Cocaine reward is reduced by decreased expression of receptor-type protein tyrosine phosphatase D (PTPRD) and by a novel PTPRD antagonist

Article abstract of DOI:10.1073/pnas.1720446115

Receptor-type protein tyrosine phosphatase D (PTPRD) is a neuronal cell-adhesion molecule/synaptic specifier that has been implicated in addiction vulnerability and stimulant reward by human genomewide association and mouse cocaine-conditioned place-preference data. However, there have been no reports of effects of reduced expression on cocaine self-administration. There have been no reports of PTPRD targeting by any small molecule. There are no data about behavioral effects of any PTPRD ligand. We now report (i) robust effects of heterozygous PTPRD KO on cocaine self-administration (These data substantially extend prior conditioned place-preference data and add to the rationale for PTPRD as a target for addiction therapeutics.); (ii) identification of 7-butoxy illudalic acid analog (7-BIA) as a small molecule that targets PTPRD and inhibits its phosphatase with some specificity; (iii) lack of toxicity when 7-BIA is administered to mice acutely or with repeated dosing; (iv) reduced cocaine-conditioned place preference when 7-BIA is administered before conditioning sessions; and (v) reductions in well-established cocaine self-administration when 7-BIA is administered before a session (in WT, not PTPRD heterozygous KOs). These results add to support for PTPRD as a target for medications to combat cocaine use disorders. 7-BIA provides a lead compound for addiction therapeutics.

Full text of DOI:10.1073/pnas.1720446115

Cocaine reward is reduced by decreased expression
of receptor-type protein tyrosine phosphatase D
(PTPRD) and by a novel PTPRD antagonist
George R. Uhla,b,c,d,e,f,g,h,i,1, Maria J. Martineza,e, Paul Paiki, Agnieszka Sulimaj, Guo-Hua Bik, Malliga R. Iyerj,
Eliot Gardnerl, Kenner C. Ricej, and Zheng-Xiong Xik
aNeurology and Research Services, New Mexico VA Healthcare System, Albuquerque, NM 87108; bDepartment of Neurology, University of New
Mexico, Albuquerque, NM 87131; cDepartment of Neuroscience, University of New Mexico, Albuquerque, NM 87131; dDepartment of Molecular
Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131; eBiomedical Research Institute of New Mexico, Albuquerque,
NM 87108; fDepartment of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205; gDepartment of Neuroscience, Johns Hopkins
School of Medicine, Baltimore, MD 21205; hDepartment of Mental Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205;
iMolecular Neurobiology, National Institute on Drug Abuse, Baltimore, MD 21224; jDrug Design and Synthesis, National Institute on Drug Abuse and
National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892; kAddiction Biology, National Institute on Drug Abuse, Baltimore, MD
l
21224; and Neuropsychopharmacology, National Institute on Drug Abuse, Baltimore, MD 21224
Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 19, 2018 (received for review November 22, 2017)
addiction-associated kinase cyclin-dependent protein kinase 5
(Cdk5) (9).
Receptor-type protein tyrosine phosphatase
D (PTPRD) is a
neuronal cell-adhesion molecule/synaptic specifier that has been
implicated in addiction vulnerability and stimulant reward by
human genomewide association and mouse cocaine-conditioned
place-preference data. However, there have been no reports of
effects of reduced expression on cocaine self-administration.
There have been no reports of PTPRD targeting by any small
molecule. There are no data about behavioral effects of any
PTPRD ligand. We now report (i) robust effects of heterozygous
PTPRD KO on cocaine self-administration (These data substan-
tially extend prior conditioned place-preference data and add
to the rationale for PTPRD as a target for addiction therapeu-
tics.); (ii) identification of 7-butoxy illudalic acid analog (7-BIA)
as a small molecule that targets PTPRD and inhibits its phospha-
tase with some specificity; (iii) lack of toxicity when 7-BIA is
administered to mice acutely or with repeated dosing; (iv) re-
duced cocaine-conditioned place preference when 7-BIA is ad-
ministered before conditioning sessions; and (v) reductions in
well-established cocaine self-administration when 7-BIA is ad-
ministered before a session (in WT, not PTPRD heterozygous
KOs). These results add to support for PTPRD as a target for
medications to combat cocaine use disorders. 7-BIA provides a
lead compound for addiction therapeutics.
PTPRD has been identified in human genomewide SNP and
CNV association studies (10–12) of several addiction-related
phenotypes, usually by clusters of nearby SNPs or CNVs that
each display association with nominal significance (10−8 < P <
10−2). Such associations began with 10K SNP studies of poly-
substance dependence (1 of 38 SNPs reported; now known to
map to PTPRD’s 3′ flank) (13) and continued with studies that
included 600K SNP associations with polysubstance dependence
(14) (1 of 119 genes), 1M SNP associations for illegal substance
abuse in African American subjects from two independent
samples (15) (1 of 97 genes), 1M SNP studies of alcohol dis-
orders in an epidemiological sample* (1 of 9 genes identified in both
European- and African-American samples), and genes identified by
SNP and CNV associations for opiate dependence (16) (1 of 3 genes).
Ability to quit smoking in clinical-trial settings displayed PTPRD as-
sociations in several studies (1 of 33 genes in ref. 17 and 1 of 116 genes
in ref. 18). PTPRD was identified by one of the top two genomewide
Significance
Substance-use disorders damage individuals and communities,
providing large societal costs. For stimulant-use disorders, there
are no Food and Drug Administration-approved medications.
Human genetic associations with common variants in the gene
encoding the receptor-type protein tyrosine phosphatase D
(PTPRD) make this cell-adhesion molecule/synaptic specifier
gene an interesting target for new addiction therapeutic agents.
We now report results of cocaine self-administration in hetero-
zygous PTPRD-KO mice, discovery that 7-butoxy illudalic acid
analog (7-BIA) inhibits PTPRD’s phosphatase with in vitro po-
tency and specificity, and discovery that 7-BIA reduces cocaine
reward in self-administration and conditioned place-preference
models. PTPRD’s phosphatase is a target for antiaddiction
medication development, and 7-BIA is a lead compound.
