Controlling cellular distribution of drugs with permeability modifying moieties

Article abstract of DOI:10.1039/c8md00412a

Phenotypic screening provides compounds with very limited target cellular localization data. In order to select the most appropriate target identification methods, determining if a compound acts at the cell-surface or intracellularly can be very valuable. In addition, controlling cell-permeability of targeted therapeutics such as antibody-drug conjugates (ADCs) and targeted nanoparticle formulations can reduce toxicity from extracellular release of drug in undesired tissues or direct activity in bystander cells. By incorporating highly polar, anionic moieties via short polyethylene glycol linkers into compounds with known intracellular, and cell-surface targets, we have been able to correlate the cellular activity of compounds with their subcellular site of action. For compounds with nuclear (Brd, PARP) or cytosolic (dasatinib, NAMPT) targets, addition of the permeability modifying group (small sulfonic acid, polycarboxylic acid, or a polysulfonated fluorescent dye) results in near complete loss of biological activity in cell-based assays. For cell-surface targets (H₃, 5HT_1A, β₂AR) significant activity was maintained for all conjugates, but the results were more nuanced in that the modifiers impacted binding/activity of the resulting conjugates. Taken together, these results demonstrate that small anionic compounds can be used to control cell-permeability independent of on-target activity and should find utility in guiding target deconvolution studies and controlling drug distribution of targeted therapeutics.

Full text of DOI:10.1039/c8md00412a

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V. L. Mariin, S. L. Koeniger, A. Baranczak, J. L. Wilsbacher, P. J. Kovar, P. E. Bacon-Trusk, M. Cheng, T. A.
Hopkins, S. T. Haman and A. Vasudevan, Med. Chem. Commun., 2019, DOI: 10.1039/C8MD00412A.
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DOI: 10.1039/C8MD00412A
MedChemComm
PAPER
Controlling Cellular Distribution of Drugs with Permeability
Modifying Moieties
Paul L. Richardson,*a Violeta L. Marin,a Stormy L. Koeniger,a Aleksandra Baranczak,a Julie L.
Wilsbacher,b Peter J. Kovar,b Patricia E. Bacon-Trusk,b Min Cheng,b Todd A. Hopkins,b Sandra T.
Haman,b and Anil Vasudevana
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Phenotypic screening provides compounds with very limited target cellular localization data. In order to select the most
appropriate target identification methods, determining if a compound acts at the cell-surface or intracellularly can be very
valuable. In addition, controlling cell-permeability of targeted therapeutics such as antibody-drug conjugates (ADCs) and
targeted nanoparticle formulations can reduce toxicity from extracellular release of drug in undesired tissues or direct
activity in bystander cells. By incorporating highly polar, anionic moieties via short polyethylene glycol linkers into
compounds with known intracellular, and cell-surface targets we have been able to correlate the cellular activity of
compounds with their subcellular site of action. For compounds with nuclear (Brd, PARP) or cytosolic (dasatinib, NAMPT)
targets, addition of the permeability modifying group (small sulfonic acid, polycarboxylic acid, or a polysulfonated
fluorescent dye) results in near complete loss of biological activity in cell-based assays. For cell-surface targets (H3, 5HT1A
,
β2AR) significant activity was maintained for all conjugates, but the results were more nuanced in that the modifiers
impacted binding/activity of the resulting conjugates. Taken together, these results demonstrate that small anionic
compounds can be used to control cell-permeability independent of on-target activity and should find utility in guiding
target deconvolution studies and controlling drug distribution of targeted therapeutics.
early in the drug discovery funnel as possible so that target-
based high-throughput approaches can be used in the hit-to-
lead and lead-optimization stages.4 The preferred choice of
target deconvolution method is often dependent on the type,
Introduction
and quantity of target present in the target cells or tissue. The
most commonly applied chemoproteomic methods rely on
photo-affinity labelling (PAL) or direct affinity pulldowns.5
Although PAL is equally applicable to membrane and cytosolic
targets,6 as well as for low-affinity targets direct from
phenotypic screens, PAL is challenging due to the usually low
cross-linking efficiency, high non-specific crosslinking to false
targets and the poor relative quantitation of crosslinking
The pharmaceutical industry faces well-documented research
productivity challenges with the discovery and development of
therapeutic agents being estimated to cost as much as $2.87
billion per new molecular entity (NME) approved.1 In the case
of clinical candidates identified via target-based screening,
attrition due to lack of efficacy resulting from poorly
understood human target validation is a significant issue.2 In
order to enhance the probability of clinical success there is
renewed interest in target agnostic phenotypic screening
approaches. While phenotypic screening more closely aligns
the drug treatment with the desired outcome, it presents
alternative medicinal chemistry challenges related to the
inherent complications of biological screening assays (e.g. off-
target effects, differential compound distribution) and low-
throughput for in vivo screening assays if used in the early
stages of drug discovery.3 Therefore, there is a strong incentive
to identify direct targets from phenotypic screening hits as
between different targets.7
chemoproteomic target
precipitation/mass-spectrometry (MS) is most successful with
highly expressed soluble targets. In addition to low
Alternatively, small-molecule
identification by affinity
abundance, multipass membrane proteins tend to be
denatured by the lysis methods used to free potential protein
targets from other cellular components and contain
hydrophobic sequences with poor MS sensitivity.8 This results
in a general loss of intramembrane target data even though
such targets represent a large proportion of targets for
approved therapeutics. Alternatively, methods appropriate for
the identification of cell-surface membrane proteins such as
ligand-directed acyl imidazole9 or GlycoCLICK require non-
permeable reagents attached to the test compounds that
result in poor identification of intracellular targets.10 Because
of the mutually exclusive nature of these methods of target
identification we sought an efficient means of determining
whether compounds had cell-surface or intracellular targets
a. Drug Discovery Science and Technologies, 1 North Waukegan Rd, North Chicago,
IL 60064, USA. E-mail: paul.l.richardson@abbvie.com.
