Targeting STAT3 anti-apoptosis pathways with organic and hybrid organic-inorganic inhibitors

Article abstract of DOI:10.1039/c9ob02682g

Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (AML) that spur efforts to identify new clinical targets and active agents. STAT3 has emerged as a potential target in resistant AML, but inhibiting STAT3 function has proven challenging. This paper describes synthetic studies and biological assays for a naphthalene sulfonamide inhibitor class of molecules that inhibit G-CSF-induced STAT3 phosphorylation in cellulo and induce apoptosis in AML cells. We describe two different approaches to inhibitor design: first, variation of substituents on the naphthalene sulfonamide core allows improvements in anti-STAT activity and creates a more thorough understanding of anti-STAT SAR. Second, a novel approach involving hybrid sulfonamide-rhodium(ii) conjugates tests our ability to use cooperative organic-inorganic binding for drug development, and to use SAR studies to inform metal conjugate design. Both approaches have produced compounds with improved binding potency.In vivoand in cellulo experiments further demonstrate that these approaches can also lead to improved activity in living cells, and that compound3aaslows disease progression in a xenograft model of AML.

Full text of DOI:10.1039/c9ob02682g

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Wang, J. O. Munoz, A. M. Stevens, A. E. Mangubat-Medina, M. J. Krueger, W. Liu, M. K. Kasembeli, J. C.
Cooper, M. I. Kolosov, D. Tweardy, M. Redell and Z. T. Ball, Org. Biomol. Chem., 2020, DOI:
10.1039/C9OB02682G.
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21 December 2017
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Targeting STAT3 anti-apoptosis pathways with
organic and hybrid organic–inorganic inhibitors
Matthew B. Minus4, Haopei Wang1, Jaime O. Munoz2, Alexandra M. Stevens2, Alicia E.
Mangubat-Medina1, Michael J. Krueger2, Wei Liu2, Moses M. Kasembeli3, Julian C. Cooper1,
Mikhail I. Kolosov5, David J. Tweardy3,6, Michele S. Redell2, Zachary T. Ball1*
1Department of Chemistry, Rice University, Houston, TX 77005 (USA).
2Baylor College of Medicine, Texas Children's Cancer Center, Houston, TX 77030 (USA).
3Department of Infectious Diseases, Infection Control and Employee Health and 6Department
of Molecule and Cellular Oncology, The University of Texas MD Anderson Cancer Center,
Houston, TX 77030 (USA).
5Department of Medicine, Baylor College of Medicine, Houston, TX 77030 (USA)
4Prairieview A&M University, Prairie View, TX, 77446 (USA)
* Corresponding Author
KEYWORDS: STAT3, Leukemia, naphthylsulfonamide, iminonaphthoquinone
1

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Abstract
Recurrence and drug resistance are major challenges in the treatment of acute myeloid
leukemia (AML) that spur efforts to identify new clinical targets and active agents. STAT3
has emerged as a potential target in resistant AML, but inhibiting STAT3 function has proven
challenging. This paper describes synthetic studies and biological assays for a naphthalene
sulfonamide inhibitor class of molecules that inhibit G-CSF-induced STAT3 phosphorylation
in cellulo and induce apoptosis in AML cells. We describe two different approaches to
inhibitor design: First, variation of substituents on the naphthalene sulfonamide core allows
improvements in anti-STAT activity and creates a more thorough understanding of anti-
STAT SAR. Second, a novel approach involving hybrid sulfonamide–rhodium(II) conjugates
test our ability to use cooperative organic–inorganic binding for drug development, and to
use SAR studies to inform metal conjugate design. Both approaches have produced
compounds with improved binding potency. In vivo and in cellulo experiments further
demonstrate that these approaches can also lead to improved activity in living cells, and that
compound 3aa slows disease progression in a xenograft model of AML.
