BACE-1 inhibitors: from recent single-target

molecules to multitarget compounds for Alzheimer’s

disease

Federica Prati, † Giovanni Bottegoni, ‡δ Maria Laura Bolognesi, § and Andrea Cavalli* ‡§

†Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life

Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, UK

‡CompuNet, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova, Italy

δHeptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire,

AL7 3AX, UK

§Department of Pharmacy & Biotechnology, Alma Mater Studiorum-University of Bologna, Via

Belmeloro 6, 40126 Bologna, Italy

KEYWORDS: Neurodegenerative disease, neurological disorder, amyloid hypothesis, tau

hypothesis, structure-based drug discovery, fragment-based drug discovery

ABSTRACT. The amyloid hypothesis has long been the central dogma in drug discovery for

Alzheimer’s disease (AD), leading to many small-molecule and biological drug candidates. One

major target has been the β-site amyloid-precursor-protein-cleaving enzyme 1 (BACE-1), with

many big pharma companies expending great resources in the search for BACE-1 inhibitors. The

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lack of efficacy of verubecestat in mild-to-moderate AD raises important questions about the

timing of intervention with BACE-1 inhibitors, and anti-amyloid therapies in general, in AD

treatment. It also suggests new possibilities for discovering BACE-1-targeted compounds with

more complex mechanisms of actions and improved efficacy. Herein, we review the major

advances in BACE-1 drug discovery, from single-target small molecule inhibitors to multitarget

compounds. We discuss these compounds as innovative tools for better understanding the

complexity of AD and for identifying efficacious drug candidates to treat this devastating

disease.

1. Introduction

In 1992, Hardy and Higgins first outlined the amyloid cascade hypothesis, suggesting that

“amyloid-beta protein (Aβ) is the causative agent in Alzheimer’s disease (AD) pathology and

that neurofibrillary tangles, cell loss, vascular damage, and dementia follow as a direct result of

this deposition”.1

Twenty-five years later, this hypothesis, in its various incarnations, still appears to be the major

inspiration for AD drug researchers. This is despite the failures of recent high-profile clinical

trials, and an increasing number of dissenting opinions concerning the effectiveness of this

hypothesis. Indeed, several recent papers have captured the attention of the field by outlining

major inconsistencies and controversies related to this hypothesis.2,3 These include the presence

of Aβ deposition in cognitively healthy elderly people, the general discrepancies between plaque

density and the severity of dementia, the lack of comprehensive nominal, functional, and

structural descriptions of oligomeric Aβ assemblies, and challenges to the validity of amyloid

animal models.4

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Nonetheless, the number of recent and ongoing clinical trials and preclinical investigations

shows that many research groups are seeking to develop small molecules that can modify the

generation, clearance, and/or toxicity of Aβ. These small molecule strategies compete with active

and passive amyloid-based immunotherapies,5 sharing with them a long history of clinical

failure.

In this respect, a recent analysis of failed phase 3 amyloidocentric agents (such as γ-secretase

inhibitors and Aβ-antibodies) points out how deficiencies in clinical trial design, as well as

liability of the pursued compounds ultimately compromised the success of the studies.6

Particularly, while some therapeutics were very unlikely to succeed due to lack of robust

preclinical data, others failed to establish adequate target engagement because of dose-limiting

site effects. Therefore, these agents did not show any clinical benefit, but neither allow to

unequivocally test and discard the mechanistic approach under investigation, as target

engagement was not measured. Hence, these considerations provide sustenance to those drug

developers and scientists who believe that the amyloid hypothesis has yet to be tested.

Big pharma’s favorite amyloidocentric AD target is the β-site amyloid-precursor-protein-

cleaving enzyme 1 (BACE-1), that is, the aspartic protease that cleaves amyloid precursor

protein (APP) at the β-secretase site. JNJ-54861911,7 CNP520,8 and LY32026269 (structure not

disclosed) are the three most recent BACE-1 inhibitors working their way through the trial

pipeline, while verubecestat 1 (MK-8931),10,11 (Figure 1) is the most advanced BACE-1 inhibitor

in clinical development. We expect other BACE-1 inhibitors to advance into clinical trials

resulting from the research described herein.

Several factors have likely contributed to the current preference for inhibiting BACE-1 rather

than Aβ aggregation. First, it is particularly challenging to develop small molecule inhibitors of

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protein-protein interactions (PPIs).12 The regions of PPIs involve large contact surfaces (1500-

3000 Å), whereas the regions of protein-small-molecule interactions are only about 300-1000 Å.

In addition, the PPI regions generally lack cavities, i.e. grooves or pockets into which a small

molecule could dock in an energetically favorable manner. The binding energy that drives

protein-protein contacts is typically distributed over a large area that lacks a defined “hotspot”

for pharmacological intervention. Taken together, these inherent limitations severely restrict the

drug-likeness of Aβ aggregation inhibitors.

Conversely and for several reasons, BACE-1 appeared (at least initially) to offer bigger

opportunities and to be a more druggable target. First, effective aspartyl protease inhibitors had

already been developed successfully in other therapeutic areas, such as hypertension and HIV.13

Second, the first crystal structure, solved in 2000,14 seemed to support straightforward structure-

based (SB) approaches. In particular, BACE-1 is a monomeric protein with a bilobal structure,

which accommodates the catalytic aspartic dyad (Asp 32 and Asp 228) between the N- and C-

terminal domains. The rather large active site is sheltered by a β-hairpin loop, known as a flap,

which forms a large and flexible portion of the binding pocket. Spurred on by rising expectations

and boosted by the discovery of different peptidomimetic inhibitors with nanomolar activity,

researchers have been seeking to develop small molecule BACE-1 inhibitors ever since.15-18

However, progress has been hampered by the rigid prerequisites for any drug that is to treat a

chronic neurodegenerative disorder and by the uncompromising nature of the BACE-1 catalytic

site (see below).15 Selectivity towards different aspartyl proteases could be an additional factor in

the attrition rate for BACE-1 drug discovery.

Thus, two decades of research have shed light on additional layers of complexity that may take

many years to explore. Nonetheless, we will argue here that the bulk of data collected still

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supports BACE-1 as a major target for AD drug discovery, both now and in the future. In

particular, we will consider new horizons such as the development of BACE-1 inhibitors with an

expanded therapeutic profile, and the use of BACE-1 inhibitors in a more promising personalized

medicine setting.

Clearly, the amyloid theory is not the only pathogenic hypothesis of a complex multifactorial

disease. Tau19 and prion20 have both been proposed to trigger AD and mediate its progression. In

addition, oxidative stress,21 neuroinflammatory22 and mitochondrial23 cascades appear to explain

the disease process and have been subject of considerable research and speculation.

To effectively confront such multifaceted and complex nature, several lines of evidence point to

multitarget compounds (often referred to as ‘multitarget-directed ligands’ (MTDLs)), which can

hit proteins in complementary pathways.24 In terms of BACE-1 inhibitors, this means developing

small molecules that can modulate both BACE-1 and other related targets. In principle, this

could considerably enhance efficacy and produce more satisfactory clinical outcomes.

In terms of personalized medicine, companion diagnostics have now entered the AD field, where

they should allow clinicians to precisely assess the benefits of anti-amyloid therapy and to

identify the “best patients” for a given treatment.25 For example, Merck is developing the BACE-

1 inhibitor 1 in conjunction with the FDA-approved amyloid PET tracer flutemetamol.

Flutemetamol will be used to identify patients who might benefit from 1 treatment and to

monitor clinical outcomes.

