Discovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods

Article abstract of DOI:10.1021/acs.jmedchem.5b01660

Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins responsible for the regulation of programmed cell death. Amplification of Mcl-1 is a common genetic aberration in human cancer whose overexpression contributes to the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through interactions with pro-apoptotic BH3 containing proteins that achieve high affinity for the target by utilizing four hydrophobic pockets in its binding groove. Here we describe the discovery of Mcl-1 inhibitors using fragment-based methods and structure-based design. These novel inhibitors exhibit low nanomolar binding affinities to Mcl-1 and >500-fold selectivity over Bcl-xL. X-ray structures of lead Mcl-1 inhibitors when complexed to Mcl-1 provided detailed information on how these small-molecules bind to the target and were used extensively to guide compound optimization.

Full text of DOI:10.1021/acs.jmedchem.5b01660

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Article
Discovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia
(Mcl-1) Inhibitors Using Fragment-Based Methods
1
Nicholas F Pelz, Zhiguo Bian, Bin Zhao, Subrata Shaw, James C. Tarr, Johannes Belmar,
Claire Gregg, DeMarco V. Camper, Craig M. Goodwin, Allison L. Arnold, John L. Sensintaffar,
Anders Friberg, Olivia W. Rossanese, Taekyu Lee, Edward T Olejniczak, and Stephen W. Fesik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01660 • Publication Date (Web): 15 Feb 2016
Downloaded from http://pubs.acs.org on February 16, 2016
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Discovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia 1 (Mcl-1)
Inhibitors Using Fragment-Based Methods
#
Nicholas F. Pelz , Zhiguo Bian , Bin Zhao, Subrata Shaw, James C. Tarr, Johannes
#!
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Belmar, Claire Gregg, DeMarco V. Camper, Craig M. Goodwin, Allison L. Arnold, John
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L. Sensintaffar, Anders Friberg^, Olivia W. Rossanese , Taekyu Lee, Edward T.
Olejniczak, and Stephen W. Fesik*
Department of Biochemistry, Vanderbilt University School of Medicine, 2215
Garland Avenue, 607 Light Hall, Nashville, Tennessee 37232-0146, USA
#
These authors contributed equally to this work.
KEYWORDS:
Fragment-based screening; apoptosis; cancer; Mcl-1; drug discovery
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ABSTRACT
Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins
responsible for the regulation of programmed cell death (PCD). Amplification of Mcl-1 is
a common genetic aberration in human cancer whose overexpression contributes to the
evasion of apoptosis and is one of the major resistance mechanisms for many
chemotherapies. Mcl-1 mediates its effects primarily through interactions with pro-
apoptotic BH3 containing proteins that achieve high affinity for the target by utilizing
four hydrophobic pockets in its binding groove. Here we describe the discovery of Mcl-1
inhibitors using fragment based methods and structure-based design. These novel
inhibitors exhibit low nanomolar binding affinities to Mcl-1 and greater than 500-fold
selectivity over Bcl-xL. X-ray structures of lead Mcl-1 inhibitors when complexed to
Mcl-1 provided detailed information on how these small-molecules bind to the target, and
were used extensively to guide compound optimization.
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INTRODUCTION
The ability of tumor cell populations to increase in number is not only dependent
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, 2
upon their rate of proliferation, but also upon their rate of attrition . Apoptosis, or
programmed cell death (PCD), is a major source of cell attrition, and the evasion of
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apoptosis is a hallmark of cancer. The cell’s decision to undergo apoptosis is regulated
by a balance of pro-apoptotic and anti-apoptotic proteins that respond to various
extracellular and intracellular stress factors, including oxygen deprivation, DNA damage,
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oncogene signaling, and cytotoxic drugs . In cells, stress can induce the oligomerization
of the pro-apoptotic proteins Bax and Bak, which leads to the permeabilization of the
outer membrane of the mitochondrion, release of cytochrome C, and the initiation of
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caspase-dependent apoptosis . Anti-apoptotic proteins such as Bcl-2, Bcl-xL, Bcl-w,
Bcl-A1 (Bfl-1), and Mcl-1 guard against PCD by sequestering their pro-apoptotic
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relatives resulting in the inhibition of apoptosis . Overexpression or up-regulation of
the anti-apoptotic Bcl-2 family proteins enhance cancer cell survival and cause the
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resistance to a variety of anti-cancer therapies . Consequently, targeting the Bcl-2 family
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of proteins represents an attractive strategy for cancer drug discovery . Indeed, this
strategy was validated by the discovery of Navitoclax (ABT-263), a potent inhibitor of
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Bcl-xL, Bcl-w, and Bcl-2 . In addition, a potent Bcl-2 selective inhibitor, Venetoclax
(ABT-199), was recently discovered to avoid the thrombocytopenia observed by
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inhibiting Bcl-xL . Venetoclax is currently in Phase III clinical trials .
