[Annual Reports in Medicinal Chemistry] Annual Reports in Medicinal Chemistry Volume 44 Volume 44 || Chapter 3 Small-Molecule Protein–Protein Interaction Inhibitors as Therapeutic

CHAPTER 3

* Chemical Sciences, Wyet

** Neuroscience, Wyeth Re

Annual Reports in MedicinISSN: 0065-7743, DOI 10.

Small-Molecule Protein–ProteinInteraction Inhibitors asTherapeutic Agents forNeurodegenerative Diseases:Recent Progress andFuture Directions

Simon N. Haydar*, Heedong Yun*,

Roland G.W. Staal** and Warren D. Hirst**

Contents 1. Introduction 51

2. Ab Aggregation and Oligomers in

Alzheimer’s Disease
52
2.1 Ab aggregation and neurotoxic oligomers
52
2.2 Small-molecule inhibitors of Ab aggregation
53
3. Tau Aggregation in Alzheimer’s Disease
58
3.1 Tau pathophysiology
58
3.2 Small-molecule inhibitors of tau aggregation
59
4. a-Synuclein Aggregation in Parkinson’s Disease
63
4.1 Biochemistry of a-synuclein aggregation
63
4.2 Small-molecule inhibitors of a-synuclein aggregation
64
5. Conclusion
65
References
66
h Research, CN 8000, Princeton, NJ 08543

search, CN 8000, Princeton, NJ 08543

al Chemistry, Volume 44 r 2009 Elsevier Inc.1016/S0065-7743(09)04403-0 All rights reserved.

51

52 Simon N. Haydar et al.

1. INTRODUCTION

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two mostcommon chronic, progressive neurodegenerative diseases affecting anestimated 10% and 1%, respectively, of the elderly population [1,2], withfinancial costs of hundreds of billions of dollars per year.

AD is characterized by the presence of extracellular parenchymaland vascular amyloid deposits containing b-amyloid peptide (Ab) andintracellular neuronal tangles composed of hyperphosphorylated tau.a-Synuclein containing Lewy bodies, spherical inclusions found in thecytoplasm of surviving neurons, are the cardinal hallmark of PD. Despitetheir distinct pathologies, these neurodegenerative diseases are increas-ingly being realized to have common cellular and molecular mechanismsincluding protein aggregation and inclusion body formation.

Compelling evidence strongly supports the hypothesis that accumu-lation of misfolded proteins leads to synaptic dysfunction, neuronalapoptosis, brain damage, and disease. However, the mechanisms bywhich protein misfolding and aggregation trigger neurodegenerationand the identity of the neurotoxic structures are still unclear. Currenthypotheses propose that, in the aggregation process, there is anaccumulation of small soluble oligomeric intermediates, which leads tothe neuropathology, whereas the large insoluble deposits that make upthe inclusion bodies might function as reservoirs of these toxic, solubleoligomers [3].

As we increase our knowledge of the role of oligomeric, fibrillar, andhigher-order molecular entities of the misfolded proteins in neurode-generative diseases, new approaches may offer themselves for ther-apeutic intervention. Over the past few years, there has been significantinterest in developing therapeutics and chemical probes that inhibit thesespecific protein–protein interactions. This effort has been hampered bythe size and the geometry of the protein interaction interface, which aredevoid of defined ‘‘pockets’’ into which a small molecule can bind in anenergetically favorable manner [4]. Despite the challenges of developingcompounds that are capable of specifically inhibiting protein–proteininteractions, there are a number of examples of small molecules thatachieve this with reasonable potency [5]. This was made possible becauseof the discovery of ‘‘hot spots’’ on the protein interaction surfaces [6].These ‘‘hot spots’’ are small regions on the protein interaction interfacethat are responsible for a disproportionate contribution to the bindingenergy of the two proteins. This review will highlight the recent progressin the development of small-molecule protein–protein interactioninhibitors that have applications in furthering the mechanistic under-standing of neurodegenerative diseases and will potentially lead to thedevelopment of rational therapeutics.

Therapeutic Agents for Neurodegenerative Diseases 53

2. Ab AGGREGATION AND OLIGOMERS INALZHEIMER’S DISEASE

2.1 Ab aggregation and neurotoxic oligomers

Substantial genetic and physiological evidence suggest that the Ab playsa central role in AD pathogenesis. Ab is a 39- to 42-amino acid peptidederived from the proteolytic processing of the amyloid precursor protein(APP) by secretases [7]. Gradual changes in the steady-state levels of Abin the brain are thought to initiate the amyloid cascade [8,9].

