Structure-Based Design of Î²-Site APP Cleaving Enzyme 1 (BACE1

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Structure-Based Design of β‑Site APP Cleaving Enzyme 1 (BACE1) Inhibitors for the Treatment of Alzheimer’s Disease Jing Yuan,† Shankar Venkatraman,† Yajun Zheng, Brian M. McKeever, Lawrence W. Dillard, and Suresh B. Singh* Vitae Pharmaceuticals, 502 W. Office Center Drive, Fort Washington, Pennsylvania 19034, United States ABSTRACT: The amyloid hypothesis asserts that excess production or reduced clearance of the amyloid-β (Aβ) peptides in the brain initiates a sequence of events that ultimately lead to Alzheimer’s disease and dementia. The Aβ hypothesis has identified BACE1 as a therapeutic target to treat Alzheimer’s and led to medicinal chemistry efforts to design its inhibitors both in the pharmaceutical industry and in academia. This review summarizes two distinct categories of inhibitors designed based on conformational states of “closed” and “open” forms of the enzyme. In each category the inhibitors are classified based on the core catalytic interaction group or the aspartyl binding motif (ABM). This review covers the description of inhibitors in each ABM class with X-ray crystal structures of key compounds, their binding modes, related structure−activity data highlighting potency advances, and additional properties such as selectivity profile, P-gp efflux, pharmacokinetic, and pharmacodynamic data.

1. INTRODUCTION Amyloid plaque formation in the brains of the aging population is tied to the pathogenesis of Alzheimer’s disease (AD) and associated dementia.1,2 This disease is a major contributor to dementia and death in individuals above age 65.2,3 Amyloid plaques are hypothesized to form from the aggregation of amyloid-β peptides (Aβ) when their levels rise above normal. The rise in the level of Aβ peptides is attributed to their increased production resulting from the sequential enzymatic processing of amyloid precursor protein (APP) by the aspartyl proteases, β- and γ-secretase.2 In late onset AD, data indicate that the increase in Aβ levels is due to decreased clearance from the brain.4 The β-secretase 1, also known as β-site APP cleaving enzyme 1 (BACE1), is predominantly expressed in the brain and is an integral membrane protein with its carboxyl terminal end associated with the membrane in endosomes.5−9 It catalyzes the first and key step in the production of amyloidβ peptides. Of all the antiamyloid approaches targeted toward treating AD, BACE1 inhibition is commonly felt to be the one most likely to succeed.10 The genetic validation with BACE−/ − knockout mice and the reduced propensity to proteolysis by BACE1 of a naturally occurring variant of APP associated with a decreased prevalence of dementia substantiated the role of BACE1 in the etiology of Alzheimer’s disease.10,11 These biological validations have provided strong evidence that agents that block the enzymatic activity of BACE1 will be efficacious in treating AD. BACE1 catalyzes the proteolytic cleavage of APP between Met and Asp residues in the sequence stretch of Lys670Met671-Asp672.12 A double mutant APP with Asn670-Leu671Asp672 sequence at the cleavage site, found in Swedish patients with early onset Alzheimer’s disease, is a more efficient substrate in vitro than the wild-type protein and is the cause for the disease in this family.12 The identification of BACE1 as © XXXX American Chemical Society

an aspartyl protease, and the subsequent expression, purification, and crystallization of the enzyme in the presence of a ligand, spurred structure-based design efforts to identify reversible inhibitors that target its active site.5−9,13,14 The success achieved in the structure-based design of inhibitors of HIV-1 protease, another aspartyl protease, spurred large scale efforts toward the design of BACE1 inhibitors in academia and the pharmaceutical industry.15 The scale of the effort behind BACE1 inhibitors is evident by noting that there are 235 crystal structures of BACE1 in the Protein Data Bank, most of them in complex with active-site directed inhibitors.16 The localization of BACE1 in the central nervous system (CNS) has posed a significant challenge in identifying low molecular weight inhibitors that exhibit potent activity in blocking the enzyme while having selectivity and good oral bioavailability, brain penetration, fraction unbound, and efficacy at a therapeutically acceptable dose. Taxonomically, BACE1 belongs to the pepsin family under the aspartyl protease superfamily and has the most sequence identity with BACE2 (52%), cathepsin D (29%), pepsin (27%), cathepsin E (27%), and renin (24%).17 Since the overall structural fold for all these proteases is similar, BACE1 inhibitors may have preference for binding to these proteases from the superfamily. Thus, the majority of the BACE1 inhibitors reported to date exhibit very little or no separation for BACE2 inhibition but exhibit varying degrees of selectivity toward the other enzymes listed above. Besides these enzymes, the inhibition of hERG activity by BACE1 inhibitors has been an additional challenge to overcome. There is evidence that the increase in the pKa and lipophilicity of the compound increases the chance of inhibiting hERG channel.18 Received: November 9, 2012

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dx.doi.org/10.1021/jm301659n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Sequence alignment of full length BACE1 sequences from human, rhesus, dog, rat, and mouse.40 The sequence numbers used in the text are offset by 60 relative to the full length sequence to be consistent with the numbering used for aspartyl proteases. Because of the availability of partial BACE1 sequence from dog, the C-terminal stretch that is available is highlighted in yellow along with all the others. The N-terminal stretch not available for dog is highlighted in cyan.

isoforms of P-gp, the concentration of compounds observed in rodent brains after oral administration is generally a predictor of their exposure in human brains.23,29 The oral bioavailability of small molecules correlates with calculated and measured physical properties.18,32−35 Similarly, some physical properties of compounds appear to be correlated with their ability to penetrate the CNS. Physical properties that impact the availability of molecules in the brain and reduce Pgp mediated efflux are molecular weight, polar surface area, log D, pKa, number of hydrogen bond donors, and chemical modifications.19,27,30,34,36,37 Also, the percent of the drug not bound to the brain tissue (the brain free fraction) is used as a guide for the capacity of a compound to exhibit measurable pharmacodynamic (PD) effects.23,30,31 The greatest challenge in progressing BACE1 inhibitors to the clinic has been the number of in vitro and in vivo criteria a compound has to satisfy in order to exhibit efficacy and safety at a therapeutically acceptable dose, since it will be given chronically and may be used prophylactically in mild AD patients. To determine the efficacy of BACE1 inhibitors in vivo, rodent (mice and rat), dog, and primate (rhesus) models can be used to measure the lowering of the Aβ peptide levels in the brain and CSF.38 Both the enzyme and substrate are well-

The location of BACE1 in the brain presents an additional challenge, since the inhibitors must cross the blood−brain barrier (BBB). The key transporter protein, P-glycoprotein (Pgp), binds to compounds that attempt to pass through the BBB and pumps them back into the blood.19,20 In general, basic compounds with increased polarity tend to be substrates of Pgp and get pumped or effluxed back into blood circulation.18,19,21−23 Several theoretical models and rules have been proposed for predicting compounds that tend to be P-gp efflux substrates, but in practice the strongest predictor whether a given compound is a P-gp substrate is derived by the experimentally determined efflux ratio (K apical→basal / Kbasal→apical).21,24−28 The efflux ratio is measured in vitro in MDCK or CaCo2 cells overexpressing P-gp or in vivo (Cbrain/ Cplasma or Cbrain/Cmuscle).26,28,29 There is a high degree of correlation between the efflux ratios measured in vitro and in vivo, which helps predict a compound’s brain permeability.21,26,29−31 Compounds with efflux ratios less than 10 in MDCK cells are likely to have good brain permeability, while ratios of 10−20 have a potential for brain permeability, and higher ratios tend to be very good substrates of P-gp and have low brain permeability. Since there is also a good correlation observed between the efflux ratios of rodent and human B