Post-GWAS drug discovery cell adhesion molecule addiction
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stimulant use disorder
ven though more than seven million Americans reported il-
licit stimulant use during a recent year (1), there are no Food
E
and Drug Administration (FDA)-approved medications for
stimulant-use disorders. Clues about novel targets for anti-
addiction medications have come from molecular-genetic studies
of human individual differences in addiction-related phenotypes
(2). Data from mouse models support several of these human
molecular-genetic findings (3–5).
Receptor type protein tyrosine phosphatase D (PTPRD) is a
target for addiction therapeutic agent development that has
been identified by modest-magnitude but replicated human
genetic associations and supported by initial conditioned place-
preference studies in mouse models (5). PTPRD is a single-
transmembrane cell-adhesion molecule/synaptic specifier that
can make homomeric and/or heterologous dimers with binding
partners expressed on membranes of adjacent neurons (6).
Binding of PTPRD by its extracellular ligands alters phos-
phatase activity of its intracellular phosphatase domain (7).
Phosphoprotein changes with KO of ptp-3, PTPRD’s Caenorhabditis
elegans homolog, support PTPRD effects on phosphorylation
of conserved tyrosines in human proteins (8) that include the
Author contributions: G.R.U., E.G., and Z.-X.X. designed research; M.J.M., P.P., A.S., G.-H.B.,
and Z.-X.X. performed research; A.S., M.R.I., and K.C.R. contributed new reagents/analytic
tools; G.R.U. and Z.-X.X. analyzed data; and G.R.U. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
1To whom correspondence should be addressed. Email: George.Uhl@va.gov.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1720446115/-/DCSupplemental.
*Jung J, Zhang H, Grant B, Chou P, American Society of Human Genetics 2017, October
17–21, 2017, Orlando, FL.
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i.v. Cocaine Self-Administration by WT and PTPRD-KO Mice. Mice were cathe-
terized via external jugular veins and allowed to press a lever for 1-mg/kg
cocaine infusions over 42 s, paired with light and sound cues under fixed-ratio
1 (FR1) schedules during the first week (SI Appendix). Mice were later pro-
vided with access to cocaine on a progressive ratio schedule that delivered
1-mg/kg cocaine infusions on the following schedule: 2, 4, 5, 6, and 9 presses
per infusion. The numbers of presses of the active lever and numbers of
infusions received were noted.
association peaks in a study of acute responses to amphetamine (19). It
was one of 156 genes identified by association with responses to
drinking during the “first five” occasions for alcohol consumption (20).
PTPRD variants that reduce expression are associated with
reduced vulnerability. Rare CNVs likely to reduce PTPRD ex-
pression are more frequent in controls than in addicted individuals
(16). There are robust genetic associations with individual differ-
ences in levels of expression of PTPRD mRNA in postmortem
brain samples (5), with 70% differences in levels of PTPRD ex-
pression in brains from major vs. minor allele carriers for common
PTPRD intron 10 SNPs. Common haplotypes marked by the
SNPs associated with lower PTPRD brain expression are associ-
ated with reduced addiction vulnerabilities (5).
These observations in humans have received initial support from
studies of mouse cocaine-conditioned place preference. Hetero-
zygous PTPRD-KO mice that express half of WT levels of PTPRD
display reduced preference for places paired with effects of the
normally highly rewarding 10-mg/kg doses of cocaine (5). Tests of
strength, motor function, anxiety, and mnemonic function reveal
no obvious confounding effects (5). However, we are aware of no
reported data concerning effects on self-administration of cocaine,
a face-valid model in which to test drug reward. In the first part of
the present report, we thus describe effects of heterozygous PTPRD
KO on cocaine self-administration under fixed and progressive ratio
schedules. These results provide substantial additional support for
PTPRD as a target for novel antiaddiction drugs.
In Vitro Assays for Characterization of PTPRD and PTPRS Phosphatase Activities
and Actions at Other Brain Sites. Production of recombinant human PTPRD and
PTPRS phosphatase fusion proteins from expressing Escherichia coli began with
PCR amplification of human PTPRD and PTPRS D1 phosphatase domain se-
quences by using appropriate oligonucleotides and cDNA prepared from human
cortical polyA+ RNA as described previously (8). These sequences were cloned
into pET-43.1 Ek/LIC inserts confirmed by sequencing, and fusion protein se-
quences expressed in BL21 cells. Transformed BL21 cells were grown, plasmid
over-expression was induced, and cells were washed and lysed to extract phos-
phatase domain fusion proteins (24) (SI Appendix). Phosphatase domain fusion
protein activity was tested by using OD405 spectrophotometric assessment of p-
nitrophenyl phosphate dephosphorylation to p-nitrophenolate. In some experi-
ments, p-nitrophenyl phosphate and 7-BIA concentrations were varied for
Lineweaver–Burke analyses. A total of 90 μL of assay mixture and preincubated
enzyme-inhibitor mix was incubated at 22 or 27 °C with OD405 assessed for
20–60 min (SI Appendix).
Receptor binding and uptake assays for 77 brain sites of action of known drugs
were performed by EUROFINS (https://www.eurofins.com) and Aaron Janowsky
under NIDA contracts as described (SI Appendix).
PTPRD is a member of a “DSF” subfamily of receptor type
protein tyrosine phosphatases that also includes PTPRS and
PTPRF. Natural product library screening has identified illudalic
acid ligands that display micromolar IC50 values (21, 22) in inhibiting
PTPRF’s phosphatase. Interest in the phosphatase “business end” of
PTPRD as a drug target is increased by nonconservative amino acid
differences between PTPRF, PTPRD, and PTPRS phosphatase
domains (23) that could allow specificity for PTPRD phosphatase
inhibitors. Although these illudalic acid analog PTPRF ligands
provide clues, no PTPRD ligand has been previously identified.