Since the elucidation of the Ab sequences [10–12], investigators haveused synthetic Ab to examine aggregation and its effects on physiology.Many in vitro studies have suggested that aggregation of Ab is essentialfor toxicity, but characterization of the Ab species that formed waslimited. However, amyloid plaque number does not correlate well withseverity of dementia [13–15], and instead, there is a stronger link betweensoluble Ab levels and the extent of synaptic loss and the severity ofcognitive impairment [16–18]. Therefore, more recent studies havefocused on various soluble forms of synthetic Ab1-40 and Ab1-42, rangingfrom monomeric to protofibrils [19].

A number of reports have described the biochemical characterization ofthe soluble Ab extracted from human AD brain. The presence of sodiumdodecyl sulfate (SDS)-stable dimers and trimers in the soluble fraction ofhuman brain and in extracts of amyloid plaques suggests that SDS-stable, lown oligomers of Ab are the fundamental building blocks of insoluble amyloiddeposits and could be the earliest mediators of neuronal dysfunction [20].Recent studies have described the physiological characterization of Abdimers isolated from AD brains that inhibit long-term potentiation (LTP), aphysiological correlate of memory, and reduce dendritic spine density innormal rodent hippocampus [21]. In addition, the dimers disrupt memory ofa learned behavior when directly injected into the brains of normal rats.

As outlined earlier, a number of Ab assemblies have been proposedto exert neurotoxic effects, but with evidence only recently emerging onwhich forms that are the pathological species in vivo, and the scarcityof structural data on the oligomers complicates a rational search forcompounds that could inhibit Ab aggregation and toxicity. Despitethese obstacles, a number of different compounds that interfere with Abaggregation, in one way or another, have been described; these arereviewed in the following sections.

2.2 Small-molecule inhibitors of Ab aggregation

2.2.1 Scyllo-inositolRecent in vitro studies with scyllo-inositol (1) have shown that it caninteract with Ab42 peptide promoting a conformational change from


random coil to b-sheet structure and stabilized it in small, nonfibrillarcomplexes, blocking fibril formation [22]. These stabilized complexeswere significantly less toxic to neuronal cell lines and primary neuronalcultures than untreated Ab42 or chiro-inositol-treated Ab42 [22].Moreover, in in vivo studies in the TgCRND8 mouse model of AD, itwas shown that compound 1 inhibited Ab aggregation, decreasedAb-induced impairments in spatial memory, reduced the cerebral Abpathology, and attenuated the rate of mortality [23]. To explore themolecular details of the inositol–Ab42 interaction, a series of scyllo-inositols were prepared in which one or two hydroxyl groups werereplaced with fluoro, chloro, methoxy, or hydrogen substituents [24].After incubation with Ab42 for 7 days, the activity of the derivativeswas measured by electron microscopy to monitor the formation of Ab42fibers. Despite the synthesis of numerous analogs, only single hydroxylsubstitutions such as 1-deoxy-1-fluoro-scyllo-inositol (2) and the disub-stituted analog 1,4-dimethoxy-scyllo-inositol (3) were shown to havesimilar activity to the parent compound 1. Compound 3 was shown toexhibit the most pronounced effect on Ab42 aggregation, in that itproduced a more homogenous population of small amorphous aggre-gates and no fibers were detected [24].

HOHO

OHOH

OHHO

1

MeOHO

OHOH

OMeHO

3

HOHO

OHOH

FHO

2

2.2.2 Tricyclic pyrones and pyridinonesHua et al. [25] used a neuronal cell line overexpressing a C-terminalfragment of APP (MC65 cells) to identify inhibitors of toxicity relatedto intracellular Ab and discovered a class of tricyclic pyrones (TP).In particular, a TP 4 that contains an adenine moiety (at N-3u) attached atthe C7-alkyl side chain of the ring system showed significant protectionin the MC65 cell assay with an EC50 value of 0.31 mM [25]. Furthercharacterization of compound 4 using surface plasmon resonance (SPR)spectroscopy, atomic force microscopy (AFM), and protein quantificationstudies showed it binds to Ab42 oligomers, inhibits Ab aggregation, anddisaggregates Ab42 oligomers and protofibrils [26]. Transgenic micetreated for 2 weeks with compound 4, administered i.c.v., resulted in 40%and 50% decreases in non-fibrillar and fibrillar Ab species, respectively.[26]. To further investigate the structure activity relationship, variousheterocycles and nitrogen-containing TP were prepared [27]. It wasconcluded that attachment of N3u-adenine at C7 side chain in compound


4 provides the strongest MC65 protective activity with an EC50 of 0.31mM.However, tricyclic pyranoisoquinolinones lacking the adenine moeity asin compounds 5 and 6 still possess protective activity with EC50 valuesof 2.49 and 1.25mM, respectively. The 6u-amino group in compound 4enhances potency but is not required for activity.