Introduction
Signal transducer and activator of transcription (STAT) proteins are intracellular signaling
proteins that mediate response to extracellular stimuli.1–3 Extracellular signaling proteins,
such as cytokines and growth factors, activate membrane-bound receptors that then recruit
and tyrosine phosphorylate the STAT3 loop domain immediately C-terminal to the SH2
domain.4 Phosphorylated STAT3 (pSTAT3) dimerizes and translocates to the nucleus where
it activates transcription of tumor survival pathways.5,6 The aberrant activation of STAT3 is
2

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a common occurrence in many cancers.6,7 Inhibition of STAT3 activity has been shown to
lead to apoptosis in cancers such as breast,8,9 head and neck,10,11 colon,12 liver,13 acute
myeloid leukemia (AML),14,15 and prostate.16 Thus, STAT3 has been implicated as a
promising protein target for the development of broad-spectrum chemotherapeutics.17
In spite of significant efforts, inhibiting STAT3 pathways has proven difficult.18 Blocking
intracellular protein–protein interactions like those that mediate STAT3 activation remains
a daunting pharmacological goal.19,20 We became interested in naphthalene sulfonamides as
anti-STAT3 compounds,4,11,14,21 and sought to understand the molecular basis of STAT3
binding, to identify their molecular target on STAT3, and to assess the anti-AML activity of
these compounds in molecular, cellular, and animal models of AML. We began this
exploration assuming that the lead compound, C188-9 (a.k.a. TTI-101) (Figure 1b),
competitively binds to the phosphotyrosine peptide binding site in SH2 domain, consistent
with previous assumptions (Figure 1a). During the course of our investigations, rhodium-
catalyzed proximity-driven covalent modification determined that modified naphthalene
sulfonamides also target a new binding site in the coiled-coil domain (CCD), and that,
importantly, binding to this site is not competed by the natural phosphotyrosine peptide
(Figure 1a, orange residue).15 In part, current research seeks to investigate the therapeutic
potential of targeting CCD.15
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Figure 1. (a) Crystal structure of a single STAT3 protein when bound as a dimer to duplex DNA.22 Nucleophilic ligands,
cysteine and methionine residues of the SH2 domain (purple) and aspartate ligands of the coiled-coil domain (blue) are
highlighted. Affinity labeling experiments with a rhodium inhibitor conjugate indicates that binding occurs at/near Phe174
(orange) in the coiled-coil domain. (b) A lead compound, C188-9 (a.k.a. TTI-101). (c) Rh-STAT3 conjugates use inorganic-
organic cooperativity to bind STAT3.
At the same time, the challenges of STAT3 inhibition provided an opportunity to explore
an unconventional approach to inhibitor development: employing rhodium–small molecule
conjugates as anti-STAT agents. Conceptually, the approach is based on cooperative organic–
inorganic binding that includes rhodium coordination to a Lewis-basic side chain near
protein binding interface (Figure 1c).22,23 We have demonstrated this concept with rhodium-
peptide conjugates, and this work provided an opportunity to explore the concept with non-
peptide inhibitors (Figure 1c). Cellular studies provided an opportunity to assess function in
living cells, as opposed to purely biophysical measurements, and these measurements shed
light on the benefits and limitations of rhodium-based approaches, compared to traditional
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small-molecule STAT3 inhibitors.
Inhibitor Synthesis
Naphthalene sulfonamides were identified as lead fragments through a combination of
screening and limited structural variation.21 Compounds with STAT3-binding activity permit
significant variation of the sulfonamide substituent (R3 in Error! Reference source not
found.), thus R3 was seen as the most straightforward site for incorporation of rhodium
centers, via target structures such as 13aa-13c. Furthermore, the C5–C8 positions of the
naphthalene ring (R4) were a largely unexplored site of variation. In the course of our studies,
we became further interested in variation of the naphthol, and in assessing the function of
redox variants of this core, such as the iminoquinone.
HO
OH
X
R4
HO
OH
R2
R1
HN
HN
SO2
R3
S
O2
linker
HN
O
O
SO2
O
O
Rh Rh
O
O
R2
13aa-13c
O
O
R3
OMe
C188-9
O
R4
R2
N
SO2
R3
Figure 2. Previously established STAT3 inhibitor motif,21 and expanded motifs discovered in this work.