Early leads targeting another important member of the family, Mcl-1, have also
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recently been reported.
Mcl-1 is of considerable interest because it is the major cause
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of resistance to Navitoclax, Venetoclax,
as well as the widely prescribed anti-cancer
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agents paclitaxel, vincristine , and gemcitabine . Also, Mcl-1 amplification is one of
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the most common genetic aberrations observed in human cancers , and its
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overexpression is implicated in a variety of cancers, including leukemia, melanoma,
lung, breast, prostate, pancreatic, ovarian, and cervical cancers.
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The Bcl-2 family of proteins, including Mcl-1, are inherently difficult to target
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because they exert their effects through a large protein-protein interface . The BH3
regions of pro-apoptotic proteins interact with Mcl-1 utilizing four hydrophobic pockets
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in Mcl-1’s binding groove
. The BH3 region of Bcl-2 family proteins is an
amphipathic helix containing four key hydrophobic residues (H1-H4: L210, L213, V216,
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V220 in Mcl-1 BH3 PDB_ID: 3MK8 or ILIF in Bim BH3 PDB_ID: 2NL9 ) that are
essential for binding to the corresponding active site pockets P1-P4 of the anti-apoptotic
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Bcl-2 family members . Mutating any of these four residues to alanine significantly
reduces binding to Mcl-1. For example, for the Mcl-1 BH3 peptide, an 800 fold
decrease is observed by mutating the H2 residue, L213 to an alanine, while mutating H3
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V216A) or H4 (V220A) causes nearly a 100 fold drop in binding affinity to Mcl-1 .
We previously described the discovery of small-molecule Mcl-1 inhibitors that
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primarily bind to the P2 pocket
. In order to obtain a more potent Mcl-1 inhibitor, we
sought to extend our molecules to occupy the entire BH3 binding interface. This was
achieved using a fragment-based approach guided by structure-based design. Our efforts
rapidly led to novel, potent Mcl-1 inhibitors that access additional pockets in the BH3
peptide binding groove. Three-dimensional structures of these new lead compounds
when bound to Mcl-1 were extremely useful for designing inhibitors with better binding
affinity and improved physicochemical properties.
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RESULTS
Identification of Fragments that Bind to Additional Sites in the Mcl-1 BH3 Pocket
In order to guide the design of analogs that could bind to the P3 and P4 sites, we
conducted a fragment-based screen of 13,824 molecules while the primary P2 site of
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Mcl-1 was saturated with compound 1. The H/ N HSQC spectrum of compound 1 (K =
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5 nM) bound to Mcl-1 served as the control spectrum from which any additional
perturbations caused by fragment binding in other parts of the BH3-peptide binding
groove could be detected.
Seven compounds 2-8 were identified that bind with millimolar affinities to Mcl-1 in
the presence of 1, (Figure 1). Hit 8 was the most potent fragment from this screen (K =
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.5 mM). All of the fragments bind to the same site based on the similar chemical shift
perturbations that were observed. Although, these fragment hits only bind weakly to
Mcl-1, a significant gain in affinity is anticipated by linking to compounds that bind to
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the P2 site .
Fragment Linker Design
Based on the chemical shift perturbations observed upon the addition of the fragment
hits, we hypothesized that these molecules were binding to the hydrophobic pocket P4
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occupied by the H4 residue of BH3-peptides . In order to reach the P4 site, we explored
the possibility of replacing the carboxylic acid of compound 1 with an acylsulfonamide,
which would provide a synthetic handle for fragment linking while retaining the acidic
functionality important for the interaction with R263. The methyl acylsulfonamide 9 was
prepared, and it exhibited a 4-fold decrease in binding affinity when compared to the
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parent acid (1). To explain this decrease in affinity, the co-crystal structure of 9 bound to
Mcl-1 was obtained (Figure 2).