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defined as “closed” when the Tyr71 side chain hydroxyl on the flap is within hydrogen bonding distance to the NH of the Trp76 side chain, which physically separates the S1 and S2′ sites. The conformation of BACE1 is defined as “open” when the flap moves away from the catalytic Asp, the hydrogen bond between the flap residues Tyr71 and Trp76 is not present, and Tyr71 does not occupy the position between the S1 and S2′ sites (Figure 2). However, there are some complexes in the

conserved across species, so there is a good exposure/response relationship across species. There is a high degree of sequence homology between the BACE1 and the APP sequences from mice (96.4%, 96.6%), rat (96.4%, 96.9%), dog (nd,39 96.8%), and rhesus (99.6%, 99.5%) versus the human isoforms.6−8,40−46 The sequence differences in the BACE1 enzyme are not near the binding site (Figure 1), and for APP, the closest residue that differs from the human sequence is at the P5′ site (Arg676Gly). There are no data to suggest that these sequence changes affect the processing of the APP by BACE1 enzyme across these species.38 Several animal models are available and are used to test the long-term efficacy of BACE1 inhibitor compounds. These include transgenic mice expressing human APP with Swedish mutations that express higher levels of Aβ peptides relative to the wild-type, which facilitates the measurement of Aβ (Tg2576).12 A transgenic mouse model expressing human wild-type APP47 and a primate model to measure the Aβ levels in the CSF using catheters implanted in the cisterna magna of rhesus monkeys48 have also been described. Normal wild-type Sprague−Dawley rats can also be used to measure the efficacy of BACE1 inhibitors with Mesoscale equipment for detection because of intrinsically low levels of Aβ in these wild-type animals.49,50 The pharmacodynamic effect of BACE1 inhibitors on the lowering of Aβ peptides in the brains of human subjects was recently shown by three different groups in human clinical trials. This review summarizes two distinct categories of inhibitors designed based on conformational states of “closed” and “open” forms of the enzyme (vide infra). The majority of inhibitors designed for the closed-form were linear and macrocyclic peptidomimetic compounds possessing potent activity against BACE1, with only a handful of them exhibiting efficacy in vivo. A series of fragment screening approaches identified inhibitors that bind to the open-form of the enzyme, which were subsequently optimized and advanced to the clinic. In each category the inhibitors are classified based on the core catalytic interaction group or the aspartyl binding motif (ABM). This review covers the description of inhibitors in each ABM class with X-ray crystal structures of key compounds, their binding modes, related structure−activity data highlighting potency advances, and additional properties such as selectivity profile, Pgp efflux, pharmacokinetic, and pharmacodynamic data.

Figure 2. Superimposed crystal structures of flap-open (PDB code 2OHU, purple) and flap-closed (PDB code 1W51, yellow) BACE1 crystal structures. Ligands have been omitted for clarity. Complete protein structures from X-ray structures are shown in panel A and close-up view of the active site is shown in panel B. The conformation of Tyr71 in the flap-open 2OHU structure (purple) breaks the hydrogen bond with Trp76 and no longer occupies the position between the S1 and S2′ sites. The figure was generated using Accelrys’ Discovery Studio graphical user interface.155

open-form like conformation with a weak or water-mediated hydrogen bond between Tyr71 and Trp76. The uncomplexed enzyme also has the flap-open conformation with the flap open wider than in the complex crystal structure (PDB codes 1W50 and 1W51). Molecular dynamics simulations suggest that both the open and closed conformations are readily accessible at room temperature which includes the conformational flexibility of Tyr71.53,54 Increased flexibility is also observed with multiple conformations in the loop located near Ser10, known as the 10s loop located near the S3 site. BACE1 in its free state has a water channel in the “special pocket” near the 10s loop site connecting the S3 site and the bulk solvent accessible through the 10s loop. It is called a special pocket because the substrate does not have a residue that occupies this site, a terminology borrowed from the one used for renin. 55 A possible conformational interplay with coupled motion between the flap and the 10s loop and ligand binding is postulated.56,57 In crystals of the free, uncomplexed enzyme, a solvent water molecule is observed between the catalytic residues Asp32 and Asp228 (PDB code 1W50), and this water is hypothesized to aid in the catalysis of the hydrolytic cleavage of the peptide bond between the Met and Asp residues of APP.51 This water is also observed in other aspartyl proteases, and the catalytic mechanism proposed for them is the basis for the proposed catalytic mechanism for BACE1.58,59 In the majority of the BACE1 crystal structures, carboxylate groups of the catalytic dyad are held almost coplanar by a network of hydrogen bonds involving main chain amide NHs (Gly34 and Thr231) and side chain hydroxyls (Ser35 and Thr231). This structure of the

2. ENZYME STRUCTURE, DYNAMICS, AND CATALYTIC MECHANISM BACE1 is a monomeric protein with the catalytic site containing the two aspartate residues 32 and 228 located between the N- and C-terminal domains. The active site is sheltered by a β hairpin loop between Val67 and Glu77 that forms a large portion of the binding pocket and provides shielding from the solvent to facilitate efficient catalysis.51 The acidic conditions optimal for the enzyme indicate that an inhibitor containing a basic amine with a pKa of 6.0 or more would exhibit potent affinity. The β hairpin loop commonly known as the “flap” is the most flexible part of the binding site assuming multiple conformational states at room temperature. A key residue in the flap, Tyr71, adopts a conformation complementary to the shape and nature of the ligand bound in the active site. The changing position of the flap relative to the catalytic dyad provides a means for the substrate and the ligands to diffuse into and out of the active site.51,52 The conformation of the protein is C



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Scheme 1. Proposed Mechanism for BACE1 Catalyzed Peptide Bond Cleavagea

a

This mechanism is consistent with those proposed for other aspartyl proteases, kinetic studies, and the X-ray and neutron diffraction data.58,59,61,62

catalytic residues has been proposed as an essential element for enzymatic activity of aspartyl proteases, and it likely applies to BACE1 as well.60−62 The bound water at the catalytic center is believed to be involved in the peptide bond hydrolysis.51 The current hypothesis is that one of the catalytic aspartyl residues acts as the general acid while the other acts as the general base.62−65 The catalytically competent enzyme is expected to be in the monoprotonated state in the active site (Scheme 1).62 The pKa values of these two Asp residues have been shown with solvent kinetic isotope studies to be 3.5 for the general base and 5.2 for the general acid.62 The enzyme exhibits optimal catalytic activity at pH 4.5 in vitro and is believed to be operating at a slightly higher pH in vivo in the lumen of the endosomes.10 The four oxygen atoms of Asp32 and Asp228 of BACE1 enzyme are not equivalent owing to their microenvironment, suggesting a possible set of distinct protonated states.57 For the purpose of inhibitor design, a qualitative analysis suggests that only the mono- and dideprotonated species are relevant.62 Protonation state could also be influenced by the nature of the ligand bound.65,66