In the second part of this report, we provide in vitro and in vivo
characterization of an illudalic acid analog as a PTPRD phosphatase
inhibitor and document this compound’s PTPRD-dependent effects
on cocaine reward. We describe: (i) synthesis and identification of a
7-butoxy illudalic acid analog (7-BIA) as an in vitro inhibitor of
recombinant human PTPRD phosphatase fusion protein with some
selectivity vs. the activity of the PTPRS phosphatase fusion protein;
(ii) failure of 7-BIA to display IC50 < 10−5 M at any of many tested
receptors, transporters, or sites of action of other drugs; (iii) toler-
ability of acute and repeated 7-BIA administration by WT C57 mice;
(iv) reduction of cocaine-conditioned place preference when 7-BIA
is administered before each cocaine conditioning session in WT
mice; (v) failure of 7-BIA administration to cause preference or
aversion for places in which mice experience this compound’s effects;
(vi) the greater numbers of WT mice (but not PTPRD heterozygous
KOs) who fail to self-administer the maximal amount of cocaine
available when well-established cocaine self-administration sessions
are preceded by administration of 7-BIA; and (vii) overall reductions
in cocaine self-administration in 7-BIA–treated WT (but not 7-BIA–
treated PTPRD heterozygous KO) animals. These results each
contribute to proof of principle that PTPRD is a druggable site for
addiction therapeutic agents and that acute modulation of PTPRD’s
phosphatase can alter stimulant reward. 7-BIA provides an attractive
scaffold for further development of PTPRD phosphatase-inhibitor
antiaddiction medications.
Synthesis of 7-BIA and Precursors. We synthesized the 7-butoxy analog of
illudalic acid [14 (boldface type added to indicate a structure), Fig. 2; syn-
thesized previously in 13 steps (22)] with modifications to three steps as a
result of low yields for bromination of acid 4, the Friedel–Crafts acylation of
5, and the deprotection of TBS ether 9 (SI Appendix). We brominated acid 4
under buffered conditions in the presence of sodium acetate in acetic acid
solution, yielding compound 5 as the sole product. We used oxalyl chloride
in the preparation of the acyl chloride intermediate in Friedel–Crafts acyl-
ation of 5, resulting in fewer byproducts. We applied mild deprotection with
tetra-n-butylammonium fluoride to yield a quantitative conversion of 9 to
alcohol 10, eliminating chromatographic purification. We purified the final
product by crystallization from diethyl ether, affording the 7-butoxy analog of
illudalic acid (i.e., 14) as white crystals with melting point of 116–117 °C.
Analytical data for all compounds agreed with reported values (22).
Acute and Repeated 7-BIA Dosing to WT C57b Mice. C57bl mice were first exposed
to acute repeated ascending i.p. dosing every 20 min, starting from 6-mg/kg
doses and ascending to 60-mg/kg doses. Other mice were administered
alternate-day doses of 60-mg/kg doses in 2-wk chronic exposure experiments.
Mice were observed to note any behavioral alterations and killed acutely and
1 wk following the last 7-BIA dose. Locomotor activity, mnemonic function
(Morris water maze), and somatosensation (pain) were also tested as described
previously (5, 25, 26). Gross pathologic evaluations were performed by a vet-
erinarian. Histopathological analyses were evaluated by veterinary pathologists
with special pathologic attention to the kidney, one of the nonneuronal/
endocrine tissues that expresses the most PTPRD mRNA in humans (27).
Effects of 7-BIA on Cocaine-Conditioned Place Preference. We administered 7-BIA
90 min before each of the two 10-mg/kg cocaine conditioning sessions. On
each of two preconditioning days, mice were allowed to explore both of the two
CPP box chambers for 20 min. On the two subsequent conditioning days, mice
were injected with vehicle or 6 or 60 mg/kg 7-BIA 90 min before administration of
10 mg/kg cocaine. A postconditioning session performed on the day following the
last conditioning session tested changes in preference for previously cocaine-paired
places. Mouse positions were tracked and stored by using ANYmaze. We also
tested preference for places paired with effects of 7-BIA alone (SI Appendix).
Effects of 7-BIA on Established Cocaine Self-Administration. WT or heterozy-
gous PTPRD-KO mice were prepared by external jugular vein catheterization
and allowed to self-administer 0.5–1 mg/kg per infusion of cocaine for at
least 20 prior sessions on FR1 and/or progressive ratio schedules. Mice that
were self-administering at least 40 (of 50 possible) times per 3-h session on
FR1 schedules were studied. Ninety minutes before the start of the sub-
sequent self-administration session, mice were pretreated with vehicle or 10
or 20 mg/kg 7-BIA (i.p.) and allowed to self-administer 1 mg/kg per infusion
of cocaine for a 3-h session. During the next sessions, mice were allowed to
Materials and Methods
Animals. Heterozygous PTPRD-KO mice and WT littermates were bred from
heterozygote × heterozygotes crosses and genotyped as described with New
Mexico VA Healthcare System (NMVAHCS) Institutional Animal Care and Use
Committee approval (5). Mice underwent self-administration testing under
auspices of the National Institute on Drug Abuse (NIDA) Animal Care and
Use Committee. Euthanasia used rapid cervical dislocation (at NVMAHCS) or
pentobarbital overdose (NIDA Intramural Research Program).
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Motivated by the convergent effects of heterozygous PTPRD
KO on conditioned place preference and self-administration
testing and the prior findings for illudalic acid analogs in studies
of PTPRF’s phosphatase and initial molecular modeling/docking
studies, we selected a 7-BIA analog (i.e., 14, Fig. 2) for synthesis
as a potential PTPRD phosphatase inhibitor. The synthetic path
shown in Fig. 2 produced >2 g of 7-BIA with an overall 17% yield
for this 13-step synthesis. The final product 14 was characterized by
NMR spectroscopy, high-resolution mass spectroscopy, combustion
analysis, and comparisons with previously reported values in the
literature. Its purity was determined to be ≥98% based on analytical
data. Mass spectrographic analyses identified some stability in 5%
DMSO solution; approximately 23% remained intact after 2 mo
storage at 4 °C. We retained amounts of purified compounds 11, 12,
and 13 to use as comparison controls.
