Chemical Chaperones as Novel Drugs for Parkinson’s Disease

TRMOME 1531 No. of Pages 14

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Review

Chemical Chaperones as Novel Drugs for Parkinson’s Disease Jordi Pujols,1,* Samuel Peña-Díaz,1 Irantzu Pallarès,1 and Salvador Ventura1,* Parkinson's disease (PD) is characterized by progressive loss of dopaminergic neurons and the accumulation of deposits of α-synuclein (α-syn) in the brain. The pivotal role of α-syn aggregation in PD makes it an attractive target for potential disease-modifying therapies. However, the disordered nature of the protein, its multistep aggregation mechanism, and the lack of structural information on intermediate species complicate the discovery of modulators of α-syn amyloid deposition. Despite these difficulties, small molecules have been shown to block the misfolding and aggregation of α-syn, and can even disentangle mature α-syn amyloid fibrils. In this review we provide an updated overview of these leading small compounds and discuss how these chemical chaperones hold great promise to alter the course of PD progression.

Highlights Orthogonal strategies such as drug repositioning, rational design, and highthroughput screening have identified molecules that target α‐synuclein (αsyn) aggregation, the central molecular event in Parkinson’s disease (PD). The anti-aggregation compounds under development target the complete spectrum of α-syn conformers through different mechanisms: monomer stabilization, prevention of dimer formation, stabilization or disruption of oligomers, avoidance of secondary nucleation, and dismantling of fibrils.

α-Syn: A Therapeutic Target in PD

Synucleinopathies are a series of heterogeneous neurodegenerative disorders that affect N10 million people worldwide. No neuroprotective or neurorestorative therapies are currently available for synucleinopathies, which include PD, multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and neurodegeneration with brain iron accumulation [1–4]. These diseases all share common neuropathological features and are characterized by the accumulation of deposits of the protein α-syn in the central and peripheral nervous systems, although the cellular and anatomical localization of the aggregates differs among the diseases [5]. PD is the most common synucleinopathy and the second most prevalent neurodegenerative disorder after Alzheimer’s disease (AD), and affects 1% of people over 60 years of age [6]. The motor symptoms of PD, including bradykinesia (see Glossary), rigidity, resting tremor, postural instability and gait disturbances, are mainly attributed to a loss of dopaminergic neurons in the substantia nigra pars compacta with concomitant dopamine deficiency in the basal glia, a key brain region for motor control [7,8]. The affected neurons exhibit α-syn cytoplasmic inclusions in the neuronal body (Lewy bodies, LBs) and/or fibrils deposited in neuronal processes (Lewy neurite, LNs) [9]. Mutations in the gene that encodes this protein (SNCA) are associated with familial cases of PD [10,11], and duplications and triplications of the SNCA gene lead to earlyonset disease [12,13]. In addition, transgenic animals expressing wild-type or mutated α-syn variants exhibit brain amyloid inclusions and develop synucleinopathy-like features [14]. These findings support a connection between α-syn and PD. Accordingly, α-syn-targeting agents are being developed in the search for a first disease-modifying therapy for PD.

α-Syn Misfolding and Aggregation

α-Syn is abundant in neuronal cells and is involved in synaptic transmission where it contributes to the release of synaptic vesicles and synaptic membrane recycling [15]. In healthy neurons, α-syn is found either bound to membranes or in a monomeric, soluble and disordered form in the cytosol, and the two species interconvert in a rapid equilibrium. It has been suggested that α-syn can also form helical tetramers in the cytoplasm [16], but this possibility remains under debate [17]. Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx

The potential of particular molecules to halt neurodegeneration in PD is already being evaluated in clinical trials. If successful, they might benefit patients suffering from other α-synucleinopathies. The development of biomarkers to allow evaluation of therapeutic efficacy will be necessary to accelerate and bring down the high cost of current clinical trials for PD.

1

Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

*Correspondence: [email protected] (J. Pujols) and [email protected] (S. Ventura).

https://doi.org/10.1016/j.molmed.2020.01.005 © 2020 Elsevier Ltd. All rights reserved.

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During pathogenesis, α-syn misfolds and self-assembles into insoluble amyloid fibrils displaying a typical cross-β-sheet fold [18]. The conformational plasticity of α-syn is dictated by its unique sequence, which can be subdivided into three distinct domains with very different physicochemical properties (Figure 1A). The N-terminal domain (residues 1–61) is highly conserved and includes five of the seven 11-residue repeats in the α-syn sequence. This region is disordered in solution, but the repeats confer an amphipathic character that assists a conformational shift into an α-helix in the presence of phospholipids and membranes [19]. This functional protein–lipid interaction might become pathological if the concentration of α-syn locally increases because, at a high protein– lipid ratio, membranes facilitate the nucleation step of α-syn amyloid formation [20]. Importantly, all identified α-syn missense mutations associated with clinical variants of PD and DLB (A30P, E46K, H50Q, G51D, A53E, and A53T) map onto this membrane-binding segment. The central region of α-syn (residues 61–95) has a hydrophobic character and is known as the nonamyloid component (NAC) because it was identified as the second major component of brain

