Human Serum Albumin-Delivered [Au(PEt3)]+ Is a Potent Inhibitor of T Cell Proliferation

Article abstract of DOI:10.1021/acsmedchemlett.7b00142

Using a modular library format in conjunction with cell viability (MTS) and flow cytometry assays, 90 cationic complexes [AuPL]ⁿ⁺ (P = phosphine ligand; L = thiourea derivative or chloride) were studied for their antiproliferative activity in CD8⁺ T lymphocyte cells. The activity of the compounds correlates with the steric bulk of the phosphine ligands. Thiourea serves as a leaving group that is readily replaced by cysteine thiol (NMR, ESI-MS). Taking advantage of selective thiourea ligand exchange, the fragments [Au(PEt₃)]⁺ and [Au(JohnPhos)]⁺ (JohnPhos = 1,1′-biphenyl-2-yl)di-tert-butylphosphine) in compounds 1 and 2 were transferred to recombinant human serum albumin (rHSA). PEt₃ promoted efficient modification of Cys34 in HSA (HSA-1), whereas use of bulky JohnPhos as a carrier ligand led to serum protein nonspecifically modified with multiple gold adducts (HSA-2) (Ellman’s test, ESI-TOF MS). HSA-1, but not HSA-2, strongly inhibits T cell proliferation at nanomolar doses. The potential role of HSA as a delivery vehicle in gold-based autoimmune disease treatment is discussed.

Full text of DOI:10.1021/acsmedchemlett.7b00142

Letter
pubs.acs.org/acsmedchemlett
Human Serum Albumin-Delivered [Au(PEt3)]+ Is a Potent Inhibitor of
T Cell Proliferation
Tyler C. Dean,† Mu Yang,† Mingyong Liu,‡ Jason M. Grayson,‡ Anthony W. DeMartino,†
Cynthia S. Day,† Jingyun Lee,⊥ Cristina M. Furdui,§ and Ulrich Bierbach*,†
†Department of Chemistry, Wake Forest University, Wake Downtown Campus, 455 Vine Street, Winston-Salem, North Carolina
27101, United States
‡Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, North Carolina 27101, United
States
⊥Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157, United States
§Department of Internal Medicine, Section on Molecular Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina
27157, United States
S
* Supporting Information
ABSTRACT: Using a modular library format in conjunction with cell viability (MTS) and flow
cytometry assays, 90 cationic complexes [AuPL]n+ (P = phosphine ligand; L = thiourea derivative or
chloride) were studied for their antiproliferative activity in CD8+ T lymphocyte cells. The activity of
the compounds correlates with the steric bulk of the phosphine ligands. Thiourea serves as a leaving
group that is readily replaced by cysteine thiol (NMR, ESI-MS). Taking advantage of selective
thiourea ligand exchange, the fragments [Au(PEt3)]+ and [Au(JohnPhos)]+ (JohnPhos = 1,1′-
biphenyl-2-yl)di-tert-butylphosphine) in compounds 1 and 2 were transferred to recombinant human
serum albumin (rHSA). PEt3 promoted efficient modification of Cys34 in HSA (HSA-1), whereas
use of bulky JohnPhos as a carrier ligand led to serum protein nonspecifically modified with multiple
gold adducts (HSA-2) (Ellman’s test, ESI-TOF MS). HSA-1, but not HSA-2, strongly inhibits T cell proliferation at nanomolar
doses. The potential role of HSA as a delivery vehicle in gold-based autoimmune disease treatment is discussed.
KEYWORDS: Gold phosphine complexes, human serum albumin, inhibitor screening, T cell proliferation
Chart 1. Structures of Gold−Phosphines
old(I)-based inorganic salts and coordination compounds
Ghave a long history of medical uses, such as treatment of
tuberculosis (TB) (sanocrysin) and rheumatoid arthritis (RA,
auranofin).1,2 Because of their poor tolerability, injectable and
oral gold(I) drugs are now only administered as second-line
disease-modifying antirheumatic drugs (DMARD) in RA
patients who have not responded to prior anti-inflammatory
treatments.1 Gold(I) drugs are reactive electrophiles that
readily undergo ligand exchange reactions with proteins and
enzymes, primarily with cysteine (Cys) and selenocysteine
(Sec) residues.1,3 Binding to, and inhibition of, multiple targets
have been implicated in the mechanism of gold(I) drugs during
various stages of RA (initiation, inflammation, tissue injury).
