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Article

Design, Optimization, and Study of Small Molecules That Target Tau

Pre-mRNA and Aﬀect Splicing

Jonathan L. Chen,^#Peiyuan Zhang,^#Masahito Abe, Haruo Aikawa, Liying Zhang, Alexander J. Frank,

Timothy Zembryski, Christopher Hubbs, HaJeung Park, Jane Withka, Claire Steppan, Lucy Rogers,

Shawn Cabral, Martin Pettersson, Travis T. Wager, Matthew A. Fountain, Gavin Rumbaugh,

Jessica L. Childs-Disney, and Matthew D. Disney *

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ABSTRACT: Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-

mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can

cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer’s disease.

Here, we describe the design of structure-speciﬁc lead small molecules that directly target tau pre-mRNA from sequence. This was

followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds

were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of

tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had

enhanced potency and selectivity for the target RNA relative to the initial hits, while also having signiﬁcantly improved drug-like

properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down

target proﬁling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons.

More broadly, this study shows that lead, structure-speciﬁc compounds can be designed from sequence and then further optimized

for their physicochemical properties while at the same time enhancing their activity.

which thereby aﬀord an improved selectivity and safety

proﬁle.²

INTRODUCTION

■

RNA plays important roles in a variety of biological processes,

including protein synthesis, gene regulation, and RNA

transport.¹Mutations in an RNA sequence can lead to cancers,

viral infections, and neurodegenerative diseases. Alternative

pre-mRNA splicing is carried out by the spliceosome and is

regulated via structured regions at intron−exon junctions that

are recognized by various splicing factors. Mutations to the

pre-mRNA sequence can lead to structural changes that

inﬂuence the ability of the pre-mRNA to be recognized by the

spliceosome. While targeting the spliceosome has been shown

to be a potential treatment for cancers, such compounds aﬀect

pre-mRNA splicing globally, which may adversely impact their

safety proﬁle.²Directly targeting a speciﬁc pre-mRNA could

inﬂuence speciﬁc splicing events by enhancing or preventing

the binding of proteins comprising the spliceosome machinery,

Current RNA-targeting modalities include antisense oligo-

nucleotides (ASOs) and small molecules. Recently, an ASO

has been approved by the U.S. Food and Drug Administration

(FDA) to treat spinal muscular atrophy (SMA) and provided a

transformative medicine (Spinraza, Biogen).³However, ASOs

have limited tissue distribution and can also stimulate the

immune system.⁴An alternative approach to target RNA is to

Received: January 20, 2020

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A

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Figure 1. Outcome of alternative splicing of exons 9 to 11 of tau pre-mRNA is regulated by a hairpin at the exon 10−intron 10 junction. (A)

Schematic of alternative splicing of exons 9 to 11 of tau pre-mRNA. A +14 C to U mutation destabilizes a hairpin at the exon 10−intron 10

junction, promoting U1 snRNP binding to the pre-mRNA, increasing exon 10 inclusion, and producing 4R tau. Binding of a ligand to the A-bulge

site stabilizes the hairpin, increasing exon 10 skipping and production of 3R tau. (B) RNA structures used in this study. Constructs consist of the

tau WT hairpin, the tau DDPAC hairpin consisting of a + 14C > U mutation, and mutants of both constructs in which a uridine is inserted across

the strand from the A-bulge to form an AU pair that serve as negative controls.

use small molecules to target three-dimensionally structured

regions. Molecular recognition of structured regions of an

RNA with a small molecule is diﬀerent than targeting an RNA

with an oligonucleotide, as the oligonucleotide targets an

unstructured region in an RNA.⁵Interestingly, the small

molecule Risdiplam, currently in clinical trials for SMA, was

identiﬁed from a phenotypic screen and aﬀects survival motor

neuron (SMN) pre-mRNA splicing by binding to a complex

between RNA and protein.⁶⁻⁸In contrast, the ASO,

nusinersen (Spinraza) aﬀects SMN pre-mRNA splicing by

targeting an intronic splicing silencer downstream of the 5′

splice site of exon 7.^9,10

In frontotemporal dementia and parkinsonism linked to

chromosome 17 (FTDP-17), a mutation in a regulatory RNA

structure in the pre-mRNA that encodes for the microtubule-

associated protein tau (MAPT, commonly referred to as tau)

causes aberrant alternative splicing and generation of a protein

isoform prone to forming ﬁbrils.¹¹In particular, this aberrant

splicing event causes the overproduction of an isoform of tau

that contains four microtubule binding domains, referred to as

4R, that aggregates. An increase in the formation of the 4R

isoform of tau is also known to play a major role in other

neurodegenerative diseases such as Alzheimer’s. In this work,

we demonstrate that small molecules can be designed to target

a structured region of tau pre-mRNA to modulate splicing.

These small molecules were designed by our lead identiﬁcation

strategy, Inforna, that is built on experimentally identiﬁed and

characterized RNA motif−small-molecule interactions.^12,13

The 3D folds within a target RNA are compared to this

database of privileged interactions, with overlap aﬀording lead

small molecule(s). This strategy has been used to identify small

molecules that target structured RNAs such as micro-

RNAs,¹⁴⁻¹⁶CUG repeats,¹⁷and an iron responsive element

(IRE) of α-synuclein mRNA.¹⁸The initial hits reported herein

for tau mRNA subsequently can be optimized to serve as useful

chemical probes of RNA function to aﬀect disease processes.

The MAPT gene contains 16 exons, among which exons 2, 3,

and 10 are alternatively spliced to yield six isoforms containing

eight constitutive exons.¹¹Overproduction of 4R tau is due to

mis-regulation of alternative splicing at exon 10 (Figure 1).¹⁹

The resulting 4R protein also has additional phosphorylation

sites that changes protein solubility and ultimately leads to

insoluble aggregates.¹⁹The tau pre-mRNA contains various

structures that regulate its biology.²⁰The FTDP-17 disease is

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caused by, for example, a C-to-U intronic mutation located 14

nucleotides (nt) downstream of the 5′ splice site of tau exon

10 (C[+14]U; commonly referred to as disinhibition−

dementia−parkinsonism−amyotrophy complex (DDPAC),

Figure 1).²¹The mutation destabilizes the pre-mRNA hairpin

structure on the exon 10−intron 10 junction, a splicing

regulatory element (SRE), thereby increasing U1 small nuclear

ribonucleoprotein (snRNP) binding and exon 10 inclusion.²²

The proportion of 4R to 3R tau is therefore above a normal

ratio in FTDP-17 patients.²¹The introduction of mutations

that thermodynamically stabilize the SRE hairpin have been

shown to reduce the 4R/3R ratio.²¹This suggests a therapeutic

strategy in which the stabilization of the hairpin by a small

molecule could be capable of inducing exon 10 skipping,

thereby reducing the overproduction of the 4R version of

tau.²¹

A small molecule that binds to the three-dimensionally

folded adenine-containing bulge (A-bulge) region of the SRE

and induces exon 10 skipping in cell-based assays was

previously identiﬁed²³by sequence-based design using the

Inforna approach.^12,13This lead chemical probe has sub-

optimal physicochemical properties. One of the major

challenges in the fast-evolving area of targeting RNA with

small molecules is to develop eﬀective strategies for lead

generation and subsequent optimization.¹³Inforna provides a

facile and potentially general route to identify small molecules

that target folded RNA structures. Herein, we describe and

implement a variety of synergistic approaches to characterize

and subsequently optimize compounds that target the 3D

structure of the tau pre-mRNA SRE. In the ﬁrst approach, we

identiﬁed all compounds that can bind to the tau SRE using

Inforna. Analysis of these compounds deﬁned a pharmaco-

phore model for ligands that bind this structure. Querying

these models for related compounds in improved physico-

chemical property space led to the identiﬁcation of new

chemical scaﬀolds for further optimization. In addition, we

solved the solution structures of a variety of compounds bound

to the SRE using NMR spectroscopy and restrained molecular

dynamics (MD), thus enabling structure-based design methods

to facilitate optimization. Each of these compounds were

assessed for aﬀecting the SRE using a suite of NMR,

biochemical, and cell-based assays. Key observations were

made with respect to (1) site-speciﬁc ligand binding to the

SRE using a compound displacement assay, (2) eﬀect on the

dynamics of the SRE upon interaction with the compound as

determined by changes in line width in NMR spectra, and (3)

changes in the molecular recognition of U1 snRNP for tau pre-

mRNA in the presence of the ligand. Armed with these

approaches, we deﬁned the structure−activity relationships

(SAR) for molecular recognition and also identiﬁed new

compounds with a variety of improved properties (Figure 2).

Ultimately, new compounds were found that target the tau pre-

mRNA A-bulge and exhibit robust functional activity in a

multitude of cell lines. Compound 1 was the previously

reported bioactive compound.²³Compounds 2 to 4 were

found in a chemical similarity search using 1 as a query

molecule, and compounds 5 to 7 were identiﬁed through hit

expansion/optimization using a pharmacophore model devised

from 1 to 4. Compound 8 was found from docking to an NMR

structure of the 4−tau RNA complex, and a hit expansion

using 8 as a query molecule resulted in the identiﬁcation of

compound 9 (details of compounds 1 to 9 are provided

below). In particular, the modulation of the tau 4R/3R ratio

Figure 2. Identiﬁcation of lead compounds that target the tau exon

10−intron 10 hairpin. Secondary and 3D structures of a splicing

regulatory element (SRE) at the exon 10−intron 10 junction were

determined by mutational analysis and NMR spectroscopy,

respectively.^19,66,78,79An Inforna-based search of a database of

RNA−small-molecule interactions that target the SRE led to the

identiﬁcation of compounds 1 to 4. Compounds 1 to 4 were then

used as starting points to identify compounds with better

physiochemical properties and potency. Pharmacophore modeling

of compounds 1 to 4, followed by a chemical similarity search and

subsequent analogue synthesis, identiﬁed compound 5 and a

piperidine amide hit that served as the starting point for the synthesis

of analogues 6 and 7. Separately, NMR spectroscopy and restrained

molecular dynamics studies were used to determine the structure of 4

in complex with the A-bulge of the hairpin. Docking of the NCI

diversity set to the tau RNA−4 complex led to 8, and hit expansion

via similarity searching around 8 aﬀorded 9. Hit expansions around

compounds 1 to 4 resulted in compounds with improved cellular

activities and drug-like properties, as indicated by central nervous

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series of small molecules that targeted the A-bulge in the exon

10 pre-mRNA hairpin. The binding of 1 to the pre-mRNA

thermodynamically stabilized its folding and modestly induced

exon 10 exclusion in the mature mRNA in cells.²³In order to

identify more potent compounds that aﬀect pre-mRNA

splicing by directly targeting the pre-mRNA, 1 was used as a

query molecule in chemical similarity searching campaigns to

identify six compounds with a Tanimoto score, or a chemical

similarity score, of greater than 90%.²⁴A chemotype analysis

was completed on these six compounds, which was used to

requery compound collections, aﬀording an additional 14

compounds (Figure S1). The 20 compounds (Figure S1) were

then tested in a luciferase reporter assay for targeting tau pre-

mRNA splicing in cells (Figure S2).²⁵The reporter genes

express luciferase in frame with exon 10 such that the

alternative splicing outcomes can be measured by luciferase

activity: the inclusion of exon 10 (4R) results in increased

luciferase activity, while its exclusion (3R) leads to decreased

luciferase activity (Figure 3). That is, when exon 10 is

included, luciferase is produced and mimics the production of

the tau 4R protein that causes FTDP-17. The eﬀect of a

compound on exon 10 splicing was studied using three

constructs: (i) wild-type (WT) tau; (ii) the DDPAC mutation,

Figure 2. continued

system multiparameter optimization (CNS MPO) scores. The higher

the CNS MPO score, the higher the probability is of achieving good

ADME (absorption, distribution, metabolism, and excretion) proper-

ties and brain penetration. A majority of CNS drugs have CNS MPO

> 4. Values of properties for Inforna compounds are averages of those

of compounds 1 to 4. Values of properties for optimized compounds

are averages of those of compounds 6, 7, and 9. Error bars indicate

SD.

was demonstrated in mouse neurons that were harvested from

a mouse model that expressed human tau pre-mRNA. To the

best of our knowledge, this is the ﬁrst small molecule identiﬁed

that promotes exon skipping by directly targeting RNA in a

neuronal cell line.

RESULTS

■

Using Chemical Similarity Searching to Identify

Compounds That Aﬀect Tau Pre-mRNA Splicing.

Previously, 1 was identiﬁed to target mutant tau exon 10−

intron 10 hairpin by querying the tau exon 10−intron 10

hairpin secondary structure against an RNA motif−small

molecule database via Inforna (Figure 2).²³This identiﬁed a

Figure 3. The eﬀects of hit compounds on tau exon 10 splicing, as assessed by a cell-based luciferase reporter assay. (A) Schematic diagram of a

mini-gene used in cell-based assays with HeLa cells transfected with a DDPAC or WT construct and treated with compounds. The hairpin at the

exon 10−intron 10 junction controls access of U1 small nuclear (sn)RNA to the splice site. In the absence of ligand at the A-bulge, the C(+14)U

mutation destabilizes the hairpin, promoting U1 snRNP loading on to the pre-mRNA and exon 10 inclusion. Ligand binding at the A-bulge site

stabilizes the hairpin and shifts the equilibrium away from unfolded RNA. The ﬁreﬂy luciferase reporter gene is in frame with exon 10 and expressed

when exon 10 is included. (B) Results of cell-based assays with HeLa cells transfected with a WT, a DDPAC, or a fully base-paired DDPAC+I17T

construct and treated with various compounds. Mutations in the DDPAC and DDPAC+I17T constructs are indicated with orange boxes. Error

bars indicate SD.