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Figure 1. α-Synuclein (α-Syn) Primary Structure, Cryo-Electron Microscopy Structures, and Atomic Models of α-Syn Strains. (A) A schematic representation of the α-syn primary structure, domains, and specific mutations involved in familial versions of Parkinson's disease (PD). Positively and negatively charged positions are shown as blue and red dots, respectively, and the positions of repeats are shown with a green line. (B) The upper panels show cryo-electron microscopy images of two α-syn fibril strains. The lower panels show the side and top views of the atomic models of the corresponding strains; the backbones are represented as ribbons and the non-amyloid component (NAC) regions are colored. In the center, the macromolecular modeling of the amyloid fibril structure for both strains is shown (adapted, with permission, from [32]).

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Glossary Bradykinesia: one of the most common symptoms of PD. It is characterized by an unusual slowness and a lack of accuracy in carrying out voluntary body movements. Cmax: maximum concentration, a pharmacokinetic parameter to evaluate the bioavailability of a particular drug in a given tissue. It allows the drug dose to be adjusted according to the therapeutic concentration window. Cross-β-sheet fold: the intermolecular stacking of parallel or antiparallel β-strands along the fibrillar axis is an amyloid hallmark. This structural disposition constitutes a thermodynamic sink that favors the formation of such structures. Dopamine replacement: pharmacological strategy aimed at treating the motor symptoms of PD by compensating for the low dopamine levels in the substantia nigra pars compacta. L-dopamine (levodopa), a precursor of dopamine, is administered in combination with carbidopa, an inhibitor of dopa decarboxylase. Dopamine agonists are emerging as an alternative therapy to L-dopa–carbidopa. Off-pathway aggregates: oligomeric or higher-order species populated during the self-assembly process that cannot evolve into fibrillar structures because of conformational misarrangements. These structures are usually not toxic and might be degraded by the cellular proteostatic machinery. Prion proteins: proteins with the ability to self-assemble and perpetuate the misfolded conformation. This phenomenon is transmissible to cells or even between organisms, and is classically linked to severe human diseases, although functional prion proteins are present in fungi and mammals. Strains: external conditions, such as pH and ionic strength, influence the formation of intermolecular contacts and the arrangement of the polypeptide backbone during the self-assembly process, resulting in structural polymorphs that differ in their transmissibility, degradation resistance, and morphology. Substantia nigra pars compacta: this subregion of the midbrain is mainly formed by dopaminergic neurons that project their axons to the dorsal striatum,

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amyloid plaques in AD [21]. This region is highly amyloidogenic when isolated and has been shown to fold into a Greek-key motif at the core of the mature fibrils (Figure 1B) [22]. In the disordered and soluble state of α-syn, this aggregation-prone region is shielded from the solvent by transient intramolecular contacts. The C-terminal domain (residues 96–140) of α-syn contains 15 acidic residues and thus has a high negative charge density. It has been reported that C-terminal truncation exacerbates α-syn aggregation and cytotoxicity, which suggests that the electrostatic repulsions exerted by this region counteract α-syn aggregation [23]. Interestingly, this domain can be phosphorylated at tyrosines 125, 133, and 136, and at serine 129, thus affecting the charge distribution and polarity of this region. In addition, C-terminal truncated forms of α-syn account for up to 30% of total α-syn in the LBs of PD patients [24]; thus, it is likely that these more aggregation-prone species play a relevant, but unknown, role in pathogenesis.

and is involved in the regulation of body movement. Target engagement: in vivo validation of the expected pharmacological activity for a given drug. It does not include efficacy parameters; however, the molecular interaction with the specific target must be confirmed.

Similarly to most amyloidogenic proteins, the formation of α-syn fibrillar structures is preceded by the assembly of the monomeric protein into small and diffusible oligomers and protofibrils (Figure 2) [25]. Multiple lines of evidence suggest that these metastable species, and not the mature fibrils, might be the main culprits for neuronal degeneration in PD [26]. These oligomeric and

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Figure 2. Schematic Representation of the Aggregation Reaction of α-Synuclein (α-Syn). The aggregation of α-syn follows a slow process that comprises different assemblies of the protein: monomeric (A), oligomeric (B), and fibrillar (C), or amorphous (D) aggregates. Accordingly, α-syn undergoes different processes during the onset and progression of Parkinson's Disease (PD): protein–lipid interaction (1), oligomerization (2), fibril elongation (3), transmission (4), seeding (5), and amorphous aggregation (6). Colored symbols illustrate the stages of α-syn aggregation that are targeted by small molecules.