b. Discovery Oncology, AbbVie Inc.
c. These authors contributed equally to development of this manuscript
† The authors declare no competing interests
‡ Electronic Supplementary Information (ESI) available: Synthetic experimental
details and extended assay results for select compounds. See
DOI: 10.1039/x0xx00000x
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attach the permeability moieties via acylation chemistry. The
driving the phenotypic response. In addition, for cell-surface
targets, we wanted to determine whether a crosslinking
moiety such as a reactive acylimidazole, or strained alkyne
responsive group could be linked to the test compound and
still reach the cell-surface in order to crosslink with
extracellular domains of potential targets.
View Article Online
permeability moieties chosen includDeOdI: 10.1039/C8MD00412A
a polysulfonated
cyanine fluorophore (SCD), a small sulfonic acid containing
group (sulfoacetic acid, SAA), and a symmetric polycarboxylate
(4-carbonyl-cyclobutane-1,2,3-tricarboxylic acid, CCT) (Fig. 2)
to give the test compounds detailed (Fig. 3). Detailed
synthetic procedures and characterization of resulting
modified compounds are described in the supplementary
There are many ways of assuring compounds remain cell-
impermeable, including attaching macromolecules such as information.
serum albumin11 and polymers such as dextran, PEG12 or
dendrimers,13 however, maintaining target-based functional
Table 1 Test compounds, targets and localization.
activity of such conjugates or complexes is unpredictable. In
other cases highly anionic fluorescent dyes have been
employed, but these dyes tend to be large molecules with
,
Compound
((+)-JQ-1
olaparib
dasatinib
FK866
Target(s)
Localization
Nucleus
Nucleus
Cytoplasm
Cytoplasm
Cell surface
Cell surface
1
2
3
4
5
6
BRD2,3,4; BRDT
PARP1, PARP2
kinases
NAMPT
H3 receptor
5HT1A and β2AR
localization/distribution properties of their own.14 15
In
addition, although dye conjugates are useful for imaging
applications their fluorescence properties interfere with the
ability to run fluorescence-based screening assays to assess
independent target engagement. We have performed
comprehensive studies (linking strategies, binding and cell-
based biological activity measurements, and for a subset of
targets (Brd, dasatinib, H3 receptor), cell-associated unbound
drug accumulation ratios - Kpuu16) of small moieties that can be
used to modify cell permeability of compounds and triage
target identification methods. By selecting ligands with a
range of nuclear, cytosolic and cell-surface targets with which
to test permeability modifier strategies we were able to
perform proof of concept studies and provide tools to apply to
our own target deconvolution and cell-disposition efforts (Fig.
1).
alprenolol
Design, synthesis and assay results for nuclear targets
The anti-inflammatory/anti-cancer thienodiazepines (Fig. 3,
Table 2) derivatives, MS417 and (+)-JQ-1 bind non-selectively
to the bromodomains of several BET-family proteins
,
20
including BRD2, BRD3, BRD4 and BRDT.19
It had been
previously shown that the ester of (+)-JQ-1 could be
converted to a PEG-linked amide while maintaining binding
to BET-family targets.21
The dPEG2-NH-modified
thienodiazepines were prepared directly from (+)-JQ-1. The
products were assayed for binding to the BRD2, BRD3 and
BRD4 bromodomains using a TR-FRET assay as previously
described.22 Likewise, the cellular activity was measured
using two cell-based assay systems: BRD4 in-cell target
engagement was shown using an engineered H1299-derived
luciferase reporter assay system, and functional BRD4
inhibition was shown in a sensitive breast cancer cell-line
Results and discussion
General design of permeability modified compounds
Ligands for these experiments were chosen based on their
known target subcellular localization patterns and included
nuclear ((+)-JQ-1 1 - Brd2, 3, olaparib 2 – PARP1), cytosolic
(dasatinib 3 - kinases, FK866 4 - NAMPT), and cell-surface (5 -
H3 receptor, alprenolol 6 - 5HT1A and β2AR) targets (Table 1).