O

O

N

N

NN

O

H

NH2

4

3′

7

1214

9′

1

10

N

OO

5

NO

OH

OMe

6

Among the various C7 side chain heterocycles prepared, noneshowed better protective activity than the adenine in compound 4. It isnoteworthy that the most potent analog prepared in this class was a2-aminopurine derivative 7 with EC50 of 0.86 mM. Replacement of theoxygen at position 2 with a nitrogen, as in compound 8, provides similarprotective activity (EC50 ¼ 0.35 mM) as that of compound 4 [27].

O

O

N

O

H

7

3′

7

1214

1

10

N

NN

NH2

NONNN

N

OH

NH2

OMe

8


2.2.3 IndolesHighly ordered p-stacking interactions between aromatic ring systems areimportant in self-assembly of complex biological and chemical supramo-lecular structures [28]. Several studies showed that aromatic interactionsmay play a critical role in early-stage amyloid formation by providingstability, as well as order and directionality in the formation of amyloidfibrils, presumably facilitated by restricted geometry of interactionbetween planar aromatic systems [29]. In particular, in amyloid peptidefragments, a high frequency of aromatic residues was noted. When thesearomatic residues were replaced with hydrophobic amino acids, adecrease in the amyloidogenic propensity was observed [30]. Gazit et al.screened various indole derivatives for their ability to prevent formation ofamyloid fibrils using fluorescence spectroscopy, AFM, and electronmicroscopy. Three inhibitors were identified: indole-3-carninol (9), 3-hydroxyindole (10), and 4-hydroxyindole (11) with IC50 values of 85, 100,and 200mM, respectively. These simple indoles effectively inhibited Abfibril formation and prevented cell death induced by 5mM Ab40 in PC12cells in culture. Curiously, analog 9 inhibited Ab fibrillization only at highconcentration, and it did not show a dose dependency on inhibition offibril formation. The inhibitory mechanism of these compounds remainsunclear; however, the authors suggest that the hydroxyl group interactswith the backbone of the peptides preventing the ability of the Ab peptideto create a p-stacking interaction, which limit the fibrillogenesis process.

NH

OH

9

NH

HO

10

NH

OH

11

Recent adsorption studies of Ab solutions on poly(tetrafluoroethylene)surfaces showed that the fluorinated surface strongly promoted a-helixre-formation [31]. A similar effect was observed with solution of Ab inCF3-containing solvents [32]. On the basis of these findings, Torok et al.[33] demonstrated the design and application of a new class of trifluoro-ethylindoles against amyloid fibrillogenesis. Analogs 12a–c showedsignificant inhibitory effect with IC50 values of 0.53, 0.23, and 0.36molinhibitor/molAb. Further structure–activity relationship demonstratedthat the CF3 and OH groups are necessary for binding to Ab peptide.It was suggested that the acidity of the hydroxyl group plays a key role inbinding to one or both lysine residues of the Ab peptide. Interestingly,removing the ester group slightly diminished the inhibitory activity.


NH

CO2EtF3C

HO

X

a; X = Clb; X = Brc; X = I

12a, b, c

Tramiprosate (3-amino-1-propanesulfonic acid; 3APS; Alzhemedt) (13)was found to maintain Ab in a non-fibrillar form, decrease Ab42-inducedcell death in neuronal cell cultures, and also inhibit amyloid deposition[34]. Treatment of TgCRND8 mice with Tramiprosate resulted in significantreduction (B30%) in the brain amyloid plaque load and a significantdecrease in the cerebral levels of soluble and insoluble Ab40 and Ab42(B20–30%) [34]. Although Tramiprosate ultimately failed in a phase IIIclinical trial, it provided a proof of concept that small-molecule inhibitorsof Ab protofibril formation may be a viable approach to AD treatment [5].