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protofibrillar α-syn forms can be transmitted from affected to neighboring healthy neurons, where they can seed the aggregation of native α-syn in a prion-like manner (Figure 2) [27,28]. This prionlike transmission provides a plausible mechanistic framework for the Braak’s hypothesis, which postulates that PD pathology may originate in synapses of the peripheral nervous system (PNS) and invade the brain via retrograde axonal transport through the vagus [29,30]. This view is consistent with the presence of pathological α-syn aggregates in the PNS of patients long before PD diagnosis [31]. Importantly, the neuron-to-neuron propagated amyloid seeds seem to exhibit a conformational diversity similar to those observed for the strains of prion protein. This diversity leads to the assembly of the endogenous protein into polymorphic fibrils that, despite sharing a common cross-β architecture, differ in their structural arrangement (Figure 1B) [32]. As in the case of the prionopathies, the specific conformational features of these strains might result in a selective preference to invade and deposit in different brain cell types. This behavior would explain why the common aggregation of α-syn might result in different pathologies. It is important to state here that, although the association of LB pathology with clinical dysfunction is well established, ~20% of people over 70 years of age display α-syn inclusions in their brains, without associated motor or memory problems. In addition, LBs also appear in unrelated neurodegenerative disorders such as AD. This has led to the hypothesis that LBs might be a way to accumulate excess α-syn in an inert compartment and are not the cause of PD [33]. This should be kept in mind in case the α-syn-centric strategies we describe in the next sections fail to demonstrate clinical efficacy.

Targeting α-Syn Aggregation

Different α-syn-targeted strategies and tools have been developed in recent years to modulate its misfolding and aggregation in the context of synucleinopathies. These strategies include: (i) SNCA gene-silencing approaches to knock out or decrease the neuronal levels of the protein, (ii) strategies to increase the clearance of soluble or aggregated α-syn by potentiating autophagic and proteasomal activities, and (iii) agents aimed at preventing the formation and/or propagation of the toxic aggregated species [34,35]. Different agents targeting the aggregation of α-syn in the central and peripheral nervous systems are also in early-stage development [36]. These include antibodies, vaccines, molecular chaperones, and small molecules. Despite their selectivity, protein-based therapeutics usually have difficulty in passing the blood–brain barrier (BBB) and may trigger collateral immunological reactions. Therefore, small molecules remain the default option for targeting brain diseases [37]. Although conventional structure-based drug design initiatives are hampered by the disordered nature of monomeric α-syn and the lack of high-resolution structures of toxic oligomers, a handful of small molecules have already shown promising activity in animal models of PD, and some have already entered into clinical trials. In the following sections we compile and dissect what we believe to be the leading smallmolecule-based initiatives to tackle α-syn aggregation in PD (Figure 3). These small molecules constitute a group of structurally diverse compounds that are discussed in terms of their molecular origin, their mechanism of action, the strategies used for their identification, and their current status of development (Figure 3 and Table 1).

Natural Compounds: Establishing the Foundation for α-Syn-Targeted Therapies for PD Natural compounds represent a major source of medications and important structural scaffolds to develop new drugs, and many of them interact specifically with biological macromolecules, mainly proteins. The ability of small molecules to interfere with α-syn aggregation was first reported in the early 2000s. Analysis of 169 molecules revealed that several natural catecholamines, 4

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Figure 3. Chemical Structures. Molecular structures of selected modulators of α-synuclein aggregation, grouped by molecular class and mechanism of action. Abbreviations: EGCG, epigallocatechin-3-gallate; LMTM, leuco-methylthioninium bis(hydromethanesulphonate).

including dopamine, L-dopa, epinephrine, and norepinephrine, were able to inhibit the formation of α-syn amyloid fibrils [38]. Oxidized derivatives of dopamine diverted the aggregation of α-syn to off-pathway aggregates. This activity seems to depend on the interaction of dopamine with the 125YEMPS129 sequence at the C terminus of the α-syn monomer [39,40]. Unfortunately, dopamine-induced oligomeric species triggered brain damage in animal models of the disease, which excluded dopamine and its derivatives as disease-modifying therapies for PD [41] while questioning the status quo of dopamine replacement as the prevailing symptomatic treatment for PD. Despite these limitations [42], the discovery of a first molecular entity modulating α-syn aggregation through a direct protein–compound interaction paved the way for future drug discovery programs. In the following years, novel naturally existing small molecules acting as inhibitors of α-syn fibrillogenesis were discovered [43–45]. Among them, we highlight the polyphenols. These molecules display several phenyl rings and hydroxyl groups that seem to act coordinately, binding aggregates through π interactions and destabilizing them by disrupting the hydrogen bond network of the β-sheet. The number of phenolic moieties and the position of the hydroxyl groups are critical for their activity. Curcumin, epigallocatechin-3-gallate (EGCG), baicalein, rosmarinic acid, kaempferol, tanshinones, tannic acid, myricetin, and quercetin all belong to this family (Box 1) [46,47]. Similarly to dopamine, many polyphenolic molecules interact with the charged and disordered C terminus of α-syn [48–50], and several of these compounds also display a catechol moiety. These compounds attenuate α-syn amyloidogenicity by facilitating intramolecular contacts and decreasing bimolecular self-assembly, thus driving the oligomerization process to the formation of nontoxic aggregates with low β-strand content, remodeling preformed toxic oligomers, or Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx

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Table 1. α-Syn Aggregation Inhibitors Validated in Animal Models of PD Name

Molecular weight

Origin

Identification

Mechanism

Animal model

Clinical phase

Refs

Curcumin

368.4

Natural

Polyphenol scaffold

Monomer stabilization Fibril disassembly

Rat

Preclinical

[35,41,51]

EGCG

458.4

Natural

Polyphenol scaffold

Off-pathway aggregation Fibril disassembly

Drosophila melanogaster, rat, monkey

Phase II

[40,44]

Baicalein

270.2

Natural

Polyphenol scaffold

Off-pathway aggregation Fibril disassembly

Mouse

N.a.a

[42,50]

Fasudil

327.8

Synthetic

Repositioning

Monomer stabilization

Mouse

Preclinical

[58]

Squalamine

627.7

Natural

Repositioning

Lipid-dependent nucleation

Caenorhabditis elegans

N.a.b

[70]

Trodusquemine

685.1

Natural

Repositioning

Lipid-dependent nucleation Seeding prevention

C. elegans

N.a.c

[71]

LMTM

287.4

Natural

Repositioning

Unknown

Mouse

N.a.b

[36,75]

CLR01

782.6

Synthetic

Rational design

Off-pathway aggregation Fibril disassembly

Mouse, Danio rerio

N.a.

[82,83]

NPT100-18A

493.6

Synthetic

Rational design

Lipid-dependent nucleation

Mouse

N.a.

[85]

NPT200-11

N.a.

Synthetic

Rational design

Lipid-dependent nucleation

Mouse

Phase I

[86]

Anle138b

343.2

Synthetic

High-throughput screening

Anti-oligomeric

Mouse

Preclinical

[94]

SynuClean-D

354.2

Synthetic

High-throughput screening

Fibril disassembly

C. elegans

N.a.

[97]

a

N.a., not available/not applicable. Phase II and III in other disorders. c Phase I in other disorders. b

Box 1. Polyphenols – Additional Information Natural polyphenols comprise N8000 compounds that can be found in some types of food (mainly vegetables and fruits), herbal beverages, and wine. They are considered to be beneficial molecules because they are potent antioxidants and anti-inflammatory agents. Accordingly, their consumption has been linked to neuroprotection and alleviation of atherosclerosis, cancer, and cardiovascular deficits [107]. They display low toxicity and can usually be administered at high doses, and have already been assayed in N300 clinical trials for different therapeutic indications. The unavoidable question is why the investigation of the therapeutic potential of these molecules in PD is discontinued when they approach the clinic if they exhibit outstanding performance in vitro and many of them demonstrate neuroprotection. The reason lies in a combination of a lack of favorable pharmacokinetics/pharmacodynamics and economic issues. First, they display poor absorption and rapid degradation upon oral administration. Second, polyphenols are metabolically unstable and are easily transformed into secondary metabolites, questioning the therapeutically relevant species and their effective concentration in the brain. Last but not least, there is an evident lack of economic interest because none of these molecules can be intellectually protected by a product patent. Polyphenolic compounds share a common structural scaffold, which includes a series of benzene rings with one to three hydroxyl, ketone, or carboxyl groups as well as structural ramifications that determine the complexity and size of the molecule. They are promiscuous inhibitors that interact with aggregation-prone regions in structurally unrelated amyloidogenic proteins. In this way, EGCG and curcumin have been shown to interfere with the aggregation of 14 different amyloidogenic proteins, including the islet amyloid protein precursor (IAPP), Aβ42, Sup35, tau, transthyretin, huntingtin, and PrP, among others [108–116]. Despite being potentially advantageous from a therapeutic point of view, this lack of specificity constitutes a barrier to their commercialization.