Chart 1. Calculated Physical Properties of 119 Representative BACE1 Inhibitors That Bind to the Closed Form of the Enzyme

3. BACE1 INHIBITOR DESIGN This review highlights key crystal structures of BACE1 inhibitor complexes for each binding mode with associated structure− activity data and the inhibitor profiles. A survey of the latest patent literature of BACE1 inhibitors was presented recently.67 The first wave of crystal structures published for BACE1 contained molecules with transition state mimics, hydroxyethylene and hydroxyethylamines, interacting with the catalytic aspartates Asp32 and Asp228. These molecules bound to the closed-form of the enzyme and filled most of the pockets in the active site that the substrate fills. The potent inhibitors of the closed form of the enzyme tended to be of high molecular weight and high polar surface area and consequently possessed poor binding efficiency68 (Charts 1 and 3). In general, these compounds suffered from poor oral absorption and low exposure in the brain due to P-gp efflux. Only a handful of literature examples in this class of inhibitors exhibit brain permeability and measurable pharmacodynamic effect in vivo. However, second generation compounds that bind to the open form of the enzyme were more druglike and penetrated the CNS having MW < 500 and lower P-gp efflux values. These compounds emerged from fragment-based screening techniques such as high throughput X-ray crystallography, NMR,

surface plasma resonance (SPR) technology, and high throughput screening. The design and development of openform inhibitors involved iterative X-ray crystallography, structure-based design, and medicinal chemistry leading to one of the most efficient binders of BACE1 (Charts 2 and 3). The inhibitors described below are classified based on the core functional group or aspartyl binding motif (ABM) that interacts with the catalytic center. 3.1. Inhibitors of the “Closed-Form” of BACE1. A large majority of inhibitors designed for the closed-form of the enzyme originating from peptidic substrate were potent but suffered from poor metabolic stability, high clearance in vivo, high efflux by P-gp, and lack of efficacy in the preclinical animal models. These molecules, mostly of high molecular weight and polarity, were optimized to reduce the MW and polarity with D



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Chart 2. Calculated Physical Properties of 92 Representative BACE1 Inhibitors That Bind to the Open Form of the Enzyme

of transgenic mice after a single intraperitoneal (ip) dose of 8 mg/kg (mpk).74

Chart 3. Binding Efficiencies of BACE1 Inhibitors Binding to (a) the Closed Form and (b) the Open Form The introduction of an aromatic ring in the P1 side chain with phenoxymethyl and benzyloxymethyl led to high MW (∼900) potent inhibitors with some exhibiting up to 1000-fold selectivity against cathepsin D (PDB codes 3DM6, 3IXK, and 3IXJ).75−78 The X-ray structures of BACE1 in complex with inhibitors such as 2 revealed that the P1 and P3 side chains occupying the S1 and S3 sites are not separate but one large contiguous pocket of the enzyme (Figure 3). This makes the S1 and S3 sites a superpocket similar to what is observed for renin.55

very little improvement in pharmacokinetic (PK) and PD properties. 3.1.1. Hydroxyethylenes (HE). As one of the transition state mimics, hydroxyethylenes evolved from statines designed for the aspartyl proteases renin and HIV1 protease.69,70 The first disclosed HE 1 (OM99-2) was designed against BACE1 exhibiting a Ki of 0.0016 μM. Its crystal structure (PDB code 1FKN), the first BACE1/inhibitor complex to be published, showed the key interaction between the hydroxyl functional group and the catalytic aspartates.13 The 8-residue inhibitor 1 spans the substrate binding pocket from S4 to S4′ subsites. This structure was used to optimize 1 to initially improve the affinity albeit with increased MW (>1600) and peptidic nature (PDB codes 1M4H, 1XN2, and 1XN3). Eventually the design process led to potent, constrained, and 100-fold selectivity versus BACE2 and >500-fold selectivity against cathepsin D. However, 44 exhibited a poor brain to plasma ratio of 0.25, preventing it from being developed further.

Another high throughput screen hit 45 from the same group showed BACE1 activity of 38 μM and cell activity of 16 μM.123 The X-ray crystal structure of 45 (PDB code 3IGB) with BACE1 showed aminoimidazole directly interacting with the catalytic site via hydrogen bonds and the two phenyl rings occupying the S1 and S2′ sites. This structure was used as the starting point for further optimization using structure-based design to generate a more efficient binder. The S3 region, directly accessible from the phenyl ring in the S1 site, provided a logical path for optimization, as exemplified by compound 46. Further optimization of compound 49 yielded compounds 50 and 51 with good in vitro potency and microsomal stability.125 Acute oral administration of these compounds at 30 mpk to Tg2576 mice resulted in a 25−30% reduction of plasma Aβ40 at the 8 h time point. However, despite low efflux ratios, these compounds provided no significant Aβ reduction in the brain. This was attributed to the low free fraction in the brain based on the in vitro measurement of binding to the brain tissue.

A structure-based effort utilizing the X-ray structural information from compound 45 led to the design of 48 and 49 with improved potency and selectivity.124,125 Interestingly, the bicyclic ABM was modified to an iminohydantoin ring, strikingly similar to compound 47 discovered by an independent group as described below. The pyrimidine ring off the phenyl occupies the S3 region and interacts with serine 229 of the protein via a water mediated hydrogen bond. The modification of the P2′ group using the difference in sequences between BACE1 and BACE2 led to several orders of magnitude improvement in selectivity over BACE2 and cathepsin D. However, these compounds did not demonstrate in vivo PD K



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Additional analogues of 49 explored led to one of the most efficient binders of BACE1. This was achieved by replacing the phenyl ring with various heterocycles at the P2′ position.126 Replacement of phenyl by pyrazole led to compounds with selectivity against BACE2 (19×) and cathepsin D (950×), albeit with a shift in potency in the cellular assay (21×). The crystal structure of 53 with BACE1 showed that its pyrazole ring in the S2′ site did not hydrogen-bond with the Tryp76 side chain. This observation led to the replacement of the pyrazole ring by a thiophene ring with an ethyl ketone in the 2-position, leading to improvements of 8-fold in enzyme potency and 170fold in cell activity (54).