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Figure 4. In vitro assays to assess compound binding to tau RNA. (A) 2-AP assay. Addition of compound quenches emission. No data are available

for 9 due to spectral overlap with 2-AP. (B) Fluorescence resonance energy transfer (FRET) FRET-based quenching (Q-)assay. (C) FRET-based

U1 snRNA assay. To the right of the schemes are plots showing correlation between each in vitro binding assay and exon 10 skipping determined at

10 μM for all compounds except 6 and 7, which were determined at 12.5 μM. Exon skipping assays were carried out with HeLa cells transfected

with a DDPAC mini-gene. Error bars indicate SD.

which causes FTDP-17 (C to U mutation at position 14); and

(iii) the double mutant, known as I17T, containing a C14U

mutation and a U insertion opposite the bulged A that served

as a cellular control because it lacks the A-bulge binding site

(Figure 1).

dose-dependent eﬀect was observed for 4 (Figure S4).

Compound 4 also had improved solubility and, therefore,

was amenable to NMR spectroscopy studies (vide infra).

The goal of the subsequent studies was to optimize the

activity of compounds that bind the exon 10−intron 10 hairpin

and rescue tau pre-mRNA splicing associated with disease. For

example, we sought to enhance drug-like properties and

therefore improve the physicochemical properties of the

compounds, enhance the window between activity and toxicity

in various cell lines, demonstrate that molecular recognition of

this RNA target can aﬀect pre-mRNA splicing in various cell

lines and settings, and broadly deﬁne tools to identify and

optimize compounds that aﬀect splicing outcomes of not only

tau but other pre-mRNAs.

Overview of NMR Spectroscopy of 4-Bound Tau RNA.

To provide insight into ligand binding and its eﬀect on the

stabilization of RNA structures, NMR spectra were collected

on the A-bulge site in complex with 4. Distance restraints

calculated from these spectra were used to model the structure

of 4 bound to the tau RNA stem mimic duplex. Stacking

interactions between 4 and the A-bulge suggest the compound

may aﬀect the emission of a ﬂuorescent analogue of adenine, 2-

aminopurine (2-AP), when 2-AP is substituted for the A-bulge.

Thus, an assay using 2-AP as a reporter to assess binding was

developed to identify other compounds that target the tau SRE

(Figure 4A). The NMR spectra and structures of 4-bound tau

RNA are discussed below in further detail.

Compounds were screened at 20 μM in the cell-based

luciferase assay with the DDPAC mutant reporter (Figure 3A).

Compounds 2 to 4 reduced luciferase activity more than 1 at

20 μM concentration, and thus a full dose response was

completed for them (Figure S3). Cells treated with 20 μM of 4

displayed a signiﬁcant reduction of luciferase (∼60% reduction,

p < 0.01), with a statistically signiﬁcant eﬀect observed at 10

μM. Cells transfected with the WT reporter also exhibited a

similar dose-dependence reduction in luciferase activity when

treated with 4. No eﬀect was observed when the compounds

were tested for aﬀecting the splicing of a tau mini gene pre-

mRNA when the A-bulge binding site was replaced with an AU

base pair, importantly showing that the eﬀect of the

compounds is selective for the A-bulge (Figure 3B).

Compound 4 was then studied in a human neuroblast cell

line, BE(2)-M17, that stably expresses a DDPAC tau exon 10

mini-gene reporter. The mini-gene contains tau exons 9, 10,

and 11 fused to NanoLuc-PEST. In contrast to the transfected

tau mini genes discussed above, exon 10 skipping (3R)

stimulates luciferase production. Compounds that simply

inhibit the translation of luciferase have no activity in this

assay. In agreement with studies in transfected HeLa cells, a

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Development of a High-Throughput Assay Using 2-

AP to Identify Small Molecules That Perturb Tau A-

Bulge Dynamics. Our biophysical studies indicate that 4

binding perturbs the dynamics of the tau A-bulge, suggesting

that a high-throughput binding assay could be developed by

substituting the A-bulge with the ﬂuorescent adenine mimic 2-

aminopurine (2-AP). The ﬂuorescence of 2-AP is dependent

upon its microenvironment (stacked vs unstacked in a helix,

for example). Indeed, 2-AP has been used previously to study

the binding of aminoglycosides to a model of the bacterial A-

site, particularly changes in the RNA’s structural dynamics.²⁶

The optimal position for 2-AP to monitor compound binding

was ﬁrst determined in models of the WT and DDPAC

hairpins. Replacing the A-bulge with 2-AP was optimal as

compared to replacing an adenine in base pairs adjacent to the

bulge, as assessed by the change induced by the binding of 4

(Figure S5).

As expected, 4 decreases the ﬂuorescence intensity of both

WT and DDPAC RNAs labeled with 2-AP in a dose-

dependent fashion (Figure S6). Fitting the resulting curves

aﬀorded EC₅₀values of 6.2 0.8 and 3.6 1.2 μM for WT

and DDPAC RNAs, respectively. Importantly, the 2-AP label

does not have a very signiﬁcant eﬀect on small-molecule

binding since K_d’s for unlabeled WT and DDPAC RNAs are

9.8 and 1.3 μM, respectively. Compound 4 was then tested

with minimal WT and DDPAC duplex constructs used for

NMR studies, but with 2-AP substitutions at the A-bulge site

(Figure S6). For the WT and DDPAC duplexes, 4 decreased

the ﬂuorescence intensity in a dose-dependent manner, with

EC₅₀’s of 0.7 0.1 and 2.1 0.4 μM, respectively (Figure S6).

The data conﬁrmed that these duplexes were good model

constructs for NMR studies of ligand−tau RNA complexes.

Using 4 as a positive control, the 2-AP binding assay was

investigated for suitability for higher throughput screening by

calculating its Z-factor, which is a statistical parameter that

quantiﬁes the dynamic range and variation in a high-

throughput screen and range from 0 to 1 (ideal).²⁷A score

of >0.5 is considered suitable for high-throughput screens. The

Z-factor for the 2-AP assay is 0.7, which indicates its suitability

for completing a screen for RNA binding and aﬀecting

dynamics.

Development of a Competitive Binding Assay That

Uses Fluorescent Quenching. An in vitro competitive

binding assay was developed to assess the binding of

compounds to the exon 10−intron 10 hairpin. In the assay,

the Q-RNA labeled at the 5′ end with a black hole quencher is

bound to a Q-probe, which consists of a ﬂuorescein amidite

(FAM)-labeled neomycin (Figure 4B).²⁹Upon complex

formation, the FAM emission intensity decreases as the

quencher and the ﬂuorophore are in close proximity.²⁸

Displacement of the Q-probe from the A-bulge by a

competitor molecule leads to the recovery of FAM

ﬂuorescence. Neomycin B was used in this binding assay

because of its favorable aqueous solubility, and additionally, an

NMR structure of the neomycin−tau RNA complex showed

that the neomycin B 5′-OH group does not form contacts with

the RNA and therefore can be functionalized to attach a

ﬂuorescent probe.^29,30The quenching assay allows for

compounds to be screened for binding that had spectral

overlap with 2-AP. The Z-factor for the quenching assay is 0.6,

which also indicates that it may be adapted as a high

throughput screening (HTS) method.

Development of a U1 snRNA Assay That Mimics

Recognition of Tau Pre-mRNA by Spliceosomal

Components. In the U1 snRNA assay, cyanine Cy3 and

Cy5 dyes are attached to the 5′ and 3′ ends, respectively, of the

exon 10−intron 10 hairpin construct (Figure 4C). The

addition of an oligonucleotide mimic of U1 snRNA causes

the hairpin to unfold and form a complex, resulting in

decreased FRET between Cy3 and Cy5. When a small

molecule is added that binds to the tau A-bulge, the hairpin is

thermally stabilized, which decreases the rate of complex

formation between U1 snRNA and exon 10−intron 10 hairpin,

as read out by a FRET decreasing rate. The U1 snRNA mimic

assay is a kinetic assay that allowed us to investigate the ability

of ligands to stabilize the exon 10−intron 10 hairpin and slow

the unfolding rate, which mimics U1 snRNA unfolding of the

same hairpin in cells and subsequent inclusion of exon 10.

After the addition of ligand, the rate of increase of Cy3

emission was measured and compared to that measured for the

free-form RNA.

The addition of 4 resulted in the lowest rate of Cy3 emission

recovery. This result is consistent with the 2-AP binding

aﬃnity assays and demonstrates that the FRET-based assay

may be used as an alternative assay for identifying compounds

that bind to a target RNA. The Z-factor for the U1 snRNA

assay is 0.6, indicating it may be adapted as a HTS method.

Primary Hit Expansion of Lead Compounds from the

Initial Inforna Hits. A hit expansion was performed based on

compounds 1 to 4 with the goal of identifying improved leads

from computational mining of small molecule collections.

Several approaches were pursued in parallel: (1) Pharmaco-

phore models were constructed using LigandScout,³²where

the training set included the four active compounds along with

16 inactive structures (See Figure S1 for structures). Shared

feature pharmacophore models were created to diﬀerentiate

the active from inactive compounds. The selected pharmaco-

phore model used in the virtual screen incorporates one

hydrophobic interaction, two H-bond donors (HBDs), two H-

bond acceptors (HBAs), one aromatic ring, and positive

ionizable areas. Positive ionizable area features were excluded

for the virtual screen to avoid retrieving highly polar molecules.

The model was screened against the Pﬁzer corporate

compound collection of approximately 3 million compounds,

and the top-ranked 1000 hits were selected by the following

process. A GPU-based implementation of the ROCS

algorithm, FastROCS,³³was used to identify compounds

from the Pﬁzer collection with high shape-based similarity to

each of the four active compounds. A TanimotoCombo³⁴

score was used to rank the compounds based on the shape and

chemical similarity between the query (four active com-

pounds) and compounds in the collection. As a result, 113 hits

with a TanimotoCombo score >1.2 were selected.³⁴A 2D

similarity search was also performed around the four active

compounds using various types of ﬁngerprints, including

ECFP_4, FCFP_4, and MOE 2D.³⁵The similarity scores

calculated using each of these ﬁngerprint methods were

normalized and combined using a mathematical rule.³⁵The

resulting hits were then ranked using the combo score, and

approximately 1000 compounds were selected for further

triaging. Compound sets from the methods outlined above

were combined and prioritized based on overall chemical

attractiveness and physicochemical properties.

Using this approach, an initial set of 305 compounds in total

were selected from the Pﬁzer compound collection and

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screened in the 2-AP assay and cell-based luciferase assay with

the DDPAC mutant reporter. This resulted in 33 putative hits

that were retested in triplicates in the 2-AP assay to inform the

selection of a subset that was further proﬁled in both in vitro

binding and luciferase assays in BE(2)-M17 cells. Among the

most interesting chemotypes to emerge from these eﬀorts was

a piperidine amide that served as the starting point for a set of

analogues, resulting in 6 and 7 as the top hits (Figure 2, Table

S5). The nature of the amide heterocycle in 6 and 7 is clearly

important for both RNA-binding aﬃnity and functional

activity, as the corresponding benzamide 10 was devoid of

activity in the in vitro assays (Table S5). In addition to the

piperidine amide series, amidine 5 was also identiﬁed from the

hit expansion, and it was also selected for detailed proﬁling.

The activities in the 2-AP assay were conﬁrmed to be 16.4 μM

for 5 and 15.2 and 16.9 μM for 6 and 7, respectively. These

results were further corroborated by demonstrating the activity

in the Q-assay and U1 mimic assay (Table S5). The translation

from in vitro binding to tau splice modulation in cell-based

assays was subsequently established by showing a dose-

dependent eﬀect in the luciferase assay with the DDPAC

mutant reporter. Compounds 5 to 7 reduced the luciferase

activity by 30% at 50, 7, and 12 μM concentrations,

respectively (Figure 3). Further conﬁrmations were also

completed in the BE(2)-M17 luciferase assay (Figure S4).

Compared with compound 4, analogues 6 and 7 have

signiﬁcantly improved central nervous system multiparameter

optimization (CNS MPO) scores (5.2 for 6, and 5.5 for 7,

compared with 2.7 for 4, on a scale from 0 to 6). The higher

the CNS MPO score is, the more likely it is that a compound

will have good brain penetration (BBP) and overall favorable

ADME (absorption, distribution, metabolism, and elimination)

and safety attributes.³⁶The CNS MPO score is calculated from

six physiochemical parameters: (1) calculated lipophilicity

(cLogP); (2) calculated distribution coeﬃcient at pH 7.4

(cLogD); (3) molecular weight (MW); (4) topological polar

surface area (TPSA); (5) number of HBDs; and (6) pK_a.³⁶In

particular, the presence of multiple HBDs can adversely impact

brain penetration. It is therefore noteworthy that compounds 6

and 7 only contain one HBD, which is a signiﬁcant

improvement relative to compounds 1 to 4. The identiﬁcation

of compounds 6 and 7 indicates that Inforna hits can serve as

valuable starting points to identify RNA modulators with

robust functional activity in cell-based assays and with

favorable drug-like properties suitable for further lead

optimization using standard medicinal chemistry approaches.