Structural data or structure-activity information was used to
guide the addition of the required linkers and permeability
moieties such that direct on-target binding was minimally
impacted in most cases. Where information to guide design
was lacking, multiple vectors for addition of the linkers were
chosen. When possible, direct on- target activity was explicitly
measured, along with PAMPA permeability17 and cell-based
assays specifically sensitive to each modified compound.
Short, discrete polyethylene glycol (dPEG) linkers with tert-
butyloxycarbonyl (Boc) protected amine head groups to couple
the ligands to the permeability modifiers were chosen to
minimize impact on physico-chemical properties of the
conjugates relative to the parent drugs (Fig. 2). Although
higher molecular weight PEG is known to prevent cellular
uptake, such short linkers have been used for linking reporters
to ligands with minimal disruption of permeability and target-
binding properties of the whole conjugate while maintaining
aqueous solubility.18 Removal of the Boc group provided a
relatively orthogonal reactive primary amine with which to
MX-1 in
a 3-day proliferation assay via CellTiter-Glo
(Promega).22 Finally, the ratio of compound in cells relative
to the compound concentration in media (Kp) and cellular
unbound drug accumulation ratio (Kpuu) were determined
(Table 2, Table S2).16 Addition of the dPEG2-NHBoc group 1a
has a consistent <5-fold impact on binding to BRD2, BRD3, or
BRD4 and also a modest impact on either cell-based assay
showing that addition of the linker is near SAR-neutral and
that the dPEG2-NHBoc minimally affects cellular uptake or in-
cell target engagement. Direct PAMPA assay results also
show minimal changes in membrane permeability from 2.24
to 6.22 x 10-6 cm/s or changes in Kpuu (1, 0.138; 1a, 0.168).
Removal of the Boc group provided 1b that maintained
binding to the bromodomains, but lost much cellular activity
(>15-fold) in each cell-based assay and decreased PAMPA
permeability to 0.015 10-6 cm/s and ~2-fold loss in Kpuu to
0.0695. Addition of the SCD modifier 1c was not tolerated in
the binding assays and resulted in complete abrogation of
cell-based activity.
The smaller CCT group 1e also
consistently impacted bromodomain binding ~20-50 fold,
whereas the smaller SAA moiety 1d had <5-fold impact on
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target binding. However, both SAA and CCT completely fluorescence interfered with the TR-FRET binding assay
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prevented cellular activity in both assays. In all cases there therefore binding of SCD conjugate 3c DtoOIt: h10e.1p03r9im/Ca8rMyDk0i0n4a1s2eA
was a larger drop in Kpuu (1c, 0.0064; 1d, 0.0219; 1e, 0.0359) targets of dasatinib were tested in an activity assay (Eurofins
It is unknown if a longer PEG-linker would have maintained DiscoveRx Kinomescan).33,34 For the primary (Abl, Src) and
binding of the CCT or SCD modified (+)-JQ-1 to the high-affinity secondary targets of dasatinib (Ki < 10 nM: BTK,
bromodomains, but given the impact longer PEG groups have CSF1R, DDR1, Fyn, Lck, PDGFRA V561D, PDGFRB, RIPK2, SlK1,
on cellular permeability, and consistent with our goals of TNK2; Table S1) the binding affinity was maintained within
finding permeability modifiers for compounds of unknown 10-fold of dasatinib itself. For four targets (DDR1, RIPK2,
mechanism, SAA appears to provide permeability SIK1, TNK2) the SCD probe 3c was >10-fold weaker than
modification of (+)-JQ-1 while minimizing effects on target dasatinib. PAMPA permeability for all but dasatinib (3, 1.49 x
binding.
10-6 cm/s) itself was significantly reduced (<0.1 x 10-6 cm/s),
even for the normally permeable dPEG2-NHBoc derivative
(3a, 0.056 x 10-6 cm/s), but was reduced to NV for 3c.
Likewise, the Kpuu values showed little change on the
addition of the dPEG2-NHBoc linker (1, 0.712; 1a, 0.654), but
dropped significantly with removal of the Boc (1c, 0.0177)
and with the anionic groups (1c, 00055; 1d, 0.0061; 1e,
0.0196). Not surprisingly, the order of Kpuu values closely
paralleled the cLogP values (Table 4). In order to evaluate
the functional consequences of the modifications, the
dasatinib sensitive K562 cell-line was incubated with each
derivative and cell-viability measured via CellTiter-Glo. In
this assay, the weakly permeable Boc protected compound
3a was only 2.6-fold less active than dasatinib, but all other
derivatives were >40-fold less active showing the functional
consequences of the permeability modifiers against this
cytosolic target.