An independent research group identified a novel isothiourea ABM through fragment based NMR screening followed by crystallographic confirmation of the solution hit (55).127 In order to reduce the likelihood of toxicity from the isothiourea group, the functional group was converted to an amidine and cyclized to generate an acylguanidine. The binding orientations of 55 and 56 are similar with their guanidine moiety aligned with the catalytic aspartates forming hydrogen bonding interactions (PDB code 3L59). The m-chorobenzyl moiety fills the S1 site in a unique fashion. The flap conformation in these structures was in the closed conformation. The addition of the isopropyl group to compound 56 leads to a flipped orientation of the ABM in compound 57, with the mchlorophenyl ring positioned in the S1′ site and the isobutyl group in the S1 site. This change results in the compound bound to the flap open conformation of the protein (PDB code 3L5B). This open conformation of the protein is common for all the molecules that present a heterocyclic ring system containing an amidine moiety and substituents in two or more subsites of the enzyme. The optimization of compound 57 led to a potent inhibitor 58 (PDB code 3L5E). An effort to remove the flexible and polar elements of 58 and improve brain permeability led to efficient binding compound 59 (PDB code 4DJU). It lost ∼300-fold potency against the enzyme but presented an ABM similar to that in 58 and provided a starting point for further optimization to improve potency, selectivity, physical properties, and consequently CNS permeability. As noted above, compound 59 is identical in structure to compound 47. A straightforward path to improve affinity of compound 59 was achieved by attaching a heteroaromatic ring to the meta position of the phenyl ring in S1 pocket to fill the adjacent S3 pocket. The X-ray structure of compound 60 (PDB code 4DJW) confirmed that it had a binding mode similar to 59.128,129 The nitrogen of the 3-pyridine points toward a special pocket at the S3 site and hydrogen-bonds with the water molecule present at 2.9 Å in the special pocket. Further optimization of the P1 and P3 substituents led to a series of potent compounds, with 61 (PDB code 4DJY) exhibiting the best potency and binding efficiency and a remarkable 350-fold

selectivity against cathepsin D. This compound had an acceptable brain to plasma ratio of 2, and at a 30 mpk acute dose it elicited a 54% Aβ reduction in CSF at 3 h postdose relative to a vehicle treated control group in wild-type rats. The demonstration of a robust and substantial PD effect without the aid of P450 or P-gp inhibitors by compound 61 was the first reported account in the literature.128,129

A fragment hit with isocytosine ABM 62 led to the identification of analogues 63 and 64.130 The crystal structure of 64 with BACE1 revealed that the 6-phenyl ring was oriented in an energetically less favorable pseudo axial conformation in the S1 site.

The introduction of a geminal methyl group at the 6-position led to an energetically favorable pseudo axial phenyl presented in the S1 pocket (65). This observation is substantiated with ab initio quantum mechanical calculations and small molecule L



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crystal structure (CSD code CPMCHX).130,131 Structure-based design led to the introduction of a flexible linker and rings filling P1 and P3 pockets and to resultant identification of the potent compound with high binding efficiency (66).

Another independent research group developed a potent series of compounds with the isocytosine ABM using amide linkers in the P3 site generating potent compounds 67 and 68.132 The amide NH was observed to form a hydrogen bond with the backbone carbonyl oxygen of Gly230 and internally hydrogen-bond with the pyridine nitrogen. In addition, the pchloro substituent on the pyridyl ring extends deep into the S3 pocket with a water mediated interaction with the protein, which contributes to the increased affinity (Figure 8). When

inhibitors along with compound 61.133 The crystal structure of BACE1 with an analogue of 69 (Figure 9) revealed that the

Figure 9. Bound conformation of an analogue of compound 69 in complex with BACE1 (PDB code 4FRS). The figure was generated using Accelrys’ Discovery Studio graphical user interface.155 Figure 8. Bound conformation of an analogue of compound 68 with an aminopiperazinone ABM (PDB code 3U6A). The figure was generated using Accelrys’ Discovery Studio graphical user interface.155

Tyr71 on the flap adopts a conformation similar to the closed conformation but is still not hydrogen-bonded to Trp76. The compound 69 exhibits improved potency and in vivo PD effects when compared to 61.

mice were given 67 by a subcutaneous dose of 20 mpk, the plasma concentrations were 0.993, 0.461, and 0.200 μg/mL with brain levels of 0.614, 0.157, and 0.21 μg/mL at 1, 2, and 4 h, respectively. The rapid decrease in plasma concentration is attributable to higher metabolic clearance, though the mechanism of clearance from the brain is not clearly understood. The introduction of a fluorine atom in the P1 phenyl group led to an improvement in potency and binding efficiency in compound 68. This change appears to have improved its metabolic stability as well, because the compound levels in the plasma and brain were 5−7 times higher with a 60 mpk dose of 68 compared to the 30 mpk dose of 67. A ring expansion of the five-membered ABM to a sixmembered iminopyrimidinone was anticipated to have a significant impact on the compound’s physicochemical properties and its conformation. In comparison to the iminohydantoin core, the iminopyrimidinone is predicted to be more basic. A second major design consideration centered on conformational differences between the five- and six-membered ring iminoheterocycle ABMs. The iminopyrimidinone ring differs from the planar iminohydantoin core in that the additional SP3 center allows puckering in the ring and introduces flexibility. Compound 69 with the iminopyrimidinone ABM and a thiophene ring in the P1 position improved its potency and possesses one of the best binding efficiencies for BACE1

A NMR based fragment screen led to the identification of yet another acyclic acylguanidine compound 70.134 Initial exploration through a solid phase library identified dichloro compound 71 with improved potency. A cocrystal structure of this M



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compound with BACE1 indicated that the dichlorophenyl moiety occupied the S1 site and substitution on this phenyl ring at the para position would fill the S3 site. The compound containing an additional basic amine in the P3 position, 73, did not exhibit a shift in the cell activity relative to that against the enzyme. Subsequently compound 73 was administered to rats at 5, 15, and 40 mpk doses via a subcutaneous route. The 40 mpk group was pretreated with 50 mpk 1-aminobenzotriazole (ABT), a pan cytochrome P450 inhibitor, to improve drug levels in the plasma. Under these conditions the compound demonstrated a dramatic decrease of Aβ in plasma with no significant lowering of Aβ in the brain or in the CSF in wildtype rats. This was attributed to low exposure of the compound in the brain and in the CSF due to high efflux by P-gp. 3.2.2. Aminoisoindoles. A scaffold hopping approach was used to modify dihydroisocytosine 74 into aminoisoindole 75

kg−1) and short half-life (0.65 h) in mice, it demonstrated a prolonged Aβ lowering effect (16 h) in the PD experiments. Thus, compound 77 displayed better PD effect than would be anticipated from its in vitro and in vivo PK properties. PK/PD modeling of the time and dose response effect data in vivo estimated that an unbound brain concentration of 0.006 μM would give 20% inhibition from baseline for 77.