Herein, by critically reviewing the literature and systematically discussing the progress made, we

hope to address the open questions regarding the validity of BACE-1 as an AD target, and to

inspire the design of novel improved inhibitors. We will first review the recent advances in the

design and discovery of BACE-1 inhibitors. We will then consider the major computational

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approaches used over the last decade to assist the design of BACE-1 inhibitors, mainly through

SB approaches made possible thanks to the existence of BACE-1 crystallographic structures. We

will conclude by reporting on MTDLs centered on BACE-1. These have the potential to become

next-generation BACE-1 inhibitors with improved efficacy in treating this devastating

neurological disorder.

2. BACE-1 inhibitors: an update

As highlighted above, the search for effective BACE-1 inhibitors for AD treatment has been a

hot topic in the past few years. The many reports in the literature have been covered by some

comprehensive reviews.15-18 Therefore, this section will focus on the most relevant medicinal

chemistry investigations carried out more recently.

Peptidomimetics were the first BACE-1 inhibitors to be reported. Despite their high in vitro

potency, they had inadequate pharmacokinetic (PK) properties, which generally made them

ineffective for treating AD. Peptidomimetics have long been a staple of BACE-1 inhibitor

research programs, with recent publications demonstrating that researchers are still interested in

them.26-29 However, the focus has mostly shifted to small molecule nonpeptide inhibitors.

Investigations into new small molecule nonpeptide inhibitors involve high-throughput, fragment-

based (FB), or virtual screenings, followed by chemical optimization. The primary goal is to

develop novel inhibitors that are smaller, with less of a peptide character, and an improved PK

profile.

However, the development of nonpeptidic BACE-1 inhibitors large enough to bind with

sufficient affinity to the enzymatic active site, yet small enough to exhibit satisfactory PK and

sufficient lipophilicity to sustain suitable brain penetration, has proven to be very challenging.16

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Initially, inhibitor size reduction resulted in a decrease in BACE-1 inhibitory potency. However,

SB drug design strategies, supported by crystallographic data, led to high affinity BACE-1

inhibitors with additional and specific drug-target interactions. Improved central nervous system

(CNS) penetration was also achieved, trough enhanced blood brain barrier (BBB) permeability

and reduced P-glycoprotein (Pgp) efflux, by properly fine-tuning compound physiochemical

properties, such as polar surface area (PSA), molecular weight (MW), lipophilicity, number of

rotatable bonds and hydrogen bond donors (HBD).18,30,31 Off-target effects also needed to be

addressed, taking advantage of structural data and reflecting on physiochemical property

balancing. Importantly, potent small molecule BACE-1 inhibitors have lately been developed to

achieve satisfactory brain penetration and robust cerebral Aβ reduction with good safety margins

in preclinical animal models. In summary, high level of understanding to develop efficient in

vivo BACE-1 inhibitors has been obtained, and recent efforts, showing how research in the field

is making strides in the right direction, are discussed below.

2.1 Aminohydantoins, aminooxazolines, aminothiazolines, and iminopyrimidinones

A key advance in nonpeptide BACE-1 inhibitor design was the discovery of 2-amino

heterocycles that engage both of the Asp moieties in the BACE-1 active site.15-18 Compared to

initial acylguanidine inhibitors, these ligands generally enabled a better physical-chemical

profile, improved brain penetrance and in vivo efficacy.

Along this line, the aminohydantoin BACE-1 inhibitor 2 (Figure 2) was recently developed by

Merck. Although 2 was potent in the enzymatic assay (Ki = 22 nM), it suffered from a

pronounced cell shift when tested in whole-cell assay (cell IC50 = 258 nM), due to Pgp efflux. It

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also showed modest oral bioavailability and lowered plasma, but not cortical Aβ40 levels in

CRND8 mice.32

Seeking to identify novel, highly soluble, and permeable BACE-1 inhibitors, Hunt et al. prepared

a collection of small, rigid, and saturated or partially saturated cores using 2-amino heterocycles.

This led to the identification of spirocyclic aminohydantoin 3 (BACE-1 IC50 = 36.8 μM) (Figure

3), which combines limited molecular flexibility with three-dimensionality,33 key factors for

permeability and solubility, respectively. The group conducted structure-activity relationship

(SAR) studies of core changes, P3 moieties, and warhead (WH) Asp-binding functional groups.

This provided compounds with double-digit nanomolar enzymatic and cell-based Aβ1-40

inhibitory potencies, but low selectivity with respect to the closely related aspartyl protease,

cathepsin D (CatD) (IC50CatD/IC50BACE = 0.2-97). (R)-enantiomers were roughly twice as potent as

the corresponding racemates, but did not consistently show increased affinity for CatD. Although

suitable alternative Asp binding moieties to eliminate efflux were not identified (the

aminohydantoin WH contributes to Pgp efflux liability as it adds two HBD and increases PSA),

good BBB permeation and Aβ reduction in cerebrospinal fluid (CSF) were observed for some

derivatives in vivo. More importantly, Hunt et al. demonstrated a

pharmacokinetic/pharmacodynamic (PK/PD) relationship between free drug concentrations in

the brain and Aβ-lowering in CSF in wild-type guinea pig and rat. Optimization of brain

exposure led to the discovery of the lead compound (R)-4 (BACE-1 IC50 = 48 nM) (Figure 3),

which significantly reduced CSF Aβ in wild-type rodents and in monkey.33

The group also incorporated a 7-tetrahydropyrane (7-THP) A-ring at the P2’ site of the chroman

scaffold, providing a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromanes.34 This

modification was envisaged to improve selectivity for BACE-1 versus CatD, due to differences

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in their active sites. They explored three different WH (aminohydantoin, aminooxazolines, and

aminothiazolines) as Asp-binding moieties, as well as SB-guided substitutions at P3 and P2′ sites

to modulate potency, selectivity, efflux and permeability (Figure 4). The aminohydantoin moiety

led to the most potent analogues, but with higher efflux ratios and lower permeabilities than the

two alternative WH. These derivatives showed good enzymatic (10-40 nM) and cellular (5-44

nM) potencies, and were 10-to-420-fold more selective for BACE-1 than for CatD. Importantly,

despite the high efflux, 5 (Figure 4) had the best coverage ratio (free brain concentration/cell

IC50) of the various 7-THP chromans in mouse PK models. Furthermore, 5 significantly reduced

CSF Aβ1-40 in guinea pig, rat, and cynomolgus (58%, 53%, and 63%, respectively).

2,3,4,4a,10,10a-hexahydropyrano[3,2-b]chromane (8-THP chroman) derivatives showed an even

better overall profile (Figure 5).35 As noted above, the aminohydantoin Asp binding moiety

correlated with high efflux, despite being the most potent. The enzymatic (41-98 nM) and

cellular (37-140 nM) potencies, and CatD selectivity (430−>2200-fold) of the aminooxazoline

and aminothiazoline analogues were dramatically enhanced by methylation at the β-position of

the 8-THP chroman core. Further optimization of the P3 moiety led to aminooxazoline 6 (Figure

5), which progressed to rat PD studies. Although 6 still showed efflux liability (efflux ratio of

3.4) and had a 7-fold lower coverage ratio in rat than 5, it showed a significant improvement in

CSF Aβ1-40 reduction (69%).