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disentangling mature fibrils. Remarkably, several polyphenols, such as curcumin, baicalein, myricetin, and EGCG, combine two or more of these activities [50–53]. Several polyphenols have been assayed in animal models of the disease. As a general trend, their administration resulted in neuroprotection and attenuation of motor deficits [46,54–59]. However, the data do not prove a direct connection between the observed protective effect and the anti-aggregation properties of polyphenols, mainly because the large majority of these studies were performed in neurotoxin-induced models of PD, and there is only weak evidence of target engagement (Box 2). Indeed, it cannot be disregarded that the antioxidant and anti-inflammatory properties of polyphenols counteract neurotoxin-induced brain damage, in addition to or instead of their anti-aggregation activities [60,61]. Curcumin and its derivatives deserve special attention because they are one of the few polyphenols that have been tested in a transgenic animal model of PD (Syn-GFP mice). The study reported motor and behavioral improvements, although target engagement remained unclear [62]. Remarkably, the neuroprotective properties of EGCG were validated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated monkeys [63], and the molecule reached Phase II clinical trials for the treatment of PDi. The study comprised 480 individuals with PD who were randomized and treated with up to 1.2 g/day of green tea polyphenols (GTPs) or placebo. After 6 months of treatment with GTPs, the unified PD rating scale (UPDRS) score, which evaluates both motor and non-motor symptoms, was significantly improved, indicating that GTPs provide mild symptomatic benefit in early untreated PD. These results illustrate the potential of natural molecules as therapeutics for PD (Box 1).

Drug Repositioning: A Shortcut for α-Syn-Targeted Therapies Drug discovery is characterized by high attrition rates that are mainly attributed to a lack of pharmacological efficacy, deficient safety profiles, or both. In addition to its intrinsic high risk, the economic investment required for such research initiatives is huge, averaging 5–10 million USD per molecule. In this context, drug repositioning offers an opportunity to reduce the risks and costs of therapeutic development. Successful therapies in cancer and in cardiovascular and neurodegenerative diseases rely on this strategy [64]. Briefly, drug repositioning consists of the identification of existing compounds that are already developed or even licensed for a different Box 2. Animal Models of PD Animal models of PD are important tools to develop and test effective pharmacological drugs. They allow the assessment of target engagement, drug efficacy, pharmacokinetic parameters, administration route, and toxicity before drugs enter Phase I clinical trials. Classically, mouse animal models of PD have been generated by chemically induced neurodegeneration of the nigrostriatal brain region. Treatment with particular neurotoxins diminishes dopamine levels, triggering some of the motor symptoms of PD [117]. Neurotoxin-induced models are optimal for testing symptomatic treatments such as dopamine replacement. However, these animals lack α-syn inclusions, and therefore they are poorly suited to test disease-modifying therapies. In addition, chemically induced neuronal damage bypasses the molecular events underlying the natural onset and progression of the disease, precluding target engagement evaluation. Most of the therapeutically relevant mammalian models of PD rely on the overexpression of the wild-type or mutated variants (A53T and A30P) of α-syn under the control of an endogenous neuronal promoter. These transgenic animals exhibit accumulation of α-syn deposits and usually present motor symptomatology, thus linking α-syn aggregates to the progression of specific pathological and behavioral traits. However, the phenotypes of these genetic models are often very different from the human condition, mainly because the aggregation of α-syn occurs simultaneously in all brain regions and is not localized in the dopaminergic neurons, which essentially remain intact. The same is true for nonmammalian transgenic models of PD, including those in Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio. Although they constitute easy-handling systems that can be used for screening purposes, the reduction of α-syn aggregates cannot be taken as a translational proof of concept. To overcome this limitation, new models of PD intend to restrict the aggregation of α-syn to the striatum by adeno-associated virus delivery of the human α-syn gene or by injecting human brain-derived or synthetic fibrils into transgenic animals. These models exhibit significant dopaminergic loss and are useful as a proof of principle for anti-aggregation compounds; however, they are still far from recapitulating the complexity of human PD.