13, Tramiprosate

H2N SO O

OH

Another recent development in Ab aggregation inhibitors was thedevelopment of Memoquin (14). This compound was shown to be amultifunctional therapy to AD, acting as an acetyl cholinesterase (AchE)inhibitor (Ki ¼ 2.6 nM), a free radical scavenger, and an inhibitor of Abaggregation [35,36].

14, Memoquin

O

O

HN

NH

NN

OO

Additional small molecules such as Congo red, curcumin, andgalantamine have been described in the literature as inhibitors of Abaggregation, recently reviewed by Hawkes et al. (2009) [37]. Thesemolecules were also found to be inhibitors of a-synuclein, and we willreport on their activity in Section 4.2.


3. TAU AGGREGATION IN ALZHEIMER’S DISEASE

3.1 Tau pathophysiology

Tau is predominantly expressed in neurons where its main function isthought to be stabilizing microtubules, particularly in axons. The taugene consists of 16 exons and alternative splicing results in six isoformsof tau protein ranging in size from 352–441 amino acids [38]. Taustabilizes microtubules by binding to them through an interaction withthe three or four microtubule-binding domains at the C-terminus of theprotein. Stabilization of microtubules by tau in neurons is importantfor maintenance of cellular morphology and transport of molecules andorganelles over long distances [39]. Binding of tau to microtubules isalso regulated post-translationally, primarily through phosphorylation,although other modifications such as glycosylation, ubiquitylation, andproteolysis have been reported for the tau protein [40]. Tau contains W80serine and threonine residues, which are potential phosphorylationsites. The phosphorylation state, which is controlled by a balance ofkinase and phosphatase activity, affects the microtubule-binding affinity.Hyperphosphorylation of tau at many sites, as seen in tauopathies, ofwhich AD is the most common, leads to reduced affinity for micro-tubules, which causes disruption of cellular trafficking leading todegeneration of synaptic terminals. This loss of function may beexacerbated by a toxic gain of function, where higher than normalconcentrations of tau increase the chances of pathogenic conformationalchanges, which in turn lead to the aggregation and fibrillization, whichmight block transport and cause cell death [40].

Phosphorylation of certain residues on tau, specifically S396 and S404,has been shown to increase the fibrillogenic nature of tau and contributeto its accumulation into paired helical filaments [41–43]. Similarly, theremoval of the C-terminus of the protein increases tau aggregation [44,45].It is thought that tau aggregation occurs in a multi-step process wherebytau is phosphorylated and dissociates from microtubules. The unboundhyperphosphorylated tau abnormally localizes to the somatodendriticcompartment of the cell, undergoes conformational changes and furtherphosphorylation. Finally, the hyperphosphorylated tau forms fibrils thataggregate into neurofibrillary tangles (NFTs) [46]. Tau aggregates alsoform in axons and dendrites, called neuropil threads. Both NFTs andneuropil threads are postulated to have a toxic gain of function. In cellmodels, tau aggregation in the cell body causes cell death [47]. In axons,neuropil threads are thought to be toxic because they might physicallyimpair transport, which would be toxic to synaptic terminals [48].

Similar to Ab, oligomeric forms of tau, which are promoted byphosphorylation [49] and are observed in aging and early AD [50], could


also be the toxic species leading to neurodegeneration. In animal models oftauopathy, there is indirect evidence that soluble, not aggregated, forms oftau are toxic: flies expressing human tau can exhibit neurodegenerationwithout fibril formation [51], and in some mouse models of tauopathy,overexpression of tau causes neuronal loss in areas without extensiveneurofibrillary pathology [52]. If oligomeric tau is toxic, formation oflarge aggregates could be viewed as protective because oligomers aresequestered into insoluble neurofibrillary pathology [3,49].

The two principal current strategies targeting tau in neurodegenera-tive disease are (i) reducing tau phosphorylation through inhibition ofspecific protein kinases [53] and (ii) anti-aggregation approaches [54].The issue of whether phosphorylation of tau precedes or follows tauaggregation remains a subject of debate, but reducing tau phosphoryla-tion is regarded by many as the preferred target, and some transgenicanimal studies have shown this to be a valid strategy [53]. In thefollowing sections, structurally diverse small-molecule inhibitors of tauaggregation are described.

