1,7-Bis(4-alaninoyl-3-methoxy-phenyl)hepta-1,6-diene-3,5-dione hydrochloride and 12e, (1E,6E)-1,7-Bis(4-valinoyl-3-methoxyphenyl)1,6-diene-3,5-dione

Table 7. Survey of metal complexes and known therapeutic classes of compounds (continued).

Compound reference

number


activity*

Refs.

13e A,B,C 20S (CT-L) 7.5 µM [174]






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hydrochloride) demonstrated a potent antiproliferative effectagainst a prostate cancer cell line [184].Krunic et al. evaluated two cyclic peptides isolated from cya-

nobacteria, scytonemides A and B, and demonstrated that in a

biochemical assay scytonemide A (Table 8, 12f) inhibited 20Sproteasome on the nanomolar scale. The compound, however,did not perform as well in a human cancer cell line, which wasspeculated to be due to the metabolic instability of the

Table 8. Survey of newly emerging classes of compounds.

Compound

reference

number


activity*

Refs.

Newly emerging classes of compounds12a See reference for structure A,C,D,E,I (T-L) 0.038 ±

0.0065 µM(PGPH) 0.035 ±0.007 µM(CT-L) 0.028 ±0.0025 µM

[178]

12b A,B,C,D,E (PGPH) 0.036 µM (A)(PGPH) 0.058 µM (B)(CT-L) 0.028 ±0.0025 µM

[178]

12c A,C (CT-L) 70% inhib.at 50 µM

[184]

12d A,C (CT-L) 70% inhib.at 50 µM

[184]

12e A,C (CT-L) 38 -- 71%inhib. at 5 -- 10 µM

[184]

12f A,C (CT-L) 0.096 µM [185]




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Table 8. Survey of newly emerging classes of compounds (continued).

Compound

reference

number


activity*

Refs.

12g A,B,C,D,E (CT-L) 2.2 ±0.3 µM (A)(CT-L) 1.9 µM

[188]

12h A,C,D,E (T-L) 9.1 µM(PGPH) 6.5 µM

[188]

12i A,B,C,D,E (CT-L) 2.5 µM(PGPH) 1.6 µM

[189]

12j A,C (CT-L) 1 � 10-3 µM [190]

12k A,C (CT-L) 7 � 10-3 µM [190]

12l A,B,C,D,E (CT-L) 0.64 µM (B)(PGPH) 0.15 µM (B)

[192]






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compound [185]. It was concluded, however, that improvementsmay be possible via chemical modifications [185]. As a follow-up to a study, where in search for novel scaffolds for proteasomeinhibition by assessing several naturally occurring acids, litho-cholic acid was identified as a potential candidate [186,187] Dangand associates synthesized and evaluated a series of lithocholicacid derivatives [188]. One of the resulting compounds(Table 8, 12g, 3a-O-(3,3-Tetramethyleneglutaryl)-lithocholicacid) inhibited CT-L activity of the 20S proteasome atIC50 = 2.2 µM in a biochemical assay as well as exhibited protea-some inhibition in a cellular assay. Another compound (Table 8,12h, 3a-O-(3-Methylenecarboxy-benzylcarbonyl)-lithocholic acid)inhibited CT-L, T-L and PGPH activities at IC50 = 1.9 µM,IC50 = 9.1 µM and IC50 = 6.5 µM respectively [188].Lansdell et al. evaluated a series of oroidin-derived

alkaloids for proteasome inhibitory activity in both biochem-ical and cellular assays and found them to inhibit the CT-L and PGPH activities of both the human 20S proteasomeand 20S immunoproteasome cores through irreversible bind-ing in the low nanomolar range. The most potent inhibitor(Table 8, 12i (-)-Palauamine) inhibited the CT-L activity atIC50 = 2.5 µM and PGPH activity at IC50 = 1.6 µM aswell as exhibiting inhibition of protein degradation incells [189].While in the process of generating a series of sterically

constrained oxazoline derivatives, Dunn and colleagues seren-dipitously discovered a series of (2S, 3R)-2-amino-3-hydroxy-butyric acid derivatives by performing a fluorogenic substrateassay followed by an intracellular activity assay [190]. Theexperiment led to potent leads (Table 8, 12j and 12k) thateventually resulted in development of a promising inhibitordelanzomib [57].Margarucci et al., in an investigation of targets of a marine

natural product with known anti-inflammatory properties [191],petrosaspongiolide M (PM), performed a chemical proteomicsstudy and identified the proteasome as a target [192]. Furtheranalysis of inhibitory potential using biochemical and cellularassays indicated PM to be a potent inhibitor with inhibitionof CT-L activity at IC50 = 0.64 µM and PGPH activity atIC50 = 0.15 µM in cells. (Table 8, 12l, petrosaspongiolideM) [192]. In a later study, Margarucci and colleagues have tested

a series of compounds belonging to petrosaspongiolide family aswell as several synthetic analogs of PM [193]. This resulted inanother compound (Table 8, 12m) that compared to PM in a puri-fied 20S activity assay at IC50 = 0.07 µM vs IC50 = 0.085 µMfor CT-L activity and IC50 = 0.07 µM vs IC50 = 1.05 µM forPGPH activity as well as showed activity in cells [193].

The variety of compounds showing promise when it comesto proteasome inhibition highlights the importance of contin-ued inquiry and interest in novel compounds and compoundfamilies that come from a variety of sources. The proteasomeis an awe-inspiring piece of molecular machinery and to tameit requires looking far beyond a small number of commonscaffolds and chemical moieties. Constantly looking for the“next big thing,” although, seemingly fruitless in some cases,is a strategy of considerable importance when it comes totargeting the proteasome.