To establish an assay for PTPRD phosphatase inhibition and to
compare results with those for the phosphatase from PTPRS, we
produced recombinant human PTPRD and PTPRS D1 phos-
phatase domain fusion proteins. We produced BL21 cells trans-
formed with pET-43.1 Ek/LIC constructs that were confirmed by
sequencing to express phosphatase domain fusion proteins with
verified human PTPRD and PTPRS phosphatases. Recombinant
human PTPRD and PTPRS fusion proteins purified from these
expressing cells converted p-nitrophenyl phosphate into para-
nitrophenolate, which provides strong 405-nM absorbance. Ac-
tivity is dependent on the amount of enzyme and time. Activity is
destroyed by boiling the purified recombinant fusion proteins.
The phosphatase activities of recombinant human PTPRD
and recombinant human PTPRS (Fig. 3 and SI Appendix, Fig.
S1) fusion proteins were each inhibited by 7-BIA. Maximal in-
hibition developed over 16–18-min preincubations, consistent
with a pseudoirreversible mechanism that has been postulated
for interactions between illudalic acid analogs and PTPRF (26).
Nonparallel Lineweaver–Burke plots (e.g., nominally non-
competitive) were compatible with a pseudoirreversible mecha-
nism of action as postulated for 7-BIA interactions with PTPRF
(SI Appendix, Fig. S2) (28). IC50 values, based on concentrations
session
Wildtype
PTPRD heterozygous knockout
session
Fig. 1. Numbers of 1-mg/kg cocaine infusions self-administered during 3-h
sessions by WT (solid line, closed symbols) and heterozygous PTPRD-KO mice
(dashed line, open circles) on experimental days shown. (Top) FR1 (1 mg/kg per
infusion) and (Bottom) progressive-ratio schedules. Mice received 1-mg/kg
cocaine infusions after completing 2, 4, 6, 9, and 12 lever presses on days 1–5,
respectively (*P < 0.05, t test).
IC4H9
Py
70oC
93%
Pd/H2
99%
K2CO3 DMF
93%
Br2
(COCl)2
AA, NaOAc
94%
SnCl4
78%
self-administer 1 mg/kg per infusion of cocaine without pretreatment. The
numbers of cocaine infusions self-administered were noted.
Results
TBSCI
NaBH4
98%
nBuLi
ClCO2CH3
Effects of reduced PTPRD expression on cocaine self-administration
supported PTPRD as a target for antiaddiction therapeutic agents.
PTPRD heterozygous KO mice took longer to establish high rates
of 1-mg/kg per infusion cocaine self-administration than WT lit-
termate controls (Fig. 1). During the last three of nine successive
exposures to 3-h sessions in which as many as 50 cocaine infusions
were available on FR1 schedules, heterozygotes self-administered
approximately half of the maximal amounts available, whereas WT
littermates self-administered approximately 70% of the maximal
available drug. Heterozygotes continued to self-administer at lower
rates than WT mice on days 6–9. There were no differences in
control responding on inactive levers during FR1 responding.
There is additional support from results of subsequent pro-
gressive ratio studies (Fig. 1). After several days’ experience with
these progressive ratio schedules, PTPRD heterozygous KO mice
displayed significantly lower break points. They therefore self-
administered approximately half the number of infusions that
were self-administered by WT littermate control mice. There were
again no differences in control responding on inactive levers.
99%
AD-mix
pTsOH
TBAF
KOH
38% over 3
steps
NaIO4
75% over 3
steps
Fig. 2. Synthetic scheme and structures, including structure for the illudalic
acid analog PRPRD phosphatase inhibitor 7-BIA (i.e., 14; further detail pro-
vided in SI Appendix).
Uhl et al.
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There is in vivo evidence for specificity. Most, if not all, of
these 7-BIA effects appear to be attributable to the compound’s
influences on PTPRD. Heterozygous PTPRD-KO mice with
extensive histories of cocaine self-administration that were pre-
treated with 7-BIA failed to obtain at least 80% of the maximally
available cocaine during only 9% of sessions.
100
80
60
40
20
0
PTPRD
PTPRS
Discussion
The present self-administration work in heterozygous PTPRD-KO
mice adds to prior results to support PTPRD as a bona fide
increasingly-“derisked” target for antiaddiction therapeutic agent
development (29). Effects on fixed- and progressive-ratio respond-
ing combine to provide good evidence for reduced cocaine reward
in heterozygous PTPRD-KO mice. Taken together with data from
conditioned place-preference and human association studies, there
is now substantial support for the idea that constitutive “trait” dif-
ferences in levels of PTPRD expression result in differences in
stimulant reward in humans and mouse models.
To test the idea that drug-induced, transient “state” differ-
ences in levels of PTPRD activity could also alter reward from
stimulant administration, we needed to identify a compound that
would alter PTPRD activities, was tolerable in vivo, and did not
induce behavioral effects that would confound data from reward
0
0.5
1
1.5
2
2.5
log10 [7-BIA] (μM)
Fig. 3. Concentration-dependent inhibition of the phosphatase activity of
recombinant human PTPRD (solid line) and PTPRS (dashed line) D1 phosphatase
domain fusion proteins by 16-min preincubation with 7-BIA at concentrations shown
(log10 × 10−6 M; note that phosphatase assays diluted 7-BIA 10-fold; SI Appendix).
of 7-BIA that were preincubated with phosphatase, were ap-
proximately 1–3 μM (PTPRD) and 40 μM (PTPRS).
There is other in vitro evidence for specificity. No tested precursor
(compounds 11–13; Fig. 2) significantly inhibited PTPRD or PTPRS
phosphatase fusion protein activity. At concentrations as high as
10−5 M, 7-BIA failed to significantly influence binding or uptake
at any of the 77 sites assessed by ligand binding or uptake assays.