in order to identify a novel ABM for targeting BACE1.135 On the basis of the crystal structure of a close analogue of 76 (PDB code 4AZY), it is reasonable to assume that the amidine group interacts with the catalytic aspartyl residues. The pyridine ring placed in the S2′ site hydrogen-bonds with the side chain of Trp76, and the pyrimidine ring occupies the S3 site. The pyrimidine nitrogen proximal to the special pocket interacts with the protein through a water mediated hydrogen bond. In an effort to reduce the pKa of the ABM, a fluorine atom was introduced in the vicinity of the exocyclic amine group leading to compounds with enhanced brain penetration. An introduction of CF2H at the P2′ position had a significant improvement in potency along with excellent oral bioavailability and a half-life of 1.5 h in mice. This compound was nominated as a clinical candidate, 76b (AZD3839, PDB code 4B05). The replacement of the pyrimidine by propynylpyridine (PDB code 4B00) at the S3 site led to improvement in potency against the enzyme and higher selectivity against hERG. In an effort to further improve selectivity against hERG, pyridine was replaced by pyridone. Pyridones demonstrated improved metabolic stability compared to pyridines across animal species. Introduction of an ethyl group on the pyridone nitrogen led to a marked improvement in potency with greater selectivity against hERG for 77.135 This compound demonstrated excellent oral bioavailability in mice (100%) and a half-life of 2.3 h. A maximum Aβ reduction of 40% in brain was observed at 1.5 h with a dose of 200 μmol/kg 77 in C57BL/6 mice. The fractions of unbound drug in plasma and brain were estimated at 0.123 and 0.014 μM, respectively, at 1.5 h after dosing. Compound 77 demonstrated a very low free fraction in brain (0.6%) and showed reduced CNS penetration. The poor penetration is partly explained by a P-gp efflux ratio of 5.1 in a MDCK-MDR1 cell-line assay. Despite a high clearance rate (150 mL min−1

Similarly, replacement of benzamidine by 4-aminoimidazole (PDB code 4B1C) led to compounds with similar potency and good selectivity against hERG.136 Replacement of pyrimidine by propargylpyridine (78) led to a significant improvement in potency while maintaining a good selectivity against hERG in this series. The introduction of the hydroxyl in compound 79 to interact with Phe108 carbonyl of the protein led to a 2-fold improvement in potency but engendered significant P-gp efflux. The P-gp efflux problem was addressed by making the pyridine regioisomer in 80 which introduced an internal hydrogen bond within the molecule and reduced the efflux from 17 to 0.5. Further SAR in this series led to identification of 81 (PDB code 4B1D), which showed a robust 40−50% Aβ reduction in the brain with an oral dose of 100 μmol/kg in male guinea pigs. N



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by ∼70% by 9 h. In these in vivo experiments equivalent levels of Aβ1−40 and Aβ1−42 reductions were observed. 3.2.4. Five-Membered Heterocycles. A screen of a compound collection identified 2-aminoquinazoline containing

3.2.3. Aminobenzthiazines. Fragment screening of about 8000 fragments with a surface plasma resonance (SPR) method yielded a fragment with a novel ABM, the 2-aminobenzthiazine in compound 82.137 The X-ray structure of 82 in complex with BACE1 showed two molecules bound in the active site. One fragment displayed interactions with catalytic aspartates, while the other fragment occupied the S1−S3 region. The positioning of the rigid flat bicyclic scaffold of 82 between the aspartates and the flap in the active site did not provide an optimal vector for attaching fragments to occupy the S1 and S3 pockets of the enzyme. Thus, the fused bicyclic ring was truncated to a thiazine ring and optionally substituted with aryl and methyl groups at the 4-position to generate the highly efficient enzyme binder 83. The presence of the methyl group geminal to the phenyl ring allows an energetically preferred axial configuration of the ring as rationalized above with compound 65. This conformation orienting the ring orthogonal to the ABM allowed for additional ring substituents like pyrimidine in the S3 pocket, providing enhanced potency, such as in compound 84. An improvement in potency while retaining enzyme binding efficiency was achieved with both 83 and 84. The fluorination of the P1 phenyl ring of 84 improved the metabolic stability for 85. Compound 85 had good affinity toward BACE1 with 10-fold selectivity over BACE2 and over 100-fold selectivity versus other related aspartyl proteases including cathepsin D. Furthermore, no shift in activity was observed in a neuronal cell assay or in an HEK cell line (IC50 = 0.3 μM). After 3 h of oral administration of 85 in APPV717F transgenic mice, it demonstrated excellent brain penetration reflected by the total brain concentrations of 1.46, 3.35, and 12.12 μg/g at 10, 30, and 100 mpk doses. The compound demonstrated a significant dose dependent reduction in Aβ, sAPPβ, and C99 fractions consistent with the levels of compound in the brain. The oral administration of 85 in beagle dogs at a dose of 5 mpk achieved a maximum plasma concentration of 1.92 μg/mL after 1 h with a half-life of 6.8 h. The maximal reduction of plasma Aβ (85%) was observed between 4 and 12 h postdose and remained below baseline after 24 h. The CSF Aβ level was reduced by 43% at the 3 h time point and was further reduced

micromolar compounds. The optimization of these hits led to identification of compound 86 (0.011 μM, PDB code 2Q15).138 Another research group screened a library of 20 000 fragments using NMR, X-ray crystallography, and SPR. This screen discovered aminoimidazole ABM containing fragments with high micromolar enzyme potency (87, 88).139 The cocrystal structure of 88 (PDB code 3MSJ) indicated that the endocylic nitrogen in the 3-position, possibly protonated, forms a hydrogen bond with Asp32. The exocyclic nitrogen also forms a hydrogen bond with both catalytic aspartates, and the alkyl hydroxyl group hydrogen-bonds to Asp228. Further modification based on structure-based modeling and comparison with 86 generated compound 89 (PDB code 3MSL). While compound 89 lacked sufficient enzyme binding potency, it served as a lead for further optimization. In an effort to identify compounds that do not get effluxed by P-gp, a novel series of compounds with a 2-aminothiazole ABM was discovered using a HTS screen.140 Further optimization of this hit led to compound 91 with a significant improvement in potency. This compound lacked activity in a cell assay, which was attributed to the weakly basic aminothiazole. Compound 91 lost activity in an enzyme assay when the pH shifted from 4.5 to 6.5, which is speculated to be due to its estimated pKa of 5.5. Replacement of the thiazole ring by an imidazole ring, with an estimated pKa of 8.3, led to compound 92 that showed improved binding activity against the isolated enzyme at pH 6.5 and in the cell. The binding mode of these compounds exemplified by a close analogue of 92 shows the aminoimidazole ABM interacting with the catalytic center (PDB code 3H0B). 3.2.5. 2-Aminoquinoline and Pyridine. A high throughput crystallographic soaking experiment with a library of about 350 fragments into BACE1 crystals identified a compound containing a 2-aminoquinoline ABM in 93. It was a weak inhibitor with an estimated potency of 2 mM but possessed a high binding efficiency.141 O



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structure with an inhibitor bound (Figure 10, PDB code 2OHU). This structure showed a hydrogen bond between the indole NH and the backbone carbonyl oxygen of Gly230 in the S3 pocket.

Figure 10. Bound conformation of a pyridine analogue of compound 97 in complex with BACE1 (PDB code 2OHU). The figure was generated using Accelrys’ Discovery Studio graphical user interface.155

Similar to the effort described above, another research group identified a 2-aminoquinoline through a fragment screen (98).142 A successful substitution of the 6-position of the quinoline ring led to the identification of a phenyl ring with improved potency (99). The X-ray crystal structure of 99 in complex with BACE1 revealed that a substitution at the ortho position of the 6-phenyl ring would present it into the S1 pocket. Introduction of chlorine, methyl, and propargyl substituents improved potency (100, PDB code 3RTH).