As shown in Figure 2, the methyl group of the acylsulfonamide of compound 9
points into the groove towards the P4 pocket. The acylsulfonamide group of 9 is next to
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R263 and maintains critical charged-charged interactions . One of the sulfonyl oxygens
is within H-bonding distance to the indole NH, which may increase the conformational
stability of the functional group when bound. The addition of the sulfonamide functional
group of 9 causes the molecule to tilt more than 2 Å away from the indole core position
of 1 (Figure 2C), which could explain the loss of binding affinity. Despite this loss in
affinity, the acylsulfonamide 9 has the advantage of providing a synthetic handle that
could be used to link to the P4 fragment hits.
To design flexible linkers between the P4 fragments and a P2 pocket binder, we
used the ternary structures of compound 10, and two of our fragment hits 2 and 8 (Figure
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). These two ternary structures reveal that fragments 2 and 8 bind to the P4 site and are
close to the methyl group of the acylsulfonamide. By superimposing the Mcl-1 BH3-
peptide onto the structures (Figure 3C), it can be seen that both fragments occupy the P4
site. The fluorinated side-chain of our tightest binding fragment (8) fits into the P4
pocket and mimics the buried methyl group of the valine residue of the Mcl-1 BH3
peptide (Figure 3B). The spacing observed in these structures suggest that a flexible
linker of three or four atoms could be used to link together compounds that bind in the
Mcl-1 P2 pocket with fragments that bind to the P4 site.
Optimization of the Fragment Linker
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Based on the two ternary structures, compounds with linkers containing two to four
atoms were designed, synthesized, and tested utilizing two different prototypical
fragments. A simple phenyl substituent was chosen to mimic the planar aromatic
fragment hits and a cyclohexyl moiety to mimic the other fragments (Table 1). Starting
from the methyl acylsulfonamide 11, with a binding affinity of 655 nM, a 2-fold affinity
gain was observed when a phenyl fragment was added using either a two-atom (12) or
three-atom (13) linker. The addition of the cyclohexyl group with the three-atom linker
as in compound 14 resulted in the greatest increase in binding affinity. However,
extending the linker by one methylene unit (15) or incorporating a basic amine (16)
caused a ten-fold decrease in potency from 14. Changing the amide connection to a
sulfonamide also reduced the binding affinity by a factor of two. These results suggest
that a three-atom linker was preferred, in agreement with the X-ray structures (Figure 3).
To separate the binding contribution of the cyclohexyl in the P4 site from the
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contributions of the flexible linker, we made compound 18. This compound exhibited a
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-fold reduction in affinity by replacing the cyclohexyl of 14 with a methyl group
suggesting that most of the added affinity of compound 14 comes from binding of the
fragment unit into the P4 pocket. Finally, incorporation of a chlorine atom at the six
position of the indole core resulted in an increase in binding affinity, as expected based
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on our previously described SAR . From these initial studies, we demonstrated that we
could link a P2 and P4 binder, increase the affinity and overcome the potency loss
inherent in replacing the carboxylic acid with an acylsulfonamide.
Before further optimizing our compounds, we obtained an X-ray crystal structure of
the linked compound 20, an analog of 19, containing a 1-naphthyl lower P2 pocket
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binder. Alignment of the new crystal structure with the previously attained ternary
structure (Figure 3A) shows a near perfect superposition (Figure 4) of the linked
compound and the separate P2 and P4 binders. The cyclohexyl ring sits directly atop
fragment 2, reaffirming P4 as a hotspot with the potential for being utilized to gain an
increase in binding affinity to Mcl-1. The linker spans a distance of 3.7 Å, almost
identical to the spacing of 3.6 Å obtained from the ternary structure (Figure 3A). Most
importantly, the binding conformation of the linked acylsulfonamide is now almost
identical to the binding pose adopted for compound 10, with only a slight rotation of the
acylsulfonamide methyl.