In the synthetic direction, the requisite 2-substituted naphthalene sulfonamides (e.g. 2a–4d,
Scheme 1) are readily available via elaboration of simple naphthalene sulfonamides (1a–1f,
Scheme 1) after sulfonamide formation from 4-amino-1-naphthol. Formation of the
sulfonamide early in the synthesis is important to limit synthetic manipulations with
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oxidatively sensitive free 4-amino-1-naphthol derivatives. Elaboration of the 2-position of
the naphthalene ring with halogens is directly achieved by electrophilic aromatic
substitution to afford arylbromides (2a-2c). Alternatively, oxidative coupling with pro-
nucleophiles (Nu–H = Ar–H, RS–H, RO–H) succeeds via the intermediacy of an
iminobenzoquinone 1aox-1fox.25 This allowed preparation of a variety of additional S-linked
(3aa-3cd) and C-linked (4a-4d) 2-substitution products.
Scheme 1. Modular synthesis of STAT3 inhibitors (a) R1SO2Cl, pyridine, MeCN, MgSO4 (32-94%) (b) Br2, CH2Cl2 (25-60%)
(c) PhI(OAc)2, acetone (d) PhSH, HCOOH (26-85%) (e) BF3·Et2O, 2-naphthol (30-70%). Yields for oxidative coupling were
taken over 2 steps. (*) Inhibitor isolated as oxidized iminonaphthoquinone.
R1 =
Br
F
OH
OH
OH
F
Br
O
a
b
MeO
MeO
F
F
F
F
NH2•HCl
NH
NH
2a-2c
O2S
R1
O2S
R1
2c
(25%)
2a
2b*
(60%)
(37%)*
1a-1f
R1 =
F
c
R2
S
HO
OH
O
OH
R2
R3
F
F
MeO
3ba
S
e
F
d
(37%)
R2 = H, R3 = H
3aa
R2 = H, R3 = H
(85%)
R3
3
N
NH
O2S
R1
NH
O
O2S
R1
O2S
R1
3aa-3cd
4a-4d
1aox-1fox
MeO
(26%) R2 = H, R3 = H
(50%) R2 = OH, R3 = H
(36%) R2 = H, R3 = OH
(35%) R2 = H, R3 = tBu
3ca
3cb
3cc
3cd
R1 =
F
F
F
O
MeO
R1 =
F
MeO
HO
HO
F
1a
O
1b
(48%)
OMe
1c
(92%)
(87%)
MeO
MeO
O
O
O
4b (61%)
4a (70%)
HO
MeO
O
O
O
HO
O
HO
O
1e
1d
(83%)
1f
(32%)
(94%)
OMe
4c (30%)
4d (48%)
In tandem, we set our sights on 8-substituted derivatives that could provide electronic and
steric perturbation of the naphthalene core, both to investigate SAR in this region and to
perturb the redox stability of inhibitor molecules. In a first approach, (Scheme 2), we
synthesized 1,8-difluoronaphthalene from 1,8-diaminonaphthalene,26 via a bis-diazonium
6

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intermediate. The 1,8-difluoronaphthalene was nitrated at the 4-position (5), and
nucleophilic aromatic substitution of the fluorine para to nitro with an alkoxide delivered
naphthyl ether, and subsequent hydrogenolysis yield 7. Efforts in this direction were limited
by safety and scalability concerns: The bis-diazonium salt was prone to spontaneous and at
times violent decomposition. As an alternative, we exploited more recent advances27 in
nucleophilic aromatic fluorination to prepare 6 from 1,5-dinitronaphthalene (Scheme 2).
This route gave better access to fluorinated naphthalene intermediates. The electrophilic
naphthalene ring 6 could be oxidized with tert-butylhydroperoxide (TBHP) in liquid
ammonia to install a hydroxyl group, affording naphthol 7. Etherification of the OH group
was next performed, providing a benzyl (8) or methyl (10) ether. At this point, the nitro
group could be reduced selectively in the presence of a platinum catalyst, and the resulting
aniline then coupled with a sulfonyl chloride to provide sulfonamide 8. Deprotection of the
benzyl group and oxidative coupling with thiophenol gave fluoride 9. Alternatively, the
methyl ethers 12a and 12b were synthesized as analogues of compounds 2a-2c.