3.2 Small-molecule inhibitors of tau aggregation

3.2.1 Phenylthiazolyl hydrazidesIn an effort to develop small-molecule inhibitors of tau aggregation,Mandelkow et al. [55,56] identified compounds related to structure 15 froma high throughput screen of a collection of 200,000 compounds. To establishthe structure–activity relationship (SAR), a series of thiazolylhydrazideswere prepared by synthetic derivatization of R1, R2, R3, and R4 [56].Structure–activity relationship of phenylthiazolyl hydrazides demon-strated that two aromatic rings at R1 and R4, a hydrophobic region onthe thiazole ring, and a hydrogen bonding acceptor on the carboxyl amideare essential for inhibitory effect of tau aggregation. Notably, compound 16showed superior potency with IC50 value of 1.6mM for inhibiting tauaggregation and reduced toxicity when tested in an N2A cell model of tauaggregation [57]. The potency of compound 16 is believed to be due to thehydrogen bonding capacity of the nitro group and to the p-stackinginteractions with the indazole group as confirmed by saturation transferdifference (STD) NMR spectroscopy experiments.

NH

HN

S

NR1

R2

R3

R4NH

HN

S

N

O

N

NH

NO2

15 16


3.2.2 Rhodanine-based inhibitorsMandelkow’s group has also described the SAR of substituted rhodanines(2-thioxothiazolidin-4-ones) [58]. Extensive SAR studies resulted in thepreparation of an interesting biphenyl rhodanine derivative 17, whichdisplayed an IC50 of 170 nM for assembly inhibition and DC50 of 130 nM fordisassembly induction. It is noteworthy that the presence of an aromaticside chain appeared necessary, supporting hydrophobic p-stacking inter-actions of this fragment [59]. Unfortunately, compound 17 and many otheranalogs showed a large discrepancy between in vitro and cell-based activity.Poor physiochemical properties were implicated as the likely cause of poorcellular results, reflecting a need for further optimization of this series.

NS

S

O

17

O

PhO

HO

3.2.3 Cationic thiacarbocyanine dyeThiacarbocyanine dye N744 (compound 18) has been shown to inhibitrecombinant tau fibrillization in the presence of anionic surfactantaggregation inducers with an IC50 value of 0.3 mM [60]. In an effort toincrease potency, a cyclic bis-thiacarbocyanine 19 was synthesized andcharacterized with respect to tau fibrillization inhibition by electronmicroscopy and ligand aggregation state by absorbance spectroscopy[61]. Data showed that the inhibitory activity of the bis-thiacarbocyanine19 was similar to a monomeric cyanine dye, but was more potent with anIC50 of 80 nM. Data reported for these two compounds further suggestthat the inhibitory activity of bis-thiacarbocyanine 19 results frommultivalency. This finding might offer an new mode of interaction fordesign of tau aggregation inhibitors [61].

S

N N

SO O

OHHO

18

S

N N

S

N

S

N

S

Br

Br

19


3.2.4 N-Phenylamines and anthraquinonesAdditional small molecule tau aggregation inhibitors were reported inthe literature derived from N-phenylamines (20) and anthraquinonessuch as daunorubicin (21) and adriamycin (22) [55,62].

HN

O

O

OHNO2N

NO2

O

O

O

OH

OH

OH

O

O

OH NH2

A B C D

O

O

O

OH

OH

OH

O

O

O

OH NH2

A B C D

HO

20 21 22

Compared to the two compound classes discussed earlier (rhoda-mines and phenylthiazolylhydrazides), the N-phenylamines displayedfar lower potencies in vitro and in cells, which precluded their use inin vivo models [54]. The b-hydroxyenone moiety in anthraquinone, alsoobserved in other inhibitor classes, such as flavonoids and naphthoqui-nones, may play a significant role in the inhibitory potency of thesechemotypes. Compounds 21 and 22 were able to inhibit the aggregationof the K19 tau construct and induced the disaggregation of preformedaggregates [55]. Substitution on the ring A in compound 21 does notappear to play a critical role of inhibitory activity. Ring D in compounds21 and 22 bearing the sugar moiety is moderately sensitive to thesubstitutions on that ring, which indicates further opportunity forstructural modifications to improve the inhibitory potency. Despite theobserved activity of anthraquinones, it should be noted that theyare known cytostatics and present a hazardous toxicological profile,which preclude them from being desired therapeutics for the chronictreatment of AD.