We tested tolerability of 7-BIA vs. DMSO vehicle in mice using
ascending-dose and repeated-dose i.p. administration. Treated
mice failed to display obvious behavioral changes that were not
seen in vehicle-injected animals. This was true of ascending doses
as high as solubility limits (providing 60 mg/kg in solutions with
some precipitate in maximally tolerated DMSO) and with 2-wk
weekday 6 mg/kg dosing. There was no evidence for any drug-
related gross pathologic processes. When sections of lung, liver,
kidney, and gut were studied by board-certified veterinary pathol-
ogists, there was no evidence for histopathology related to acute
ascending or 2-wk repeated administration.
6
5
4
3
2
1
0
*
veh-veh
coc-veh
coc-7BIA
We then tested effects of pretreatment before each cocaine-
conditioning session on preferences for places paired with 10 mg/kg
cocaine (5). Pretreatments with 6 mg/kg i.p. 7-BIA 90 min before
each conditioning session significantly reduced preference for
cocaine-paired sides (Fig. 4). We confirmed significant levels of 7-
BIA/metabolites in acetonitrile extracts of brains of 7-BIA–treated
mice killed 90 min after 7-BIA injections by using mass spectro-
graphic analyses. There was no significant difference in locomotor
activity measured during the cocaine conditioning sessions pre-
ceded by vehicle vs. those preceded by 7-BIA injection. Pairing
environments with 7-BIA itself provided no evidence for rewarding
or aversive properties for this compound.
We next tested effects of 7-BIA administration before cocaine
self-administration sessions. The 7-BIA pretreatments reduced
cocaine self-administration in highly experienced WT mice that
were lever-pressing to self-administer near-maximal available
cocaine (50 1 mg/kg infusions on FR1 schedule) during ≥20 prior
sessions. The 7-BIA pretreated mice failed to obtain at least 80%
of available cocaine during 52% of sessions, compared with the
18–22% of sessions during which vehicle or nonpretreated mice
failed to obtain at least this fraction of the maximal available
cocaine (P = 10−4, t test). There were thus significant mean
differences in self-administration (Fig. 4). Mice also failed to
self-administer at least 80% of available cocaine during 31% of
self-administration sessions that took place 1–2 d following single
7-BIA treatments, suggesting some persistence of the com-
pound’s effects. There were again no significant effects on lo-
comotor activity recorded when the mice were experiencing
effects of cocaine with 7-BIA vs. vehicle pretreatments.
50
45
40
35
30
25
20
*
*
None Vehicle 7-BIA 10 7-BIA 20 7-BIA
mg/kg mg/kg KOs
treatment
Fig. 4. (Top) Cocaine-conditioned place preference in mice with vehicle (black)
and 7-BIA (gray; >6 mg/kg) pretreatment before each conditioning session
(*P = 0.03, t test; n = 12; Values normalized. 1 represents time (+/− SEM) in
seconds spent (during postconditioning–preconditioning test sessions) in con-
trol mice which were pretreated prior to each conditioning session with saline
vehicle for cocaine and DMSO vehicle for 7-BIA). (Bottom) Well-established
cocaine self-administration is reduced significantly by 7-BIA pretreatments
(90 min before session start) in WT but not in PTPRD heterozygous KO mice
(n = 12–126 sessions; 13 WT and 6 heterozygous KO mice; *P < 0.05).
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models. Our identification of 7-BIA as a PTPRD phosphatase
There are limitations that may render 7-BIA a lead compound,
inhibitor and our establishment of modest, if any, in vivo 7-BIA but perhaps not a drug. 7-BIA displays limited solubility; mouse i.p.
behavioral or pathological toxicities allowed us to use this com- dosing greater than 20 mg/kg is thus problematic. The slow in vitro
pound in vivo for assays of cocaine reward. Cocaine provides ro- onset of PTPRD phosphatase inhibition is compatible with the
bust place preference when paired with a specific environment pseudoirreversible mechanism of action that has been postulated
during two conditioning sessions. 7-BIA treatment before each of for illudalic acid analog interactions with PTPRF, perhaps pro-
these conditioning sessions reduces preference for cocaine-paired viding another limitation (21). We have documented levels of 7-
environments. These results support efficacy of transient PTPRD BIA/metabolites in brains of mice killed at times when 7-BIA ad-
phosphatase modulation in reducing cocaine reward.
ministration provides behavioral effects, but we have only limited
Cocaine maintains self-administration at high rates. In WT mice information about 7-BIA’s cellular permeability. Although there is
with well-established cocaine self-administration, 7-BIA pretreat-
ments reduce self-administration. These 7-BIA effects are re-
versible and are not found in heterozygous PTPRD-KO mice.
some selectivity for PTPRD phosphatase inhibition compared with
PTPRS, even greater selectivity may be desirable. A motor-neuron
phenotype reported in mice with homozygous KO of PTPRD and
These self-administration data reinforce conditioned place pref- PTPRS (but not in heterozygotes) provides caution (31). We have
erence results to support the idea that transient inhibition of
PTPRD’s phosphatase can reduce cocaine reward. Elimination of
7-BIA’s effects in heterozygous KOs support the idea that much or
identified preservation of approximately 23% of intact compound/
salts in preliminary mass spectrographic analyses of 7-BIA stored in
DMSO solution for 2 mo at 4 °C; improved stability would also be
all of 7-BIA’s in vivo action result from effects on PTPRD. Cocaine desirable. We have characterized in vitro time course, IC50 esti-
reward is reduced by transient reductions in PTPRD activity.