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Guided by the two ternary and linked compound co-crystal structures, we tested
analogs of our fragment hits 2-7 (Table 2). When compared to compound 14 (K = 118
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nM), incorporation of indole or benzofuran moieties (21-25) produced no improvement in
binding affinity, regardless of the point of attachment. In an effort to combine the spatial
characteristics of the indole and cyclohexyl fragments, a chiral indoline moiety was
incorporated. Compound 26 containing a 2-(S)-indoline had better binding affinity to
Mcl-1 than other fused bicyclic derivatives. Installation of the chlorine in the 6-position
further improved the binding affinity. The 2-(R)- indoline enantiomer 28 exhibited the
lowest K within the series (28 nM) with a 2-fold improvement compared to the
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benchmark cyclohexyl analog 19. These results suggest that a non-planar geometry at the
linking position is preferred.
Directly attaching pyrrole fragments 5 and 6 as exemplified in compounds 29 and 30
provided similar affinities as the cyclohexyl group of 14. Removal of the pyrrole N1
substitution resulted in a significant reduction in inhibitory activity against Mcl-1 as
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shown in compound 31. However, this loss in affinity was fully recovered by capping
the pyrrole NH with the methyl group (32), which resulted in a lower K than the analogs
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aromatic N1 substitutions of fragment hits 5 and 6 are unnecessary for binding. Both
compounds 33 and 34, which contain a furan as the P4 unit, were also potent Mcl-1
inhibitors, and the 2-furanyl attachment was preferred at this site over the 1-position.
Substitutions based on analogs of the tightest binding fragment 8 were also tested.
Initial linking of the 2-(1H-pyrazol-3-yl)phenol moiety in 35 increased binding affinity
by a factor of two over the simple methyl-acylsulfonamide 11. Reducing the fragment to
a phenyl ring in compound 13 had little effect on affinity which suggests that the 5-6
linked aromatic scaffold of hit 8 may not be essential for binding at the P4 site. This is
consistent with the co-crystal structure where the pyrazol of 8 is positioned outside of the
pocket. To mimic the fluorinated side chain of fragment 8, we tested simple fluorinated
phenyl analogs 36-38. These analogs showed a two to four-fold enhancement in affinity
over the parent 13 (K = 430 nM) with the meta-fluoro isomer 37 being the most potent.
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Attempts to extend the fluorine substituents further into the P4 hydrophobic pocket
produced trifluoromethyl derivatives 39-41. These compounds also improved the affinity
for Mcl-1 compared to 13 while showing a different positional preference for the
substitution. Unlike fluorine, substitution of the phenyl with a CF at the ortho-position
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(
39, K = 18 nM) is four to six times more potent than the meta- (40) and para-isomers
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41), respectively. These results suggest that introducing a fluorine atom at the correct
location in the P4 pocket can enhance affinity, and that the per-fluorinated ethyl group of
fragment 8 is likely the main contributor to its binding at this site.
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To test this hypothesis, we explored the effect of adding small aliphatic groups at the
P4 site. Compound 42, containing a methyl P4 moiety, was used as the benchmark to
further investigate this SAR. Adding a trifluoromethyl in 43 showed only a marginal
improvement in binding affinity. However, extension of the carbon chain by an ethyl
(44) or iso-propyl (45) group resulted in a two to three fold enhanced affinity for Mcl-1.
The inclusion of extended branched alkyl groups (46, 47) was found to significantly
increase the binding affinity. These SAR trends can be rationalized by a close
examination of the ternary structures of fragment 2 and 8 and the linked compound 20
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(Figure 5), in which the sp3-hybridized C4 cyclohexyl methylene of 20 aligns with the
planar indole and pyrazole units of the fragments. These data suggested that removal of
the C4 methylene would likely improve shape complementarity with the pocket. The
concept was applied in the design of compound 46, which showed a 4-fold improvement
in affinity when compared with the cyclohexyl analog 19. Finally, the most potent
inhibitor 47 (K = 10 nM) in this series was obtained by extending the branching point of
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the P4 moiety of 45 by one methylene, which led to an overall 7-fold affinity
enhancement. This substituent and its position is similar to that observed for the valine in
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the Mcl-1 BH3 peptide .