Other aminooxazoline derivatives have recently been reported as BACE-1 inhibitors.36-39 The

aminooxazoline xanthene 7 (Figure 6) has good BACE-1 enzymatic (2 nM) and cellular (25 nM)

potency, as well as overall favorable properties. It showed robust CSF and brain Aβ-lowering

activity in a rat PD model, but QTc prolongation in cardiovascular models, which is consistent

with in vitro activity on the cardiac hERG potassium channel (hERG Ki = 0.66 μM).36 Indeed,

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hERG inhibition has frequently been associated with BACE-1 inhibitors. In fact, there is

evidence that the increase in the pKa and lipophilicity, which is required for efficacious BACE-1

inhibitors, increases the chance of hERG inhibition.40 Hence, reducing these two parameters is a

common approach for reducing hERG affinity. However, it is known that decreasing the pKa of

the Asp-binding motif generally decreases BACE-1 potency, while introduction of polarity

increases PSA, which correlates with Pgp recognition. A balance of low Pgp efflux, hERG, and

BACE-1 potency was achieved by introducing polarity at the P2′ site of 7 and simultaneously

reducing the PSA of the P3 group.36 Introducing a fluorine in position 4 of the xanthene ring also

improved BACE-1 enzymatic and cellular potencies (5-10-fold).36 The enhanced BACE-1

potency within the series was attributed to a favorable hydrogen-bond interaction between the 4-

fluoro atom and NH Trp76 of BACE-1. Thus, introducing a nitrogen atom at the 3- or 4-position

of the xanthene core was investigated to similarly engage NH Trp76.37,38 This modification

boosted BACE-1 potency and reduced hERG binding affinity, but elevated the Pgp efflux ratio.

Optimizing the P3 and P2’ groups resulted in several compounds with improved in vitro

profiles.37,38

The development of 3-aza-4-fluoroxanthenes was also explored.39 These lead-optimization

approaches resulted in compounds with robust reductions in CSF and brain Aβ levels in a naïve

rat PD model. In particular, compounds 8, 9 (AMG-8718), 10, and 11 (Figure 6) had excellent in

vitro profiles (BACE-1 IC50 = 0.3, 0.7, 0.9, 0.3 nM, respectively; cell IC50 = 4, 4.4, 21.1, and 4

nM, respectively) and good in vivo PD responses in rats. These compounds exhibited modest

hERG activity (8, hERG Ki = 3.28 μM; 9-11, hERG Ki 10 μM), and significantly reduced

potential for QTc prolongation in cardiovascular safety models in dog (8, 9 and 11) and monkey

(10).36-39 10 and 11 also produced robust and sustained reductions in CSF and brain Aβ in a non-

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human primate PD model. They showed low in vivo clearance and good oral bioavailability in

preclinical species.38,39 Unfortunately, 9’s drug development was discontinued due to toxicity

issues. It elicited retinal toxicity leading to thinning of the outer nuclear layer of the retina in a

one month rat toxicity study to support first-in-human trials.41 Although the origin of the toxicity

was not established, it was speculated it was likely an off-target effect related to inhibition of

other proteases. Mandal et al. recently reported on novel bicyclic iminopyrimidinones42 to

optimize the in vivo profile and address the off-target liabilities (cytochrome CYP3A4 and

hERG inhibition) of a previous series of fused pyrrolidine iminopyrimidinone represented by 12

(Figure 7).43 Starting from 12, the group improved the ancillary profile and CNS properties by

truncating the P3 cyano-phenyl group (to reduce lipophilicity and hERG inhibition) and

optimizing the fluoropyrimidine moiety binding in the S2′−S2″ region (to modulate BACE-1

affinity and address CYP3A4 inhibition). They subsequently optimized the P1 thienyl ring to

improve affinity and selectivity. This resulted in the 2,4-difluorophenyl analogue 13 (BACE-1 Ki

= 5 nM, cell IC50 = 14 nM) (Figure 7), which robustly lowered CSF and cortex Aβ40 in both rats

and monkeys.

Importantly, by occupying the S1 and S2′-S2″ subpockets, 13 has a molecular shape that is novel

among those S1-S3-binding BACE-1 inhibitors that can potently lower central Aβ in nonrodent

preclinical species.

2.2 Aminothiazines

The FB-derived aminothiazine 14 (LY2811376, Figure 8) developed by Eli Lilly showed well-

balanced PK profiles and reduced CSF Aβ levels in different animal species.44 It was also the

first molecule to demonstrate sustained brain Aβ reduction in healthy volunteers. However,

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clinical trials were discontinued due to preclinical retinal toxicity, potentially related to the off-

target CatD inhibition.44

Compound 14 exhibited only modest BACE-1 inhibitory activity (IC50 = 0.24 μM). Therefore,

increasing potency was considered as one strategy for widening the therapeutic window with

respect to the observed toxicity. In this regard, by targeting the flap backbone of the BACE-1

active site, Wu et al. discovered the 6-dimethylisoxazole-substituted biaryl aminothiazine 15

(Figure 8), with 34-fold improved BACE-1 inhibitory activity (IC50 = 8 nM) over 14.45 Despite

its potency, 15 produced only modest in vivo activity in rodents, due to poor brain penetration

and metabolic instability mainly related to oxidation of the pyrimidine ring. Both hurdles were

overcome with the development of the pyrazine-carboxamide 16 (Figure 8). The pyrimidyl

moiety of 15 was replaced with a carboxamide, and the 5,6-diflurophenyl substituent in the S1

pocket was optimal in terms of activity and metabolic stability. Optimization of the amide side

chain in the S3 pocket resulted in 16, which exhibited potent BACE-1 inhibitory activity (IC50 =

0.7 nM), and robust brain Aβ reduction in rat dose-response studies.45 An alternative approach to

addressing 15’s two major issues was to develop S3-truncated heteroaryl substituted

aminothiazines.46 Pyrimidine truncation boosted brain penetration (by reducing MW and PSA)

and metabolic stability (by removing a metabolic soft spot). Compound 17 (Figure 8) showed

improved BBB permeability and, despite its moderate enzymatic activity (IC50 = 0.15 μM), it

demonstrated significant brain Aβ reduction in rodents. 17 was relatively stable in rat and mouse

liver microsomes, but unstable in human microsomes, due to oxidation of the methyl group(s) of

the isoxazole ring. 17’s metabolic stability was addressed by replacing the 6-dimethylisoxazole

ring with a pyrimidine ring. This led to 18 (Figure 8) with good brain permeation and metabolic

stability, and enhanced in vivo activity in rodents.

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An alternative approach to widening 14’s therapeutic window was to improve BACE-1

selectivity versus CatD. In this respect, Butler et al. recently reported on a reductionist approach,

truncating 14 to just the necessary pharmacophore, then evaluating opportunities to improve

CatD selectivity using SB drug design, without impacting CNS penetration.47 The group

developed a series of aminothiazine-THP fused derivatives, carrying a small lateral chain to

engage an induced pocket (S2’) formed by movement of the protein flap. These derivatives were

efficiently selective, maintaining the desired PK profile, as exemplified by 19 (Figure 8). The

compounds displayed robust efficacy in lowering both peripheral and central Aβ loads in vivo in

mice and dog models. In addition, chronic treatment in aged PS1/APP mice decreased the

number and size of Aβ-derived plaques. Most importantly, a 2-week exploratory toxicology

study of 19 revealed no retinal toxicity.

Despite its excellent overall properties, 19's weak in vitro potency against BACE-1 (IC50 = 1.07

μM), meant a high human dose was projected ( 500mg/day), which prevented its further

investigation. A new generation of novel heterocycle-substituted analogues of 19 was designed

based on structural information. Particularly, as the fluoromethyl group of 19 was found to

suboptimally fill the S2’ pocket, it was postulated that positioning a heteroaryl substituent in this

pocket could improve potency, while maintaining the overall lipophilicity and alignment of

ADME properties. This led to potent, low-clearance, CNS-penetrating BACE-1 inhibitors,

represented by 20 in Figure 8 (BACE-1 IC50 = 52 nM, cell IC50 = 4n M).48 However, further in

vitro profiling with human recombinant CYPs showed that CYP2D6 significantly contributed to

20’s metabolism, causing drug-drug interaction (DDI) concerns.

SB-guided efforts to reduce 20’s cytochrome CYP2D6 metabolism issues resulted in highly

potent derivatives, with reduced risk for DDI, central efficacy, and improved hERG therapeutic

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margins. In particular, due to their promising overall profiles, compounds 21 and 22 (Figure 8)

have advanced to long-term safety studies, with the results expected in due course.