This work provides development and in vivo characterization of
a lead compound that is specifically targeted to PTPRD, a cell-
adhesion molecule/synaptic specifier relevant to addiction. Docu-
mentation of 7-BIA–induced changes in cocaine-conditioned place
preference and self-administration solidifies 7-BIA as a lead com-
pound for a novel antiaddiction therapeutic strategy. These phar-
macological results, in turn, increase validation of PTPRD as a target
for novel addiction therapeutic agents. Our development of improved
chemical synthetic pathways that increase yields of 7-BIA, expression
vectors for production of recombinant PTPRD and PTPRS phos-
phatase fusion proteins, purification of these PTPRD and PTPRS
phosphatase fusion proteins, and development and validation of
phosphatase assays using these fusion proteins provide background
work that allowed in vitro characterization of 7-BIA as a PTPRD
ligand and phosphatase inhibitor. We identified lack of gross toxicity
in mice, a precondition to interpretable in vivo work. In vivo studies
then identified 7-BIA’s ability to reduce cocaine reward in condi-
mates, and Lineweaver–Burke analyses for 7-BIA, but have not
performed full kinetic analyses of this inhibition. As we identify 7-
BIA analogs that provide more selective PTPRD phosphatase in-
hibition and other desirable properties, fuller in vitro kinetic
analyses as well as fuller in vivo pharmacodynamic, pharmacoki-
netic, and toxicologic analyses will be important.
Other cautions about PTPRD as a target for novel therapeutic
agents come from associations between (i) total, lifelong human or
mouse PTPRD KO and cognitive difficulties (5, 32) and (ii) findings
that rare variation likely to dramatically alter PTPRD expression can
promote or inhibit cancer formation or properties of cancer cells (33,
34). Neither humans nor mice with moderate differences in levels of
PTPRD expression display any cognitive abnormalities. Neither we
nor others have detected any tumors in studies of hundreds of mice
with 50% reductions in PTPRD expression at ages up to 6 mo. There
is no evidence that common human haplotypes that drive 70% in-
dividual differences in expression alter cancer propensities. We nev-
ertheless need to be mindful of the possibilities that even intermittent
tioned place preference and self-administration settings in WT mice. pharmacological modulation of PTPRD activity might conceivably
There is evidence for substantial specificity of 7-BIA effects. In
exert side effects on carcinogenesis, cognition, or other phenotypes.
Neuronal processes of PTPRD-expressing neurons grow when
vitro support for specificity includes: (i) lack of PTPRD phospha-
tase inhibition from synthetic precursors, (ii) lack of 7-BIA effects their PTPRD is allowed to make homomeric bonds with PTPRD
on assays for 77 tested brain targets, (iii) >30-fold greater potency
at PTPRD than at PTPRS phosphatases, and (iv) reported
(22) >100-fold greater 7-BIA potency at PTPRS than at PTPRC,
PTPN1, PTPN2, DUSP22, CDC25A, or CDC25B phosphatases. In
vivo, ablation of 7-BIA effects on cocaine reward in heterozygous
PTPRD-KO mice provides positive evidence for specificity, making
it unlikely that the reduced cocaine reward reported is a result of 7-
BIA’s abilities (22) to inhibit PTPRS or PTPRF’s phosphatases.
We have identified no in vivo 7-BIA behavioral effect in WT mice;
there is thus no behavioral evidence for behaviorally important
nonspecific effects. Nevertheless, future studies that seek alter-
ations in 7-BIA effects in mice with reduced expression of PTPRS
or PTPRF can provide additional behavioral controls.
Binding of PTPRD by its extracellular ligands is likely to alter
phosphatase activity of its key intracellular “D1” phosphatase do-
main (7). Interest in the phosphatase “business end” of PTPRD as a
drug target was increased by nonconservative amino acid differ-
ences between PTPRD and its other subfamily members, PTPRF
and PTPRS† (23).We have targeted the activity of PTPRD’s D1
domain in the present work. Prior studies have documented virtu-
ally perfect correlations between illudalic acid potencies in inhibit-
ing phosphatase activities at PTPRF D1 vs. D1+D2 constructions
(30). Our in vitro D1 domain results fit with our in vivo 7-BIA ef-
ficacy and selectivity results. Nevertheless, future studies of the ef-
fects of 7-BIA and related illudalic acid analogs on PTPRD’s D1+
D2 domain proteins could add support for the mechanism of action
of this compound.
expressed by adjacent cells (35). There are also heteromeric PTPRD
ligands, including slit- and trk-like proteins, IL-1 receptor-like and
accessory proteins, leucine-rich repeat and fibronectin type III
domain-containing proteins, and netrin G ligand 3 (36–39). Ventral
midbrain, striatal/accumbens, cerebral cortical, reticular thalamic,
and other circuits that express PTPRD mRNA in likely dopamine,
acetylcholine, glutamate, and GABA neurons (40) are thus likely to
develop and adapt differently when they constitutively express
PTPRD at differing levels. Lifelong differences in PTPRD activity,
including those conferred by common human PTPRD intron 10
haplotypes or by heterozygous KOs, are thus likely to influence
structures and functions of neuronal processes and synapses in brain
regions that include those linked to rewarding effects of stimulants
and other addictive substances. Lifelong differences in PTPRD
activity that could function by providing differences in neuronal
connectivities are now strongly linked to differences in vulnerability
to stimulant effects by associations with mouse cocaine place pref-
erence (5), mouse cocaine self-administration, and human ratings of
positive stimulant effects (19).
The current work provides evidence that acute and transient
alterations in PTPRD activity influence stimulant reward. 7-BIA
administration 90 min before cocaine conditioning or self-
administration sessions produces substantial reductions in place
preference and self-administration that are nearly as large as those
provided by heterozygous KO (5). Data from self-administration
supports partial reversal of these acute 7-BIA effects by the time
of the next self-administration session. Such relatively rapid time
courses focus our thinking on acute changes in phosphorylation
status of proteins within PTPRD-expressing neurons. Studies of
C. elegans KOs of its PTPRD homolog, ptp-3, elucidate candidate
PTPRD-modulated phosphotyrosine homologs in 45 human proteins
†Jung J, Zhang H, Grant B, Chou P, American Society of Human Genetics 2017, October
17–21, 2017, Orlando, FL.
Uhl et al.