Olaparib 2, an approved cancer therapeutic, inhibits the
nucleus localized DNA repair enzymes poly (ADP-ribose)
polymerase-1 and -2 (PARP) at very high affinity.23
Replacement of the cyclopropylcarbonyl group with a linker
and fluorophore maintains on-target binding.24 Starting from
des-cyclopropylcarbonyl olaparib we prepared the
permeability modified derivatives 2a-e as shown in the
supplementary material. These probes were tested via TR-
FRET PARP1 ligand-displacement assay and CellTiter-Glo-
based cell-viability assays in insensitive (DLD1) and sensitive
(DLD1 BRCA2-/-) cell-lines as previously described (Table 3).25
The PAMPA permeability of olaparib itself was good, 3.37 x
10-6 cm/s, but most of the modified derivatives were
significantly less permeable with most of the derivatives
giving no measureable values (NV). The addition of the
dPEG2-NHBoc 2a, Boc-removal 2b and even SCD addition 2c
had only modest effects on PARP1 binding, however SAA 2d Nicotinamide phosphoribosyltransferase (NAMPT) is
a
and CCT 2e adduction impacted direct binding substantially primarily cytosolic protein and potential oncology treatment
with a >10-fold and >50-fold loss in TR-FRET affinity target because of its key role in maintaining cellular NAD+
respectively. Also, from the cell proliferation data it appears levels via salvage synthesis.35 Recently, several NAMPT
that the PARP inhibition sensitive cell-activity correlates very inhibitor series have been described36 including our own
closely with direct target binding values, with very little isoindoline urea series which have shown target-specific killing
correlation with any permeability modification by the anionic of cancer cells.37 For NAMPT we designed our probes based
head group. However, the limited dynamic range of BRCA2-/- on the known NAMPT inhibitor FK866 4 modified with an
modified DLD1 cell-line and/or the extremely high binding aminomethyl functional group 4f38 prepared from the
affinity of the compounds for PARP1 may be masking any truncated precursor.39 Acylation with the dPEG2-NHBoc linker
underlying permeability effects for this particular target.
gave the Boc protected intermediate 4a which was
deprotected with TFA to give the free amine 4b. Addition of
the modifying groups was accomplished via acylation 4c-e (Fig.
3). Measured PAMPA permeability of FK866 was quite high (4,
Design, synthesis and assay results for cytosolic targets
Dasatinib 3 is a marketed drug for the treatment of chronic 39.5 x 10-6 cm/s), but decreased significantly with addition
myelogenous leukemia and Philadelphia chromosome- ofthe aminomethyl group (4f, 0.09 x 10-6 cm/s), rebounded
positive acute lymphoblastic leukemia.26 Beyond the primary somewhat with the dPEG2-NHBoc (4a, 1.2 x 10-6 cm/s) and
Bcr-Abl and Src family tyrosine kinases, dasatinib has many became unmeasureable when the modifiers were added (4c-
,
28
additional molecular targets.27
Previously described 4e, NV). The probes were screened via a TR-FRET ligand
modified versions of dasatinib guided the design of displacement assay (Table 5) with the binding affinity of the
permeability modified probes that maintain binding to many probes improved by the presence of the NAMPT cofactor 5-
,
29
targets involved in biological activity.27
To des-ethoxy phosphoribosyl-1-pyrophosphate (+PRPP versus -PRPP) as
dasatinib 3f, prepared as described,30 was added the dPEG2- expected for this compound series.37 Activity in the binding
NHBoc linker via alkylation chemistry to give the desired assay was impacted <3-fold for the dPEG2-NHBoc compound
conjugate 3a which was partially converted to the remaining 4a, but then decreased with addition of SCD (4c, 23.5-fold, -
probes as shown in supplementary material. These were PRPP; 6.5-fold, +PRPP), SAA (4d, 47.5-fold, -PRPP; 39.5-fold,
tested in an in vitro TR-FRET kinome screening panel31 using +PRPP) or CCT (4e, 59.8-fold, -PRPP; 29.5-fold, +PRPP). Except
methods generally described32 with data shown (Table 4, for SCD, the cell-based data was even more striking in the
Table S1, Fig. S1). For several of the tested kinases, the SCD NAMPT-inhibitor sensitive PC3 cell-line. Both SAA 4d and CCT
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4e caused complete loss of cell-killing activity as determined important for cell-surface alprenolol targets, PAMPA
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-6
by CellTiter-Glo assay,40 but 4c maintained cell-killing generally permeability for alprenolol (6, 10.9 x 10 cm/s) decreased to
DOI: 10.1039/C8MD00412A
in-line with the loss of binding affinity and not clearly related <0.1 x 10-6 cm/s for 6.1a-e. Ligand binding to 5-HT1A was
to permeability. As expected, the non-permeability modifier modestly affected by addition of the thioether ester 6.1f, ~3-
compounds 4f, 4a and 4b maintained some cell-killing activity. fold). Addition of dPEG2-NHBoc 6.1a had minimal additional
The smaller anionic groups SAA and CCT appear to be more binding effects but addition of the permeability modifiers
consistent in preventing cellular uptake and killing of NAMPT impacted binding (6.1c, 18.9-fold; 6.1d, 15.2-fold; 6.1e, 35.2
inhibitor sensitive PC3 cells.
fold). The impact of the modifications on 5-HT1A antagonist
activity was quite drastic, with signicant losses with addition of
the thioether linked ester (6.1f, 2.9-fold). The Boc-dPEG2-
linked material was nearly equal to alprenolol itself (6.1a, 0.64
µM), but the other versions were all completely inactive in the
functional antagonist assays. The impact on β2AR radioligand
displacement was minimal (<5-fold) for all modifications
except CCT (6.1e, 24.6-fold) and likewise for the functional
antagonist IC50 except CCT (6.1e, 5.27-fold) consistent with
the modifications being tolerated at β2AR.