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Scheme 2. Synthesis of 5-fluoronaphthylsulfonamides (a) i) HBF4 (aq) NaNO2 ii) molten KHF2 (2-20%) (b) HNO3, NaNO2
(60%) (c) BnOH, NaH, CH2Cl2 (61%) (d) Pt/C, NaBH4 (99%) (e) CsF, DMSO, 100 °C (23%) (f) NH3, TBHP, NaOH, THF (70%)
(g) BnBr, Cs2CO3, CH2Cl2 (75%) (h) i) Pt/C, NaBH4 ii) 4-methoxybenzenesulfonyl chloride, pyridine, MgSO4 (62%) (i) Pd/C,
H2, (j) PhI(OAc)2, PhSH, (CF3)2CHOH (37%) (k) MeI, K2CO3, DMF (84%) (l) i) Pd/C, NaBH4 ii) R2SO2Cl, pyridine, MgSO4 (45-
89%) (m) Br2, CH2Cl2 (56-75%)
F
F
F
F
NH2 NH2
b
a
NO2
5
c,d
F
OH
S
F
OBn
F
OH
F
NO2
g,h
f
i,j
e
HN
OMe
SO2
HN
NO2
k
NO2
S
O2
NO2
6
7
8
9
OMe
F
OMe
F
OMe
F
OMe
F
OMe
F
OMe
MeO2C
O
MeO2C
O
Br
Br
l
m
l
m
HN
p-Tol
HN
p-Tol
HN
HN
S
O2
S
O2
NO2
S
O2
S
O2
12a
11a
10
11b
12b
Several structures with different ester functional groups serve as anchor points for
rhodium conjugation (4a, 4b, 4c in Table 1). General methods that have been previously
reported allow for facile cooperative rhodium(II) binding to neighboring residues that
strongly coordinate to metal centers, such as methionine or histidine, allowed 50-500 fold
increases in potency in other systems.23,24,28 The presumptive target of naphthalene
sulfonamide inhibitors, the SH2 domain, contained several such residues (Figure 1a, purple).
However, the coiled coil domain—later identified as another target of naphthalene
sulfonamide inhibitors—is devoid of histidine, cysteine, and methionine residues. This
discovery allowed us to assess the suitability of cooperative inhibition with rhodium
conjugates toward binding sites that lack such strong metal-binding sites. (Figure 1a, blue).
We chose to append esters onto the C4 substituent of the naphthalene ring (Error! Reference
8

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source not found.), a region of the inhibitor which was previously found to be tolerant to
steric and electronic variations.21 We prepared several rhodium conjugates from ester-
containing intermediates by acidic hydrolysis followed by metalation with a heteroleptic
rhodium reagent containing labile trifluoroacetate groups (Table 2). Unfortunately, Rh-
naphthylsulfonamide conjugates could not be made by traditional procedures that involve
refluxing the carboxylate inhibitors in benzene. These conditions proved to be too harsh for
the organic starting materials. Instead methodologies previously developed by our group
were used to successfully couple these small molecules to rhodium.15
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Table 1. Preparation of rhodium conjugates by carboxylate exchange with heteroleptic rhodium trifluoroacetates.
HO
OH
HO
OH
1) HCl, acetone
2) Rh2(R)3(tfa)
iPr2NEt
R1
HN
HN
linker
S
linker
S
CF3CH2OH
CO2Me
O
O
O2
O2
O
O
Rh Rh
O
O
13aa –13ac,.13ae – 13c
R2
O
O
R3
HO
OH
HO
OH
HO
OH
HN
S
CO2Me
HN
S
NH
O2
O2
SO2
1) HCl, acetone
2) cis-Rh2(OAc)2(tfa)2
iPr2NEt
O
O
O
O
Rh Rh
O
O
O
R2
O
CF3CH2OH
13ad
R3
linker
R1
R2
yield (%)
63
CO2Me
entry
R3
product
1
2
CO2Me
13aa
13ab
Me
Me Me
nBu nBu
4a
nBu
15
13ac
13ad
13ae
13af
21
15
42
15
3
4
5
6
fluorescein
Me Me
Me Me
Me Me
n.a.