However, in order to improve the ligand binding efficiency of these compounds, a series of amides substituted at the 3position of the quinoline were pursued (101 and 102).

The X-ray structure of 93 with BACE1 indicated the proximity of the hydrophobic S1 pocket. Further SAR development of 93, with an attempt to occupy S1 pocket, led to the identification of a compound with micromolar enzyme potency, 94. An in silico substructure analysis identified a 2,3diaminopyridine of 95 as an alternative hit with an IC50 of 40 μM. On the basis of the X-ray structure of 2,3-diaminopyridine with BACE1 (PDB code 2VA7), a series of analogues were designed that presented aromatic rings in S1 and S3 pockets (96 and 97).

The cocrystal structure of 102 with BACE1 enzyme (PDB code 3RTN) revealed that the methyl group of the o-tolyl group occupies the S1 site while the cyclohexyl portion of the amide occupies the S2′ subsite with the amide hydrogenbonding with the carbonyl oxygen of Gln34. The X-ray structure of 102 in conjunction with in vitro efflux data of related analogues led to the design of a potent, brain penetrant compound 103. A 60 mpk acute dose of 103 showed 42% reduction of Aβ in the CSF at the 2 h time point in rats. A high throughput screen by another group identified aminoquinazolines (104, 105) with a submicromolar affinity toward BACE1.138 An optimization effort improved the potency and led to the discovery of 106. The X-ray structure of 106 (PDB code 2Q15) coincidentally revealed a binding mode similar to that of the aminoquinoline 103.

Replacing the pyridine ring by an indole in 96 (PDB code 2OHT) led to a 4-fold improvement in BACE1 binding potency. Addition of a benzyloxy group to 96 in the P2′ position led to compound 97 with a 20-fold improvement in potency against BACE1. The crystal structure of an analogue of 97, which has a pyridine in place of the phenyl ring in the P2′ position, was the first publicly disclosed open-form BACE1 P



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3.2.6. Unique Binders. A novel series of compounds containing a spiropiperidine iminohydantoin moiety, as in 113, were identified through high throughput screening.144 The X-ray crystal structure of 114 with BACE1 (PDB code 3FKT) revealed a unique binding mode with the piperidine basic amine interacting with the catalytic water molecule held tightly between the aspartates. The optimization of these compounds primarily focused on the P2 or P3 group. These compounds exhibited moderate P-gp efflux ratios in vitro and in vivo, and compound 115 showed a good brain to plasma ratio of 0.44 and brain concentration of 1.7 μM after 30 mg/kg ip dose in mice. The lack of efficacy of this compound in transgenic mice expressing human APP was attributed to very low free fraction in the brain. A similar compound 116 (PDB code 4FM7) exhibited potent inhibition of BACE1 and excellent brain permeability.145 Administration of 116 to wild-type FVB mice produced a significant, dose-dependent reduction in central Aβ40 levels at 100 and 300 mpk doses. 3.2.7. Cyclic Basic Amine. The use of cyclized systems containing a basic amine such as pyrrolodine, piperidine, and piperazine has been successfully implemented in the design of renin inhibitors, but this strategy has not worked well for the BACE1 enzyme.55

A series of oxazolines were evaluated that target the catalytic aspartates, similar to other cores from compounds that work in the CNS.143 The X-ray structure of 108 showed that the phenyl ring on the right-hand side was orthogonal to the S2′ hydrophobic pocket. The orientation of the phenyl ring was altered by tying the P1 and P2′ rings (109) which allowed for introduction of groups that can occupy the S2 pocket.

This modification led to very potent compounds in the xanthane series. A 100-fold boost in potency was seen when the biphenyl oxazine compound 110 was constrained to yield xanthane, a highly efficient binder 111. Further replacement of the methoxy group by an isobutyloxy group on the prime side led to an additional 100-fold boost in potency and increase in enzyme binding efficiency (112, PDB code 4FRK). Compound 112 demonstrated acceptable PK properties when dosed intravenously in rats at 2 mpk with a clearance of 1.08 L h−1 kg−1 and a t1/2 of 3.2 h. When dosed orally at 10 mpk, compound 112 had an oral bioavailability of 31% with plasma AUC of 7.29 μM·h. After an oral dose of 30 mpk in wild-type rats, this compound demonstrated a brain to plasma ratio of 1.1 at 4 h with an Aβ reduction of 45% in brain and 76% in CSF. Similarly, at 4 h after an oral dose of 100 mpk it demonstrated an Aβ reduction of 63% in brain and 81% in CSF.

An exception to this was the recent disclosure of a pyrrolidine ring spirocyclized to an indolinone ring in 117 (PDB code 3UDH) presenting a novel class of BACE1 inhibitors.146 The millimolar binding spiropyrrolidine was discovered through a high throughput crystallographic screen by soaking 4-compound mixtures from a fragment library into Q



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stability, poor bioavailability, and high P-gp efflux and as a result do not lower Aβ in the brains of animal models in a majority of the cases. In contrast compounds designed for the open form of the enzyme generally exhibit lower molecular weight and lower polar surface area. These compounds are druglike with novel basic heterocyclic rings binding to the catalytic aspartates. These inhibitors need a minimum of two specificity pockets filled to exhibit good affinity toward the enzyme and thus possess higher binding efficiency, unlike the closed-form inhibitors that need a minimum of three specificity pockets filled to possess detectable affinity. The lack of the need to hydrogen-bond to the backbone NHs of the flap appears to prevent the necessity of having an amide bond in the molecules in order to display significant activity. The nature of the pocket in open form thus appears to be more “druglike” than the closed form and provides an advantage in generating low molecular weight and moderately polar compounds that are able to pass through the blood−brain barrier and enter the CNS and exhibit efficacy.

crystals of the BACE1 enzyme. This screen, followed by iterative crystallographic soaking and refinement, yielded a basic secondary amine in the spirocyclic-pyrrolidine ring interacting with the catalytic aspartates of BACE1 in an orientation similar to the way piperidines and piperazines are observed to interact with renin’s catalytic center.147−149 The optimization of compound 117, feasible only into the prime side from the pyrrolidine ring because of the binding mode, led to compounds with micromolar affinity and an efflux ratio less than 7.0 in MDCK cells. The most potent compound 118 (4 μM, PDB code 3UDQ), with a sulfone containing bicyclic ring system in the S2′ site, interacted with the backbone NH of Thr72 on the flap and had an efflux ratio of 6.8.