Design, synthesis and binding results for cell-surface targets
There are presently four known histamine-responsive G-
protein coupled receptor subtypes (H1, H2, H3, H4),41 of which
two are well-established therapeutic targets (H1 – allergic
rhinitis and H2 – gastric acid secretion).42 More recent work
has attempted to target the H3 receptor resulting in the
discovery of several very potent antagonists, including a series
of quinolone H3 inverse-agonists used as the basis of modified
probes here.43 Previous work showing that pyrazole nitrogen
linked dPEG2 versions of quinolone H3 receptor ligands 5
maintained high affinity for the full-length human H3
receptor10 allowed the design of the permeability-modified
series 5a-e (Fig. 3). Although all the probes maintained high
affinity (<30 nM) Ki for the human H3 receptor as determined
by CEREP agonist ligand displacement assay there was
significant variability in relative affinity depending on the
identity of the modifier, but much less variability in functional
readouts with the exception of dPEG2-NH2 5b, which showed a
50-fold loss in activity (Table 6). Relative to the original ligand
5, addition of the dPEG2-NHBoc 5a caused a 33-fold loss in
binding which was restored on removal of the Boc group 5b.
The permeability modifiers also impacted binding with CCT 5e
causing a >50-fold binding loss while the impact of SAA 5d
(12.4-fold) and SCD 5c (4.2-fold) were more modest. As
expected for this series of compounds based on an antagonist
compound, they were all inactive in the functional agonist
assay except the dye-labeled 5c (88 nM EC50). For this series
of H3 ligands, preventing uptake into cells via permeability
modifiers had quantitative, but not qualitative impact on
binding to the cell-surface receptor highlighting the utility of
the modifiers to verify cell-surface target location.
For alprenolol, there exists a second accessible vector for
attaching the permeability modifiers by replacing the isopropyl
amine group with alkyl linkers as had been done previously for
making fluorescent alprenolol derivatives.47
Based on
published information, we used an extended dPEG4 linker for
this vector in order to maintain affinity at β2AR. Synthesis of
the modified derivatives was accomplished based on
modification of published methods to give the desired
products 6.2a-e. Again, addition of the linker itself decreased
PAMPA permeability to <0.1 x 10-6 cm/s for 6.2a-e. 5-HT1A
binding of the dPEG4-NHBoc and dPEG4-NH2 derivatives 6.2a-b
was unaffected (<2-fold) relative to 6, but was more subtle
with the permeability modifiers added (6.2c, 132-fold; 6.2d,
12.1-fold; 6.2e, 82.4-fold). In contrast, functional antagonist
activity was drastically affected for all modifications tried,
although alprenolol itself was only weakly active in this assay.
For β2AR, the impact of permeability modifiers for the amine
vector probes caused substantial loss of binding. The impact
of alprenolol modification on β2AR binding was modest for
dPEG4-NHBoc 6.2a (7.4-fold), but quite extreme with the
anionic groups added (6.2c, >1000-fold; 6.2d, 150-fold; 6.2e,
>1000-fold), but given the extremely high affinity of 6 for β2AR
(0.5 nM) the dynamic range for β2AR is far greater than for 5-
HT1A where alprenolol is weaker (7, 21.6 nM). Functional
antagonist activity for β2AR was impacted in a parallel manner
(6.2a, 2-fold; 6.2b, 30.3-fold; 6.2c, 151-fold; 6.2d, 25.3-fold
and 6.2e, >300-fold).
Alprenolol 6 is a high-affinity non-selective β2-adrenergic
receptor (β2AR)44 and serotonin receptor (5-HT1A, 5-HT1b)
antagonist.45 Sufficient SAR and target structural information
was available to design linked, modified versions of alprenolol
that were anticipated to maintain on-target affinity dependent
on linker position and linker length.46,47 Adaptation of previous
work demonstrating that alprenolol could be attached to a
thiol modified affinity resin via an ene-reaction to purify β2AR46
led to the design of an ester modified alprenolol 6.1f which
was synthesized using a photocatalyzed thiol-ene reaction on
the LOPHTOR flow instrument.48,49 Hydrolysis of the ester and
addition of the dPEG2-NHBoc linker via amidation of the
resulting carboxylic acid allowed the subsequent production of
the permeability modified series as described for the other
probes 6.1a-e. Consistent with previous probes, but less
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DOI: 10.1039/C8MD00412A
Table 2 (+)-JQ1 1 and derivatives cLogP, PAMPA permeability, unbound drug accumulation ratio (Kpuu), TR-FRET binding (BRD2, BRD3,
BRD4) and cell-based data (MX-1 CellTiter-Glo, H1299 luciferase reporter assays).