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indication [65]. Accordingly, the safety of these molecules is guaranteed because all of them have undergone Phase I clinical trials. Taking advantage of this rationale, fasudil, squalamine, trodusquemine, and leuco-methylthioninium bis(hydromethanesulphonate) (LMTM) have been repurposed as therapeutic candidates for PD. Fasudil is an inhibitor of human Rho kinase [66], whose overactivation is responsible for several types of vascular disorders [67]. This small molecule has been approved to treat cerebral vasospasm in China and glaucoma in Japan [68]. This compound crosses the BBB and was shown to induce neuroprotection in MPTP-intoxicated mice and increased the survival of dopaminergic neurons, an effect that was initially attributed to its activity as an inhibitor of brain Rho kinase [69]. A subsequent study demonstrated that fasudil interacts with the aromatic side chains of Tyr133 and Tyr136 at the C terminus of monomeric α-syn. Fasudil binding to α-syn decreases the nucleation and elongation constants of the in vitro aggregation reaction and reduces intracellular accumulation of α-syn in a cellular model of the disease, all in a concentration-dependent manner [70]. The administration of the molecule to A53T α-syn transgenic mice demonstrated target engagement, with a significant reduction in α-syn deposits and cognitive rescue, although motor improvement was poor. Squalamine and trodusquemine are a pair of steroid-polyamine molecules originally isolated from the dogfish shark Squalus acanthias because of their antimicrobial activity [71,72]. Remarkably, they account for more than 15 clinical trials, including a Phase III trial to test squalamine efficiency in the treatment of age-related macular degeneration [73]. These compounds behave as cationic lipids that interact with the inner leaflet of the plasma membrane [74,75] and destabilize protein– membrane contacts [76]. Polyamines can also interact with the negatively charged C terminus of α-syn [77]. Thus, it was not surprising that both compounds were able to interfere with the oligomerization of α-syn at the membrane moiety by restoring its random coil conformation, promoting its dissociation, and reducing the amount of membrane-bound oligomers, thus helping to avoid subsequent lipid-induced α-syn aggregation [78,79]. Their activity was confirmed in a neuronal cell model, where they alleviated mitochondrial dysfunction. In addition, both molecules reduced the number of intracellular α-syn aggregates and increased motility and lifespan in a Caenorhabditis elegans model of PD. However, only trodusquemine can bind to the surface of fibrils and interfere with secondary nucleation events, which makes this compound more effective. In addition, in contrast to squalamine, trodusquemine can cross the BBB and is better suited for therapeutic indications. An additional case of successful repositioning is methylthioninium chloride (MTC), also known as methylene blue. This phenothiazine, together with its reduced stable LMTM variant, is an inhibitor of tau protein aggregation [80,81] and has reached Phase III clinical trials as a therapy for AD [82]. Based on this evidence, MTC and LMTM were also validated as potent inhibitors of α-syn in a cellular model of PD as well as in the L58 and L62 wild-type α-syn transgenic mice lines, where their administration reduced brain α-syn aggregates [83]. Despite the lack of mechanistic information on the nature of the protein–drug interaction, the fact that LMTM enhances both autophagy and mitochondrial respiration [84], while displaying optimal toxicological and pharmacokinetic properties, makes this molecule a particularly interesting candidate for further clinical study in PD. It is likely that the safety profiles of the above-described drugs will allow them to enter the clinic very soon.

Rational Drug Design: De Novo Discovery of α-Syn Aggregation Inhibitors

The intrinsically disordered nature of α-syn and the complexity of the aggregation process preclude the use of conventional drug discovery strategies which typically exploit 3D protein 8

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structures to design drugs that dock into protein binding pockets or protein–protein interaction sites [85–87]. In the case of α-syn, rational discovery usually targets protein residues involved in the weak noncovalent intermolecular contacts that initiate α-syn dimerization and/or oligomerization. CLR01 and NPT100-18A are two structurally unrelated small molecules that illustrate the success of the approach. The molecular tweezer CLR01 displays a curved structure that creates a concave negatively charged cavity. This design allows CLR01 to bind specifically to exposed Lys side chains through transient interactions. CLR01 has been shown to efficiently inhibit several amyloidogenic proteins [88,89] and was able to block α-syn aggregation in a dose-dependent manner at substoichiometric ratios [90]. In addition, mature α-syn fibrils became dismantled after incubation with a 10-fold excess of CLR01. The primary site of interaction for CLR01 is well defined and involves Lys10 and Lys12 at the N terminus of α-syn. The compound reduces the inherent toxicity of α-syn aggregates in cellular models and in a transgenic zebrafish model of PD. The intracerebroventricular and subcutaneous administration of CLR01 in a transgenic mouse model of PD (Thy1-αsyn) attenuated motor dysfunction. However, it did not reduce aggregated α-syn levels [91]. CLR01 exhibits favorable pharmacokinetics and toxicity profiles [92], but the lack of demonstrable target engagement in a mammalian model of the disease might limit its further development. Another remarkable example of rational design is the NPT100-18A molecule from Neuropore and its lead derivative NPT200-11 that were developed in partnership with UCB Pharma [93,94]. NPT100-18A, a compound aimed at interfering with α-syn dimerization, was developed on the assumption that α-syn oligomerization could be initiated by the formation of dimers on membrane surfaces [95–97]. Molecular dynamic simulations suggested the existence of a pharmacophore responsible for dimerization that comprises residues 96–102 at the α-syn C terminus [96,97]. NPT100-18 is the best performer from a chemical library of 34 peptidomimetic compounds targeting the predicted protein–protein interaction region. NPT100-18A precludes the formation of α-syn mature aggregates in vitro and releases monomeric α-syn by destabilizing oligomers formed in a membrane-mimetic environment. NPT100-18A reduces the accumulation of aggregates and their subsequent toxicity in primary neuronal cells overexpressing α-syn. Administration of this molecule to E57K α-syn transgenic mice revealed neuroprotection in several brain regions, including the neocortex, hippocampus, and striatum, as a result of an overall decrease in the aggregated and oligomeric species of α-syn. Unfortunately, no concluding results were obtained for the substantia nigra [93]. Despite encouraging target engagement and symptomatic amelioration, NPT100-18A pharmacokinetic properties were far from optimal, with poor BBB permeability and a low concentration of the bioactive molecule in the brain. In an effort to bypass these hit limitations, Neuropore developed the lead compound NPT200-11, a nonpublic structure, that increased the Cmax value and the brain plasma exposure ratio by 129- and 66-fold, respectively. This allowed the attainment of optimal brain concentrations in mice after an oral administration of 10 mg/kg. This pharmacokinetic optimization did not significantly affect target engagement or symptomatic amelioration [94]. Notably, in 2016, NPT200-11 successfully completed a clinical Phase I trial involving 55 healthy participantsii.