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(8) including Cdk5 and the signal transducer and activator of tran- vulnerability to develop restless leg syndrome (44), individual dif-
scription STAT3 (38). Cdk5 encodes a kinase that is regulated by its
own phosphorylation and is strongly implicated in phosphorylation-
induced changes in the properties of the MAPT microtubule-
associated protein tau that is involved in neuroadaptive and
maladaptive processes (41). PTPRD-mediated changes in the
phosphorylation of STAT3 Y705 phosphotyrosine alter the ability
of this transcription factor to homodimerize, move into the nu-
cleus, and change transcription of other genes (36). Acute changes
in tyrosine phosphorylation provide plausible mechanisms for
influencing drug reward; both Cdk5 and STAT3 activities have
been implicated in addictions (42, 43).
There is thus now a substantial rationale for optimizing 7-BIA–
related structures and advancing improved PTPRD phosphatase
inhibitors to clinical testing for substance use disorders. Success in
this work will result in 7-BIA–related compounds that inhibit
PTPRD phosphatase with increased solubility and stability; oral
bioavailability; favorable biodistributions; increased specificities;
lack of apparent behavioral, biochemical, or histopathological
toxicities; lack of worrisome off-target effects; and beneficial
effects on stimulant-conditioned place preference and self-
administration. Success in these goals will also support studies of
the use of PTPRD ligands in other phenotypes that have been
associated with human PTPRD variation, including effects on
ferences in densities of neurofibrillary neuropathology conditions
in postmortem brains of individuals with Alzheimer’s disease (24),
and mood instability (45). Success may also motivate identification
of small molecules that can allow adult pharmacological modula-
tion of other cell adhesion molecules [e.g., CDH13 (3)] that are
candidates to play roles in addiction-related connectome signaling.
We initially focused on stimulant-use disorders in the present
work because (i) combined human and mouse data are strongest for
stimulants, (ii) focus on one drug class will focus human phase II
clinical trials, and (iii) there are no FDA-approved medications for
these disorders. Nevertheless, human genetic data support the ef-
fects of reduced PTPRD function on disorders of use of opiates,
alcohol, and other addictive substances. It thus seems likely that
PTPRD phosphatase inhibitors will ultimately display broad appli-
cability in disorders of use of at least several addictive substances.
ACKNOWLEDGMENTS. The authors thank Drs. A. Newman, D. White, H. Davis,
J. Acri, I. Montoya, W. Wang, and T. Prisinzano for providing help and support.
This work was supported by the Biomedical Research Institute of New Mexico
(G.R.U., M.J.M.), Veterans’ Health Administration (G.R.U.), National Institutes
of Health (J.D., A.S., K.C.R.), National Institute on Drug Abuse (A.S., I.M., K.C.R.,
G.R.U., Z.-X.X.), and National Institute on Alcohol Abuse and Alcoholism (A.S.,
I.M., K.C.R.).
1. Substance Abuse and Mental Health Services Administration (2016) SAMHSA Results
from the 2015 Survey of Drug Use in Households (Center for Behavioral Health Sta-
tistics and Quality, Rockville, MD).
2. Uhl GR, et al. (2008) Genome-wide association for methamphetamine dependence:
Convergent results from 2 samples. Arch Gen Psychiatry 65:345–355.
3. Drgonova J, et al. (2016) Cadherin 13: Human cis-regulation and selectively-altered addic-
tion phenotypes and cerebral cortical dopamine in knockout mice. Mol Med 22:537–547.
4. Drgonova J, et al. (2015) Altered CSMD1 expression alters cocaine-conditioned place
preference: Mutual support for a complex locus from human and mouse models. PLoS
One 10:e0120908.
23. Hendriks WJ, Pulido R (2013) Protein tyrosine phosphatase variants in human he-
reditary disorders and disease susceptibilities. Biochim Biophys Acta 1832:1673–1696.
24. Chibnik LB, et al. (2017) Susceptibility to neurofibrillary tangles: Role of the PTPRD locus
and limited pleiotropy with other neuropathologies. Mol Psychiatry 23:1521–1529.
25. Hall FS, Sora I, Uhl GR (2001) Ethanol consumption and reward are decreased in mu-
opiate receptor knockout mice. Psychopharmacology (Berl) 154:43–49.
26. Moriya Y, et al. (2015) Sex differences in the effects of adolescent social deprivation
on alcohol consumption in μ-opioid receptor knockout mice. Psychopharmacology
(Berl) 232:1471–1482.
27. Tissue Expression of PTPRD. Summary. The Human Protein Atlas. Available at www.
proteinatlas.org/ENSG00000153707-PTPRD/tissue.
28. Inhibition of Enzyme Activity. Available at www.csun.edu/∼hcchm001/5enzyme.pdf.
Accessed November 14, 2017.
5. Drgonova J, et al. (2015) Mouse model for PTPRD associations with WED/RLS and
addiction: Reduced expression alters locomotion, sleep behaviors and cocaine-
conditioned place preference. Mol Med 21:717–725.
6. Um JW, Ko J (2013) LAR-RPTPs: Synaptic adhesion molecules that shape synapse de-
velopment. Trends Cell Biol 23:465–475.
7. Takahashi H, Craig AM (2013) Protein tyrosine phosphatases PTPδ, PTPσ, and LAR:
Presynaptic hubs for synapse organization. Trends Neurosci 36:522–534.
8. Mitchell CJ, et al. (2016) Unbiased identification of substrates of protein tyrosine
phosphatase ptp-3 in C. elegans. Mol Oncol 10:910–920.
9. Rosen LG, Rushlow WJ, Laviolette SR (2017) Opiate exposure state controls dopamine
D3 receptor and cdk5/calcineurin signaling in the basolateral amygdala during reward
and withdrawal aversion memory formation. Prog Neuropsychopharmacol Biol
Psychiatry 79:59–66.
10. Ishiguro H, et al. (2006) NrCAM in addiction vulnerability: Positional cloning, drug-
regulation, haplotype-specific expression, and altered drug reward in knockout mice.