As noted above, Eli Lilly discovered 1444 and, more recently, the furo[3,4-d][1,3]thiazin-2-amine

picolinamide LY2886721 (23 in Figure 9), a potent BACE-1 inhibitor that reached Phase 2

clinical trials to treat early AD.49 Ely Lilly researchers then used a FB approach to target

topologically complex polycyclic aminothiazines as BACE-1 inhibitors. Starting from the

millimolar bicyclic thiazine fragment hit 24 (Figure 10), identified in a high concentration

BACE-1 enzymatic assay, they obtained the FB-derived compounds 25 and 26 (Figure

10).50Compounds 25 and 26 showed poor potency in vitro (BACE-1 IC50 = 1.83 and 2.25 μM,

respectively) and in vivo (lack of Aβ brain reduction was consistent with low cerebral exposure).

However, key SAR hypotheses could be tested with these compounds. These tests revealed that

S3-binding could remarkably increase the potency of this series, but that conformational

constraint would not lead to further enhancements.

Bristol-Myers Squibb recently developed a novel series of more easily accessible furo[2,3-

d]thiazine isosteric analogues of 23, represented by 27 and 28 in Figure 9 (BACE-1 IC50 = 20

and 37 nM, respectively). 27 and 28 showed cellular activity comparable to that of the parent

compound 23 and good in vitro permeability. However, they demonstrated low exposure and

only modest reduction in Aβ brain levels in mice.51

2.3 Iminothiadiazinanes dioxide

Merck recently reported on the discovery and characterization of a novel iminothiadiazinane

dioxide class of BACE-1 inhibitors, represented by the advanced clinical compound 1 (Figure

1).11 Starting from the elaboration of initial isothiourea fragment screening hits,52 subsequent

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search for heterocycle isothiourea isosteres, and extensive SAR exploration around the

iminothiadiazinane dioxide core, Merck developed the highly optimized inhibitor 1. Based on the

potent in vitro and vivo iminopyrimidinone BACE-1 inhibitor 29 (BACE-1 Ki = 1.7 nM),53 and

seeking to identify novel Asp-binding cores that could offer unexplored intellectual property

space, the iminothiadiazinane dioxide 30 (BACE-1 Ki = 2.4 nM) was developed (Figure 11).

Although 30 (cell IC50 = 55 nM) displayed weaker cellular potency than 29 (cell IC50 = 11 nM), it

was significantly more potent in vivo, probably due to the enhanced CNS penetration imparted

by the iminothiadiazinane dioxide core. In an acute rat PD assay, 30 afforded robust reductions

of CSF and cortex Aβ40 levels. Despite the favorable PD profiles, 29 and 30 were not developed

further due to metabolic issues and modest CatD selectivity (21- and 47-fold, respectively).

These two liabilities were overcome by SB optimization of the P3 and P1 binding motifs, leading

to the discovery of 1 (BACE-1 Ki = 2.2 nM). Compound 1 was highly selective for BACE-1

over other aspartyl proteases, with the exception of BACE-2, and potently inhibited Aβ

formation in cell-based assay (cell IC50 = 2.1 nM). Compound 1 showed a good PK/PD profile in

preclinical species, profoundly lowered CSF and brain Aβ levels after acute and chronic

administration to rats and monkeys, and was advanced to phase 1 clinical trial to assess safety,

PK and PD in healthy volunteers and mild to moderate AD patients.10 Single and multiple doses

were generally well tolerated and produced reductions in Aβ levels in the CSF of both healthy

human subjects and AD patients. In 2012, 1 entered into phase 2/3 clinical trial in people with

mild to moderate AD (EPOCH trial), and in 2013 a phase 3 trial for prodromal AD (APECS

trial) was also started. While EPOCH has been recently discontinued due to lack of efficacy,

APECS will continue to address the question of timing of intervention of BACE-1 inhibition in

AD (results are expected by February 2019).54

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2.4 Iminohydantoins

Merck recently reported SB drug design efforts around the high-throughput screening hit,

spiropiperidine iminohydantoin 31 (BACE-1 IC50 = 22 μM, cell IC50 > 30 μM) (Figure 12).55

This led to compounds with more than 200-fold and 90-fold improved BACE-1 enzymatic and

cell-based potency, respectively. This remarkable achievement was made possible by adding a

strategically placed methyl group on the piperidine ring. This methyl group maintained the

required pKa of the N-piperidine nitrogen, filled a small hydrophobic pocket, and stabilized the

best conformation for optimized binding to the receptor.56 Importantly, derivative 32 (BACE-1

IC50 = 6 nM, cell IC50 = 63 nM) (Figure 12) also showed good selectivity versus CatD (IC50 =

16.3 μM), and Aβ reduction in CSF in mice and monkeys.

2.5 Aminoxazines

Rombouts et al. recently reported on novel 6-F-3-amino-1,4-oxazines for the development of

BACE-1 inhibitors with improved CNS permeability.57 Lead optimization efforts fine-tuned the

amidine pKa and BACE-1 primary activity, resulting in compound 33 (BACE-1 IC50 = 7.6 nM;

cell IC50 = 8.1 nM) (Figure 13). Compound 33 was orally bioavailable, centrally active, and

robustly lowered brain and CSF Aβ levels in mouse and dog models, respectively. However,

brain penetration still needs to be balanced against metabolic stability and cardiovascular safety

(i.e. hERG inhibition).

2.6 Aminoquinolines

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The potent SB-designed aminoquinoline derivative 34 (BACE-1 IC50 = 0.8 nM) was recently

developed by linking the known BACE-1 inhibitor 35 (BACE-1 Kd = 140 nM)58 with the S3

binding fragment 36, identified in a 19F-NMR second-site fragment screen (Figure 14).59

Compound 34 exhibited a 350-fold increase in BACE-1 affinity, while maintaining reasonable

ligand efficiency and gaining much improved selectivity over CatD with respect to the parent

compound 35.

2.7 Macrocyclic acyl guanidines

The acyl guanidine chemotype is well-known in BACE-1 inhibitor development. Boy et al

recently reported on a macrocyclization strategy to improve the in vitro potency and Pgp liability

of parent acyclic acyl guanidine inhibitors.60 Compound 37 (Figure 15) showed good in vitro

potency (BACE-1 Ki = 3.2 nM, Cell IC50 = 140 nM), with robust peripheral Aβ-reduction in a

wild-type mice model. However, further effort is still required to lower Pgp efflux and enhance

CNS penetration, thereby achieving meaningful central exposure.

2.8 Miscellaneous

Recently reported in silico studies have led to the identification of new series of 4-oxo-1,4-

dihydro-quinoline-3-carboxamides,61 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-

dihydropyridines,62 allylidene hydrazinecarboximidamides,63 sulfonyl-amino-acetamides,64 and

(3S,4S)-4-aminopyrrolidine-3-ols65 with micromolar BACE-1 inhibitory activity. These can be

considered as suitable starting points for further investigation.

3. SB approaches to discovering BACE-1 inhibitors

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BACE-1 has been extensively characterized at the structural level. The enzyme presents three

peculiar features that should be taken into account during drug discovery. First, the BACE-1

binding site is very extended (roughly 1000 Å3 in size) and displays an elongated shape over 20

Å in length along the main axis.66 Usually, this pocket is conveniently described as a collection

of sub-sites, which display varying physico-chemical local environments. Recently, a

combination of docking studies and molecular dynamics (MD) simulations was used to

investigate the selectivity of four BACE-1 inhibitors with respect to closely related proteases

(BACE-2 and CatD).67 Simulations showed that these compounds were too bulky to fit in the

comparatively narrow binding pocket of BACE-2 and, conversely, could not establish stable

interactions within the wider pocket of CatD.