Clinician’s Corner α-Syn is an intrinsically disordered protein involved in synaptic transmission. The aggregation of α-syn into amyloid fibrils is the molecular hallmark of Parkinson’s disease (PD) and is responsible for the onset and progression of the disease, causing the degeneration of brain dopaminergic neurons. Interfering with α-syn aggregation is considered to be one of the best strategies for a disease-modifying therapy for PD. Small molecules that target this process in the brain have demonstrated therapeutic efficacy in different animal models of PD, and some of these molecules have already entered clinical trials. Drug repositioning, rational design, and high-throughput screening strategies have all identified active molecules to tackle the α-syn aggregation in PD. The mechanism of action of chemical chaperones is diverse and ranges from α-syn monomer stabilization to disruption of preformed amyloid assemblies. This multiplicity of strategies can potentially be combined to treat both the early and late stages of PD. There is an urgent need for effective biomarkers that can assess the impact of chemical chaperones on the prodromal development of PD without a need for complex endpoint evaluations of motor, behavioral, and mental capacities.

Expanding the Molecular Repertoire: High-Throughput Screening of Large Drug-like Collections A powerful alternative for the discovery of novel active molecules is high-throughput screening of large collections of chemically diverse compounds. These initiatives necessarily rely on optimized assays that attempt to minimize time and cost per test while preserving specificity and sensitivity. The stochastic nature of protein aggregation impacts on experimental reproducibility and thus Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx

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compromises hit selection by this type of 'brute force' approach. Notably, by optimizing α-syn aggregation protocols and/or integrating new detection methods, several groups managed to overcome this limitation and discovered a set of novel α-syn aggregation inhibitors [98–101]. The following presents the examples of Anle138b and SynuClean-D. Anle138b was discovered after two consecutive rounds of screening. A first assay evaluated the capacity to inhibit prion protein (PrP) amyloid assembly, and 3,5-diphenyl-pyrazole (DPP) was the most active compound among ~20 000 chemically diverse drug-like compounds [102]. Keeping the DPP scaffold, the authors designed a set of 150 molecules in a structure–activity relationship (SAR) effort intended to balance brain permeability and anti-aggregation activity. Anle138b was selected as the leading compound of this initiative. This molecule does not interact with the α-syn monomer but instead binds to a hydrophobic pocket in oligomeric assemblies, interfering with β-sheet formation. Remarkably, the hydrophobicity of the binding site impacts on the fluorescence emission spectrum of Anle138b, which allows its use to detect early-stage aggregates, with potential implications for PD diagnosis [103]. The therapeutic performance of Anle138b was evaluated in three different PD mouse models where oral administration of the compound ameliorated PD-related symptoms including motor activity, gut motility, weight, neuroprotection, and survival. In the A30P transgenic model, Anle138b was shown to reduce α-syn aggregation in the brain, thus linking this phenotype to the observed neuroprotection. Importantly, a therapeutic effect was also observed in symptomatic late-stage rodents, suggesting that Anle138b might be used not only for prevention or in early-stage patients but also for symptomatic cases of PD [104]. Moreover, preclinical analysis suggests that the molecule is innocuous, even after long-term and high-dose treatment. In addition, it displays adequate pharmacokinetics, brain distribution, and pharmacodynamic properties. Thus, it is likely that we will soon see clinical trials for this promising compound. SynuClean-D (SC-D) is a small molecule discovered after screening N14 000 different drug-like compounds [99]. The assay identified molecules that were able to reduce the nucleation or elongation rates in an α-syn amyloid fibril kinetic assay conducted in 96-well microtiter plates. The inhibitory activity of selected compounds was further validated using orthogonal light scattering and transmission electron microscopy. SC-D interacts with aberrant α-syn assemblies without recognizing the functional and soluble form of the protein [105]. The molecule inhibits up to 70% of the aggregation of α-syn into amyloid fibrils in vitro and is active against both the wild-type protein and variants associated with familial PD. SC-D inhibits aggregation at substoichiometric concentrations and impedes the propagation of α-syn amyloid aggregates in protein misfolding cyclic amplification assays (PMCAs). Computational data suggest that the compound docks into the inner cavity of α-syn fibrils and, indeed, disrupts up to 50% of one-week-old amyloid fibrils, the disrupted material being innocuous for neuronal cells. SC-D itself is not toxic to neuronal cells, including primary cultures of cortical neurons at fivefold the active concentration, and it increases by twofold the number of neuronal cells devoid of α-syn aggregates. The efficacy of SC-D has been assayed in two different C. elegans models of PD. In the first model, treatment with SC-D reduced by twofold α-syn intracellular aggregates and rescued muscular paralysis, increasing animal mobility. In the second model, treatment with SC-D increased by fourfold the percentage of animals with a complete set of fully functional dopaminergic neurons. The neuroprotective activity of SC-D is the highest reported in this animal model. Overall, SC-D seems to act at different points in the pathogenic cascade mediated by αsyn aggregation; however, its pharmacokinetic and pharmacodynamic properties should be evaluated before it can be considered as a reliable candidate for clinical studies.