CF3
CF3 CF3 CF3
7
13b
13c
37
8
Me
Me
Me Me
Me Me
O
4b
CO2Me
8
CO2H
4c
SPR analysis
Potency was initially assessed by surface plasmon resonance (SPR), allowing
quantification of binding and straightforward comparison to other studies.21 Variation of the
R1 and R2 groups provided additional insight into the structural landscape of affinity. A
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perfluorinated ring within compound 2a—suggested by the appearance of this group in
another STAT-binding compound,8,29 results in a 3-fold increase in potency from C188-46
(Table 2). When this change was combined with variation at the C2 position, significant gains
in affinity were observed. Compound 3aa was found to be 10-fold more potent than 2a. Being
inspired by this increase in potency, we made different analogues of the inhibitors. In general
inhibitors with thiophenyl in the C2 position displayed greater potency. Even when the
perfluorinate ring in the R1 position was replaced with another alkyl or aryl group, the
potency remained similar. In addition, it seems the thiophenyl ring is functionally tolerant to
hydroxylation and alkylation. Inhibitors (3cb-3cd) were only slightly less potent than their
parent compound (3ca). In summary, SPR analysis demonstrates that molecular recognition
of the inhibitor to STAT3 is dependent on the 1,2,4 substitution on the naphthalene ring. This
is highlighted by the fact that smaller molecules without the sulfonamide-like moiety (14-16)
still inhibit STAT3-phosphopeptide binding.
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Table 2. Inhibition of STAT3 phosphopeptide binding. Inhibitive potency (IC50) was determined by established SPR and/or
a cellular STAT3 phosphorylation assay.
SPR
pSTAT3
SPR
pSTAT3
SPR
(IC50, µM)
OH
NH
(IC50, µM)(IC50, µM)
(IC50, µM) (IC50, µM)
OH
14
HO
OH
4b
Br
Br
Br
2c
0.1
72
68
Br
8
8.6
20
O2S
NH
O2S
NH2
15
MeO2C
3aa,
MM-206
CO2Me
O
OH
S
Br
HO
OH
0.7
8.7
4c
NH2
16
Br
50
NH
F
20
O2S
F
F
NH
O2S
NO2
F
HO
OH
c188-9
F
S
CO2H
OH
3ba
OH
9
F
4
S
NH
0.8
12
NH
O2S
11.7
2
O2S
NH
O2S
OMe
OMe
OH
NH
c188-19
S
OMe
OH
3ca
S
12b
2
F
OMe
0.9
7.3
Br
O2S
NH
O2S
5.7
5
F
MeO2C
NH
c188-46
OH
O2S
Br
3cb
OH
NH
OH
S
O
CO2Me
24
NH
17
2
OAc
NH
O2S
>100
O2S
S
MeO2C
F
c188-47
OH
F
Br
O2S
3cc
OH
F
S
F
F
49
NH
F
OH
18
O2S
NH
4
4.6
O2S
O
Me
O
OMe
MeO2C
O
O
2a
OH
S
OH
Br
3cd
4
S
NH
F
8
NH
O2S
F
F
NH
O2S
1
16
33
O2S
F
F
F
MeO2C
F
F
F
19
F
OTf
2b*
O
N
HO
4a
S
Br
OH
5
26
0.2
25
NH
NH
O2S
O2S
O2S
MeO2C
OMe
OMe
12

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Naphthalene sulfonamide oxidation
The 1,4-substitution pattern of hydroxyl-naphthalenesulfonamides renders the core
structure susceptible to oxidation. Some synthetic intermediates (i.e. 1b, 1c, and 1f) indeed
proved quite prone to air oxidation and subsequent decomposition. In vivo oxidation could
be a decomposition pathway, but it is also possible that oxidized iminoquinone species (e.g.