3.2.5 Polyphenols, phenothiazines, and porphyrinsTaniguchi et al. [63] reported three classes of compounds (phenols,phenothiazines, and porphyrins) that were able to inhibit aggregation ofhuman tau 46. Polyphenols such as compound 23 shared the SARdescribed in the previously section for anthraquinones with character-istic b-hydroxyenone moieties. Phenothiazines possess a tricycliccore that incorporates sulfur and nitrogen atoms on the central ringsystem. They possess positively charged atoms and hydrophobic


aromatic groups similar to the benzothiazole inhibitors describedpreviously. The charge-neutral phenothiazines are reported to have goodblood–brain barrier permeability. The planarity and aromaticity of thecentral heterocyclic core appears to play a critical role of tau aggregationinhibitory activity. In particular, compound 24 (MTC, methylthioniniumchloride) also known as methylene blue was reported as a potent in vivotau aggregation inhibitor with sub-micromolar potency in cells [64,65].Data from the phase II clinical trial with methylene blue reported asignificantly lower rate of decline of cognitive functions compared with aplacebo (81%, po0.0001). Although these data are preliminary andrequire further confirmation, it does present a promising approach forthe management of AD and a potential proof of concept for the strategyof inhibiting tau aggregation.

O

OHHO

HO

HOO OH

OH

23

S

N

N N

24

Porphyrins such as compound 25 are the only organometalliccompounds that bind differently than other reported tau aggregationinhibitors. The inhibitory activity depends on a central metal (iron or zinc)as phthalocyanine compound lacking the central metal has a weakerinhibitory potency [63]. It has been suggested that the coordination ofthe metal center with histidine on the protein–protein interface plays asignificant role in tau aggregation inhibition [66].

N

N

N

N

HOOCC2H4

HOOCC2H4

Fe

25


4. a-SYNUCLEIN AGGREGATION IN PARKINSON’S DISEASE

4.1 Biochemistry of a-synuclein aggregation

a-Synuclein is a small 140 amino acid, natively unfolded protein with littlesecondary or tertiary structure whose aberrant aggregation has beenlinked to the etiology of PD. a-Synuclein can assume either an a-helicalconformation upon binding to lipids and membranes or a b-sheetconformation upon aggregation at high concentrations, elevated tem-perature, agitation, or in the presence of metals, simple alcohols, ordetergents [67,68]. Increases in b-sheet content are associated withformation of small soluble oligomers, larger protofibrils, and the macro-molecular fibrils. Dimers are the smallest oligomeric aggregates withlimited b-sheet structure and have been proposed to act as seeds in thenucleation-dependent aggregation of a-synuclein [69,70]. Both oligomersand protofibrils have increased amounts of b-sheet structure comparedwith monomeric or lipid bound a-synuclein. The predominantly observedstructure of the protofibrils is globular, although they are also able to formrod-like filaments as well as annular rings that can insert into lipidmembranes enabling leakage of small molecules [67]. These annularprotofibrils have been implicated in the pathogenesis of PD by the virtuethat the A53T and A30P mutations either increase the propensity ofa-synuclein to form annular protofibrils or stabilize them. The annularprotofibrils can insert into lipid membranes and enable leakage of ions,neurotransmitters, and small dyes [67]. Although these properties makethe oligomers and protofibrils attractive targets, their size and morpho-logical continuum present a tremendous logistical hurdle for assaydevelopment and structure activity relationship analysis.

Eventually, a-synuclein aggregates into insoluble fibrils that have avery high b-sheet content. It is the insoluble fibril that is deposited inLewy bodies in the brains of patients suffering from PD, which are thedefining pathological hallmark of the disease. A long-standing issuehas been whether Lewy bodies are markers of a neurodegenerativeprocess or a protective mechanism, serving as a means to sequester toxicoligomers and protofibrils [3]. Still, fibrils, as the end point of aggregationand the hallmark of pathological PD, have been targeted extensively inattempts to discover small-molecule inhibitors of a-synuclein aggrega-tion as a treatment to slow down or halt the progression of PD.