These include lysosomal and extracellular cathepsin proteases
involved in antigen processing and matrix degradation in
diseased tissues, respectively.4,5 Gold(I) complexes, especially
those bearing lipophilic phosphine (PR3) ligands, have also
shown potent antiproliferative activity in cancer cell lines and
antitumor activity in vivo. Auranofin (Chart 1) is currently
being evaluated for the treatment of chronic lymphocytic
leukemia (CLL) in phase II clinical trials.6 The mechanism of
action of gold−phosphine compounds is thought to involve
inhibition of the mitochondrial and cytoplasmic thioredoxin/
thioredoxin reductase (Trx/TrxR) systems, triggering oxidative
stress and cell death.7,8 Like other metallopharmaceuticals,
these compounds often have a narrow therapeutic window
because they fail to discriminate between cancer cells and
normal cells. To address the unfavorable drug-like properties of
gold(I) compounds, more tolerable entities based on new
ligand and prodrug designs, as well as targeted forms of delivery
have been devised.7,9
To identify new suppressors of T lymphocyte activation,
proliferation, and function during undesired autoimmune
Received: March 28, 2017
Accepted: April 21, 2017
© XXXX American Chemical Society
A
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Chart 2. Phosphines and Thioureas Used to Assemble a Modular Library of Gold(I) Complexes
response, such as systemic lupus erythematosus (SLE), we
recently investigated the immunosuppressive activity of mixed
gold(I)−phosphine−thiourea complexes.10 In these com-
pounds gold is linearly coordinated by the thiourea sulfur of
a 9-aminoacridine ligand or structurally related derivatives, and
a trans-ligand including (pseudo)halides and phosphines.11
Gold(I) complexes of the acridinylthiourea derivative 1-[2-
(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU)
were originally designed as derivatives of Pt-ACRAMTU, the
prototype of a potent class of DNA-targeted platinum−acridine
hybrid anticancer agents.12,13 The dicationic phosphine-
modified complex, [Au(PEt3)(ACRAMTU)](NO3)2 (1), in-
hibited T cell proliferation at concentrations an order of
magnitude lower than had previously been observed in human
cancer cells by disrupting the mitochondrial Trx/TrxR
system.10 It also demonstrated potent immunosuppressive
activity in mice but was mildly toxic to the test animals when
injected intraperitoneally.10 Targeting the redox balance of T
cells with gold-based therapies to treat autoimmune disorders is
an unexplored concept. In a search for new derivatives with
improved selectivity and reduced toxicity, we have now
investigated structure−activity relationships of this pharmaco-
phore by screening 90 gold(I)−phosphine analogues using a
modular library format. Specifically, we wanted to assess how
bulky phosphines affect the reactivity and antiproliferative
activity of this chemotype. We discovered that transfer of
[Au(PEt3)]+ in one of the most potent derivatives to Cys34 in
human serum albumin (HSA) via thiourea−thiol exchange
produced a metal-modified protein that strongly inhibited T
cell proliferation.
Scheme 1. Library Assembly for Screening Gold(I)−
a
Phosphine−Thiourea Complexes
a
Reagents and conditions: (a) 1.1 equiv of P1−P9, DCM, r.t., 1 h; (b)
1.1 equiv of L1−L9, DMF, r.t., 30 min.
compounds, eight electron-rich dialkyl−aryl phosphines14 of
varying steric hindrance (“Buchwald phosphines”, P1−P8)
were introduced in place of PEt3 (P9). The goal of replacing
PEt3 in compound 1 with bulkier phosphine ligands was to
enhance the metal’s selectivity (and potentially tolerability) by
reducing its chemical reactivity. As ligands trans to phosphine,
eight thioureas containing N-heterocyclic chromophores of
varying basicity, borrowed from our extensive library of
ACRAMTU derivatives, were introduced: 9-aminoacridines
L1, L2, L5 (pKa ≈ 9−10, DNA intercalating),15,16 1,2,3,4-
tetrahydro-9-aminoacridine (tacrin) L3 and -4-aminoquinoline
L4 (pKa ≈ 9−10, nonintercalating),17,18 9-aminoacridine-4-
carboxamides L6 and L7 (pKa ≈ 8),19 and 9-substituted L8
(pKa ≈ 4, nonintercalating).20 In addition, the simple derivative
1,1,3,3-tetramethylthiourea (tmtu, L9) was also studied. The
rationale behind testing the current set of thioureas was to
establish the structural requirements for L as a carrier group.