5. CLINICAL TRIALS The efficacy achieved by 85 in preclinical studies allowed it to be progressed to human clinical trials. The phase I trial design involved a single ascending dose of 85 in healthy human subjects. Both blood and CSF samples were drawn over time to measure levels of drug and Aβ in the plasma and CSF.137 Overall, significant dose-dependent reductions in the levels of Aβ40 and Aβ42 in the plasma and CSF were observed. The 30 and 90 mg doses showed a significant mean reduction of Aβ40 and Aβ42 compared with the placebo at 7 h postdose and achieved a maximum reduction at 12−14 h. A single dose of 90 mg of 85 resulted in a significant dose dependent reduction of plasma Aβ40 (80% at 7 h) and a mean reduction of 64% over the first 24 h. Overall 85 was well tolerated in the clinical trials with no serious adverse events reported. These findings provided the first proof-of-concept in humans of the pharmacodynamic effect of Aβ lowering in the CSF by blocking the enzymatic activity of BACE1. In parallel to the phase I studies with 85, a 3-month rat toxicological study revealed a cytoplasmic accumulation of finely granular autofluorescent material dispersed within the retinal epithelium and less prominently within neuronal and glial cells in the brain at 30 mpk dose. A subsequent study using 85 in BACE1−/− knockout mice demonstrated that the findings observed in the retinal epithelium and brain were not a BACE1 mechanism based toxicological effect. However, these findings halted the progression of the clinical study of 85. A subsequent follow-up of the patients’ eye examination revealed no damage to the retina up to 6−10 months past the completion of the phase I study. The following compounds for which structures have not been disclosed have successfully completed phase I clinical trials. A second Lilly compound, LY2886721, recently completed phase I clinical trials in healthy human volunteers.151 This compound was administered in both single-ascending and multiple-ascending doses at 12, 40, and 60 mg over 7 days in mild-to-moderate Alzheimer’s patients. This compound was slated to enter phase II clinical trials in patients suffering from mild-to-moderate AD using the 15 and 35 mgs doses with a target reduction of 30% and 60% in CSF Aβ, respectively. A successful phase I clinical study was also completed with MK-8931 in single-ascending and multiple-ascending doses of

An additional class of pyrrolidines was discovered through a HTS screen by another independent group, identifying compounds like 119.150 An optimization of 119 led to compound 120. The X-ray crystal of 120 complexed with BACE1 (PDB code 3UFL) indicated that the terahydronapthalene occupied the S1 site. The introduction of a nitrogen atom into the tetrahydronapthalene ring and the addition of the chlorine atom adjacent to it improved the potency in the cell assay for compound 121. It demonstrated good selectivity against other aspartyl proteases with good bioavailability due to low clearance. The conformation of the enzyme bound to these spiropyrrolidines appears to differ depending on the substitution pattern on the scaffold. Interestingly, compound 117 appears to bind to the closed form of the enzyme; however, compound 120 binds to the open form. This could be due to the subtle difference in the scaffold and substitution pattern on the ABM.

4. SUMMARY OF BINDING MODES The flexible conformation of the active site flap in the BACE1 enzyme has allowed the design of two distinct categories of inhibitors that target either the closed form or the open form. The presence of a hydrogen bond between residues Tyr71 and Trp76 defines the conformation of BACE1 as closed, and the lack of this hydrogen bond defines it as open with a few exceptions. The inhibitors that have been designed to target the closed form of the enzyme have largely been of high molecular weight and high polar surface area. These compounds exhibit an essential interaction with the catalytic aspartates Asp32 and Asp228, hydrophobic aromatic systems in P1 and P3, and polar functional groups in the P2 positions. The prime side substitutions contained largely hydrophobic substituents. In all the compounds, hydrogen bonds to the backbone NH of the flap are essential to exhibit activity. Though these compounds are potent in vitro, in general they suffer from poor metabolic R



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12, 40, and 60 mg over 7 days in mild-to-moderate AD patients.152 This compound, upon successful completion of phase I, was designated to start phase II−III clinical trials with the recruitment of mild-to-moderate AD patients to receive 15 and 35 mg of the compound with a target of 30% and 60% reduction in CSF Aβ in a 26-week study. A third compound, E-2609, also completed phase I clinical trials in healthy human volunteers receiving single-ascending and multiple-ascending doses over a period of 14 days.153 There have been four other compounds that entered phase I clinical trials, RG-7129, HPP-854, AZD-3839;154 however, data from these studies have not been disclosed.

Shankar Venkatraman has been with Vitae Pharmaceuticals since 2009. Currently, as a Director of Chemistry, he manages the BACE1 inhibitor project. Prior to joining Vitae, he worked at Merck for 8 years as a Senior Research Fellow, working on a wide variety of therapeutic targets. He was a key inventor for MK-0617, a VLA4 antagonist for multiple sclerosis. He started his career at Axys Pharmaceuticals (Celera Genomics) and made numerous significant contributions to the cathepsin K project for osteoporosis. Shankar obtained his Ph.D. degree at University of Minnesota, followed by doing postdoctoral stints at University of Minnesota and University of Kansas. Yajun Zheng is a Senior Research Fellow in Computational Drug Design at Vitae. He received a Ph.D. in Organic Chemistry from the University of Texas at Austin under M. J. S. Dewar and M. A. Fox. After several years of postgraduate research at Penn State, PA (with K. M. Merz), University of California (with J. A. Leary and T. C. Bruice), and Pacific Northwest National Lab, he joined DuPont in 1997 as a computational chemist. In 2007, he joined Vitae. His research interests include the areas of protein structure−function−dynamics relationship, enzyme catalysis, organic reaction mechanism, molecular modeling, and drug design.

6. SUMMARY AND SCOPE In this account we used available BACE1/inhibitor complex crystal structures and associated structure−activity data to describe their binding modes highlighting specific binding interactions, and the affinity improvements achieved for them. Also, description of the physical−chemical properties and their relationship to in vitro and in vivo properties such as metabolic stability, P-gp efflux, pharmacokinetics, and pharmacodynamics were presented. The open form of the BACE1 enzyme appears to offer a more druglike binding cavity than the closed form of the enzyme. As a result, the inhibitors targeting the open form are of low molecular weight, high binding efficiency, low polar surface area with improved metabolic stability, P-gp efflux, and oral bioavailability and demonstrate Aβ lowering in animal models and in human clinical trials. The availability of X-ray crystal structures of fragments and multiple lead molecules has facilitated the identification of diverse classes of potent BACE1 inhibitors. However, significant challenges exist in optimizing the lead molecules to enhance selectivity against cathepsin D/E, hERG, and other targets while maintaining good efflux and pharmacokinetic properties for a safe and efficacious drug. These challenges have limited the number of compounds successfully entering clinical trials and progressing to long-term human studies. The successful completion of phase I human clinical trials by three different research groups with LY2886721, MK-8931, and E-2609 and the initiation of later stage clinical trials bodes well for the development of small-molecule therapeutics that target the BACE1 enzyme to treat Alzheimer’s disease. The outcome of phase II and III clinical trials from these three lead candidates will be much anticipated. Hopefully, BACE1 inhibitors will enhance cognitive end points and lead to a safe and effective disease-modifying therapeutic for patients suffering from Alzheimer’s disease.