Many molecules have been shown to inhibit fibril formation(curcumin, Congo red, epigallocatechin gallate, peptide-mimetics, andnon-steroidal anti-inflammatories) including the flavonoid, baicalein, andthe neurotransmitter, dopamine [71–77]. More detailed studies with thelatter two molecules reveal that while these compounds are inhibiting


fibril formation, they are also stabilizing oligomers/protofibrils [77]. In thecase of dopamine, the aggregates are much more stable than theoligomers/protofibrils produced under more ‘‘conventional’’ conditions,in that they are SDS stable. Caution should also be exercised whentargeting disruption of fibril b-sheet as the tertiary structure of manyproteins contains b-sheets. If inhibitors of fibril formation are to bedeveloped as a therapeutic for PD, appropriate screens should bedeveloped to assess their ability to stabilize soluble oligomers/protofibrils,to assess the toxicity of any stabilized species and the ability to disrupt theb-sheet structure in other proteins.

4.2 Small-molecule inhibitors of a-synuclein aggregation

Conway et al. [77] have shown that catecholamines such as compound 26can inhibit the formation of a-synuclein fibrils by stabilizing oligomericintermediates. Li et al. [78] reported that the oxidation state of cate-cholamine affected the inhibitory activity. As such, dopaminochrome (27),one of the oxidation products of dopamine, was shown to be more potentat inhibiting a-synuclein fibril formation than the parent dopamine [78,79].This also raises the possibility that the protein may be covalently modifiedby the dopaminochrome or dopamine under oxidative conditions.

HO

HO26

NH

O

O

27

NH

Various polyphenolic compounds such as flavonoids have also beenshown to be effective inhibitors of a-synuclein aggregation [80]. One ofthese inhibitors is baicalein, 28, isolated from the Chinese skullcap plant(Scutellaraia baicalensis), has been shown to directly bind to a single site ona-synuclein with submicromolar affinity and, as such, inhibit formationof fibrils through the stabilization of oligomers [71,81]. It has beensuggested that the quinone oxidation by-product (29) of baicalein isresponsible for the observed inhibitory activity.

O

OOHHO

HO

28

O

OOHO

O

29


Other small-molecule inhibitors of a-synuclein have been identified inthe literature such as Congo red.[80]. Polyphenolic compounds suchas rifampicin, curcumin, and tetracycline are capable of inhibiting botha-synuclein [79] and Ab aggregation [82] in a concentration-dependentmanner with some potency (IC50o10 mM). Li et al. [83] point outthat many of these polyphenolic anti-fibrillogenic compounds haveantioxidant activities and readily oxidize in the presence of atmosphericoxygen to form quinones and thus may all act through oxidative modifi-cation of peptides. This modification, formation of quinone adductsor formation of Schiff-base, may act to inhibit fibril formation byconstraining the peptides to a conformation not compatible with thetight orderly packing of b-sheets found in fibrils, but at the sametime stabilizing soluble oligomers. Although some of these compoundshave been demonstrated to protect cells against a-synuclein over-expression, in agreement with the biochemistry of a-synuclein aggrega-tion [84], many reports either attribute the efficacy of the compoundsto metal chelation and antioxidant activity or do not show themechanism to be inhibition of protein aggregation. Furthermore,caution in pursuing these polyphenolic and catecholamine compoundsas aggregation inhibitors is urged, however, as one long-standinghypothesis postulates that it is actually the soluble oligomers, notthe insoluble fibrils that are neurotoxic in PD. Still, these polyphenolic/anti-oxidant types of compounds appear to share a commonmechanism: oxidation-dependent modification of a-synuclein, whichinhibits fibril formation. If the issues, raised earlier, are resolved in thedrug discovery process, then small-molecule inhibitors of a-synucleinaggregation could be key therapeutics for PD [82]. However, in general,it has been noted from the current literature that there are fewreports and limited efforts to develop structural activity relationshiparound the compounds listed earlier, suggesting that drug discoverystrategies to identify small-molecule inhibitors of a-synuclein arestill evolving.

5. CONCLUSION

In summary, the literature reviewed in this chapter imply two generalassumptions regarding the inhibition mechanism of amyloid proteinfibril formation by small molecules: (a) specific structural conformation isnecessary for b-sheet interaction and stabilization of the inhibition–protein complex; (b) aromatic interaction between the inhibitor moleculeand the aromatic residues in the amyloidogenic sequence, potential‘‘hot spots,’’ may direct the inhibitor to the amyloidogenic core blocking


the protein–protein interaction. These assumptions are highly relevantfor future design of small-molecule inhibitors as therapeutic agentsfor the treatment of amyloid-associated diseases. In conclusion, drugdevelopment for AD and PD remains highly active, and there is arealistic expectation that new therapies will be evaluated in clinical trialsin the near future.

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