PAMPAa
10-6 cm/s
2.24
TR-FRET IC50 (µM)c
Cell EC50 (µM)d
b
Compnd
(+)-JQ1, 1
1a
1b
1c
1d
1e
Modifier
--
Boc
H
cLogP
4.87
4.15
2.34
-2.96
-1.42
1.67
Kpuu
BRD2
BRD3
0.023
0.042
0.007
>20.5
0.078
0.456
BRD4
MX-1
0.144
0.526
2.48
9.11
>10
Changee
1
H1299
0.064
0.718
8.49
>10
Changee
1
11.2
>50
>50
>50
0.138
0.168
0.019
0.035
0.016
>45
0.144
0.526
2.48
9.11
>10
6.22
0.0146
NV
NV
NV
3.65
17.2
>50
>50
>50
0.0695
0.0064
0.0219
0.0359
SCD
SAA
CCT
0.078
0.997
>10
>10
>10
>10
>50
aNV (no value) indicates no permeability detected. bKpuu, cTR-FRET IC50 and dCell EC50 values are reported as the geometric mean from at
least two duplicate runs. eChange indicates ratio of assay results relative to (+)-JQ1
Table 3 Olaparib 2 and derivatives cLogP, PAMPA permeability, TR-FRET binding (PARP1) and cell-based data (DLD wt, DLD BRCA2-/-
CellTiter-Glo).
PAMPAa
10-6 cm/s
3.37
0.081
NV
0.00119
NV
NV
TR-FRET Ki (µM)b
PARP1
Changed
Cell EC50 (µM)c
DLD (BRCA-/-)
0.10
0.20
0.70
0.60
0.60
1.10
7.80
Compnd
2
2f
Modifier
--
truncated
Boc
H
SCD
cLogP
1.236
1.057
2.123
0.315
-4.894
-1.519
-0.354
DLD (wt)
19.0
>100
>100
>100
70.0
Changed
1
2
7
6
6
11
>50
0.000787
0.000376
0.00192
0.00148
0.00056
0.0106
1
0.48
2.43
1.88
0.71
13.4
>50
2a
2b
2c
2d
2e
SAA
CCT
53.0
>100
NV
0.0415
aNV (no value) indicates indicates no permeability detected. bTR-FRET Ki and cCell EC50 values are reported as the geometric mean from at
least two duplicate runs. dChange indicates ratio of assay results to olaparib 2
Table 4 Dasatinib 3 and derivatives cLogP, PAMPA permeability, unbound drug accumulation ratio (Kpuu), TR-FRET binding (Abl, Src), and
cell-viability data (K562 CellTiter-Glo).
PAMPAa
10-6 cm/s
1.49
0.0371
0.0065
0.056
TR-FRET Ki (nM)c
Cell IC50 (µM)d
K562
Changee
b
Compnd
3
3f
3a
3b
3c
3d
3e
Modifier
--
truncated 2.459
Boc
H
cLogP
2.379
Kpuu
Abl
0.457
3.30
0.418
0.103
0.35g
0.49
Changee
1
Src
Changed
1
0.712
0.654
0.0177
0.00055
0.00606
0.0196
0.397
0.295
0.449
0.785
0.261
0.37f
0.851
0.925
0.000638
1
7.2
1.52
2.66
0.88
1.25
2.88
3.14
NAe
NAf
2.57
>50
45.7
>50
>50
4.144
2.336
0.915
0.225
0.766
1.07
1.11
0.00164
0.62783
0.02913
0.11976
0.18259
SCD
SAA
CCT
-5.143 NV
-1.767 0.0274
-0.603 0.0122
0.507
aNV (no value) indicates indicates no permeability detected. bKpuu, cTR-FRET Ki and dCell IC50 proliferation values reported as the geometric
mean from at least two duplicate runs. eChange indicates ratio of assay results relative to dasatinib 3. fNA (not available) indicates
compound not tested in this assay. gSCD fluorescence interferes with TR-FRET assays, assay run with Eurofins DiscoverX Kinoscan
Table 5 NAMPT inhibitor 4 and derivatives cLogP, PAMPA permeability, TR-FRET binding , and cell-based data (PC3 CellTiter-Glo).
PAMPAa
TR-FRET Ki (µM)b
Cell Viability IC50 (µM)c
PC3
Compnd
Modifier
cLogP
10-6 cm/s
NAMPTd
(-PRPP)
0.006
0.055
0.014
0.060
0.141
0.285
0.359
Changee
NAMPTd
(+PRPP)
0.0002
0.0011
0.0002
0.0019
0.0013
0.0079
0.0059
Changee
Changee
4
4f
4a
4b
4c
4d
4e
--
2.991
1.943
2.834
1.030
-4.18
-0.803
0.361
39.5
0.092
1.2
0.016
NV
NV
NV
1
9.2
1
5.5
1
9.5
6.5
39.5
29.5
0.0060
0.0302
0.0307
0.2963
0.1968
7.2252
7.3261
1
5.0
5.1
49.5
32.9
>1000
>1000
CH2-NH2
Boc
H
SCD
SAA
CCT
2.33
10.0
23.5
47.5
59.8
aNV (no value) indicates indicates no permeability detected. bTR-FRET Ki and cCell IC50 proliferation values reported as the geometric mean
from at least two duplicate runs. dNAMPT (-PRPP), NAMPT (+PRPP) indicates the assay run without and with 200 µM 5-phosphoribosyl-1-
pyrophosphate. eChange indicates ratio of assay results relative to dasatinib 4.