Table 2, 2b*) may represented an active species, with naphthalenesulfonamides serving as a
pro-drug. Iminoquinone structures might serve as reactive electrophiles for covalent
inhibition pathways, and in this sense they might putatively share features in common with
other known STAT3 inhibitors, such as LY530 and BA-TPQ,31 with structures consistent with
electrophilic reactivity. Significant decomposition of C188-9 was observed within 4 hours in
aqueous buffer under air (Figure S1), and crude NMR investigations showed new peaks
consistent with a iminoquinone. In one case, an iminoquinone intermediate 2b* (Table 2)
was stable enough to be isolated, characterized, and tested. This iminoquinone also
demonstrated significant binding affinity in our SPR-based assay, suggesting that
iminoquinone intermediates may contribute to at least some function in vivo.
To further probe these issues, we prepared a brief series of C188-9 analogues with an explicit
goal of improving lifetime in media. Although some structural changes abrogated binding, a
few did show promising binding by SPR, including fluoro-ether 12b and triflate 19 (Table 2).
Interestingly, O-acylation of the naphthol hydroxyl group has a significant negative effect on
binding in the case of 17, but substitution on oxygen is tolerated in other cases (e.g. 12b, 18,
and 19). Unlike the parent C-188-9, fluoro-ether 12b and triflate 19 are indefinitely stable in
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aqueous buffer in air. The binding potency of 12b and 19 suggests that oxidation and/or
accessing an iminoquinone structure is not required for binding or activity.
Inhibition of intracellular phosphorylation
The more promising molecules (3aa-3ca) were tested in intracellular STAT3
phosphorylation assays. To extrapolate intracellular potency, Kasumi-1 human AML cells
were treated with differing concentrations of inhibitor (Figure 3a). Cells were incubated
with granulocyte-colony stimulating factor (G-CSF) to induce STAT3 phosphorylation.32
After incubation, the cells were lysed and levels of STAT3 phosphorylated on tyrosine 705
(pSTAT3) were quantified with a fluorescent pSTAT3 antibody. Although 3aa, 3ba, and 3ca
performed similarly by SPR (Table 2), their ability to inhibit STAT3 phosphorylation in cells
was markedly different. This suggests that their molecular recognition of STAT3 in an
isolated system like SPR is similar, however, in a protein-rich redox-buffered environment
these inhibitors no longer behave the same. It is important to note that differences in small
molecule specificity and cellular uptake can also affect the intracellular potency of the
inhibitors. Nevertheless, 3aa was clearly the most potent small-molecule pSTAT3 inhibitor.
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We wanted to ensure that the molecule 3aa also inhibits inducible STAT3 phosphorylation
in other AML cell lines (Figure 3b). Following a 30-minute treatment with 3aa prior to G-CSF
stimulation, all three of the AML cell lines tested (HL-60, MOLM-13, and Kasumi-1) exhibited
dose-dependent decreases in STAT3 phosphorylation with IC50 0.8–1.9 μM. This
demonstrates that the anti-pSTAT3 activity of 3aa can be seen across different AML cell lines.
To further evaluate the therapeutic potential of 3aa, we confirmed its ability to induce
apoptosis in different AML cell lines and primary tumor cells from pediatric AML patients.
Cells were incubated with different doses of each inhibitor and subsequently incubated with
fluorescent Annexin V. Annexin V binds to apoptotic cells, and FITC-Annexin V conjugates
allow quantification of apoptosis by fluorescence assisted cell sorting (FACS). A 24-hour
treatment with 3aa increased apoptosis in all AML cell lines and primary samples tested
(Figure 3c). In contrast, acute lymphoblastic leukemia (ALL) cell lines—which do not have
upregulated STAT3 activity33—were much less sensitive to apoptosis induction (Figure 3c,
KOPN-8 and RS4-11). These findings are is consistent with the idea that 3aa inhibits STAT3
phosphorylation in AML cell lines and induces apoptosis through that mechanism of action.