Concluding Remarks Despite decades of investigation, PD remains incurable, and only symptomatic therapies are available. We have compiled a battery of promising small molecules that target the presumed 10

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Outstanding Questions How should chemical chaperones be administered early in the course of the disease to modify progression? Do natural polyphenols act as aggregation inhibitors, antioxidants, or both? Is there room to optimize their pharmacokinetic and pharmacodynamic properties? The mechanism of action of some inhibitors relies on their interaction with monomeric α-syn. Would these inhibitors compromise the natural function of α-syn and thus promote a loss-of-function phenotype even though they block the formation of toxic aggregates? In the absence of biomarkers that report on the early stages of PD, would small molecules that act by stabilizing monomeric α-syn or targeting oligomers be useful at all? Genetic variations and post-translational modifications tune α-syn aggregation. How will the molecules under development manage these alternative variants of the protein? Can a single molecule be active against all the different amyloid polymorphs that α-syn can populate? Would we need to develop personalized medicine initiatives to treat synucleinopathies? How do human cells manage the residual aggregates generated in offpathway reactions or after the disruption of mature amyloid fibrils? Are natural proteostatic mechanisms sufficient to handle such sticky macromolecular structures? Can molecules that target different αsyn conformations be converted into a combined therapy suitable for both the prodromal and late stages of PD? What role does the gut play in PD? Can the disease, or at least some of its symptoms, be treated with drugs that target α-syn aggregation in the gastrointestinal tract? Are chemical chaperones specific for α-syn, or can they also be employed to treat other protein conformation diseases?

Trends in Molecular Medicine

origin of the disease, the aggregation of α-syn (Figure 3 and Table 1). Their mechanism of action is strongly influenced by the way in which they have been discovered: rationally designed molecules usually target the monomeric or early oligomeric states, whereas compounds identified by highthroughput screening tend to interact with late-state assemblies, suggesting that these assays have an intrinsic bias towards molecules interacting with aggregated species. The structural scaffolds that confer anti-aggregation activity to chemical chaperones seem to share some common features. These molecules usually contain a planar hydrophobic core formed by aromatic rings that interact with apolar exposed regions in α-syn assemblies. This core is frequently coated with polar projections that interfere with hydrophobic packing and disrupt intermolecular hydrogen bonds, thus acting as protective gatekeepers. However, these polar groups tend to limit the ability of the molecules to cross the BBB, and a compromise between anti-aggregation activity and brain bioavailability is usually required at the 'hit to lead' stage. However, increasing evidence suggests that the spread of α-syn pathology from the peripheral to the central nervous system may be an important etiological factor in PD [106], and effective compounds that have been discarded because of their low BBB permeability may have a second chance in therapy. Indeed, the nonpublic structure ENT-01, a molecule developed by Enterin, which targets the enteric neuron and displaces α-syn from the intracellular membranebinding site, has been shown in a Phase IIa study not only to restore bowel function of patients but also to improve neuropsychiatric symptomsiii, suggesting that PD might be treated via the gut. This and other important questions for further research are highlighted in the Outstanding Questions (see Outstanding Questions). Because α-syn misfolding and aggregation occur early in the pathology, targeting these processes with chemical chaperones before extensive neurodegeneration has occurred seems to be an optimal strategy to treat PD. However, in the absence of objective biomarkers that report on the efficacy of these small molecules in human subjects, target engagement cannot be demonstrated and, accordingly, long, large, and very expensive trials are necessary to prove their therapeutic potential. Clearly, biomarker and drug development efforts should go hand by hand if we want to target PD at its early stages, before it becomes incurable. Acknowledgments This work was funded by the Spanish Ministry of Economy and Competitiveness (grant BIO2016-78310-R to S.V.) and by the Institución Catalana de Investigación y Estudios Avanzados (ICREA; grant ICREA-Academia 2015 to S.V.).

Resources i

https://clinicaltrials.gov/ct2/show/NCT00461942

ii

https://clinicaltrials.gov/ct2/show/NCT02606682

iii

https://clinicaltrials.gov/ct2/show/NCT03781791

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