Flexibility is the second relevant feature to consider when designing BACE-1 inhibitors. The

BACE-1 binding pocket is very flexible, as flexibility is instrumental to catalysis, and induced-fit

effects are to be expected. As of today, querying the Protein Data Bank for the human BACE-1

UniProt Accession ID (P56817) returns 355 unique hits.68 Possibly due to the abundance of

crystal structures combined with its pharmacological relevance, BACE-1 is considered an ideal

case study for investigating the role of receptor flexibility in docking and virtual screening.69-71

Cosconati and colleagues implemented an advanced ensemble docking strategy relying upon an

energy-weighted set of descriptors averaged over multiple conformations of BACE-1.72 Their

method identified novel chemotypes displaying BACE-1 inhibitory activity in a prospective

virtual screening campaign. Selected examples 38-40 are reported in Figure 16.

The third relevant structural element is the enzyme’s catalytic machinery: the aspartic dyad

formed by Asp32 and Asp228 and located at the center of the binding site. Since contacting the

dyad is fundamental for inhibitory activity, several studies have attempted to identify the

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protonation state of the aspartic acid residues conducive to tighter interactions.73,74 Ligands

interacting with the aspartic dyad by means of chemically different groups preferentially interact

with different protonation states. Different protonation states alter the local electrostatic

environment and differently positioned hydrogen atoms in neutral side chains alter hydrogen

bond networks. A mismatch between a certain chemotype and the dyad titration state can

compromise the reliability of ligand affinity predictions. Barman and Prabhakar systematically

investigated the effect of eight rearrangements of the dyad in self-docking simulations carried

out on eight diverse ligands.75 For each chemotype, they identified a protonation state that

returned the best results in terms of reproducing the native pose and the binding energy. Even

when the bound conformation was reproduced by multiple dyad rearrangements, one often

emerged as the most favorable. This confirmed the existence of a clear link between dyad

protonation and ligand bound conformation. Dominguez and colleagues further extended the idea

of improving docking accuracy by modulating dyad protonation, extending pKa calculations to

all titratable residues in the BACE-1 binding site.76 Their results showed a small but significant

improvement with respect to protocols where side chains are assigned default states. Bottegoni et

al. considered five protonation states for the dyad in a virtual screening protocol, which was

purposefully designed to identify multitarget fragments endowed with activity at BACE-1 and

the glycogen synthase kinase 3GSK-3, another known target for AD treatment).77 Fragment-

dyad protonation binding scores were systematically evaluated and each compound was assigned

the best generated score. Interestingly, the only fragment endowed with dual activity at both

targets, 41 (Figure 16) preferentially interacted at the BACE-1 variant displaying a di-

deprotonated dyad.

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In the above studies, protein flexibility and dyad protonation were considered almost

independent issues. In Kacker et al., we combined docking, quantum-mechanical (QM)

calculations, and MD to investigate the roles played by dyad protonation and protein flexibility

in order to identify novel BACE-1 inhibitors by virtual ligand screening.78 Our results

demonstrated that the two variables had to be considered together in order to obtain significant

enrichment in terms of the number of active compounds and their chemical diversity. This may

explain why, until recently, computational methods, which focused on only one of these two

aspects at a time, did not play a pivotal role in BACE-1 inhibitor identification.79 Indeed, MD is a

technique whose role in drug discovery has been steadily growing in recent years. Since the

pioneering work of Park and Lee, MD has been used, either by itself or in combination with QM

techniques, to investigate dyad protonation.80 More recently, constant pH MD (CpHMD)

simulations produced relevant insights into the crosstalk between protonation states and protein

conformations. The original CpHMD implementation includes pH as an additional

thermodynamic term in classic MD.81 Changes in the protonation state of titratable residues were

introduced along the trajectory by a Metropolis Monte Carlo (MMC) procedure. CpHMD can be

embedded within a replica exchange framework, allowing the exchange of pH between adjacent

replicas.82,83 This significantly improves sampling, generating much more efficiently a converged

thermodynamic ensemble in both conformational and protonation spaces. Constant pH replica

exchange MD (pH-REMD) was used to elucidate, at the molecular level, the role of pH in

substrate and inhibitor binding.84 BACE-1 exists in multiple conformational states. At its

working pH (between 2.5 and 4.5), the catalytically active conformation is the most populated.

Values of pH outside this range shift the thermodynamics equilibrium toward self-inhibited, non-

catalytically competent conformers. In another study, Kim and colleagues used constant pH-

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REMD to calculate pH-dependent binding free-energy profiles associated with selected BACE-1

inhibitors.85 They reported how free-energy-calculation errors exceeding 8 kcal/mol can be

caused by incorrectly assigning the protonation state of both the catalytic dyad and other

titratable residues surrounding the catalytic machinery. Constant pH-REMD was also used to

retrospectively explain a puzzling affinity difference between two structurally related BACE-1

inhibitors.86 Even relatively high-resolution crystallographic structures of the bound complexes

could not account for the difference in potency, since the two molecules interacted with the

enzyme with comparable orientations and contacting the same residues. Simulations explained

how, at BACE-1’s working pH range, the structural differences between these compounds, while

very subtle, were still pronounced enough to stabilize different protonation states in the dyad,

altering the surrounding H-bond pattern and ultimately accounting for the different potency.

In more advanced stages of drug discovery, the research focus shifts from identifying novel

compounds to optimizing the identified hits. BACE-1 inhibitors have been successfully

investigated using protocols that estimate the relative binding free-energy variation associated

with a structural modification of a molecule.87 The idea is to explore the chemical space around

an interesting scaffold to identify those substituents which lead to improved affinity or, more

generally, a better pharmacological profile. At this stage, using these more computationally

demanding methods is acceptable because, on average, there are fewer molecules to investigate.

Ciordia and colleagues used free-energy perturbation (FEP) to investigate a series of amidine-

containing spirocyclic BACE-1 inhibitors.88 Targeting specific subpockets at BACE-1 binding

site, they first calibrated the FEP procedure on retrospective data to identify the best trade-off

between sampling thoroughness and correlation with experimental values of the simulation

output. The authors emphasized the importance of accomplishing such calculations within an

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acceptable timeframe for a drug discovery campaign, particularly in an industrial setting. They

then applied the tuned protocol to predict new compounds. After synthesis and biological

evaluation, the predicted potencies for the synthesized spiroaminodihydropyrroles (exemplified

by 42 in Figure 16) were in fairly good agreement with the experimental results. In addition to

computational time, FEP calculations are also limited because reliable predictions cannot be

achieved for more radical modifications. To overcome this, Keränen and colleagues recently

reported a “Core Hopping FEP+” approach that was successfully applied to a series of

acylguanidine-bearing BACE-1 inhibitors (exemplified by compound 43 in Figure 16).89

Comparing the computational outcome with experimental results, the match was very accurate

for local substitutions and, while slightly less accurate, still encouraging when more radical

scaffold modifications were attempted. Interestingly, when investigating the effect of sampling

in obtaining converged calculations, multiple short runs returned more accurate results than a

single, more extended trajectory.

In summary, while success stories have been episodically described and several new hit

compounds have been reported, the prospective application of computer-assisted drug discovery

methods to BACE-1 remains challenging. This multifaceted target severely limits the reliability

of standard computational hit-discovery and optimization protocols. In line with a general and

widespread trend in drug discovery,87,90-92 more complex and time-consuming methods based on

MD, QM, and enhanced sampling approaches,90so far applied in an almost exclusively

retrospective fashion, will play an increasingly important role in the prospective development of

new BACE-1 inhibitors.

4. Multitarget compounds centered on BACE-1

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MTDLs are (usually) small organic molecules that can simultaneously modulate more than one

target. They are gaining increasing attention from the medicinal chemistry community because

they can show superior therapeutic profiles relative to conventional single-target molecules,

particularly for treating complex and multifactorial diseases, including neurological disorders.