Our structural data (Figure 5) suggested that the flexible ethylene portion of the
linker could be replaced by a 5-6 membered ring to connect the P4 moiety to the
acylsulfonamide. This modification would reduce the number of rotatable bonds and
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could improve permeability and other drug like properties of the molecules . Various
rigid linker groups were tested (compounds 49-52) and it was found that incorporation of
cyclic aromatic groups resulted in higher affinities for Mcl-1 than the analogous methyl
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acylsulfonamides 9 and 11 (Table 3). To rationalize the improved affinity of the cyclic
linker units, a co-crystal structure of compound 49 bound to Mcl-1 was obtained (Figure
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49
50
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53
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59
60
acylsulfonamide group maintains the critical charged-charged interaction with R263
similar to the methyl analog 9 (Figure 6A). Unlike the methyl acylsulfonamide 9, the
conformation of the P2 binding unit in 49 adopts the higher affinity pose that was
observed in our original acid analog 1 (Figure 6B). These observed conformational
changes likely explain the improvement in affinity for 49 and 50. All of the Ar groups
listed in Table 3 gave similar affinities.
To further improve the binding affinity of the acylsulfonamide series, we
investigated the effect of substitutions on the indole core. In previous work, it was shown
that substitutions at the 7-position resulted in enhanced binding affinities for 2-indole
1
4
acids . To investigate if this was also true for our phenyl acylsulfonamides, we added
both 5 and 6-membered heteroaryl groups to the 7-position of the indole. As shown in
Table 4, the unsubstituted pyridine-3-yl group in 53 caused a marginal increase in affinity
while analogs 54 and 55 containing a methyl group next to the indole linking position
exhibited 10- and 26-fold enhanced potency against Mcl-1 relative to 49. These results
strongly suggested that substitutions on the 7-aryl group could enhance binding by
adopting a conformation where the methyl group points towards the P3 site much like the
14
earlier indole acids . This hypothesis was supported by compound 56 where an
orthogonal conformation of the 3,5-di-Me-1H-pyrazole group to the indole core would be
favored to relieve the steric congestion exerted by the two methyl groups. We next tested
13
if 6-Cl analogs would give a further synergistic boost in affinity . Compounds 57-59
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were prepared and when tested all of the 6-Cl analogs exhibited binding affinities below
the level able to be accurately determined in our Fluorescence Polarization Anisotropy
(FPA) assay conditions based on the concentration of Mcl-1 and the affinity of our
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peptide based FITC-Bak probe.
For compounds 56-59 (Table 4), with affinities higher than could be determined
in our original assay, we measured their K ’s in the presence of 1% Fetal Bovine Serum
i
(FBS). This approach assesses the effect of serum protein binding on the inhibitory
3
2
activity of the ligand to Mcl-1 , and affinities measured under the condition can be a
good predictor of activity in cellular assays. This approach has been used in other
1
4
33
studies of Mcl-1 and other Bcl-2 family members as a predictor of efficacy in cell
assays. Using this assay for ranking, we found that the 6-Cl group further enhanced the
series, with compound 59 having the highest potency in the presence of 1% FBS.
The binding mode of 49 (Figure 6) suggested that substitutions on the 4-position
of a phenyl or 5-position of a furan could be used to reach the P4 pocket. Because the
amide group of N260 sits along the path to P4, we decided to test polar linking groups.
Compounds 60-61, having a furan-5-carboxylic acid moiety, were prepared to test the
hypothesis. We found that their binding affinities were still well below the level able to
be accurately determined in our FPA assay. To understand their binding mode, a
structure of 60 complexed to Mcl-1 was determined (Figure 7). As expected, the di-
methyl pyrazole adopts a nearly orthogonal conformation to the indole core, and the 6-Cl
is in the P2 pocket adjacent to helix 4. In addition, the newly introduced carboxylic acid
is positioned in the groove within H-bonding distance to the amide NH of N260
explaining the observed enhancement in the binding affinity. It can also be seen in the
1
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structure that the NH of the pyrazole points out of the binding pocket, and thus
methylation of the pyrazole in 61 did not affect Mcl-1 affinity. These results suggest that
an additional group could be introduced at the NH of the pyrazole to improve the drug-
like characteristics of the inhibitors or to generate a small molecule probe for biochemical
assays without reducing binding to Mcl-1. Unfortunately, Both 60 and 61 do not show
measurable permeability in the PAMPA assay, which is likely due to having two acidic
functional groups.