After thoroughly investigating our 3aa in the in vitro models, we moved to investigate its
potency in in vivo mouse models. STAT3-dependent cyctotoxicity in apoptosis assays
suggested at least a half-log dosing window. Mice were injected intravenously with MV4-11
human AML cells. After two weeks, mice were treated for another 2-4 weeks with 3aa.
Results from in vivo imaging, bone marrow and spleen harvesting indicate that 3aa increases
mouse survival by decreasing the expansion of MV4-11 leukemia cells (Figure 3 d–f).
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Figure 3. Portions of this figure were adapted from a preliminary report.15 Compound 3aa inhibits G-CSF-induced STAT3
phosphorylation, induces apoptosis in human AML cells, and slows disease progression in a xenograft model of AML. (a)
Histogram of decrease in pSTAT3 in Kasumi-1 cells. (b) 10 inhibits G-CSF-induced pSTAT3 in multiple AML cell lines.
Legend values indicate IC50 values. Mean ±SD for n = 3. (c) Apoptosis quantified in Annexin V-FITC-labeled cells treated
with 10 for 24 h. Spontaneous apoptosis in untreated cells was subtracted to yield the % apoptosis attributable to drug.
Data shown for an AML cell line (MV4-11, HL-60) and primary patient-derived AML cells (p198) compared to ALL cell lines
(KOPN-8, RS4-11) as a negative control. (d-e) NSG mice were injected iv with 106 MV4-11. ffluc AML cells at day 0. After
two weeks, mice received compound 10, 30 mg/kg (n=8), or vehicle (n=6), ip daily 5 d/week for 4 weeks (weeks 2-6). (d)
Luminescence images one week after treatment. Colorized signal intensity indicates amount of active disease, from low
(blue) to high (red). (e) The percent of MV4-11 cells in bone marrow and spleen was evaluated by flow cytometry at the
time of euthanasia. *P<0.05 (f) Disease burden was measured non-invasively by bioluminescence weekly. Each line
represents the trajectory of an individual mouse. Mice were euthanized when moribund or at the end of 8 weeks.
Rhodium conjugates as STAT3 inhibitors
After garnishing some promising results in the traditional small molecule studies with 3aa,
we shifted our focus to the biological efficacy of small molecule-rhodium conjugates. Again,
we used SPR to screen inhibitor potency (Table 3). Rhodium-inhibitor conjugate 13aa, 13ac,
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and 13b, showed potency increases over their analogous esters. Compared to previous
studies with other binding sites, this affinity gain is rather modest, and is presumably
consistent with at most very weak binding to side chains such as carboxylates (i.e. residue
Asp 170 near the coiled-coil binding site) rather than stronger binding to histidine or
methionine. A previous study found that sterically demanding carboxylates improved the
decomposition half-life of rhodium complexes,34 but the sterically crowded rhodium
complex 13ac had potency similar to other rhodium conjugates. The divalent structure 13ad
did demonstrate moderately improved STAT3 affinity (0.1 μM).
It seemed possible that a more Lewis-acidic rhodium center could benefit from more
potent binding to peripheral residues.35 The coiled-coil domain does have several
carboxylate side chains near the ligand-binding site (Figure 1a, blue), including Asp170, and
we reasoned that carboxylates, while weaker ligands than histidine or methionine, might be
sufficient ligands for a metal center with increased Lewis acidity. Alteration of the ancillary
ligands on rhodium allows tuning the Lewis acidity of the metal center. An electron-poor
variant, 13ae did show improved binding to STAT3 (IC50 = 0.04 μM, Table 3): compound
13ae is 100× more potent than C188-9, and 25× more potent than the nearly isosteric
rhodium complex 13aa. This effect may be quite subtle: The analogous tris-trifluoroacetate
complex (13af) exhibited a more modest affinity enhancement (IC50 = 0.2 μM, Table 3),
perhaps reflecting a subtle balance of competitive water binding.34
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Table 3. SPR analysis of binding for carboxylate-containing and rhodium-conjugate compounds.