Indeed treating Alzheimer’s, Parkinson’s, and other neurodegenerative diseases has been one of

the major focuses of multitarget drug discovery strategies in the last twenty years.24 From

Melchiorre et al’s pioneering study93 through to recent MTDLs that interfere with AD’s amyloid

and tau cascades,94-96 many papers have hypothesized that innovative multitarget strategies could

address the complexity of neurodegenerative diseases, which remain a huge unmet medical need

worldwide. However, none of these compounds has progressed beyond the preclinical phase,

demonstrating that MTDLs create a big challenge in terms of both discovery and development.

BACE-1 is a rather difficult target for SB design, mainly because the site is quite solvent-

exposed. This requires a good compromise between hydrophobic and hydrophilic moieties in

order to achieve good affinity, good residence time, and an appropriate PK profile. Additionally,

experimental evidence has shown that different functional groups present on diverse BACE-1

inhibitors can stabilize the dyad in different protonation states, thus altering the dyad’s overall

charge. This makes SB design of BACE-1 binders even more challenging.78 In terms of

designing BACE-1-based MTDLs, it can be very demanding to identify small molecules that can

inhibit BACE-1 in combination with a second (or a third) target. Indeed, it is not easy to design

and optimize small molecules for two targets that are often unrelated, genetically and

biologically. Furthermore, it is well known that pharmacokinetics has been quite an issue in

BACE-1 inhibitor field,17 further challenging the discovery of multitarget compounds centered

on BACE-1. Finally, from the in vivo evaluation standpoint, usually multitarget compounds can

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be particularly complex to investigate, and systematic experimental protocols have to be defined

to properly profile them, also in terms of comparison with drug combinations. For these reasons,

the literature contains just a few reports of MTDLs centered on BACE-1, and these molecules

have mainly been evaluated in vitro by means of biochemical and cell-based experiments. Here,

we review and discuss these examples from the medicinal chemistry perspective. We first

consider dual-target inhibitors modulating BACE-1 and another AD-related target. Then, we

discuss multitarget compounds where more than two targets are simultaneously hit. We will

discuss molecules that inhibit BACE-1 and have other “general” and often target-unrelated

mechanisms, such as antioxidation, radical scavenging, and metal chelation.

A comment is eventually required on the scaffold selection in multitarget drug discovery, as

many compounds can show multiple mechanisms of action in vitro, mainly because of the

promiscuity and wide reactivity of the chemical groups they carry. In these circumstances, there

exist the actual risk that such multitarget compounds are simply promiscuous molecules,

sometimes resulting in pan-assay interference compounds (PAINS) rather than in clean and

rationally designed multitarget compounds.97 This issue should be carefully considered in

polypharmacology-based drug discovery and well-established protocols should be devised to

properly design, discover, and develop multitarget molecules. This aspect is even more relevant

for CNS-directed candidates, as the lead optimization phase should comply with a suitable drug

metabolism and PK profile for CNS, while preserving affinity towards two or more targets.

4.1 Dual BACE-1/acetylcholinesterase (AChE) inhibitors

The acetylcholinesterase (AChE) enzyme was the first target within the cholinergic hypothesis to

be extensively investigated in the search for novel AD drug candidates. Anti-AChE compounds

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are still the only drugs approved for treating AD. Despite their limited efficacy, they can

alleviate symptoms in the early symptomatic phase of the disease.98 From a multitarget drug

discovery standpoint, it was rather obvious that the pharmacophoric functions responsible for

AChE inhibition might be combined with functional groups thought to interact with BACE-1. In

2008, Piazzi et al. reported a novel series of coumarin-derivatives that were potent and balanced

dual BACE-1/AChE inhibitors.99 These compounds were derived from AP2238 (44 in Figure

17), one of the first ever AChE inhibitors that was rationally designed to bind both the internal

and peripheral anionic sites of AChE.100 This peculiar feature allowed 44 to also interfere with

AChE-induced A aggregation, making it an MTDL with a rather unique mechanism of action.

Starting from pharmacophore 44, Piazzi et al. substituted one of its two methoxy groups with a

halophenylalkylamidic group, obtaining rather large compounds endowed with a dual

mechanism of action. Indeed, the potency against AChE was fairly well-preserved, while gaining

activity against BACE-1. In particular, 45 (Figure 17) showed a fairly balanced biological profile

against the two targets, with an IC50 of about 181 nM against AChE and 150 nM against BACE-

1.

In 2014, seeking to identify a novel class of dual BACE-1/AChE inhibitors, the same group

screened an internal library of benzophenone derivatives, with promising AChE-inhibiting

profiles, for activity against BACE-1.101 Most of these compounds were inactive. However, a

couple showed single-digit micromolar activity against BACE-1, but with a remarkably reduced

potency against AChE. This example demonstrates that discovering and, more importantly,

optimizing MTDLs is likely one of the biggest challenges in multitarget drug discovery. Indeed,

optimizing hits against one target often significantly reduces potency against the other(s). This

makes MTDL lead optimization a rather complicated and often frustrating process. Similarly, in

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2015, Rampa et al. reported a series of compounds based on the indanone structure, where

chemical modifications on the common core could switch from BACE-1 to AChE inhibition and

vice versa. This further demonstrates the complexity of optimizing dual-target compounds

against such ancestrally divergent biological targets.102

Following the hybrid approach, Zha et al. very recently designed, synthesized, and biologically

evaluated more than 20 tacrine-benzofuran compounds as dual inhibitors. Of these, 46 (Figure

17) showed subnanomolar potency against AChE and single-digit micromolar inhibition against

BACE-1, further highlighting the complexity of balancing the biological profiles of multitarget

inhibitors.103

In the same year, Costanzo et al. published a promising study involving a series of donepezil-

derivatives as dual AChE/BACE-1 inhibitors. In addition to reporting a sustainable multistep

protocol for synthesizing a set of donepezil derivatives, the authors tested the new molecules for

selectivity against AChE (versus BuChE). Given donepezil’s mild activity against BACE-1, they

also tested these molecules for BACE-1 inhibition. Compound 47 (Figure 17) was slightly less

potent than the parent compound against AChE (Ki = 29 nM versus 10 nM of donepezil), with a

promising inhibiting profile against BACE-1 (less than 20% of residual enzymatic activity at 1

M concentration). This compound could indeed be a promising candidate for developing

AChE/BACE-1 multitarget inhibitors to treat AD.104

In 2009, Zhu et al. used a slightly different strategy to rationally design a series of

BACE-1/AChE inhibitors. In particular, they rationally combined moieties responsible for AChE

inhibition with those able to modulate BACE-1. This led to three series of derivatives with a

dual-target mechanism of action.105 Reviewing all the data published by the authors, it is

interesting to note that most of the compounds showed single-target activity, and that slight

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chemical modifications allowed them to switch from pure AChE inhibitors to pure BACE-1

inhibitors, with a few genuine dual-target compounds. Fine-tuning the single-target and dual-

target activities can deepen our understanding of the SARs for each compound class. This should

allow a more rational and accurate engineering of next-generation BACE-1/AChE dual-target

inhibitors.

For the final examples of dual BACE-1/AChE inhibitors, we mention two studies where the

authors obtained compounds with rather complex mechanisms of action. These molecules

modulated BACE-1, AChE, and additional targets. In 2009, Camps et al. designed and

discovered a series of quinoline-chlorotacrine hybrids with a rather complex biological profile.

These molecules inhibited AChE, by binding at the enzyme’s internal and peripheral anionic

sites.106 As such, these hybrid compounds also counteracted the AChE-induced A aggregation.