5. Expert opinion

What should be immediately obvious is the diversity of chem-ical structures that can inhibit the proteasome. In this review,we catalogued more than a dozen different classes of mole-cules. Interestingly, activities of the classes display a commonrange from one group to another with low micromolar tomid-nanomolar being the frame of inhibitory potency; at leastas far as IC50s are concerned. A common motif in drug dis-covery is the screening-to-optimization approach, which isalso discussed in this manuscript. Clearly, the proteasome isamenable to inhibition by a wide variety of scaffolds andmotifs. This is likely due to several factors: i) the active sitessignificantly differ in their recognition pockets, ii) the activesites are very large as they accommodate peptides allowingsubstantial surface area for inhibitor binding, and iii) the cat-alytic THR1, common to each of the proteasome’s threeactivities, is a ready target for chemical modification. Whilethese factors can contribute to difficulty in isolating a givencompound’s mode of action, the process of identifying alead compound is relatively straight forward and such effortstend to be successful. However, characterizing and optimizinga compound (in regards to mechanism, specificity, andpotency) can be difficult. This leads to a situation where

Table 8. Survey of newly emerging classes of compounds (continued).

Compound

reference

number


activity*

Refs.

12m A,B,C,D,E (CT-L) 0.07 µM (B)(PGPH) 0.07 µM (B)

[193]




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identification of an inhibitor is relatively easy compared tocreating a lead candidate suitable for clinical trials.

Several major issues present themselves when designingproteasome inhibitors. The first is whether to target the 20Sor 26S complex. Assays examining the 26S are more difficultthan those looking at the 20S particle [9]. However, it is the26S that is found within the cell and, as has been shownwith the HIV protease inhibitor ritonavir, there are occasionswhere an inhibitor of the 20S can actually be an activator ofthe 26S via a not yet fully understood mechanism [165,194,195].The next question is whether or not to target one or moreof the activities of the proteasome. The chymotrypsin-like activity is a common target, especially in anticancerresearch, as inhibition of this activity is strongly correlatedwith induction of apoptosis in cancer cells. However, inhibi-tion of all three activities may be necessary for potency andto fully interrupt protein degradation. To this end, modelingwill be an important strategic approach since the three sitesare similar in their catalytic regions but differ greatly withintheir substrate recognition regions, as should be anticipated.

Tissue targeting, particularly in regards to cancer, is a majorissue in proteasome inhibition. Bortezomib, the first clinicallyapproved proteasome inhibitor, demonstrates significant tox-icity. Many of the off-target effects are likely derived fromthe reactive “warhead” of the compound, but it should alsobe appreciated that many tissues rely on proteasome activity.It has been reported that normal cells are “resistant” to apo-ptosis caused by proteasome inhibition. However, this doesnot render those cells immune to proteasome inhibition orresistant to other off-target effects that may arise due to thepresence of warheads or the need for higher or longer dosesof inhibitor. Therefore, tissue targeting and high potencymay be more important and will provide better protectionfrom toxic effects. Obviously, increased potency is desirablein order to reduce load on a patient but this is offset by thetrend that covalent or irreversible inhibitors, while morepotent, have reactive groups that increase the risk of off-target effects. Thus, tissue targeting may be the primarymeans of avoiding toxicity especially in cancer therapeutics.After all, if an inhibitor is going to kill cancer cells and istargeted specifically to cancer tissues then toxicity fromsecondary action is less consequential.

With this in mind, different avenues to specifically targetcells or tissues of therapeutic interest should be carefullyexplored. In the case of cancer, its hallmark of accumulatingcopper seems an obvious path to targeted therapy by coppercomplexing compounds. The ligands of such complexes alonecould be designed to have minimal toxicity in the absence ofcopper. In addition, the concentration of copper would dic-tate the concentration of active complexes; thus, reducingconcentration of active compounds in healthy cells to belowtoxic levels. Moreover, a copper complex carrying a reactivegroup would require a lesser dose of the drug upon deliveryreducing the concentrations of active complexes in healthycells even further.

Promising results achieved with low-toxicity naturalcompounds and their derivatives should also be noted.Even though these compounds may not lead us to thenext big thing in proteasome inhibition as a therapy, theiruse as a preventative measure vis-a-vis diet can help lowerincidence of disease. This is extremely valuable in thelong term.

In addition to developing known tried and true scaffolds,the approach of screening large libraries should not be over-looked. The diversity of proteasome inhibitors is evident inthis review, however chemical space is infinitely large andcasting the net wide will, undoubtedly, yield novel com-pounds that may develop into promising clinical candidates.Use of modern high-throughput techniques in combinationwith virtual screening will allow researchers to interrogate amuch larger body of compounds.

Development and discovery of proteasome inhibitors hasbeen steadily gaining momentum. As more investigators puttheir efforts toward conquering this target and strike that per-fect balance of potency, toxicity, and bioavailability, the nextfew years may result in more successful clinical candidatesand therapeutics.

Declaration of interest

This work was supported by the Chemical Biology Corefacility at the Moffitt Cancer Center and Research Instituteand the University of South Florida College of Artsand Sciences.



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AffiliationYuri Pevzner1,2, Rainer Metcalf2,

Melanie Kantor2, Desiree Sagaro2 &

Kenyon Daniel†3

†Author for correspondence1University of South Florida,

Chemistry, 4202 E Fowler Ave,

Tampa, FL 33612, USA2Moffitt Cancer Center and Research Institute,

Screening and Modeling Unit,

Chemical Biology Core,

12902 Magnolia Dr., Tampa,

FL 33612, USA3Moffitt Cancer Center, Screening and Modeling

Unit, Chemical Biology Core,

12902 Magnolia Dr., Tampa,

FL 33612, USA

E-mail: [email protected]

Y. Pevzner et al.


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Recent advances in proteasome inhibitor discovery

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