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Brian M. McKeever has been the Director of the Structural Biology Group at Vitae Pharmaceuticals since 2005. He is responsible for the production of target proteins for structure−function studies, in-house activity and binding assays, and the delivery of X-ray crystal structures of liganded or inhibited complexes for discovery and experimental validation of our in-house structure-based drug design work. Prior to joining Vitae, Brian was a Senior Research Scientist at Merck & Co. for 21 years, working on a wide variety of therapeutic targets, three of which led to the development of Trusopt, Crixivan, and Vioxx. He received his Ph.D. in 1993 from the Department of Molecular Biology and Biochemistry at State University of New York at Stony Brook under the guidance of Dr. Raghupathy Sarma. Lawrence W. Dillard is Senior Director of Discovery Chemistry at Vitae Pharmaceuticals where for the past 10 years he has managed chemistry efforts in the company’s renin and β-secretase programs. Prior to Vitae, he was a founding scientist of Pharmacopeia, Inc., where he spent 10 years working in a variety of collaborations targeting a wide range of therapeutic areas. He holds a Ph.D. in Synthetic Organic Chemistry from Duke University, NC, and was a Postdoctoral Fellow at Columbia University, NY, working in the laboratories of Professor W. Clark Still. Suresh B. Singh has been the Director of Computational Drug Design at Vitae Pharmaceuticals since 2004. He is responsible for the development and application of the structure-based design software program, Contour, to drug discovery projects. He is a co-inventor of the renin and 11β HSD-1 inhibitors that are currently in clinical development. He also manages the design efforts in the BACE1 inhibitor program. Prior to joining Vitae, Suresh was a Research Fellow at Merck for 8 years and at Wyeth for 3 years, working on a wide variety of therapeutic targets. He received is Ph.D. in Computational Chemistry from New York University under the guidance of Nicholas Geacintov and Suse Broyde and held a postdoctoral fellowship with the late Peter Kollman at University of California, San Francisco.

AUTHOR INFORMATION

Corresponding Author

*Phone: 215-461-2048. E-mail: [email protected]. Author Contributions †

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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Biographies Jing Yuan has been a medicinal chemist at Vitae Pharmaceuticals since 2002. She is a co-inventor of the renin inhibitor that is currently in clinical development. She also has been a key member of the BACE1 inhibitor program. Jing received her Ph.D. in Organic Chemistry from Temple University, PA, and held a postdoctoral fellowship with Jeffrey Winkler at University of Pennsylvania.

ABBREVIATIONS USED

BACE1, β-site amyloid precursor protein cleaving enzyme 1; Aβ, amyloid β; Cat D, cathepsin D; Cat E, cathepsin E; APP, amyloid precursor protein; MDCK, Madin−Darby canine kidney; ABM, aspartate binding motif; MW, molecular weight; log D, logarithm of the buffer−octanol partition coefficient; S



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Prabu-Jeyabalan, M. M.; Thayer, K.; Schiffer, C. A. Molecular basis for drug resistance in HIV-1 protease. Viruses 2009, 2, 2509−2535. (16) RCSB PDB. http://www.rcsb.org/pdb. As of Mar 4, 2013, at 2.27 p.m. EST, there are 235 BACE1 X-ray structures using the key word BACE1 followed by selecting entries with 95% sequence similarity to BACE1. (17) Percent identity of the target sequences was calculated with BACE1 sequence (accession code P56817) from NCBI using sequence database search program BLASTP (version 2.2.27) against the nonredundant protein sequences database with keyword organism=Homo sapiens (taxid:9606) on Jan 27, 2013 (http://blast. ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM= blastp&BLAST_PROGRAMS=blastp&QUERY=P56817.2&LINK_ LOC=protein&PAGE_TYPE=BlastSearch). (18) Gleeson, M. P. Generation of a set of simple, interpretable ADMET rules of thumb. J. Med. Chem. 2008, 51, 817−834. (19) Hitchcock, S. A.; Pennington, L. D. Structure−brain exposure relationships. J. Med. Chem. 2006, 49, 7559−7583. (20) Broccatelli, F.; Larregieu, C. A.; Cruciani, G.; Oprea, T. I.; Benet, L. Z. Improving the prediction of the brain disposition for orally administered drugs using BDDCS. Adv. Drug Delivery Rev. 2012, 64, 95−109. (21) Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L. J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W. Passive permeability and P-glycoprotein-mediated efflux differentiate central nervous system (CNS) and non-CNS marketed drugs. J. Pharmacol. Exp. Ther. 2002, 303, 1029−1037. (22) Fridén, M.; Winiwarter, S.; Jerndal, G.; Bengtsson, O.; Wan, H.; Bredberg, U.; Hammarlund-Udenaes, M.; Antonsson, M. Structure− brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids. J. Med. Chem. 2009, 52, 6233−6243. (23) Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in central nervous system drug discovery. J. Med. Chem. 2013, 56, 2−12. (24) Seelig, A.; Landwojtowicz, E. Structure−activity relationship of P-glycoprotein substrates and modifiers. Eur. J. Pharm. Sci. 2000, 12, 31−40. (25) Penzotti, J. E.; Lamb, M. L.; Evensen, E.; Grootenhuis, P. D. J. A computational ensemble pharmacophore model for identifying substrates of P-glycoprotein. J. Med. Chem. 2002, 45, 1737−1740. (26) Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.; Morgan, J. B.; Webster, L. O.; Serabjit-Singh, C. S. Rational use of in vitro Pglycoprotein assays in drug discovery. J. Pharmacol. Exp. Ther. 2001, 299, 620−628. (27) Ha, S. N.; Hochman, J.; Sheridan, R. P. Mini review on molecular modeling of P-glycoprotein (Pgp). Curr. Top. Med. Chem. 2007, 7, 1525−1529. (28) Doran, A.; Obach, R. S.; Smith, B. J.; Hosea, N. A.; Becker, S.; Callegari, E.; Chen, C.; Chen, X.; Choo, E.; Cianfrogna, J.; Cox, L. M.; Gibbs, J. P.; Gibbs, M. A.; Hatch, H.; Hop, C. E. C. A.; Kasman, I. N.; LaPerle, J.; Liu, J.; Liu, X.; Logman, M.; Maclin, D.; Nedza, F. M.; Nelson, F.; Olson, E.; Rahematpura, S.; Raunig, D.; Rogers, S.; Schmidt, K.; Spracklin, D. K.; Szewc, M.; Troutman, M.; Tseng, E.; Tu, M.; Van Deusen, J. W.; Venkatakrishnan, K.; Walens, G.; Wang, E. Q.; Wong, D.; Yasgar, A. S.; Zhang, C. The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1a/1b knockout mouse model. Drug Metab. Dispos. 2005, 33, 165−174. (29) Yamazaki, M.; Neway, W. E.; Ohe, T.; Chen, I. W.; Rowe, J. F.; Hochman, J. H.; Chiba, M.; Lin, J. H. In vitro substrate identification studies for P-glycoprotein-mediated transport: species difference and predictability of in vivo results. J. Pharmacol. Exp. Ther. 2001, 296, 723−735. (30) Tang, C.; Kuo, Y.; Pudvah, N. T.; Ellis, J. D.; Michener, M. S.; Egbertson, M.; Graham, S. L.; Cook, J. J.; Hochman, J. H.; Prueksaritanont, T. Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid concentrations in rhesus monkeys. Biochem. Pharmacol. 2009, 78, 642−647.

PSA, polar surface area; po, per oral; iv, intravenous; mpk, milligrams per kilogram; PDB, Protein Data Bank

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