SPR IC50 (uM)
R1
R2
R3
entry
1
cmpd
13aa
ester Rh(II)
25
25
1
Me
Me Me
nBu nBu
nBu
2
0.6
13ab
13ac
13ad
13ae
25
25
25
1
3
4
5
fluorescein
Me Me
Me Me
Me Me
0.1
0.04
n.a.
CF3
6
7
CF3 CF3 CF3
25
8
0.2
1
13af
13b
Me
Me
Me Me
Me Me
8
50*
0.3
13c
*SPR IC50 of the carboxylic acid derivative 4c was measured.
Next, we examined the cellular activity of the lead rhodium conjugate on STAT3
phosphorylation and the induction of apoptosis in Kasumi cells (Figure 4). To provide a
useful comparison, rhodium conjugates were directly compared against C188-9 and
compound 4a, an isoelectric organic analogue without the capacity for coorperative organic–
inorganic binding. In STAT3 intracellular phosphorylation assays (Figure 4b), the
electronically-perturbed rhodium complex 13ae again exhibited significantly improved
activity. The Kasumi cells were exceptionally resistant to both C188-9 and 4a, while 13ae
inhibited STAT3 phosphorylation at lower micromolar potencies. In all, this is the first
example of rhodium complexation enhancing the potency of any intracellular inhibitor in
cellular assays andthese results indicate that rhodium conjugation is a viable strategy for
increasing the potency of a biological probe for intracellular assays.
In order to see if this increase phosphorylation inhibition translated to induction of
apoptosis, we compared 13ae, 4a, and C188-9 in an Annexin assay (Figure 4c). The rhodium
complex 13ae was more potent than both C188-9 and 4a, which is in agreement with the
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phosphorylation inhibition results. Therefore the fact that 13ae consistently outperformed
C188-9 in these in vitro assays suggests further development of rhodium conjugates is
warranted.
Figure 4. (a) Small molecules inhibit STAT3/phosphopeptide (p1068) binding. STAT3 phosphopeptide binding was
measured with established SPR protocols.21 (b) Kasumi cells were treated with G-CSF and then with inhibitors. Cells were
analyzed for phosphorylated STAT3 (pSTAT3). (c) Apoptosis was measured 24 h after treatment of AML cells with STAT3
inhibitors.
Naphthalene sulfonamides are a useful class of probes to alter STAT3 function.
Optimization of the sulfonamide core allowed development of 3aa, an inhibitor with
improved binding potency, and anti-STAT3 activity in cells. Some of the naphthalene
sulfonamides also display in vivo activity in relevant cancer models.11,36 For example,
pentafluoro compound 3aa, significantly decreases tumor progression in a mouse model of
AML. Anti-phosphorylation activity correlates well with apoptosis induction in tumor
models for the naphthalene sulfonamide compound class, again consistent with a specific
STAT3-driven mode of action. Overall, optimization of the naphthalene sulfonamide core has
resulted in modest gains in potency and activity, perhaps due to innate features of the coiled-
coil binding site, which lacks a deep ligand-binding pocket. This limited optimization success
led us to pursue tandem efforts to explore rhodium conjugates as potential solution to vexing
inhibitor-development problems. The substantial increase in potency observed with
rhodium complex 13ae indicates that significant gains in binding affinity are possible, even
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without proximal strong metal-binding residues (histidine, methionine, cysteine), by
perturbing the electronic structure of the rhodium center in favor of increased Lewis acidity.
Perhaps most importantly, the improvement in STAT3 binding carries through to cell-based
assays, supporting our previous conclusions that rhodium complexes can enter and act
within living cells. Complexes such as 13ae may serve as probe compounds with unique
specificity; although many important questions remain, especially regarding in vivo
application of these compounds.
Acknowledgements
M.B.M. was supported by a Ruth L. Kirchstein National Service Award (NIH F31CA180696).
We acknowledge support from the NIH (5R21CA170625), from the Robert A. Welch
Foundation Research Grant C-1680 (Z.T.B.), the National Science Foundation (CHE-1904865,
Z.T.B.), the Gillson Longenbaugh Foundation (M.S.R.), the Virginia and L.E. Simmons Family
Foundation, and the MD Anderson Foundation (D.J.T).
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