Using the 4−WT Tau RNA Structure and 2-AP Assay

to Identify Novel Small Molecule Binders That Inform a

Complementary Hit Expansion. As the in vitro 2-AP assay

is suitable for screening small molecules for binding to tau

RNAs, small molecules from the NCI diversity set³⁷that were

ﬁrst triaged by docking were studied. Brieﬂy, small molecules

from the diversity set were docked into the structure of the 4−

WT tau RNA complex and scored for binding. Glide docking

scores were computed for each docked structure from free

energy terms that favor binding, such as hydrogen-bonding and

hydrophobic interactions, and terms that hinder binding, such

as ligand strain and water desolvation.³⁸Compounds with a

Glide docking score <−9 were considered hits. A subset of

these compounds was commercially available (n = 15) and

carried forward in binding assays with tau RNAs. Of the

compounds (10 out of 15), 67% can be categorized into three

chemotypes: ellipticines, bis-quinoliniums, and phenothiazines,

which are known RNA binders. In particular, an ellipticine

binds to r(CGG)^expand inhibits its association with DGCR8Δ

(K_d= 76 4 nM); bis-quinoliniums bind to and stabilize RNA

G-quadruplexes, and a phenothiazine, acetopromazine, binds

to the A-bulge loop in HIV-1 TAR RNA (K_d= 270 nM).³⁹⁻⁴¹

The 15 compounds were screened for binding to 2-AP-labeled

WT and DDPAC RNAs. Within these compounds, the most

active compound, 8, decreased 2-AP emission with an EC₅₀of

4.8 μM and was active in the cell-based luciferase assay with an

IC₅₀of around 10 μM (Figure 3 and Table S5).

To verify that these compounds were selective for the A-

bulge, as compared to other bulges, competitive binding assays

between 2-AP-labeled DDPAC RNA and unlabeled RNAs with

each single nucleotide bulge were completed.⁴²These data are

summarized in Table S6. For 5 to 7, saturable binding is only

observed for the RNA containing the A-bulge, with IC₅₀s

ranging from 12 to 15 μM. Likewise, 4 showed a ∼10-fold

selectivity for the RNA with an A-bulge over a C-bulge, with

no saturable binding observed for RNAs with G- or U-bulges

(up to 200 μM concentration). Compound 8 was ∼7-fold

selective for the A-bulge (IC₅₀= 3.2

0.6) over the other

bulges (IC₅₀’s ranging from 19 to 26 μM). Thus, all ﬁve

compounds show selectivity for the A-bulge, as compared to

other single nucleotide bulges.

Using 8 as a starting point, the Pﬁzer corporate compound

collection of approximately 3 million compounds was again

searched using a combination of the 2D similarity and

FastROCS³³methods described above along with consid-

eration for CNS physicochemical properties. Ultimately, about

200 compounds were selected for screening, leading to the

identiﬁcation of 9 as a promising lead (Figure 2). Compound 9

exhibits single-digit micromolar activity in the quenching and

U1 assays with K_dand IC₅₀values of 4.9 and 2.1 μM,

respectively, and it resides in a good CNS property space, as

indicated by the CNS MPO score of 4.4. The activity in the 2-

AP assay could not be established due to compound emission

interfering with the 2-AP signal.

The improved biochemical potency of 9 translated into

robust and dose-dependent functional activity in the cell-based

luciferase assay, with an IC₅₀of approximately 10 μM. More

importantly, 4 and 8 have signiﬁcant cellular toxicity at 50 μM,

whereas 9 has no toxicity even at 100 μM (Figure S7). The

larger window between toxicity and activity indicates greater

biocompatibility of 9. Finally, 5 to 9 were also tested and had

no eﬀect on the splicing of the A-bulge-mutated tau mini-gene

in transfected HeLa cells (Figure 3B).

Interestingly, there is a good correlation between the EC₅₀,

K_d, or IC₅₀values of all compounds determined by the

diﬀerent assays and percentage of exon 10 skipping, with

correlation coeﬃcients of 0.6 to 0.7 for each assay (Figure 4).

However, factors such as cellular uptake and solubility can

aﬀect a compound’s biological activity, resulting in a modest

diﬀerence in in vitro K_dor EC₅₀values relative to cellular IC₅₀

values.³¹Among the three in vitro assays, the U1 snRNA mimic

assay gives the best correlation with biological activity (R =

0.7), demonstrating this assay best reﬂects events that happen

in the biological environment.

Thermodynamic Stability of Tau RNAs in Complex

with Compounds 4−9. Collectively, the biochemical and

cellular assays suggest that compounds 4 to 9 thermally

stabilize the tau RNA. To provide experimental evidence that

this is the case, we completed optical melting experiments for

the DDPAC, WT, and DDPAC+I17T constructs in the

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Figure 5. Target proﬁling in cells using Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP). (A) Scheme of Chem-CLIP to react

with an RNA target in a cell to validate target engagement of small molecules. (B) The compounds used in Chem-CLIP and competitive Chem-

CLIP (C-Chem-CLIP). (C) The Chem-CLIP probe of bioactive ligand 9 (compound 11) reacts with tau pre-mRNA in LAN5 cells, whereas

control probe 12 does not. C-Chem-CLIP shows that when increasing concentrations of 9 are preincubated with LAN5 cells, the pull-down of tau

pre-mRNA with a constant concentration of 11 is diminished in a dose-dependent manner. (D) Compound 11 reacts with tau pre-mRNA in WT

and DDPAC mini-gene-transfected HeLa cells, whereas 12 does not. Preincubation of 9 diminished pull-down of 11 in C-Chem-CLIP. Compound

11 does not pull-down I17T mutant tau pre-mRNA in transfected cells. Collectively, Chem-CLIP pull-down of 11 and C-Chem-CLIP studies show

that target engagement is dependent on the presence of the 3D structure of the RNA target, as mutations abate pull-down. Error bars indicate SD.

** P < 0.01 and *** P < 0.001, as determined by a two-tailed Student t-test.

absence or presence of compounds (Figure S17). Among the

unbound constructs, the DDPAC RNA had the lowest melting

temperature (T_m), and the WT RNA had the highest T_m. The

T_mof WT and DDPAC increased the most in the presence of

compounds 4, 8, and 9, with greater eﬀects on the DDPAC

RNA than the WT RNA. These results are consistent with

binding aﬃnities determined from the three in vitro binding

assays and activities in the cell-based assays. In agreement with

in vitro binding assays, compounds 5, 6, and 7 increased the T_m

of the WT and DDPAC RNAs less than the higher aﬃnity

binders 4, 8, and 9. Piperidine amides 6 and 7 increased the T_m

of WT and DDPAC RNAs similarly to each other. In the

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DDPAC+I17T construct, only compound 8 signiﬁcantly

increased T_mand stabilized the RNA, in agreement with the

lower selectivity observed in cell-based luciferase assays and

RT-qPCR. Taken together, each compound binds to the WT

and DDPAC tau RNA constructs and stabilizes the DDPAC

construct to a greater degree than the WT construct.

Photoprobe 11 only reacted with tau WT and DDPAC RNA

with an IC₅₀value of about 10 μM, while not capturing the

I17T mutant. In contrast, 12, due to lacking an RNA-binding

module, did not label any RNAs in this experiment (Figure

S12). C-Chem-CLIP experiments were completed with 9 to

compete with 11 for the RNA binding sites and thereby reduce

the labeling of tau pre-mRNA with photoprobe 11. The IC₅₀of

9 in C-Chem-CLIP experiments was close to 10 μM, further

bolstering 11 and 9 binding to RNA targets with a similar

aﬃnity.

Testing Compounds for Aﬀecting Tau Pre-mRNA

Alternative Splicing in Cells by RT-qPCR and Western

Blot. Next, we veriﬁed that the compounds with activity in

vitro and in cell-based luciferase assays are functioning at the

RNA level by measuring levels of 3R and 4R mRNA isoforms

by RT-qPCR.²¹In these experiments, a Vivo-Morpholino ASO

complementary to the exon 10−intron 10 junction was used as

a positive control (4R-to-3R ASO) and was expected to inhibit

exon 10 inclusion.²¹Compounds 4 to 9 were tested in HeLa

cells expressing a WT or a DDPAC mini-gene as well as for

their eﬀects on endogenous tau in LAN5 neuroblastoma cells.

Indeed, all six compounds reduced the 4R/3R ratio (4R tau

mRNA expression divided by 3R tau mRNA expression)

(Figure S8). Given that 9 is one of the most active compounds

in all assays, we measured the change in endogenous tau 4R

and 3R protein levels in LAN5 cells by Western blot.

Compound 9 and the 4R-to-3R ASO both demonstrated a

signiﬁcant reduction of the 4R/3R ratio (4R tau protein

abundance divided by 3R tau protein abundance), without

changing total tau expression (Figure S9). To summarize the

results of the hit expansions compared with initial Inforna hit

compounds 1 to 4, the follow-up leads 6, 7 and 9 have

dramatically increased CNS MPO scores, from an average of

3.0 to 4.8 (Figure 2), as well as modestly enhanced cellular

activity. Considering that CNS drug candidates usually have

CNS MPO scores over 4,³⁶the identiﬁcation of 6, 7, and 9

starting from Inforna hits followed by subsequent hit

expansions and analogue synthesis indicates that this approach

can be a valuable lead generation strategy to identify drug-like

leads for directly targeting RNA.

We then used Chem-CLIP and C-Chem-CLIP to study

target engagement in LAN5 neuroblastoma cells (Figure 5C)

and transfected HeLa cells (Figure 5D). A 4-fold enrichment

of tau pre-mRNA was observed with 10 μM of 11 but not 12

in the pulled-down fraction, as measured by RT-qPCR

compared with the amount of tau pre-mRNA before pull-

down (Figure 5C and 5D). The C-Chem-CLIP experiment

was performed by preincubating cells with an increasing

concentration of parent compound 9, followed by treatment

with a constant concentration of 11. Similar to in vitro C-

Chem-CLIP studies, the pull-down of tau pre-mRNA by 11

was inhibited in a dose-dependent manner when RNA targets

were preoccupied by 9 (signiﬁcantly at 10 μM; Figure 5C and

5D). Again, 12 did not pull down any of the RNA targets.

Importantly, there was no signiﬁcant enrichment by 11 in A-

bulge-mutated tau-mini-gene-transfected HeLa cells, which

conﬁrms that the binding site of 11 is also the A-bulge, the

same as parent compound 9 (Figure 5D). We then measured

the enrichment of mature tau mRNAs, which do not contain

the A-bulge, mRNAs containing iron responsive elements

(IREs) with single nucleotide bulges, GAPDH, and β-actin

mRNAs by 11 to assess its selectivity for the tau A-bulge

(Figure S14). GAPDH and β-actin mRNAs were chosen as

they represent highly abundant cellular RNAs. Compound 11

did not enrich these mRNAs, conﬁrming it is selective for the

tau exon 10−intron 10 junction in tau pre-mRNA.

Demonstrating Binding of Compound 9 to Tau pre-

mRNA in Cells Using Chem-CLIP. Given that 9 is one of the

most active compounds in all assays, we sought to proﬁle its

binding to tau pre-mRNA in cells. By studying the complex of

9 with the WT tau RNA, we designed compound 11 to further

assess the in vitro and cellular target engagement of 9 by using

Chemical Cross-Linking and Isolation by Pull-down (Chem-

CLIP) (Figure 5A) to study the compound occupancy.^14,43−47

Competitive Chem-CLIP (C-Chem-CLIP), a complementary

approach, was also used to evaluate the binding of parent

compound 9 directly by competing for the binding of RNA

targets with the Chem-CLIP probe 11. On the basis of the

binding mode of 9 as established by NMR spectroscopy

studies (vide infra), no stacking interaction was contributed by

the cyclohexane of 9. Therefore, it provides a site that can be

modiﬁed with cross-linking modules. We introduced a

diazirine module for photo-cross-linking with biomolecules in

proximity and an alkyne puriﬁcation module for pull-down

with streptavidin beads after click-chemistry-mediated con-

jugation to a biotin azide reporter (Figure 5B).⁴⁸A control

compound 12 lacking the RNA-binding module was also

synthesized (Figure 5B). Chem-CLIP probe 11 had a similar

activity to 9 in in vitro and biological assays, indicating that the

introduction of the diazirine alkyne module has a limited eﬀect

on compound binding to the target RNA (Figures S10 and

S11, Table S5). The ability of 11 and 12 to react with the tau

hairpin RNAs was assessed in vitro (Figure S12 and S13).