Surprisingly, they also inhibited BACE-1. In particular, compound 48 (Figure 17) was about 14

nM against AChE and also blocked almost 80% of BACE-1 activity at 2.5 µM concentration,

pointing to an IC50 in the submicromolar range. In addition, 48 inhibited both AChE-induced and

spontaneous A aggregation in the high micromolar range. Overall, its complex biological

profile suggested 48 as a promising MTDL, acting at different key points in complementary

neurodegenerative cascades of AD. Additionally, this compound crossed the BBB, despite its

large size and rather high MW.

In 2011, Huang et al. designed and discovered a series of quinoxaline derivatives, which also

showed a complex multitarget biological profile.107 These compounds showed BACE-1

inhibition and/or AChE inhibition. Some were also histamine H3 receptor antagonists. The

cholinergic hypothesis provides the rationale for this additional biological activity. Blocking this

presynaptic receptor can increase ACh levels in the synaptic cleft via a different mechanism than

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classical AChE inhibition. The activated presynaptic histamine H3 receptor decreases the release

of ACh from cholinergic neurons. Antagonists of this receptor can thus increase ACh levels,

likely producing a synergistic effect for treating AD. Of the 17 quinoxaline analogues in the

series, most showed activity against one or two targets. Only one compound (49 in Figure 17)

modulated (i.e. inhibited and antagonized) all three selected targets. Similarly, Viayna et al.

recently reported a new series of rhein-huprine hybrids with a rather complex pharmacological

profile, being AChE/BACE-1 dual inhibitors and dual antiaggregating compounds for Aβ42 and

tau.108

This further highlights the complexity of discovering, optimizing, and developing MTDLs.

Pragmatically speaking, we envisage that a well-designed MTDL drug discovery program will

involve the rational and accurate selection of two targets, whose modulation provides a true

synergistic effect against a given disease. This effect can be pharmacologically demonstrated at

an early stage by using selective binders of the two selected targets. Then, the chemical design

step should be conducted very carefully to allow modifications on the identified scaffold(s) that

are not detrimental for either target. SB approaches could be remarkably helpful in these very

early steps. A clear understanding of the SARs could help in rationally selecting modifications to

achieve balanced MTDL lead candidates. There should be sufficient room (in terms of chemical

tractability, size, and MW) for lead optimization, so that pharmacokinetics and toxicology

profiles can be carefully considered, and chemical modifications made to improve the profile of

the new compounds without losing affinity for either target. FB drug discovery would be

particularly appropriate here. This is because fragments can intrinsically be endowed with a

polypharmacological mechanism of action, and because there is more room for chemical

modifications on fragments than on larger lead-like compounds.

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A comment is here required on the possible clinical utility of dual AChE/BACE-1 inhibitors in

AD. Actually, the lack of efficacy of 1 in mild-to-moderate AD suggests that it is too late in the

disease process for BACE-1 inhibition to be effective. Conversely, this patient cohort could be

symptomatically treated with AChE inhibitors, despite the well-known moderate efficacy of

these inhibitors in the clinical practice. Indeed, the two targets can play a role in two well-

separated phases of the disease progression. We may therefore be confronted with a scenario

where a dual AChE/BACE-1 could lead to a rather superfluous anticholinesterase activity in

prodromal AD patients, and to a clinically useless inhibition of BACE-1 in mild-to-moderate AD

patients. The two drugs could conversely be used separately in different phases of the disease,

with clear different mechanisms of action tackling different pathological pathways in the AD

progression. The most recent clinical trials have greatly helped in better detangling the role of

different pathways and molecular mechanisms in the disease progression and therapeutic

treatments.

4.2 Dual BACE-1/GSK-3 inhibitors

While BACE-1 is one of the key enzymes within the amyloid cascade, GSK-3 is one of the

kinases responsible for phosphorylation of the tau protein, which then detaches from

microtubules to form neurofibrillary tangles. This is AD’s second key histopathological

hallmark. Indeed, one strategy for treating AD could be inhibiting both enzymes, possibly

producing a downstream synergistic effect. With this in mind, we recently discovered and

developed a class of fragments bearing a triazinone skeleton as dual BACE-1/GSK-3

inhibitors.94,95 In detail, exploiting a ligand-based approach, we combined the pharmacophoric

functions responsible for binding to the enzymes. These were i) the cyclic amide group that binds

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to kinases, and ii) the guanidine moiety that, when positively charged, binds to the aspartic dyad

of BACE-1. Merging these functional groups led to a series of triazinone derivatives with a

balanced micromolar affinity for the two targets. Most of the new derivatives had a very low

MW. This prompted us to assess the pharmacokinetic profile of one selected compound.

Compound 50 (Figure 18) showed good bioavailability (66%) upon oral administration in mice,

and good BBB penetration. Indeed, at the very low dose of 3 mg/kg, 50 showed a brain

concentration of 0.62 M after 30 minutes. Although below the in vivo target engagement levels,

this represents an attractive starting point towards a promising lead optimization campaign.

Interestingly, cellular data showed that this multitarget compound class also had anti-

neuroinflammatory and neurogenic effects. This may have been due to GSK-3 modulation or

the simultaneous inhibition of both targets. Subsequent in vivo data will ultimately demonstrate

whether the molecule’s profile makes it suitable for progression through the drug discovery and

development pipelines.

Taking a different approach to dual BACE-1/GSK-3 inhibitors, Di Martino et al. developed

MTDLs by exploiting the versatility of the natural compound, curcumin.96 Using a SB approach

to modify this privileged molecule, suitable moieties were mounted onto the curcumin

framework to confer the new molecules with the ability to bind to both enzymes. The series of

curcumin derivatives was fairly potent against BACE-1 and GSK-3but, for most derivatives,

the two actions were not properly balanced. For example, 51 (Figure 19) showed nanomolar

inhibition of BACE-1, but single-digit micromolar inhibition of GSK-3.

Interestingly, some of these compounds induced the NAD(P)H:quinone oxidoreductase 1

(NQO1). This enzyme is responsible for the endogenous conversion of quinones to

hydroquinones. By counteracting the oxidative stress that is a common feature of AD, this could

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translate to a potential neuroprotective effect. Indeed, a recent longitudinal study demonstrated

that air pollution from traffic could be an important risk factor for vascular dementia and AD.109

Exposure to air pollution can lead to chronic oxidative stress, which is a risk factor in the

pathogenesis of AD.110 NQO1 could thus be an intrinsic detoxifying and antioxidant weapon for

tackling oxidative stress. Molecules that can induce this enzyme are potential antioxidant drug

candidates for AD treatment. This intriguing mechanism was originally reported by some of us

in 2007, when investigating the polypharmacological profile of memoquin.111 In particular, we

demonstrated that the antioxidant effect of this quinone-bearing compound was due to the

hydroquinone species, which could be obtained through the enzymatic activity of NQO1 in vivo.

This concept was confirmed and extended in a subsequent study of memoquin in vivo. We

showed that sulphorafane, a potent natural inducer of NQO1, significantly increased the

antioxidant and neuroprotective effects of memoquin and other quinone-bearing compounds.112

According to the “mitochondrial dysfunction hypothesis” in AD, exploiting this detoxifying and

antioxidant mechanism could be one innovative strategy for tackling AD’s pathophysiology. The

curcumin-based derivatives developed by Di Martino et al. were therefore endowed with a rather

complex polypharmacological profile thanks to their dual inhibition of BACE-1 and GSK-3 and

their positive antioxidant effect as inducers of the NQO1 enzyme. It should however here be

mentioned that curcumin, as well as other natural compounds, could be endowed with a highly

promiscuous profile,97,113,114 and therefore further experimental evidences should be collected to

clearly demonstrate that such compounds specifically modulate the intended targets, rather than

showing a PAINS-like profile.

In conclusion, the papers by Prati et al. and Di Martino et al. point the way to a systematic

development of dual BACE-1/ GSK-3 inhibitors. These two key enzymes are involved in AD’s

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two major pathological cascades (amyloid and tau) of AD. The resulting dual inhibitors may be

endowed with pharmacological profiles that are superior to those of classic single-target

molecules.