While ACRAMTU-based ligands alone proved to be signifi-
cantly less cytotoxic than their Pt and Au complexes in all solid
and hematological tumor models studied,11,16 one goal of the
screen was to establish whether the intercalator can be replaced
with non-DNA binding, less genotoxic moieties. For compar-
ison, the nine chlorogold(I) precursors, [AuClP#] (with L10 =
chloride), were also included in the screen.
Using a previously established synthetic scheme,10 we
generated mixed gold(I)−phosphine−thiourea complexes
from nine phosphine ligands (P) and nine thiourea ligands
(L) (Chart 2). The reactions involved substitution of the
tetrahydrothiophene (tht) and chloro ligands in [AuCl(tht)] in
two consecutive reactions steps by P and L (Scheme 1).
The nine precursor complexes [AuClP#] were isolated and
fully characterized prior to assembling the second step (Scheme
1, step b) on 96-well plates using microscale reactions.
Complete conversion to the desired complexes [AuP#L#]n+
(# = 1−9; n = 1, 2; denoted below as P#−L#) was confirmed
by 31P NMR spectroscopy for selected samples. Reactions were
used directly as stock solutions in a colorimetric cell viability
assay. To produce the desired structural diversity in this set of
To assess the inhibitory effect of the 90 gold(I)−phosphine
+
̈
analogues, purified naive CD8 T cells were treated with test
compounds at concentrations of 150 and 600 nM for 60 min at
37 °C, stimulated with αCD3 and αCD28 antibodies, and
incubated for another 72 h. The specific increase in T cell
proliferation post stimulation in the presence of test compound
relative to untreated control was then determined using a
colorimetric (MTS) cell viability assay. Analysis of the
inhibition data across the entire library shows that the activity
of the compounds is dominated by the nature of the phosphine
B
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ACS Medicinal Chemistry Letters
Letter
ligands (P). At a concentration of 150 nM (Figure 1), PEt3
(P9) and the sterically least hindered phosphines among the
contains a single reactive free thiol, Cys34, is now being used as
a versatile injectable delivery platform to improve the safety and
efficacy of therapeutics and diagnostics.22 Because of its well-
documented high affinity for gold(I) agents,1 we wanted to
address whether the serum protein is able to mediate the
inhibitory properties of our gold(I)−phosphines in T cells, or if
it merely serves as a nucleophilic sink that traps the thiophilic
metal. The procedure involved (i) treatment of rHSA with gold
compound, (ii) determining the level of gold-modified cysteine
using Ellman’s test, (iii) size-exclusion chromatography to
remove released ACRAMTU ligand and desalt the sample, (iv)
quantification of total protein recovered using Bradford assay,
and (v) characterization of the products by electrospray
ionization time-of-flight mass spectrometry (ESI-TOF MS,
Figure 2). Compound 1, containing the sterically less hindered
Figure 1. Prescreening results for the 90 gold(I)−phosphine−thiourea
complexes at a fixed concentration of 150 nM of test compound in a
modular high-throughput format in CD8+ T cells. Compound activity
is expressed as percent increase in metabolic activity relative to isotype
vehicle-treated control cells. Inhibition data has been sorted by
phosphine component. Each circle represents a specific library
member, P#−L#. The two compounds selected for further study,
P1−L1 and P9−L1, are highlighted with arrows.
dialkyl−aryl derivatives, P1 and P2, produced the most active
complexes with inhibition levels of greater than 50% compared
to the vehicle-treated control. By contrast, the compounds
modified with P3−P8, which contain substituted biphenyl
moieties, showed significantly reduced activity. Within each of
the nine sets of complexes the inhibitory activity varies
depending on the nature of L, suggesting that the thiourea
ligands modulate, to some extent, the activity of the complexes
(Figure 1). However, no trends in the relative contribution to
the overall activity from L across the nine sets of complexes
seem to exist and no collectively applicable structure−activity
relationships (SAR) emerged. At 600 nM test compound, all
derivatives except for those containing the bulkiest phosphines
(P3, P4, P5, and P8) lead to complete suppression of T cell
proliferation (data not shown). By contrast, complexes based
on the latter four phosphines showed no dose response in this
assay.