Eﬀect of Compounds on Other RNAs Containing the

Tau A-Bulge. As a further test of selectivity, we studied the

eﬀect of 7 and 9 on other RNAs that contain the same A-bulge

at tau exon 10-intron 10 junction. We identiﬁed ten such

RNAs in a database of all secondary structural elements found

in human microRNA precursor hairpins (Figure S15).⁴⁹

Treatment of LAN5 cells with 7 or 9 had no eﬀect on the

expression level of any of the RNAs studied (Figure S15). This

could be due to a variety of factors, including: (i) the next

nearest neighbor base pairs of the A-bulge in these sequences

diﬀer from those in the tau exon 10 hairpin, which may aﬀect

small molecule recognition; (ii) expression level; and (iii) in

nine of the ten miRNAs, the A-bulge is located in a

nonfunctional sites, i.e., not in a Drosha or Dicer processing

site.

Compound 9 Aﬀects Tau Pre-mRNA Splicing in hTau

Mice Primary Neurons. In addition to transfected and

neuroblastoma cell lines, we evaluated 9 in cultured primary

neurons extracted from htau transgenic mice. Brains from htau

mice pups were isolated and, after removal of the meninges,

the neurons from the cortex were isolated and cultured in

precoated plastic plates (Figure 6).⁵⁰After 15 days in vitro

(DIV), the cultured neurons were treated with 9, and the 4R/

3R tau ratio was measured by RT-qPCR after 2 days, similar to

studies in LAN5 cells. [Note that gentamicin was added to the

culture medium at a ﬁnal concentration of 4.2 nM. To verify

that gentamicin did not aﬀect tau exon 10 splicing, LAN5 cells

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dynamics simulations to generate a structure of the complex.

The results of 1D and 2D NMR spectra of unbound tau RNAs

and 4-bound WT tau RNA are described in Supporting

Information.

Overall Structures of the Unbound RNA and WT Tau

RNA−4 Complex. The overall structure of the unbound WT

tau RNA stem mimic, as determined by NMR spectroscopy, is

shown in Figure 8A and described in Supporting Information.

An ensemble of 20 structures of the WT tau RNA−4 complex

was generated by using restrained molecular dynamics and 182

NOE distance restraints (Figure S27 and Table S10). The

average root-mean-square deviation (RMSD) for the structures

is 1.92 Å (Table 1), indicating good convergence. The bound

RNA structure consists of an A-form helix (Figure 8B). All of

the canonical base pairs on either side of the A-bulge form,

consistent with the NMR data (Figure S22). The average

C1′−C1′ distances among all base pairs range from 10.3 to

10.8 Å. The A-bulge is positioned in the minor groove,

consistent with the break in the NOESY walk at residue A6

and the loss of NOEs from C5H2′ to A6H8 and A6H2′ to

G7H8 (Figure 8B). The closing GC pairs are stabilized by

stacking interactions with 4, and their formation is consistent

with observed C-amino to G-imino NOEs (Figures 8, 9, and

S20). However, the C16H1′ to G17H8 NOE for the bound

structure was not observed, most likely due to the broadening,

spectral overlap, or increased distances due to the stacking of 4

between these nucleotides. The helix is overwound at the

5′G4C5/3′U18G17 step. The G4−U18 pair has a relatively

large propeller twist, base pair opening, and shear (see

Supporting Information). As expected from the NOEs, the

compound stacks between the neighboring bulge base pairs.

Relatively large buckle values were observed for the G3−C19

and G7−C16 pairs (see Supporting Information). A hydrogen-

bonded network helps to stabilize the structure. Speciﬁcally,

hydrogen bonds occur in the major groove between the C5

amino protons and N3 of 4 and the C5 amino nitrogen and

H02 of 4. These interactions may provide sequence speciﬁcity

for the binding of 4 to the A-bulge loop ﬂanked by the GC

pairs. Considerable stacking among the fused aromatic rings of

G7, 4, and G17 suggests that these stacking interactions

stabilize the complex. Altogether, the binding of 4 to the RNA

results in the formation of a complex largely stabilized by the

hydrogen-bonding and stacking interactions.

1D NMR Spectra of RNAs Bound to Compounds 5−9.

An analysis of the bioactive compounds 5 to 9 revealed three

diﬀerent scaﬀolds. Among these scaﬀolds, RNA−ligand NOEs

were not obtained for compounds with a 4-phenylpiperidine

core (compounds 6 and 7). To investigate the diﬀerential

binding of these scaﬀolds to the RNAs, NMR spectra were

acquired on each compound in complex with tau RNAs.

WaterLOGSY spectra were acquired on 5 to 9 in complex with

the WT and DDPAC duplex constructs (Figures S29 to S33

and Tables S1A and S1B) and, as a control, on the compounds

alone to determine whether indications of binding may result

from aggregation. The results of these experiments, in addition

to the 2D NMR spectra of 5 and 9 bound to WT tau RNAs,

are summarized in Supporting Information.

Overall Structures of the WT Tau RNA Complexed

with Compounds 5 and 9. Ensembles of 20 structures were

generated using 258 and 173 distance restraints for the tau

RNA−5 and tau RNA−9 complexes, respectively, including 13

intermolecular restraints between the tau RNA and 5 and 11

intermolecular restraints between the tau RNA and 9 (Figure

Figure 6. Eﬀect of 9 on the pre-mRNA splicing outcome of tau exon

10 in primary neurons harvested from human tau transgenic mice

(htau mice). (Top) Primary neurons were extracted and cultured

from htau mice brains as described in Experimental Section. Total

RNA was extracted after 48 h with treatment of DMSO (vehicle), 4R-

to-3R ASO, or a Scrambled ASO control (“Scrambled”). The 4R/3R

ratio, which was calculated as 4R tau mRNA expression divided by 3R

tau mRNA expression, was measured by RT-qPCR. (Bottom)

Compound 9 downregulated 4R/3R ratio dose-dependently, up to

50%. The 4R-to-3R ASO decreased 4R/3R ratio by 80%, whereas the

scrambled ASO had no eﬀect. Error bars indicate SD.

were separately treated with gentamicin at 0.4, 4, and 40 nM,

and the 4R/3R tau ratio was measured after 2 days (Figure

S16). No eﬀect on tau splicing was observed at any of the

concentrations tested.]

Compound 9 demonstrated dose-dependent downregulation

of the 4R/3R ratio, up to 50%, showing that the bioactivity of

9 is not limited to transfected or neuroblastoma cells but is also

observed in primary cortical neurons (Figure 6). Compound 9

is an initial lead molecule that emerged following a single hit

expansion around 8. Future SAR studies are likely to give rise

to second generation compounds with improved potency that

may enable testing directly in htau transgenic mice.

Studies of RNA-Small Molecule Complexes. Our NMR

studies of 4 bound to a model of the tau exon 10-intron 10

junction enabled us to develop models of other compounds

bound to the RNA, providing important insight into molecular

recognition. Below, we discuss additional details about the 4-

tau RNA complex as well a summary of our NMR studies of

complexes with 5−9. In brief, NMR studies were commenced

by acquiring ligand-based WaterLOGSY spectra⁵¹on the

RNA−small molecule complexes to conﬁrm the binding of the

small molecule to the RNA and the solubility of the complex

(Figure 7). 1D ¹H NMR spectra were then collected to

identify the chemical shift and line width changes to the imino

proton resonances as an additional indication of small-

molecule binding. If these spectra indicate binding, then 2D

¹H NMR spectra were acquired to assign the RNA and small

molecule resonances and calculate distance restraints from

intramolecular and RNA−small molecule NOEs. Finally, these

distance restraints were applied to restrained molecular

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Figure 7. Scheme for NMR-based determination of structures of RNA−small molecule complexes. First, a WaterLOGSY spectrum was acquired on

1

the RNA−small molecule complex to assess binding and solubility of the complex. Next, 1D H spectra were acquired to identify shifts in

resonances that are expected to occur upon small-molecule binding. 2D NMR spectra were then acquired to assign resonances and calculate

distance restraints, which were used to model the RNA−small molecule complex via restrained MD simulations. Compound 5 was presented as an

example in this scheme.

S28 and Tables 1, S11, and S12). The average RMSD for the

WT tau RNA−5 and −9 complexes is 1.11 and 1.37 Å,

respectively (Table 1), indicating good convergence. In each

complex, all of the base pairs are consistent with the 1D and

2D imino proton data (Figure S40 and S41). The average

helical rise and twist are similar for both WT tau RNA−5 and

−9 complexes and consistent with an A-form helical structure

of RNA, similar to that observed for the 4-bound complex (see

Supporting Information). The average C1′−C1′ distances for

the complexes range from 10.5 to 10.8 Å for all residues, which

is also consistent with A-form helices. In each complex, the A-

bulge is displaced into the minor groove (Figure 8). In the 5-

bound structure, the displaced A-bulge appears to stack within

the minor groove, and the 9-bound structure has the A-bulge

partially displaced, with the hydrogen-bonding surface of the

adenine pointing out of the minor groove. In the 5-bound

structure, the C7 methyl group is oriented toward the minor

groove and is supported by observed NOEs between the

methyl protons and the sugar protons of the adjacent base

pairs to the A6-bulge (Figures 8 and S38). The 9-bound

structure, with the 9-C7 methoxy group oriented toward the

minor groove and the 9-C16 methyl group in the major

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Figure 8. NMR solution structures of unbound and bound WT tau duplex model. (A) Structure of the free-form WT tau duplex model. (B)

Structure of the 4-bound WT tau RNA. (C) Structure of the 5-bound WT tau RNA. (D) Structure of the 9-bound WT tau RNA. Structures were

calculated by restrained simulated annealing. In panels (A) to (D), views of the binding site residues are depicted with stick representations for the

RNA and sphere representations for compounds. (E) Overlay of unbound and 4-, 5-, and 9-bound structures. Compounds 4, 5, and 9 stack

between the C5−G17 and G7−C16 closing base pairs. The helices in the 5- and 9-bound structures appear to be more tightly wound to form

interactions with the compounds.

Table 1. Structural Reﬁnement Statistics of Unbound and Bound WT Tau Duplexes

unbound

RNA

4-bound

RNA

5-bound

RNA

9-bound

RNA

no. of restraints

all distance restraints, including hydrogen bonds

all NOE restraints

intraresidue

191

150

69

79

2

N/A

41

182

258

173

141

68

62

2

7

221

121

79

2

146

75

57

2

sequential residues

long range RNA (excluding 4−, 5−, or 9−RNA)

intramolecular 4, 5, or 9

intermolecular 4−, 5−, or 9−RNA

hydrogen bonds

6

1

13

37

0

11

27

105

41

0

dihedral

0

RMSD of experimental restraints

distance (Å)

dihedral (deg)

3.8 × 10⁻³

N/A

2.8 × 10⁻³

N/A

4.2 × 10⁻³

N/A

2.1 × 10⁻³

RMSD of structures for heavy atoms (Å)

all residues, excluding 4, 5, or 9

all residues and 4, 5, or 9

outer helices (residues 1−3, 9−14, and 19−21)

4, 5, or 9

1.79 0.31

N/A

2.01 0.40

N/A

1.92 0.49

1.91 0.47

1.92 0.61

1.06 0.38

1.53 0.37

1.11 0.25

1.12 0.25

1.02 0.31

0.50 0.33

0.85 0.17

1.37 0.36

1.41 0.34

1.40 0.47

1.08 0.60

1.14 0.34

Bulge loop and inner base pairs, including 4, 5, or 9 for bound structure (residues 4−8 and

0.84 0.19

15−18)

Bulge loop and closing pairs, including 4, 5, or 9 for bound structure (residues 5−7 and 16−17) 0.63 0.19

1.43 0.39

0.97 0.22

1.23 0.40

groove, is also supported by NOEs between the methyl groups

and the adjacent base pairs of the A-bulge (Figures 8 and S39).

The methoxy groups of 5 and the tertiary amine of 9 lie in the

major groove and generally do not form hydrogen-bonding

interactions with the RNA. In the 5-bound structure, overlaps

between the quinolone ring of 5 and C5 and G17 bases suggest

that the complex is primarily stabilized by stacking interactions

(Figure 9). Additionally, van der Waals interactions form

between the methoxybenzene group and the RNA. In the 9-

bound structure, overlaps between the aromatic rings of 9 and

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Figure 9. Schematic diagrams and 3D structures of intermolecular interactions between the WT tau duplex model and compounds 4, 5, and 9 in

NMR structures. (A) Interactions in the tau RNA−4 complex. (B) Interactions in the tau RNA−5 complex. (C) Interactions in the tau RNA−9

complex. In the 3D structures, 4, 5, and 9 are colored orange, green, and blue, respectively, while the RNA is colored gray. Gray dashed lines in the

3D structures represent hydrogen-bonding and stacking interactions; van der Waals interactions are not shown.

C5, G7, and G17 bases suggest that stacking appears to

primarily occur between 9 and these bases (Figure 9). The N-

methylcyclohexylamine group of 9 appears to form van der

Waals interactions with the wall of the major groove. The

buckle values for the C5−G17 and G7−C16 base pairs are

results of the NMR data and structural calculations, both 5 and

9 displace the A-bulge and stabilize the RNA by stacking

between the adjacent base pairs.

Comparison of the Unbound and Bound Structures.