Neuropsychopharmacology 31:572–584.
29. Uhl GR, Drgonova J (2014) Cell adhesion molecules: Druggable targets for modulating
the connectome and brain disorders? Neuropsychopharmacology 39:235.
30. Yang X, et al. (2005) Discovery of novel inhibitor of human leukocyte common
antigen-related phosphatase. Biochim Biophys Acta 1726:34–41.
31. Uetani N, Chagnon MJ, Kennedy TE, Iwakura Y, Tremblay ML (2006) Mammalian
motoneuron axon targeting requires receptor protein tyrosine phosphatases sigma
and delta. J Neurosci 26:5872–5880.
32. Choucair N, et al. (2015) Evidence that homozygous PTPRD gene microdeletion causes
trigonocephaly, hearing loss, and intellectual disability. Mol Cytogenet 8:39.
33. Yu X, et al. (2017) Protein tyrosine phosphatase receptor-type δ acts as a negative
regulator suppressing breast cancer. Oncotarget 8:98798–98811.
34. Woodward EL, et al. (2017) Genomic complexity and targeted genes in anaplastic
thyroid cancer cell lines. Endocr Relat Cancer 24:209–220.
11. Fowler CD, Lu Q, Johnson PM, Marks MJ, Kenny PJ (2011) Habenular α5 nicotinic
receptor subunit signalling controls nicotine intake. Nature 471:597–601.
12. Jackson KJ, et al. (2010) Role of alpha5 nicotinic acetylcholine receptors in pharmaco-
logical and behavioral effects of nicotine in mice. J Pharmacol Exp Ther 334:137–146.
13. Liu QR, et al. (2005) Pooled association genome scanning: Validation and use to identify
addiction vulnerability loci in two samples. Proc Natl Acad Sci USA 102:11864–11869.
14. Liu QR, et al. (2006) Addiction molecular genetics: 639,401 SNP whole genome as-
sociation identifies many “cell adhesion” genes. Am J Med Genet B Neuropsychiatr
Genet 141B:918–925.
15. Drgon T, et al. (2011) “Replicated” genome wide association for dependence on il-
legal substances: Genomic regions identified by overlapping clusters of nominally
positive SNPs. Am J Med Genet B Neuropsychiatr Genet 156:125–138.
16. Li D, et al. (2015) Genome-wide association study of copy number variations (CNVs)
with opioid dependence. Neuropsychopharmacology 40:1016–1026.
17. Uhl GR, et al. (2007) Molecular genetics of nicotine dependence and abstinence:
Whole genome association using 520,000 SNPs. BMC Genet 8:10.
18. Uhl GR, et al. (2008) Molecular genetics of successful smoking cessation: Convergent
genome-wide association study results. Arch Gen Psychiatry 65:683–693.
19. Hart AB, et al. (2012) Genome-wide association study of d-amphetamine response in
healthy volunteers identifies putative associations, including cadherin 13 (CDH13).
PLoS One 7:e42646.
20. Joslyn G, Ravindranathan A, Brush G, Schuckit M, White RL (2010) Human variation in
alcohol response is influenced by variation in neuronal signaling genes. Alcohol Clin
Exp Res 34:800–812.
35. Wang J, Bixby JL (1999) Receptor tyrosine phosphatase-delta is a homophilic, neurite-
promoting cell adhesion molecular for CNS neurons. Mol Cell Neurosci 14:370–384.
36. Yamagata A, et al. (2015) Structure of Slitrk2-PTPδ complex reveals mechanisms for
splicing-dependent trans-synaptic adhesion. Sci Rep 5:9686.
37. Yoshida T, et al. (2012) Interleukin-1 receptor accessory protein organizes neuronal
synaptogenesis as a cell adhesion molecule. J Neurosci 32:2588–2600.
38. Kwon SK, Woo J, Kim SY, Kim H, Kim E (2010) Trans-synaptic adhesions between
netrin-G ligand-3 (NGL-3) and receptor tyrosine phosphatases LAR, protein-tyrosine
phosphatase delta (PTPdelta), and PTPsigma via specific domains regulate excitatory
synapse formation. J Biol Chem 285:13966–13978.
39. Lin Z, Liu J, Ding H, Xu F, Liu H (2018) Structural basis of SALM5-induced PTPδ di-
merization for synaptic differentiation. Nat Commun 9:268.
40. Allen brain atlas images (2018) Available at http://mouse.brain-map.org/. Accessed
November 14, 2017.
41. Bhounsule AS, Bhatt LK, Prabhavalkar KS, Oza M (2017) Cyclin dependent kinase 5: A
novel avenue for Alzheimer’s disease. Brain Res Bull 132:28–38.
42. Takahashi S, et al. (2005) Increased activity of cyclin-dependent kinase 5 leads to attenua-
tion of cocaine-mediated dopamine signaling. Proc Natl Acad Sci USA 102:1737–1742.
43. Chen JX, et al. (2017) Activation of TLR4/STAT3 signaling in VTA contributes to the
acquisition and maintenance of morphine-induced conditioned place preference.
Behav Brain Res 335:151–157.
44. Earley CJ, Uhl GR, Clemens S, Ferré S (2017) Connectome and molecular pharmacological
differences in the dopaminergic system in restless legs syndrome (RLS): Plastic changes
and neuroadaptations that may contribute to augmentation. Sleep Med 31:71–77.
45. Ward J, et al. (2017) Genome-wide analysis in UK Biobank identifies four loci asso-
ciated with mood instability and genetic correlation with major depressive disorder,
anxiety disorder and schizophrenia. Transl Psychiatry 7:1264.
21. Ling Q, et al. (2008) Illudalic acid as a potential LAR inhibitor: Synthesis, SAR, and
preliminary studies on the mechanism of action. Bioorg Med Chem 16:7399–7409.
22. Ling Q, et al. (2010) Synthesis and LAR inhibition of 7-alkoxy analogues of illudalic
acid. Yao Xue Xue Bao 45:1385–1397.
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