5. Conclusions and perspectives

While submitting this paper, we learned about compound 1’s lack of efficacy in mild-to-

moderate AD.115 Thus, we are again faced with profound questions concerning the amyloid

hypothesis along with the timing of BACE-1 inhibition, which remains one of the critical points

for properly developing novel drug candidates based on this hypothesis. We are forced to ask

ourselves: should we continue to develop BACE-1 inhibitors? Will it be worth the time, effort,

and cost? How large is the impact of the proper patient stratification and the clinical trial design?

It barely seems logical to persist in developing molecules that inevitably fail. Indeed, these

questions will probably find answers in the still ongoing phase 3 clinical development of 1 in

prodromal AD.

At this point, there is obvious appeal in the view that AD scientists in academia and industry

should learn from failures, both their own and those of others. Thus, a more appropriate question

may be: what have we, as a community, learned in the last 20 years of discovering and

optimizing BACE-1 inhibitors?

First, nowadays we know how to merge the structural requirements for potency and protease

selectivity with the strict need for BBB permeation, oral bioavailability, low metabolic clearance,

and tolerable toxicity.

Second, we have corroborated the power of FB approaches in CNS drug discovery: the case of 1

nicely exemplifies how an NMR screen can provide a weak fragment-hit that can be further

optimized into a nanomolar protease inhibitor. Other examples have since followed.10,11,94,95

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Third, we have successfully incorporated the concepts of personalized medicine into drug

discovery and clinical trials. In the future, this could streamline the drug development process

and enable targeted therapies (once developed) to be delivered appropriately. In this respect, we

expect very interesting data to be produced by testing BACE-1 inhibitors in early-onset familial

AD patients. Indeed, they may be the ideal stratified patient population who will benefit most

from a “pure” anti-amyloid therapy. From this rational stratification, we could then perhaps

better understand whether BACE-1 inhibitors could be useful for non-familial AD patients.

Fourth, we have been reminded again of something that we already knew:24 the unfathomable

complexity of AD. Perhaps, the amyloid hypothesis is not sufficient to explain this intricate

multifactorial pathogenesis of AD. A feasible solution may thus be afforded by a well-designed

MTDL that can synergistically inhibit BACE-1 and modulate a second interrelated AD target.

The examples discussed herein point to the power of this approach and highlight the still-

undiscovered potential and the complexity of such molecules in AD drug discovery and

development.

AUTHOR INFORMATION

Corresponding Author

*E-mail: andrea.cavalli@iit.it

Author Contributions

The manuscript was written with contributions from all authors. All authors have approved the

final version of the manuscript.

ACKNOWLEDGMENT

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We thank the University of Bologna and the Italian Institute of Technology for financial support.

We thank Grace Fox for copyediting and proofreading.

ABBREVIATIONS

CatD, cathepsin D; CpHMD, constant pH molecular dynamics; FB, fragment-based; FEP, free-

energy perturbation; GSK-3glycogen synthase kinase 3 MMC, Metropolis Monte Carlo;

MTDLs, multitarget-directed ligands; NQO1, NAD(P)H:quinone oxidoreductase 1; pH-REMD,

pH replica exchange molecular dynamics; PPIs, protein-protein interactions; QM, quantum-

mechanical; SB, structure-based; 7-THP, 7-tetrahydropyrane; 8-THP, 8-tetrahydropyrane; WH,

warhead.

BIOGRAPHIES:

Federica Prati received her Master’s degree in Chemistry and Pharmaceutical Technologies

from the University of Bologna in 2010. In 2013, she was a Visiting Scholar at the University of

Dundee Drug Discovery Unit (DDU), and in 2014 she was awarded the Ph.D. in drug discovery

from the University of Genova, in collaboration with the Istituto Italiano di Tecnologia

(Genova). She is presently working as Open Lab-Marie Curie fellow at the Tres Cantos

medicines development campus in GlaxoSmithKline, in close partnership with the DDU of

Dundee.

Her research interests focus on medicinal chemistry and drug discovery. Particularly, she

contributed to the development of small active molecules, through fragment-based and multi-

target approaches, against cancer, tuberculosis, neurodegenerative and neglected tropical

diseases.

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Giovanni Bottegoni is a scientist and an entrepreneur. He holds a Ph.D. in pharmaceutical

sciences (University of Bologna, Italy, 2006) and a master in healthcare management (SDA

Bocconi, Italy, 2014). For two years, he was post-doc at The Scripps Research Institute in La

Jolla, California (USA), and then he joined the Istituto Italiano di Tecnologia (Genova, Italy)

first as a post-doc and later as a team leader. In 2014, he co-founded BiKi Technologies, a start-

up company that commercializes software solutions for medicinal chemistry and, for two years,

was CEO of the company. He currently works as a senior scientist for Heptares Therapeutics

(Welwyn Garden City, UK). He is the author of over 50 publications and co-inventor in several

patents. In 2015, he was awarded the DCF prize for medicinal chemistry by the medicinal

chemistry division of the Italian chemical society.

Maria Laura Bolognesi is Professor of Medicinal Chemistry and Director of the Chemistry and

Pharmaceutical Technologies degree program at the University of Bologna. Her research spans

the design, synthesis and pharmacological investigation of small molecules in the

neurodegenerative and neglected tropical disease therapeutic areas. She has a track record of

more than 140 publications in high-ranked scientific journals, including patents and patent

applications, and more than 50 invited talks worldwide. She was awarded a Distinguished

Visiting Professor Fellowship at the Complutense University of Madrid in 2009, and a PVE

Fellowship at the University of Brasilia in 2014. She is also an Associate Editor of Journal of

Medicinal Chemistry.

Andrea Cavalli is Professor of Medicinal Chemistry at the University of Bologna and Research

Director at the Istituto Italiano di Tecnologia, where he is also Deputy Director for Computation

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& Data Science. His research interests are in the field of computational drug discovery. In

particular, he has developed and applied computational approaches to accelerate the discovery of

drug-like compounds in several therapeutic areas, including cancer, neurodegenerative diseases,

and neglected tropical diseases. Prof. Cavalli is the author of more than 200 publications in high-

ranked journals, inventor of several PCT international patents, and has delivered numerous

invited lectures and seminars. In 2003, he was awarded the “Farmindustria Prize for

Pharmaceutical Research”. He is co-founder of the high-tech startup company, BiKi

Technologies.

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Figure 1. Advanced Merck’s BACE-1 inhibitor 1.

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Figure 2. Aminohydantoin 2.

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Figure 3. Spirocyclic aminohydantoins 3 and 4.

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Figure 4. Spirocyclic aminohydantoin 5.

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Figure 5. Spirocyclic aminooxazoline 6.

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Figure 6. Spirocyclic aminooxazolines 7-11.

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Figure 7. Iminopyrimidinones 12 and 13.

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Figure 8. Aminothiazines 14-22.

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Figure 9. Aminothiazines 23, 27 and 28.

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Figure 10. Aminothiazines 24-26.

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Figure 11. Iminopyrimidinone 29 and iminothiadiazinane dioxide 30.

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Figure 12. Spiropiperidine iminohydantoins 31 and 32.

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Figure 13. Aminoxazine 33.

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Figure 14. Aminoquinoline 34 design rationale.

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Figure 15. Cyclic acyl guanidine 37.

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Figure 16. Examples of BACE-1 inhibitors (38-43) discovered employing computer-assisted drug discovery methods.

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Figure 17. Dual binding site AchE inhibitor 44, and dual BACE-1/AChE inhibitors 45-49.

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Figure 18. Dual BACE-1/GSK-3 triazinone inhibitor 50.

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Figure 19. Dual BACE-1/GSK-3 curcumin inhibitor 51.

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TOC Graphic

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