The bound and unbound structures consist of A-form helices

with the same canonical base pairs. The average C1′−C1′

distances among all base pairs in the bound and unbound

spectra are similar, but the 4-, 5-, and 9-bound structures are

slightly less compressed than the unbound structure and have

average helical rise values that are closer to typical A-form

RNA (2.8 Å) than the unbound structure.⁵²The average

13.8

8.0 and −25.1

9.7°, respectively. In the 5-bound

structure, relatively large buckle values of 20.5 8.5 and −12.2

10.5° are observed for the C5-G17 and G7-C16 base pairs,

respectively. The helix is overwound at the 5′G4C5/

3′U18G17 steps for both the 5- and 9-bound structures,

with similar helical twist values for both structures. From the

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helical twist values of the 5- and 9-bound structures are

approximately that of the unbound structure (33.7°), while

that of the 4-bound structure is slightly lower (31.0°). The

major groove at the 5′G4C5/3′U18G17 and 5′G7U8/

3′C16A15 steps widened upon the binding of 4 (see

Supporting Information). In comparison, less widening of

the major groove occurs at these steps upon the binding of 5

(see Supporting Information). The binding of 9 also resulted

in less widening of the major groove width at these steps than

the binding of 4 (see Supporting Information). Minor

conformational changes of the G4−U18 pair occurred upon

the binding of 4, as evident from the relatively small changes in

propeller twist and base pair opening; however, the magnitude

of the buckle increased by 6.9°. At the same time, greater

changes occurred in the conformation of the closing G7−C16

pair, with a considerable decrease in the propeller twist, while

the buckle increased in both that pair and the G3−C19 pair

(see Supporting Information). In the 5-bound structures, the

propeller twist and base pair opening of the G4-U18 pair

changed by 11.3 and 9°, respectively, while the magnitude of

the buckle increased by 8.3°. In the 9-bound ensemble of

structures, the values for the propeller twist, base pair opening,

and magnitude of the buckle of the G4−U18 pair changed by

10.7, 8.4, and 7.8°, respectively. Relative to the 4-bound

structures, smaller changes in the propeller twist and buckle

occurred at the G7−C16 pair of the 5-bound structures upon

compound binding (see Supporting Information). A larger

change in the buckle occurred at this base pair in the 9-bound

structures (see Supporting Information). These data indicate

that the binding of 4, 5, or 9 to the helix induces helical

structure changes, especially in the closing base pairs of the

bulge loop, that facilitate stacking, van der Waals, and/or

hydrogen-bonding interactions between the ligand and the

RNA.⁵³These interactions are facilitated by a balance of the

hydrophobic and hydrophilic groups that maintains the

solubility and membrane permeability.⁵⁴In particular, the

number of HBDs and HBAs in 5 and 9 are outside of the range

for which poor absorption is expected (over ﬁve and 10,

respectively), while the molecular weights of 4, 5, and 9 are

less than 500 g/mol.⁵⁵These interactions may contribute to

the increased selectivity of the drugs for the target and thus

reduce the toxicity associated with oﬀ-target binding.

bioactivity, and solubility, allowing for NMR structure

determination of the complex. On the basis of this structure,

an in vitro ﬂuorescence-based binding assay was developed and

then employed in two separate strategies to screen for

compounds with improved physiochemical properties and

activity. One strategy consisted of constructing a pharmaco-

phore model using the Inforna hits, followed by hit expansion

and analogue synthesis, which led to 6 and 7. The second

strategy consisted of molecular docking of the NCI diversity

set, testing of the top scoring compounds, and then hit

expansion, which led to 9. Accounting for these and other

parameters, the average CNS MPO score increased to 4.9 for

lead compounds 6, 7, and 9 from 3.2 for the initial Inforna hits,

1 to 4. Among the optimized compounds, 9 was the most

potent in cellulis and bound to the target RNA with the greatest

aﬃnity. Furthermore, 9 was more selective for the A-bulge site

and less toxic than its parent compound 8. Thus, it was carried

forward in Chem-CLIP studies to assess for target engagement.

The C-Chem-CLIP studies using 11 showed that 9 selectively

inhibited pull-down of the tau pre-mRNA in a dose-dependent

manner in LAN5 cells expressing endogenous tau and in

transfected HeLa cells expressing the A-bulge-containing

hairpin. Separately, 9 decreased levels of 4R tau mRNA

relative to 3R tau mRNA in humanized mouse primary

neurons in a dose-dependent manner.

Compound selectivity was demonstrated via thermal melting

experiments, miRNA expression proﬁling, and Chem-CLIP. In

the thermal melting experiments, 4−9 stabilized the tau hairpin

structures, with melting temperatures correlating with the

binding aﬃnities and bioactivities of the small molecules. The

2-AP competitive assays and thermal melting experiments also

showed that the compounds selectivity bound to and stabilized

constructs containing the A-bulge over RNAs lacking the A-

bulge or other bulges. MiRNA expression proﬁling with

compounds 7 and 9 showed that these compounds were

selective against microRNAs containing A-bulge motifs in the

same sequence context as in the tau exon 10 hairpin. In Chem-

CLIP studies, photoprobe 11 selectively enriched pull-down of

tau pre-mRNA over other mRNAs. Taken together, this study

highlights two complementary approaches, both rooted in the

evaluation of an RNA structure, that have successfully resulted

in the identiﬁcation of superior chemical matter, as evident by

improvements in bioactivity, selectivity, physicochemical

properties, and cell viability.

The compounds identiﬁed in this study generally consisted

of fused aromatic rings that stabilize the exon 10 hairpin by

stacking interactions. Compound 4 is a derivative of

dibenzothiopene, itself an analogue of carbazole, while 8 and

9 contain ellipticine cores, and 5 contains a quinoline core.

Indeed, DNA-targeting drugs based on these cores primarily

act via DNA intercalation.⁵⁷⁻⁵⁹Although intercalators may

bind nonspeciﬁcally to RNA,^60,61cross-peaks between the tau

WT NMR construct and compounds 4, 5, and 9, in particular

asymmetric compounds 5 and 9, in NMR spectra support a

limited number of binding modes. This indicates structure-

speciﬁc recognition of the A-bulge site by these compounds,

which are discussed in further detail below. However, the lack

of strong evidence that compounds 6 and 7 bind to the tau

RNA constructs under NMR conditions indicates that, for

some compounds, no direct correlation may be observed

between the binding interactions to the NMR constructs and

in vitro or in cellulis activity.

DISCUSSION

■

Previous studies have rescued aberrant tau pre-mRNA splicing

using ASOs, demonstrating that the pre-mRNA hairpin can be

targeted for therapeutic beneﬁt.²¹The success of this ASO led

to the investigation of small-molecule interactions with the tau

pre-mRNA hairpin. In 2000, G. Varani’s group reported the

NMR solution structure of a complex between neomycin and

the tau SRE²⁹and then later the structure of the tau SRE

bound to mitoxantrone (MTX), a more selective compound

identiﬁed from a high-throughput screen.⁵³Using dynamic

́

combinatorial chemistry, Lopez-Senin et al. identiﬁed addi-

tional ligands that stabilize the hairpin, likely by binding

around the adenine bulge.⁵⁶While these molecules have been

shown to bind to the tau exon 10−intron 10 junction hairpin,

the compounds did not rescue aberrant splicing.

The ﬁrst compound, 1, that improved aberrant tau pre-

mRNA splicing was identiﬁed from a structure-based search of

compounds via Inforna that targets the 5′ splice site. An

Inforna search using this compound as a query led to

compound 4, which demonstrated improved binding aﬃnity,

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The NMR structures of 4, 5, and 9 in complex with WT tau

RNA show that these compounds share features with the

pharmacophore model. The hydrogen bonds that the two

guanidines of 4 form with the closing C5 cytosine of the A-

bulge are consistent with the two HBDs and HBAs of the

amidines in the pharmacophore model. Similarly, the amidine

of 5 forms hydrogen bonds with the phosphates of the A-bulge

and adjacent cytosine. In contrast, 9 does not form hydrogen

bonds with the RNA. All three compounds contain fused ring

systems that form stacking interactions with the closing base

pairs of the A-bulge, consistent with the predicted stacking

interactions by the aromatic heterocyclic indole of the

pharmacophore model. Taken together, these results indicate

that stacking interactions are necessary to stabilize the tau

RNA−small molecule complex, while hydrogen-bonding

interactions may enhance selectivity.

The recognition of the A-bulge site by 4 appears to be driven

by hydrogen-bonding interactions between the guanidinyl

groups of 4 and the closing nucleobases, while the aromatic

portion of 4 forms stacking interactions with the closing base

pairs. These hydrogen bonds lead to a speciﬁcity in drug

binding over intercalation alone. Compound 5 forms hydro-

gen-bonding interactions with the phosphate backbone of C5,

2′-OH group of A6, and nucleobases of C16 and G17, while its

methoxybenzene group appears to ﬁll the region of the binding

cavity across the strand from the A-bulge and is positioned to

form van der Waals interactions with the C16 nucleotide

opposite of the A-bulge (Figure 9B).⁶²In the 9−RNA

complex, the N-methylcyclohexylamine of 9 ﬁlls the major

groove of the RNA helix and forms van der Waals interactions

with the G7, U8, A15, and C16 nucleotides. In addition to the

enthalpic gains from the van der Waals interactions, ﬁlling a

binding cavity with substituents that form such interactions can

bring about entropic gains from desolvation that displaces

water molecules from the binding cavity and allows them to

join bulk water.^62,63In comparison to the 4-bound complex,

the RNA in the 5- and 9-bound complexes appears to be more

tightly wound to form more interactions with each compound,

as evident from their greater helical twist values relative to that

of the 4−RNA complex (vide supra). The shape complemen-

tarity between 5 and 9 and the RNA confers structural

speciﬁcity, which has been known to lead to less toxicity

associated with oﬀ-target binding.⁶⁴

compounds is 5, which forms up to six hydrogen bonds with

the target RNA and van der Waals interactions with its

methoxybenzene group in the major groove but to a lesser

degree than the N-methylcyclohexylamine group of 9.

However, its smaller fused ring system compared to 4 or 9

results in fewer stacking interactions with the RNA. Taken

together, compounds 5 and 9 contain a balance of hydrophilic

and hydrophobic groups to maintain drug-likeness while

achieving target selectivity via diﬀerent binding modes based

on their available functional groups.

The NMR structure of the unbound stem mimic (Figure 8)

exhibits similar structural features as those of the 25-nt WT tau

hairpin previously reported by Varani et al.⁶⁶In these

structures the adenine is either partially stacked on the C4-

G14 base pair or slightly displaced into the minor groove. The

bound stem mimic complexes have similar features to the

MTX-bound WT hairpin reported by Zheng et al.⁵³MTX, 4,

5, and 9 form stacking interactions with the GC pairs adjacent

to the A-bulge. In all structures, the A-bulge is displaced from

the helix, retaining the anti conformation. For the MTX- and

4-bound structures, the adenine adopts a more external

position, while in the 5- and 9-bound structures, the adenine

is within the minor groove. NMR data supporting the degree

to which the adenine is displaced include the absence of

intramolecular NOEs to the A-bulge in the 4−RNA spectrum

and presence of H6/H8 to H1′ connectivities through the A-

bulge in the 5− and 9−RNA spectra. The structures of the

bound 4, 5, and 9 have fewer hydrogen bonds than those

observed for the bound MTX. A second set of NOEs

corresponding to the unbound conformation are observed

among RNA residues around the binding site in both the MTX

and 4-bound complexes, suggesting that the RNAs exist in an

equilibrium of bound and unbound conformations. Such

NOEs were not observed in the 5- or 9-bound complexes,

which may suggest a faster exchange between the bound and

unbound conformations. The 4- and MTX-bound complexes

share similar structural features, with predominant stacking

between the adjacent base pairs, while the 5- and 9-bound

complexes exhibit less stacking but have more interactions

within the major groove. The greater amount of stacking of 4

relative to 5 or 9 may result in slower dissociation, resulting in

both bound and unbound conformation NOEs in the 4-bound

complex.

Selectivity may be gained from the substituents that ﬁll the

binding cavity if they do not ﬁt into smaller binding pockets of

oﬀ-target molecules, decreasing the oﬀ-target binding aﬃnity.⁶⁵

While adding charged groups that form electrostatic

interactions may increase the selectivity at the target site, it

can lead to poor oral bioavailability and membrane

permeability.⁵⁴In contrast, adding hydrophobic moieties to

drugs may lead to solubility problems.⁵⁴Therefore, a balance

between the hydrophobic and hydrophilic nature of a drug

must be achieved for the drug to be transported across

membranes.⁵⁵Among these compounds, 4 has the most HBDs

and HBAs (6 and 8, respectively), while 9 has the fewest (2

HBDs and 6 HBAs, respectively) and 5 is in between (4 HBDs

and 5 HBAs). Thus, target recognition by 4 is primarily

achieved by hydrogen-bonding interactions and stacking

interactions between the closing base pairs of the A-bulge. In

contrast, the absence of target-speciﬁc hydrogen-bonding

interactions by 9 may be compensated by stacking interactions

and van der Waals interactions formed by its N-methylcyclo-

hexylamine group in the major groove. Intermediate to these

The NMR structures of the compounds bound to the tau

SRE, together with the binding assays, show that compounds

that target the tau SRE can have diﬀerent binding modes.