Figure 2. Deconvoluted ESI-TOF mass spectra of rHSA (calculated
molecular weight based on amino acid sequence is 66 437 Da) (A),
and rHSA reacted with 1.33 equiv of [Au(PEt3)]+ (MW 315.13) (B)
or 1.75 equiv of [Au(JohnPhos)]+ (MW 495.38) (C).
To shed light on the reactivity of the pharmacophore with
biologically relevant thiols, we studied the interactions of two
derivatives with glutathione (GSH): P9−L1 (compound 1),
which was confirmed to be highly active at submicromolar
inhibitory concentrations consistent with those reported
previously,10 and P1−L1 (compound 2) containing 1,1′-
biphenyl-2-yl)di-tert-butylphosphine (“JohnPhos”) ligand,
which also demonstrated exquisite activity in the prescreen
(Figure 1). (P1−L1 was resynthesized on a preparative scale
and fully characterized; see the Supporting Information.) 31P
NMR spectra recorded of the reactions of P9−L1 and P1−L1
with 2 equiv (excess) of GSH in PBS buffer (pH 7.6) show a
single new peak for each complex at 38.4 and 63.5 ppm,
respectively, consistent with a mixed phosphine/thiol coordi-
nation of gold(I) in both cases.21 Likewise, positive-ion
electrospray ionization mass spectra (ESI MS) of the reaction
mixtures confirm that the thiourea ligands in P9−L1 and P1−
L1 have been replaced by GSH sulfur (Supporting
Information). Only in reactions of P9−L1 were trace amounts
of free phosphine (oxide) observed, corroborating the
selectivity of the ligand exchange reaction.
phosphine ligand PEt3, was able to modify >85% of the Cys34
free thiol groups present in the sample of rHSA. Under the
same conditions, compound 2 containing bulky “JohnPhos”
produced a maximum of 40% gold-phosphine modified Cys34.
Attempts to increase the yield of the latter adduct by applying a
larger excess of 2 were unsuccessful and instead led to rHSA
molecules containing multiple [AuJohnPhos]+ fragments
(Figure 2C). These findings suggest that reaction of the
bulky [Au(JohnPhos)]+ moiety in compound 2 with Cys34
may be sterically disfavored and result in nonselective transfer
of gold to alternative, more accessible amino acid residues, such
as methionine or histidine.23
Using carboxyfluorescein succinimidyl ester (CFSE) labeled
CD8+ cells in conjunction with flow cytometry analysis, we
compared the effects of compound 1, compound 2, rHSA·
[Au(PEt3)] (HSA-1), and rHSA·n[AuJohnPhos] (HSA-2) on
T cell proliferation. Tetrazolium-to-formazan reduction in
colorimetric cell viability assays measures cell metabolic activity
and often under- or overestimates the antiproliferative potential
of drugs.24 To account for this shortcoming and to determine
the true number of viable, proliferating T cells, we used an assay
Next, we studied the reactions of compounds 1 and 2 with
recombinant human serum albumin (rHSA). HSA, which
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based on direct cell counting. Purified, labeled CD8+ cells were
treated with vehicle, rHSA, gold complexes, or the correspond-
ing modified protein samples for 60 min, activated with the
appropriate antibodies for 72 h, and analyzed for CFSE
fluorescence (Figure 3). When treated with 400 nM compound
One important role of the lipophilic phosphine carrier
ligands is to facilitate uptake across membranes and, as a
component of cationic Au complex ions, lead to gold
accumulation in the mitochondria. The extent to which gold
drugs are mobilized from plasma proteins, with which they
rapidly associate while in circulation, and the exact mechanisms
of intracellular gold trafficking and targeting of Sec residues in
TrxR are unknown. A critical observation in our system is that
HSA-1 maintains the same high activity as the parent
compound, 1. This is an important finding since previous
clonogenic cell culture assays reported by Snyder et al. have
shown that serum albumin reduces auranofin’s cytotoxicity,27
presumably by sequestering the drug’s active gold component.