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DOI: 10.1039/C8MD00412A
Table 6 CLogP, CEREP H3 receptor cell-based data for the H3 receptor ligand 5 and derivatives.
PAMPAa
CEREP Human H3 Assay Datab
Compnd
Mod. cLogP
10-6 cm/s
Binding
Antagonist
Ki (µM)
0.00045d
0.0015
0.0007
0.0019
0.0056
0.0254
Changec
Functional
Antagonist
IC50 (µM)
0.043
0.041
0.001
0.013
0.045
0.15
Changec
Functional
Agonist
EC50 (µM)
>3
5
--
Boc
H
3.436
4.510
2.702
1.13
0.873
0.0246
1
33
1
5a
5b
5c
5d
5e
0.95
0.02
0.3
1.0
3.5
>3
>3
0.088
>3
>3
1.6
4.2
12.4
56
SCD
SAA
CCT
-7.488 0.284
-1.402 NV
-0.238 0.0131
aNV (no value) indicates indicates no permeability detected. bCEREP H3 antagonist binding, functional antagonist and functional agonist
assay results. cChange indicates ratio of assay results relative to 5. dBinding affinity determined by published radioligand displacement
assay.51
Table 7 CLogP, CEREP 5-HT1A and β2AR receptor cell-based binding data for alprenolol 6 derivatives based on thio-ene reaction.
PAMPAa
10-6 cm/s
CEREP 5-HT1A
Ki (µM) Changed Func.
Antag.
CEREP β2ARc
b
Compnd Modifier
cLogP
Changed
Ki (µM) Changed Func.
Changed
Antag.
IC50 (µM)
IC50 (µM)
6
--
2.652
2.431
3.018
1.210
-6.269 NV
-2.893 NV
10.9
0.0216 0.38
0.0561
0.0489 0.871
0.3090 6.32
0.630
2.900
0.640
>10
>10
>10
0.217
1
0.22
>5
>5
>5
0.0005 0.033
0.015
0.0032 0.21
0.0029 0.19
0.0260 1.73
0.0164 1.09
0.0786 24.6
0.0033
0.072
0.022
0.019
0.096
0.086
0.380
0.046
1
0.31
0.26
1.33
1.19
5.27
SCH2COOMe
6.1f
6.1a
6.1b
6.1c
6.1d
6.1e
0.0436
0.0189
NV
1
1
Boc
H
SCD
SAA
CCT
0.925
0.740
1.72
18.9
15.1
35.2
-1.729 0.0035
>10
>5
aNV (no value) indicates indicates no permeability detected. bCEREP 5-HT1A radioligand displacement assay and functional antagonist
assays. cCEREP β2AR radioligand displacement and functional antagonist assays. dChange indicates ratio of assay results relative to 6.1f
Table 8 CLogP, CEREP 5-HT1A and β2AR receptor cell-based binding data for alprenolol 6 derivatives based on on epoxide opening reaction.
b
PAMPAa
10-6 cm/s
CEREP 5-HT1A
Ki (µM) Changed Func.
Antag.
CEREP β2ARc
Compnd Modifier
cLogP
Changed
Ki (µM) Changed Func.
Changed
Antag.
IC50 (µM)
IC50 (µM)
6
--
Boc
H
SCD
SAA
CCT
2.652
2.761
0.954
-6.526 NV
-3.150 0.0046
-1.986 0.0012
10.9
0.035
0.032
0.0216
0.0241 1.12
0.0358 1.66
2.86
0.261
1.78
1
0.630
>1
>1
>1
>1
1
0.0005
0.0037 7.4
0.0103 20.6
0.547
0.752
0.512
1
0.0033
0.0069
0.10
0.50
0.0834
>1
1
6.2a
6.2b
6.2c
6.2d
6.2e
--
--
--
--
--
2.09
30.3
151
25.3
303
132
12.1
82.4
1094
150
1024
>1
aNV (no value) indicates indicates no permeability detected. bCEREP 5-HT1A radioligand displacement assay and functional antagonist
assays. cCEREP β2AR radioligand displacement and functional antagonist assays. dChange indicates ratio of assay results relative to 6.
intermediate dPEG2-NHBoc and modified conjugates. For
phenotypic assay hits with unknown targets this information
may not be available so more substantial work in deriving
Conclusions
linked intermediates may be necessary to maintain potency
with such less optimized systems. In the cases here the
activity of the dPEG2-NHBoc derivatives was generally
maintained regardless of subcellular localization of target(s),
but for phenotypic assay hits that may not be the case.
Secondarily, these results show that all the anionic
permeability modifiers are able to effectively prevent
While originally conceived as a means to allow rapid binning of
phenotypic screening hits into cell-surface and intracellular
targets, the data from this extensive test set is more nuanced.