While the 2-AP assay is a measure of the displacement of the

A-bulge from the helix, the U1 assay is a measure of the

stabilization of the hairpin by small molecules. These

diﬀerences in the binding modes may explain why 9 has a

similar K_dvalue to 4 in the U1 assay yet does not signiﬁcantly

displace the A-bulge from the helix. The diﬀerent binding

modes for stabilizing the tau SRE are evident when comparing

the MTX- and neomycin-bound tau SRE structures reported

by Varani’s group, where MTX binds by the displacement of

the A-bulge from the helix, while neomycin binds to the major

groove with a minor displacement of the A-bulge.^29,53,65

Understanding these aspects of binding, intercalation, and

presence in the major groove and how they control splicing are

key to developing therapeutics against the tau SRE.

Comprehensive studies of these compounds can promote a

better understanding of how to chemically control a pre-

mRNA splicing outcome. Small molecules that aﬀect SMN

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pre-mRNA splicing for treatment of SMA facilitate exon

inclusion by stabilizing a SMN2 pre-mRNA−protein complex.

Wang et al. showed that one of these compounds, SMN-C2,

binds to SMN2 splicing activators FUBP1 and KSHRP and

increases their aﬃnity with the exon.⁴⁷In contrast, Zhang et al.

identiﬁed a small molecule that inhibited G-quadruplex-

dependent exon inclusion by disrupting RNA G-quadruplex

structures near the splice site and inhibiting their association

with the splicing factor hnRNPF.⁶⁷Together, these studies

demonstrate that small molecules can be used to target pre-

mRNAs and inﬂuence alternative splicing outcomes. A deeper

understanding of how these molecular recognition events aﬀect

this process and an understanding how to target these

structures to aﬀect exon inclusion or exclusion could therefore

be broadly important.

sequently be optimized using traditional medicinal chemistry

approaches.

EXPERIMENTAL SECTION

■

Compounds. Compounds 1 to 4 and 8 (Figure 2) were obtained

from the National Cancer Institute (NCI). Compounds 5 to 7 and 9

and 10 (Figure 2) were obtained from Pﬁzer. A second batch of

compound 9, in addition to compounds 11 and 12, were synthesized

as described in the Supporting Information.

Cell Culture. HeLa cells were maintained in DMEM with 10%

fetal bovine serum (FBS) and 1% GlutaMax at 37 °C with 5% CO₂.

Cells were discarded after 40 passages. The BE(2)-M17 cells were

stably transfected with a Nanoluc-PEST reporter in frame with tau

exons 9−11, resulting in an increase in the signal when exon 10 is

excluded. Stably transfected cells were selected for by using Zeocin.

Cells were grown in DMEM/F12 50:50 mix with 10% FBS, 1%

Glutamax, and 100 μg/mL Zeocin at 37 °C in a 5% CO₂atmosphere

and were passaged no more than 10 times according to the suggested

protocol from ATCC. LAN5 cells were grown in RPMI 1640 medium

supplemented with a 1× penicillin/streptomycin solution (Corning)

and 20% FBS at 37 °C with 5% CO₂. The protocol for neuron cell

culture is described below (htau Mice Primary Neuron Experiments).

Luciferase-Reporter Assay to Study Exon 10 Alternative

Splicing. The exon 10 mini-genes, both WT and DDPAC, fused to

ﬁreﬂy luciferase, have been previously described.²³HeLa cells were

transfected with WT, WT+I17T, DDPAC, or DDPAC+I17T tau

minigenes with Lipofectamine 2000 (Life Technologies) or JetPrime

(Polyplus Transfection) per the manufacturers’ recommended

procedures. For large-scale compound screening, HeLa cells (75

mm dish) were batch transfected and then trypsinized from the

surface. The cells were resuspended in growth medium, transferred to

384-well plates using a WellMate dispenser (Thermo Scientiﬁc), and

then treated with compound (transferred using a Beckman pintool in

100 nL volumes) for 18 h at 37 °C and 5% CO₂. Compound

treatment was completed in the same fashion for both transfected and

stably expressing cell lines. The activity of hit compounds was

assessed by completing a dose−response analysis in 96-well plates.

After the treatment period, the cells were washed with 1× DPBS,

and then lysed with 10 mM sodium phosphate buﬀer (pH 7.8)

supplemented with 0.2% Triton 100-X (1× TLysis Buﬀer). The lysed

cells were incubated with a buﬀered solution of ﬁreﬂy luciferase

substrate (200 mM Tris-HCl, pH 8, 15 mM MgSO₄, 0.1 mM EDTA,

25 mM dithiothreitol (DTT), 1 mM ATP, 0.2 mM coenzyme A, and

200 μM luciferin) for 10 min, and then luminescence was measured

with a Biomek FLx800 plate reader (1.0 s integration time).⁶⁸

Cell Viability. Cells were treated with compound as described

above. After the removal of the compound-containing growth

medium, the cells were washed with 1× DPBS and incubated in

growth medium containing 10% WST-1 cell proliferation reagent

(Roche) or CellTiter Fluor (Promega) for 40 min. The absorbance of

the solution was measured at 450 and 650 nm with a SpectraMax M5

plate reader (Molecular Devices) for cells treated with WST-1. The

background signal at 650 nm was subtracted from the WST-1 signal at

450 nm for analysis. For cells treated with CellTiter Fluor, the

ﬂuorescence was measured with 390 nm excitation and 505 nm

emission wavelengths. Cells treated with a compound were compared

to untreated cells to determine the eﬀect of the compound on cell

viability.

CONCLUSIONS

■

In this work, 4 was identiﬁed from a chemical similarity search

of lead compound 1 and has improved activity for in inducing

the exclusion of tau exon 10. Compound 4 and other initial

hits from chemical similiarty searching were subsequently used

as starting points to identify leads with improved drug-like

characteristics via two complementary strategies. In the ﬁrst

approach, pharmacophore modeling of 1 to 4 and substructure

and similarity searching followed by analogue synthesis led to

piperidine amides 6 and 7. Amidine 5 was also identiﬁed from

the hit expansion approach and selected for proﬁling. The 2-

AP, Q-, and U1 snRNA mimic assays provided complementary

means to identify screening hits and conﬁrmed that these

compounds indeed bind to the tau hairpin construct.

Translation of in vitro biochemical activity to a functional

eﬀect on splice modulation was demonstrated using luciferase-

based cell assays and RT-qPCR experiments. The second hit

generation approach relied on the docking of ligands from the

NCI diversity set to the NMR-derived structure of 4 bound to

the tau pre-mRNA. A select set of compounds were

subsequently screened in the in vitro assays, resulting in the

identiﬁcation of 8.

Hit expansion around 8 aﬀorded compound 9, which

demonstrated a dose-dependent reduction of the ratio of 4R-

to-3R tau mRNA levels in mouse primary neurons.

Furthermore, compound 9 reduced the pull-down of tau pre-

mRNA in cells in Chem-CLIP experiments using photoprobe

11, thereby demonstrating target engagement in cells. Detailed

NMR experiments were performed to characterize the binding

of 4, 5, and 9 to the tau hairpin. NMR spectroscopy and

restrained simulated annealing of these compounds in complex

with a model of the WT tau RNA stem showed that they

stabilize the tau hairpin by stacking and hydrogen-bonding

interactions with base pairs adjacent to the A-bulge. This work

demonstrated that a combination of structure-based drug

design, in vitro screening, thermal melting, and cell-based

assays can be used to identify compounds with drug-like

properties that target tau pre-mRNA and reduce levels of 4R

tau in FTDP-17. This strategy may be used to identify and

optimize compounds that target other incurable diseases

caused by aberrant pre-mRNA splicing. Furthermore, the

ability to improve activity and drug-like properties of hits

obtained from the Inforna platform bodes well for designing

structure-speciﬁc ligans from sequence, aﬀording lead com-

pounds that target RNA. It is conceivable that drug-like hits

identiﬁed using the strategies described herein can sub-

Quantitative Reverse Transcription PCR (RT-qPCR). HeLa

cells were seeded in 60 mm dishes and incubated for 12−16 h at 37

°C. The cells were transfected with WT, WT+I17T, DDPAC, or

DDPAC+I17T luciferase reporter minigenes using JetPrime (Polyplus

Transfection) per the manufacturer’s recommended protocol and

incubated for 4 h. After transfection, HeLa cells were collected;

reseeded in 24-well plates at a 0.6 × 10⁵cell/well density; treated with

compounds, a Vivo-Morpholino 4R-to-3R ASO (Gene Tools, LLC)

against the exon 10−intron 10 junction with the sequence 5′-

TGAAGGTACTCACACTGCCGC-3′, or a scrambled control; and

incubated for 48 h. LAN5 cells were seeded in 24-well plates at a 1 ×

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10⁵cell/well density, treated with the same compounds or

oligonucleotides, and incubated for 48 h. Total RNA was extracted

from the cells and analyzed via RT-qPCR to determine the levels of

3R and 4R tau mRNA. Cells were washed with 1× DPBS buﬀer and

then lysed and extracted using the Quick-RNA MiniPrep kit (Zymo

Research) following the manufacturer’s protocol. The concentration

of the extracted RNA was measured with a Nano Drop 2000 (Thermo

Fisher Scientiﬁc). The cDNA was generated from 150 ng of the total

RNA using qScript (Quanta Bio) following the manufacturer’s

recommended protocol. The qPCR reactions consisted of SYBR

Green 5× PCR mix (Invitrogen), 200 nM primers, and 10 ng of RT

product. Each biological replicate was tested in three technical

replicates. The levels of 18S rRNA were monitored as the endogenous

control for relative quantiﬁcation (see Table S14 for primers). The

primers for RT-qPCR in transfected HeLa cells and LAN5 cells are

provide in Table S14.

EC

⁵⁰[C_t] + [compound] + [RNA]]

K_d

1

θ =

[EC₅₀+

2[RNA]

2

l

o

i

j

y

z

EC₅₀

j

z

− mjEC₅₀

+

+ [C_t] + [compound] + [RNA]z

j

z

o

j

k

K_d

{

n

0.5

|

o

− 4[compound][RNA]} + A

o

(2)

~

where θ is the percentage of 2-AP tau RNA bound, [RNA] is the

concentration of 2-AP-labeled tau RNA, EC₅₀is the EC₅₀of 2-AP-

labeled tau RNA and compound, [compound] is the concentration of

compound, C_tis the concentration of competing, unlabeled RNA, K_d

is the competitive dissociation constant of competing, unlabled RNA,

and A is a constant.

Western Blot. The protein expression of 4R, 3R, and total tau was

analyzed in LAN5 cells after treatment of 4R-to-3R ASO, compound

9, or DMSO vehicle for 48 h. The cells were gently washed twice with

ice-cold 1× DPBS and lysed in 2% SDS lysis buﬀer containing

protease (539137, Millipore) and phosphatase inhibitor cocktails

(P5726, Sigma), followed by brief sonication. After quantifying the

protein concentration using a BCA protein assay kit (Thermo Fisher

Scientiﬁc, Waltham, MA), an equal amount of protein from each

sample was separated in 15% SDS-PAGE (GenScript, Piscataway, NJ)

and transferred onto polyvinylidene diﬂuoride (PVDF) membranes.

Nonspeciﬁc binding was blocked with 5% (w/v) nonfat dry milk for 1

h at room temperature, and the membranes were probed overnight

with primary antibodies speciﬁc for 4R tau (1:2000, 05-804 from

MilliporeSigma), 3R tau (1:5000, 05-803 from MilliporeSigma), and

total tau (1:5000, 46687S from Cell Signaling). β-Actin (1:10000,

3700S from Cell Signaling) was used as a protein loading control.

After three 10 min washes with 1× TBST (Tris-buﬀered saline, 0.5%

Tween 20), the membranes were incubated in horseradish peroxidase

(HRP)-conjugated secondary antibodies (1:10000) for 1 h at room

temperature and washed three times with TBST. Immunocomplex

signals of each protein were then detected using an enhanced

chemiluminescence detection system (ECL, PerkinElmer, Waltham,

MA). The abundance of protein in each sample was determined based

on the band intensity from an ImageJ analysis and then normalized to

β-actin bands.

2-AP Assay on Hit Expansion Library. An aliquot of 10 μM 2-

AP-labeled WT or DDPAC hairpin was folded in 1× Assay Buﬀer.

BSA was added to the RNA solution to a ﬁnal concentration of 40 μg/

mL. Aliquots of 16 μL containing 0.5% DMSO and 50 μM of 4 in a

1× Assay Buﬀer were separately dispensed into empty wells of assay-

ready plates as negative and positive controls, respectively. Then, an

equal volume of 1× Assay Buﬀer was dispensed into the other wells of

the 384-well plates using an Aurora liquid handler. Plates were

incubated for 15 min at room temperature and preread on a Tecan

plate reader using 310 nm excitation and 380 nm ﬂuorescence

emission wavelengths to investigate the compound internal

ﬂuorescence. Next, 4 μL of an RNA solution was then dispensed

into the plates. Plates were incubated for 30 min at room temperature

and read on the Tecan plate reader using the same wavelengths

described in 2-AP Assay.

Quenching Assay. An aliquot of 2 μM quencher-labeled RNA

(Q-RNA) with a sequence corresponding to the DDPAC hairpin was

folded in a 1× Assay Buﬀer. BSA and Q-probe were added to the

RNA solution to ﬁnal concentrations of 40 μg/mL and 10 nM,

respectively. The Q-RNA and Q-probe solution was incubated at

room temperature for 15 min. Compound was added to the solutions,

and serial dilutions were performed on the RNA−compound

solutions. After 30 min of incubation at room temperature, the

ﬂuorescence was measured using a BioTek FLx800 plate reader with

485 nm excitation and 535 nm emission wavelengths.