However, the same study also provided support for a
mechanism by which the [Au(PEt3)]+ moiety of auranofin is
exchanged between, and transported by, multiple membrane-
bound and cytoplasmic Cys-containing proteins until it reaches
its pharmacological target. Prior work by Pellom et al.28 has
demonstrated that activated T cells upregulate surface free
thiols (SFT) following stimulation prior to entering S phase in
the cell cycle. Thus, a shuttle mechanism involving membrane
thiols may provide a cellular uptake mechanism for [Au-
(PEt3)]+ in HSA-1 that would not require endocytosis of HSA-
gold. Inefficient transfer of gold from ACRAMTU to Cys34 in
HSA, and from HSA to SFT, caused by the bulky JohnPhos
group may explain why compound 2 and HSA-2 are relatively
less effective mediators of T cell inhibition. Mobilization of
[Au(PR3)]+ from HSA and transfer to cellular targets appears
to be an important prerequisite for antiproliferative activity in T
cells. The results from this study suggest that HSA may not
only play an important role as an endogenous mediator of T
cell inhibition but may also have a potential use as a
biocompatible delivery system for the systemic treatment of
autoimmune diseases with gold-based drugs.
Figure 3. Pretreatment with compound 1 or HSA-1 effectively inhibits
T cell activation and proliferation. (A) Results of the flow cytometry
experiments. Effect of 400 nM 1, 2, HSA, HSA-1, and HSA-2 on
CFSE fluorescence in unstimulated CD8+ cells and CD8+ cells
activated with αCD3 and αCD28 antibodies. (B) Percent proliferating
cells based on loss of CFSE fluorescence in histograms for vehicle and
treatment groups. The mean and standard deviation for three
independent experiments are plotted. For microscopic images of
cells treated with HSA and HSA-1, see the Supporting Information.
1 or HSA-1 at the same concentration (based on protein),
virtually no change in the fluorescence signal was observed after
stimulation of the cells with antibodies, indicating that
proliferation of activated T cells was completely inhibited. By
comparison, compound 2 was a significantly weaker inhibitor
than expected from the prescreening data, potentially
confirming the limitations of the MTS assay. HSA-2 and
HSA alone had no effect on cell proliferation.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00142.
The current study sheds light on the SAR of a class of mixed-
ligand gold(I) complexes and on their mechanism of action.
Using a modular screening platform, we demonstrated that the
ability of the pharmacophore to reduce T cell viability depends
on the steric hindrance of the phosphine ligand. [Au(PEt3)]+,
the classical mitochondriotropic cation in gold(I)-based drugs,
inhibits proliferation at the submicromolar concentrations
tested, both as a component of compound 1 and when
delivered as HSA-1. ACRAMTU appears to serve as a readily
exchangeable carrier ligand, similar to the thioglucose ligand in
auranofin,25 and does not appear to be essential for the
inhibitory activity of 1 in CD8+ cells. We took advantage of the
thiourea−thiol ligand exchange chemistry to modify cysteine
thiol in rHSA with [Au(PEt3)]+. ACRAMTU proves to be an
ideal transfer ligand for generating gold(I)-modified protein: (i)
the acridine derivative is protonated at physiological pH,
rendering the compounds 2+ charged and highly water-soluble;
(ii) ACRAMTU has a UV−vis signature at 413 nm, which
allows easy detection and precise spectrophotometric quanti-
fication; (iii) [Au(PEt3)]+ transfer from ACRAMTU to cysteine
thiol proceeds quantitatively and without generating side
products. Unlike auranofin’s thioglucose ligand, ACRAMTU
released in the process does not compete with thiol binding nor
reverse it, or cause decomposition of the [Au(PEt3)]+ moiety.26
Experimental procedures, details of product character-
ization, and supplementary figures (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: bierbau@wfu.edu.
ORCID
Ulrich Bierbach: 0000-0001-5658-7662
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
Funding
This work was funded in part by a SPARK grant from Wake
Forest Innovation and Commercialization Services, the Arnold
and Mabel Beckman Foundation, and by the Comprehensive
Cancer Center of Wake Forest Baptist Medical Center (NCI
Cancer Center Support Grant P30CA012197). The HRMS
spectra were acquired on a Thermo Scientific LTQ Orbitrap
instrument, which was purchased with funds provided by the
NSF (grant award number 947028).
D
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
T.C.D. gratefully acknowledges support through a Beckman
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