Our test cases were all high-potency compounds for which
there was SAR/structural data to drive the design of the
6 | J. Name., 2012, 00, 1-3
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engagement of intracellular targets, with the small sulfonic cells/well in 96-well white plates (Corning 3610) in 90 µL of growth
View Article Online
acid (SAA) and polycarboxylic acid (CCT) being most consistent, media. Compounds were serially diluted inDloOgI:in10c.r1e0m39e/nCt8sMinD0D0M41S2OA
whereas the polysulfonic fluorophore (SCD) impacts from 10,000 µM to 0.01 µM, and then further diluted 100X in RPMI
permeability independent direct target binding, as well as media. Addition of 10 µL of the compound solution to the cell wells
causing interference in fluorescence-based assays (dasatinib resulted in final compound concentrations of 10,000 nM to 0.01
TR-FRET). The effect of the modifications on the biophysical nM. Cell plates were incubated with compound for 3 days and cell
unbound drug accumulation (Kpuu) values matched the viability determined using CellTiter-Glo viability assay detection of
biological results for the subset of targets tested (Brd, total intracellular ATP.
dasatinib, H3 receptor), though the relevance for cell-surface
targets is unclear. For cell-surface targets (H3, 5-HT1A, β2AR)
NAMPT TR-FRET binding and cell-based assays
binding potency, and functional activity was impacted by the
Equilibrium TR-FRET binding assays measuring displacement of a
modifiers to such an extent that it would not be possible to
fluorescent OG488-labeled version of compound 4b was run
conclude that loss of activity with modifier addition means the
essentially as described.50 CellTiter-Glo viability assays (PC3 cell-
target is intracellular. However, we can conclude that if
line, originally from ATCC and authenticated by STR analysis) were
run as previously described (Table 5).43
addition of one of these anionic modifiers maintains cellular
activity, the target is very likely cell-surface. The impact on
agonist compounds for cell-surface targets was not a topic
covered in this work. Future work in this area will explore the
effect of permeability modifiers on compounds known to act
on transporters or are uptaken by endo/pinocytosis as that
could complicate interpretation of the results. In addition,
direct comparison to macromolecular conjugates used for
controlling cellular uptake will be explored.
Intracellular compound binding and accumulation
Unbound compound accumulation ratios (Kpuu) consisting of
compound binding (fu, HEK) and intracellular accumulation (Kp) was
determined in HEK293 cells (from ATCC) similar to previously
described methods.16 Modifications to the referenced method and
details on the analytical methods can be found in the
supplementary material under Detailed Analytical Methods.
Experimental
Acknowledgements
Synthesis of linked and linked permeability modified compounds
Detailed synthetic procedures and characterization of all newly
described compounds in the supplementary material.
We thank Michael Curtin for providing the NAMPT compound
4f; Anita McGreal and Frank Wagenaar for assistance with
compound purification; Gilbert Diaz for coordinating CEREP
screening; and Henning Stöckmann for critical advice and
editing on the manuscript; Rick Yarbrough, Jan Waters, David
Whittern and Ian Marsden for assistance in interpretation of
NMR spectra.
Physicochemical property determinations
Calculated n-octanol/water partition coefficients (cLogP) were
determined using ChemDraw Professional™.
Bromodomain TR-FRET binding and cell-based assays
Disclaimers
TR-FRET binding assays for BRD2, BRD3 and BRD4; MX-1 Cell-Titer
Glo proliferation, and H1299-luciferase reporter assays were run as
previously described (Table 2).22
All authors are employees of AbbVie. The design, study
conduct, and financial support for this research were provided
by AbbVie. AbbVie participated in the interpretation of data,
review and approval of this publication.
PARP1 TR-FRET binding and cell-based assays
TR-FRET binding assays for PARP1; CellTiter-Glo cell-viability assays
[DLD (wt), and DLD (BRCA-/-)] were run as previously described
(Table 3).25
Kinase TR-FRET assays
TR-FRET binding assays for primary (Abl, Src; Table 4) and secondary
targets (Table S1, Figure S1) were run as previously described.28
Notes and references
Dasatinib analog cell viability assays
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Controlling Cellular Distribution of Drugs with Permeability Modifying
Moieties
Paul L. Richardson,*a Violeta L. Marin,a Stormy L. Koeniger,a Aleksandra Baranczak,a Julie L. Wilsbacher,b Peter J. Kovar,b
Patricia E. Bacon-Trusk,b Min Cheng,b Todd A. Hopkins,b Sandra T. Haman,b and Anil Vasudevana
Anionic moieties can be used to control the cell-permeability of drugs and used to select the appropriate
target identification method for phenotypic screening hits.
Modifier dPEGx-Linker
Ligand
H
N
O
O
Cell-surface
targets
1,3
Semi-Permeable
H
Permeable
O
Intracellular
targets
Permeable
O
Semi-permeable
Impermeable
Impermeable
O
HO2
C
O
O
-O3
S
Sulfocyanine dye
HO2
C
CO2
H