2-AP Assay. Sequences corresponding to the WT and DDPAC

hairpins that contain a 2-AP substitution at the A-bulge site (2-AP-

labeled WT/DDPAC hairpin) were folded by heating the complex to

70 °C for 3 min in a 1× Assay Buﬀer (0.185 M NaCl, 8 mM

NaH₂PO₄, pH 6.3) and slowly cooling to room temperature. Bovine

serum albumin (BSA) was added to the RNA solution to a ﬁnal

concentration of 40 μg/mL. All binding assays were carried out in

triplicate in 96- or 384-well half area plates at room temperature with

a Tecan plate reader (gain, 100; integration time, 40 μs).

Fluorescence excitation and emission wavelengths were set at 310

and 375 nm, respectively. Dissociation constants were ﬁt to eq 1:¹⁵

U1 snRNA Mimic Assay. A 200 nM aliquot of dual-labeled RNA

with a sequence corresponding to the DDPAC hairpin was folded in a

1× Assay Buﬀer at 65 °C for 5 min and then slowly cooled to room

temperature. BSA was added to the solution to a ﬁnal concentration

of 40 μg/mL. Compounds were then incubated with the RNA

solutions at room temperature for 15 min. A U1 snRNA mimic with

the sequence 5′-GGUACUCAC-3′ was added to the solution to a

ﬁnal concentration of 20 μM. The ﬂuorescence intensity of Cy3 was

measured as a function of time (in kinetics mode) for 20 min using a

Cary Eclips ﬂuorescence spectrophotometer, with excitation and

emission wavelengths of 532 and 568 nm, respectively.

Molecular Docking. The NCI diversity set comprising 1974

I = I₀+ 0.5Δε(([FL]₀+ [RNA]₀+ K_t)

̈

compounds was prepared in LigPrep (Schrodinger, Inc.). Brieﬂy, the

− (([FL]₀+ [RNA]₀+ K_t)²− 4[FL]₀[RNA]₀)^1/2

)

ionization in the pH range of 5 to 9, tautomers, and stereoisomers

options were used to calculate all the possible chemical structures.

The 3947 compounds generated from these calculations were

minimized using an OPLS 2005 force ﬁeld. An NMR model of the

tau A-bulge structure bound to 4 was prepared by removing the

bound ligand, assigning a bond order, and adding the missing

(1)

where I and I₀are the observed ﬂuorescence intensities in the

presence and absence of RNA, respectively, Δε is the diﬀerence

between the ﬂuorescence intensity in the presence of an inﬁnite

concentration of RNA and the absence of RNA, [FL]₀and [RNA]₀

are the concentrations of small molecule and RNA, respectively, and

K_tis the dissociation constant.

Competitive binding assays were completed using a previously

published protocol.¹⁸Brieﬂy, compounds 4−8 were incubated with 2

μM 2-AP-labeled tau DDPAC hairpin RNA and with increasing

concentrations of competitive DDPAC RNAs containing a C-bulge, a

G-bulge, or a U-bulge substitution. The concentration of each

compound corresponded to their EC₅₀determined by 2-AP binding

assays (Table S5). The change of the ﬂuorescence intensity was ﬁt to

eq 2:

̈

hydrogen atoms. Glide (Schrodinger, Inc.) docking experiments were

completed in the HTVS mode with no constraint. The grid size for

the docking was set to 20 × 20 × 20 Å³around the center of the 4

position. The ﬁnal ligand poses with a Glide docking score of −9 or

better were selected for further analysis (n = 32).

In Vitro Chem-CLIP and C-Chem-CLIP. Approximately 10000

counts of ³²P 5′-end-labeled tau WT, tau DDPAC, tau WT+I17T, or

tau DDPAC+I17T RNA were folded in 100 μL of a 20 mM HEPES

buﬀer at 60 °C for 5 min. After cooling to room temperature,

dilutions of 11 or 12 were added and incubated at 37 °C for 30 min.

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Alternatively, for C-Chem-CLIP, dilutions of competing parent

compounds, 9, were incubated with RNA for 30 min before adding

11 or 12. The reaction mixture was then irradiated with 365 nm UV

light in a UV stratalinker for 10 min. A click mixture (0.5 μL of

disulﬁde biotin azide (Click Chemistry Tools), 10 mM; 1 μL of

CuSO₄, 10 mM; 1 μL of THPTA, 50 mM; and 1 μL of sodium

ascorbate, 250 mM) was then added to each reaction and incubated at

37 °C for 2 h. Next, 10 μL of streptavidin beads (Dynabeads MyOne

streptavidin C1 beads (Thermo Scientiﬁc)) was added to the reaction

for pull-down of cross-linked RNA for 10 min. Unreacted RNA was

removed by placing reaction tubes on a magnetic separation rack. The

RNAs cross-linked to a small molecule remain attached to the beads

via a biotin−streptavidin interaction. The click mixture buﬀer was

then collected, and the beads were washed three times with wash

buﬀer (10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 4 M NaCl, 0.2%

Tween-20). The wash buﬀer was collected and mixed with the click

mixture buﬀer. The amounts of radioactivity in the buﬀer and on the

beads were quantiﬁed with a Beckman Coulter LS6500 liquid

scintillation counter. The percentage of RNA captured was

determined by using the radioactivity on the beads divided by the

total radioactivity in the buﬀer and on the beads.

Cellular Chem-CLIP and C-Chem-CLIP. LAN5 cells were grown

to ∼80% conﬂuency as monolayers in 100 mm dishes. The HeLa cells

were transfected, as described above in Luciferase-Reporter Assay to

Study Exon 10 Alternative Splicing, and grown to ∼80% conﬂuency as

monolayers in 100 mm dishes. The cells were then treated with 10

μM 11 or 12 for 6 h. For C-Chem-CLIP, dilutions of competing

parent compound, 9, were incubated with cells for 12 h before adding

11 or 12. After treatment, the cells were washed with 1× DPBS, and

then 10 mL of ice-cold 1× DPBS was added before irradiation with

365 nm UV light in a UV stratalinker for 10 min. The cells were

scraped, and the total RNA was extracted using TRIzol LS reagent

(Invitrogen) per the manufacturer’s protocol. Approximately 20 μg of

the total RNA was then incubated with a click mixture in 25 mM

HEPES buﬀer at 37 °C for 2 h, as described in In Vitro Chem-CLIP

and C-Chem-CLIP. The total RNA was cleaned up by RNA Clean XP

beads (Beckman Coulter) according to the manufacturer’s protocol.

Pull-down by streptavidin beads and the washing steps were

performed as described in In Vitro Chem-CLIP and C-Chem-CLIP.

After washing, the RNA on the beads was cleaved using a 1:1 mixture

of TCEP (200 mM) and K₂CO₃(600 mM) with shaking at 37 °C for

30 min. The reaction mixture was quenched with one volume of

iodoacetamide (400 mM) with shaking at room temperature for 30

min. The RNA in the buﬀer was collected by using a magnetic

separation rack and was cleaned up by RNA Clean XP beads. RT-

qPCR, as described above (Quantitative Reverse Transcription PCR),

was performed on RNA before and after pull-down using the forward

primer −5′-GGAAGTGGTGTGAGTGCGTACAC-3′ and the re-

verse primer −5′-CACCTTCAGCCAACTTCCAATG-3′. The rela-

tive fold enrichment of tau pre-mRNA was measured using eq 3:

inactivated (Invitrogen, 10082139); 10 mL of Glutamax-I (Invitrogen,

35050061); and 100 μL of gentamicin (Invitrogen, 15710064).

Feeding Medium. Feeding medium contains the following: 490

mL of Neurobasal-A (Invitrogen, 10888022), 10 mL of Glutamax-I

(Invitrogen, 35050061), and 100 μL of gentamicin (Invitrogen,

15710064). Note, add 2% B27 just before feeding (Invitrogen,

17504044).

Miscellaneous Reagents. Miscellaneous reagents include the

following: poly-^D-lysine (PDL; Sigma, P7280), Trizma Tris base

(Sigma, T1194), and Papain suspension (Worthington, LS003124).

Culture plates (Corning CellBIND surface microplates) were

precoated with 0.1 mg/mL PDL, which was dissolved in Tris buﬀer

overnight or for 2 h at 37 °C. Next, 1 mL per pup of dissection

medium was placed on ice. Then, 5 mL per pup of plating medium for

the disassociation step, with the required volume of feeding medium

for plating, and 1 mL per pup of dissection medium with 1% Papain

were prewarmed at 37 °C. The cortices from P0 htau mouse pups

(B6.Cg-Mapttm1-(EGFP)Klt Tg(MAPT)8cPdav/J, Jackson Labora-

tories) were removed and placed in ice cold dissection medium for

dissection and removal of meninges. Then, the dissected cortices were

placed into ice-cold dissection medium. The tails of P0 htau mouse

pups were collected for further genotyping. After dissection, cold

dissection medium was aspirated, and the warmed dissection

medium/Papain solution was added. The solution was placed at 37

°C for 20 min, and then, one volume of plating medium was added to

stop Papain activity. Meanwhile, the PDL solution in precoated plates

was aspirated. The plates were washed with 1× DPBS with added

feeding medium and incubated at 37 °C. The medium in dissection

tubes was carefully aspirated. The remaining tissue was carefully

washed three times with plating medium. After washing, 2 mL of fresh

plating medium was added. The tissue was dissociated by gently

triturating through a 5 mL serological pipet three times and then

through a P1000 pipet tip three times. Then, 1 mL of the supernatant

was transferred to a new 15 mL tube, and then, 1 mL of fresh plating

medium was added. Trituration was repeated three to six times

through the P1000 tip until all chunks of tissue were dissociated, and

1 mL of the supernatant was transferred every time. The cell mixture

in new tube was diluted to 10 mL with plating medium and then run

through a 40 μm cell strainer. Then, 10 mL of cells was spun down at

1900 rpm for 4 min. The cell pellet was resuspended in 1 mL of fresh

plating medium and then diluted to the required density in feeding

medium +2% B-27. The neuron cells were plated in prewarmed

plates. Approximately 50% of the medium was changed every 3−4

days with feeding medium +2% B-27. P0 htau mouse pups were

genotyped before compound treatment. After 15 days, the neuron

cells were treated with 9 and Vivo-Morpholino ASO and were

scrambled control and incubated at 37 °C for 48 h. The total RNA

was extracted and analyzed via RT-qPCR, as described above

(Quantitative Reverse Transcription PCR). Primers used for the

evaluation of mouse neuron cells are provided in Table S14.

Optical Melting Experiments. The thermal stabilities of WT,

DDPAC, and DDPAC+I17T RNA constructs were analyzed by

optical melting experiments with and without compounds. The RNAs

were heated to 95 °C and slowly cooled to room temperature. The

samples were then cooled to 15 °C upon the addition of or in the

absence of compound and then heated to 85 °C at a rate of 1 °C/min.

The absorbance was monitored at 260 nm. The melting curves were

ﬁtted with MeltWin 3.5.⁶⁹

Design and Preparation of RNA Samples for NMR. All RNA

constructs were purchased from GE Healthcare Dharmacon, Inc.

Oligoribonucleotides that form the duplex, including r(5′-CCGG-

CAGUGUG-3′) + r(5′CACACGUCGG-3′) to mimic the stem of the

WT tau SRE and r(5′-CCGGCAGUGUG-3′) + r(5′CAUAC-

GUCGG-3′) to mimic the stem of the DDPAC tau SRE, were

deprotected and desalted with PD-10 columns according to the

manufacturer’s recommended protocol. Equimolar amounts of the

sequences were combined in 400 μL of NMR buﬀer (10 mM

KH₂PO₄/K₂HPO₄and 0.05 mM EDTA (pH 6.0)) to make three

separate samples. The ﬁnal concentrations of RNA in the samples

were 250, 400, 500, or 700, including 20 μL of D₂O in each sample to

before pull‐down−ΔC_tafter pull‐down)

relative fold enrichment = 2^−(ΔC

t

(3)

where ΔC_t^{before pull‑down}is the diﬀerence between the C_tvalues for the

RNA of interest and a housekeeping gene (18S) in total RNA from

cells and ΔC_t^{after pull‑down}is the diﬀerence between the C_tvalues for the

RNA of interest and the same housekeeping gene in RNA after pull-

down.

hTau Mice Primary Neuron Experiments. The details

pertaining to media preparation are presented in the following.

Dissection Medium. The dissection medium consisted of the

following: 430 mL of culture-grade H₂O (Fisher Scientiﬁc,

SH3052902), 50 mL of 10× HBSS without Ca²⁺and Mg²⁺

(Invitrogen, 14185052), 10 mL of HEPES (Invitrogen, 15630080),

5 mL of pyruvate (Invitrogen, 11360070), 5 mL of glucose solution

(Thermo, A2494001), and 100 μL of gentamicin (Invitrogen,

15710064).

Plating Medium. Plating medium is comprised the following: 465

mL of Neurobasal (Invitrogen, 21103049); 25 mL of FBS, heat

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provide a lock signal. RNA samples were reannealed by heating to 80

°C for 3 min and were slowly cooled to room temperature to form

duplexes.

experiments with compounds 9, 11, and 12; the eﬀects

of compounds 7 and 9 on microRNA expression; the

eﬀect of gentamicin on relative amounts of 4R and 3R

Tau mRNA in LAN5 cells assessed by RT-qPCR; the

eﬀect of compounds on the thermodynamic stability of

NMR Spectroscopy. Shigemi NMR tubes (Shigemi, Inc.) were

used for all NMR experiments. NMR spectra were acquired on Bruker

Avance III 600, 700, and 850 MHz spectrometers equipped with

cryoprobes. The 1D spectra and 2D NOESY and DQF-COSY spectra

with diﬀerent mixing times were acquired at 281.5 and 298 K on

samples in 95% H₂O/5% D₂O and 100% D₂O alone or in the

presence of 4−9. 1D ¹H NMR spectra were acquired on either WT or

DDPAC tau hairpin stem mimic duplexes alone. Each compound was

then titrated into the ﬁnal compound/RNA molar ratios of 0.0, 0.5,

1.0, 1.5, and 2.0. WaterLOGSY (water-ligand observed via gradient

spectroscopy) experiments were carried out on samples of the WT tau

stem mimic duplex mixed with each compound to determine the

formation of the RNA−small molecule complexes.⁷⁰⁻⁷²Samples for

WaterLOGSY experiments contained 15 μM RNA and 300 μM

compound for a 1/20 RNA/compound ratio. WaterLOGSY spectra

were phased to give negative signals for negative NOEs with

water.^71,72Samples for 2D NMR spectroscopy consisted of 700 μM

free-form WT tau RNA, to which 4 was added to a ﬁnal concentration

of 700 μM for studies of the 4−RNA complex. Additionally, 2D

spectra were acquired on a separate sample containing 400 μM WT

tau RNA and 600 μM 5 or 9. For 1D and 2D spectra of samples in

95% H₂O/5% D₂O, an excitation sculpting sequence⁷³was applied

during acquisition to suppress the water signal. Proton chemical shifts

were referenced internally to the frequency of water. Then, 2D NMR

spectra were processed with NMRPipe,⁷⁴and the resonances were

assigned with SPARKY.⁷⁵

Methods for Obtaining NOE Restraints and Structure

Calculations. NOESY cross peaks were integrated assuming a

Gaussian distribution, and the volumes were converted to distances

using H5−H6 pyrimidine peaks as the reference. Distance restraints

were created by setting the lower and upper bounds to 0.5 Å the

calculated distance for each peak. Peaks with potential spin diﬀusion

were given upper bounds of +1.0 Å. Interbase (H6/8-H6/8) restraints

were assigned between 3.5 and 5.0 Å and 5.0 and 6.5 Å based on their

relative intensity at a mixing time of 400 ms. All imino protons

involved in GC and AU base pairs exhibited cross peak patterns

indicative of WC base pairing, and therefore, standard WC base pair

restraints were applied for these pairs. The G4−U18 mismatch was

not restrained in the calculations. Structures were calculated by

restrained molecular dynamics using AMBER14 or AMBER16.⁷⁶The

system was heated to 2000 K over 10 ps and then cooled to 0 K,

totaling 35 ps in implicit solvent using the 99SB and GAFF force

ﬁelds. Charges were assigned to 4, 5, and 9 using the AM1-BCC

method model. NMR restraints were applied at the peak temperature.

From a single initial structure of free-form or 4-bound WT tau RNA,

100 calculations were performed with randomized initial trajectories.

For the 5- and 9-bound WT tau RNA duplex, 60 calculations were

performed with randomized initial trajectories. The top 20 structures

were selected by their adherence to the NMR data. Helical and base

pair properties were extracted with X3DNA.⁷⁷

1

Tau RNA constructs; H titration and WaterLOGSY

1

NMR spectra with the compounds; 2D H NOESY

NMR spectra of unbound and bound WT stem mimic

duplexes; synthesis of compounds 5−7 and 9−11;

summary of chemical shifts changes of WT and DDPAC

1

stem mimic duplexes induced by compounds; H NMR

chemical shifts of unbound and bound stem mimic

duplexes; K_d, EC₅₀, or IC₅₀(μM) values for compounds

determined from in vitro binding assays for compounds

binding to RNA constructs; NOE restraints used for

modeling of unbound and bound stem mimic duplexes;

primers of RT-qPCR, Chem-CLIP, and microRNAs

(PDF)

Unbound RNA (PDB)

4-Bound tau WT RNA contruct (PDB)

5-Bound tau WT RNA contruct (PDB)

9-Bound tau WT RNA contruct (PDB)

(XLSX)

Accession Codes

NMR chemical shifts for the unbound and 4-, 5-, and 9-bound

tau WT RNA construct were deposited in the Biological

Magnetic Resonance Data Bank as entries 30697, 30698,

30699, and 30700, respectively. Atomic coordinates for the

unbound and bound tau WT RNA constructs were deposited

in the RCSB Protein Data Bank as entries 6VA1, 6VA2, 6VA3,

and 6VA4, respectively.

AUTHOR INFORMATION

Corresponding Author

■

Matthew D. Disney − Department of Chemistry and

Neuroscience, The Scripps Research Institute, Jupiter, Florida

33458, United States; orcid.org/0000-0001-8486-1796;

Email: Disney@scripps.edu

Authors

Jonathan L. Chen − Department of Chemistry and

Neuroscience, The Scripps Research Institute, Jupiter, Florida

33458, United States; orcid.org/0000-0001-7907-9732

Peiyuan Zhang − Department of Chemistry and Neuroscience,

The Scripps Research Institute, Jupiter, Florida 33458, United

States

Masahito Abe − Department of Chemistry and Neuroscience,

The Scripps Research Institute, Jupiter, Florida 33458, United

States

Haruo Aikawa − Department of Chemistry and Neuroscience,

The Scripps Research Institute, Jupiter, Florida 33458, United

States

Liying Zhang − Pﬁzer Worldwide Research and Development,

Cambridge, Massachusetts 02139, United States

Alexander J. Frank − Department of Chemistry & Biochemistry,

State University of New York at Fredonia, Fredonia, New York

14063, United States

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/jacs.0c00768.

■

sı

Analysis of 1D and 2D NMR spectra of unbound and

bound RNAs; set of 20 compounds identiﬁed by Inforna

and screened in cell-based luciferase assay; the eﬀect of

compounds on expression of luciferase in cell-based

assays and on relative amounts of 4R and 3R Tau

mRNA assessed by RT-qPCR; binding assays of 4 with

2-AP substituted RNA constructs; cell toxicity data for

compounds assessed by the cell viability reagent; protein

modulation with compound 9 and 4R-to-3R ASO

treatment; results of Chem-CLIP and C-Chem-CLIP

Timothy Zembryski − Department of Chemistry &

Biochemistry, State University of New York at Fredonia,

Fredonia, New York 14063, United States

Christopher Hubbs − Department of Chemistry and

Neuroscience, The Scripps Research Institute, Jupiter, Florida

33458, United States

S

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(4) Gallego, J.; Varani, G. Targeting RNA with small-molecule

drugs: Therapeutic promise and chemical challenges. Acc. Chem. Res.

2001, 34 (10), 836−843.

HaJeung Park − Department of Chemistry and Neuroscience,

The Scripps Research Institute, Jupiter, Florida 33458, United

States

Jane Withka − Pﬁzer Worldwide Research and Development,

Cambridge, Massachusetts 02139, United States

Claire Steppan − Pﬁzer Worldwide Research and Development,

Groton, Connecticut 06340, United States

Lucy Rogers − Pﬁzer Worldwide Research and Development,

Groton, Connecticut 06340, United States

Shawn Cabral − Pﬁzer Worldwide Research and Development,

Groton, Connecticut 06340, United States

Martin Pettersson − Pﬁzer Worldwide Research and

Development, Cambridge, Massachusetts 02139, United States

Travis T. Wager − Pﬁzer Worldwide Research and

Development, Cambridge, Massachusetts 02139, United States

Matthew A. Fountain − Department of Chemistry &

Biochemistry, State University of New York at Fredonia,

Fredonia, New York 14063, United States

Gavin Rumbaugh − Department of Chemistry and

Neuroscience, The Scripps Research Institute, Jupiter, Florida

33458, United States

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opportunities at the intersection of chemistry, biology, and medicine.

J. Am. Chem. Soc. 2019, 141 (17), 6776−6790.

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X.; Moon, Y. C.; Trifillis, P.; Welch, E. M.; Colacino, J. M.; Babiak, J.;

Almstead, N. G.; Peltz, S. W.; Eng, L. A.; Chen, K. S.; Mull, J. L.;

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function and longevity in mice with spinal muscular atrophy. Science

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Servais, R.; Jain, M.; Deng, L.; Bullock, C.; McLellan, M.; Schuierer,

S.; Murphy, L.; Blommers, M. J.; Blaustein, C.; Berenshteyn, F.;

Lacoste, A.; Thomas, J. R.; Roma, G.; Michaud, G. A.; Tseng, B. S.;

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Fishman, M. C.; Dietrich, W. F.; Dales, N. A.; Sivasankaran, R. SMN2

splice modulators enhance U1-pre-mRNA association and rescue

SMA mice. Nat. Chem. Biol. 2015, 11 (7), 511−517.

Jessica L. Childs-Disney − Department of Chemistry and

Neuroscience, The Scripps Research Institute, Jupiter, Florida

33458, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.0c00768

(8) Ratni, H.; Ebeling, M.; Baird, J.; Bendels, S.; Bylund, J.; Chen, K.

S.; Denk, N.; Feng, Z.; Green, L.; Guerard, M.; Jablonski, P.;

Jacobsen, B.; Khwaja, O.; Kletzl, H.; Ko, C. P.; Kustermann, S.;

Marquet, A.; Metzger, F.; Mueller, B.; Naryshkin, N. A.; Paushkin, S.

V.; Pinard, E.; Poirier, A.; Reutlinger, M.; Weetall, M.; Zeller, A.;

Zhao, X.; Mueller, L. Discovery of Risdiplam, a selective survival of

motor neuron-2 (SMN2) gene splicing modifier for the treatment of

spinal muscular atrophy (SMA). J. Med. Chem. 2018, 61 (15), 6501−

6517.

Author Contributions

^#These authors contributed equally to this work.

Funding

This work was supported by Pﬁzer, the National Institutes of

Health (R01-GM097455, DP1-NS096898, and P01-NS099114

to M.D.D, and S10-OD021550 to The Scripps Research

Institute), the Tau Consortium and Rainwater Charitable

Foundation (to M.D.D.), and the Huntington’s Disease

Society of America (J.L.C.).

(9) Ottesen, E. W. ISS-N1 makes the First FDA-approved drug for

spinal muscular atrophy. Transl. Neurosci. 2017, 8, 1−6.

(10) Singh, N. N.; Howell, M. D.; Androphy, E. J.; Singh, R. N. How

the discovery of ISS-N1 led to the first medical therapy for spinal

muscular atrophy. Gene Ther. 2017, 24 (9), 520−526.

Notes

The authors declare the following competing ﬁnancial

interest(s): M.D.D. is a founder of Expansion Therapeutics.

(11) Brunden, K. R.; Trojanowski, J. Q.; Lee, V. M. Y. Advances in

tau-focused drug discovery for Alzheimer ’s disease and related

tauopathies. Nat. Rev. Drug Discovery 2009, 8 (10), 783−793.

(12) Disney, M. D.; Winkelsas, A. M.; Velagapudi, S. P.; Southern,

M.; Fallahi, M.; Childs-Disney, J. L. Inforna 2.0: A platform for the

sequence-based design of small molecules targeting structured RNAs.

ACS Chem. Biol. 2016, 11 (6), 1720−8.

ACKNOWLEDGMENTS

■

The authors thank Samuel Hasson (Pﬁzer) and Lyn Jones

(Pﬁzer) for initiating the project and Xiangming Kong (The

Scripps Research Institute) and Chunhua Yuan (The Ohio

State University) for assistance with NMR experiments.

Minimization by simulated annealing and molecular dynamics

simulations were performed with computational resources

provided by The Scripps Research Institute, State University of

New York at Fredonia, and Florida Atlantic University. This

study made use of the Campus Chemical Instrument Center

NMR facility at The Ohio State University.

(13) Velagapudi, S. P.; Gallo, S. M.; Disney, M. D. Sequence-based

design of bioactive small molecules that target precursor microRNAs.

Nat. Chem. Biol. 2014, 10 (4), 291−297.

(14) Costales, M. G.; Hoch, D. G.; Abegg, D.; Childs-Disney, J. L.;

Velagapudi, S. P.; Adibekian, A.; Disney, M. D. A designed small

molecule inhibitor of a non-coding RNA sensitizes HER2 negative

cancers to Herceptin. J. Am. Chem. Soc. 2019, 141 (7), 2960−2974.

(15) Velagapudi, S. P.; Cameron, M. D.; Haga, C. L.; Rosenberg, L.

H.; Lafitte, M.; Duckett, D. R.; Phinney, D. G.; Disney, M. D. Design

of a small molecule against an oncogenic noncoding RNA. Proc. Natl.

Acad. Sci. U. S. A. 2016, 113 (21), 5898−5903.

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