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60
This information was used to make a new high affinity small molecule probe 62
by linking a fluorescein label (2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid) to the
NH of the pyrazole of 60 through a flexible spacer unit. Indeed, this probe exhibited a Kd
of 0.46 nM in FPA based equilibrium binding assay as a function of Mcl-1 concentration
(
Figure 8a). This probe is 37-fold more potent than the Bak peptide based probe (K = 17
d
nM) used in our first assay. The corresponding parent 60 and 61 were tested in
competitive binding experiments to test their ability to displace the probe 62 from Mcl-1
and showed K ’s of 0.36 and 0.78 nM (Figure 8b), respectively, that were in good
i
agreement with K of the labeled probe. The new probe has excellent sensitivity, aqueous
d
solubility, and chemical stability for a FPA binding assay and was the easiest to
implement in our workflow and was thus used to determine the affinities for all
subsequent potent Mcl-1 inhibitors.
Compounds 63-75 shown in Table 5 represent Mcl-1 inhibitors that contain all of
the features that we found to increase affinity in our exploratory SAR including an
optimized indole P2 unit and incorporation of P4 fragment with a rigid aromatic linker.
The first set of compounds 63-69 was constructed by connecting beneficial P4 binders
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6
identified from the earlier studies, to the 3 position of the phenyl linker with an amide
group. The attachment position and spacing are consistent with the Mcl-1 co-crystal
structure of compound 49 (Figure 6B). These compounds exhibited potent inhibitory
0
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activities beyond the detection limit of the Bak peptide probe, and had K ’s in the range
i
of 42-92 nM in the presence of 1% FBS. When binding affinities were measured using
the new small molecule probe 62, we found that the SAR trends found on less elaborated
scaffolds were mostly conserved when combined in the compounds of Table 5. The
increase in affinity from the parent core in 50 to the 7-tri-methyl-pyrazole analog 59 was
further compounded by addition of P4 binders. The high affinity of compound 66 (K =
i
2
.9 nM) showed 6-fold improvement in potency when compared to the parent 59, and the
3-furanyl group in 35 was also one of the optimal P4 substituents in the flexible linker
series.
Amide analogs of 61, compounds 70 and 71, were designed based on the Mcl-1
co-crystal structure with 60 (Figure 7). P4 site fragments were connected using a reverse
amide linker to preserve the beneficial H-bond found between the terminal acetate of 60
and N260. Indeed, compound 70 bound to Mcl-1 with high affinity (K = 2.4 nM) to
i
confirm the effectiveness of the new linker unit. Compound 71 containing the 4-pyridyl
P4 moiety showed 3.5-fold weaker Mcl-1 affinity than the phenyl analog 70, however in
1
2
the fact, compound 71 maintained the highest affinity among all examples in Table 5 in
1
% FBS suggesting that serum protein binding for the series could be minimized by the
-pyridyl moiety.
4
Our earlier SAR also suggested a pyridyl linker could be as effective as a phenyl
group (compound 49 vs. 51) and has better pharmaceutical properties. Therefore,
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compounds 72-75 were prepared to test the feasibility of a 4-pyridyl linker by connecting
P4 groups through an amine bridge. These compounds were also found to be very potent
Mcl-1 inhibitors with compounds 73 and 75 exhibiting K ’s of 1 nM.
i
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59
60
These results suggest that the rigid aromatic linker effectively delivers a P4 binder
with enhanced conformational stability while maintaining a high affinity conformation at
the P2 site. Indeed, compounds 63-75 have substantial affinity improvements (up to 17
fold in the presented examples) compared to the parent phenyl acylsulphonamide 59.
These compounds still have high protein binding and poor permeability (e.g. compound
-6
7
0, 72-75 have Pampa permeability < 3.0 10 cm/s) and these two factors likely
contribute to their high micromolar EC in cell viability studies (data not shown).
50
Although these compounds are potent Mcl-1 inhibitors, earlier work on Bcl-2 family
12
15
inhibitors including Mcl-1 indicate that picomolar inhibitors with low protein binding
is necessary for robust cell activity. In the supplementary material, we show evidence of
activity in a pull down assay in cell lysates. This experiment does not depend on cell
permeability and protein binding (Figure S1 Supplementary material).
Selectivity profile over Bcl-xL
The selectivity of compounds for Mcl-1 over Bcl-xL was tested using the anti-
apoptotic protein Bcl-xL in the Bak FPA assay (Table 5). As can be seen in Table 5, all
of the tested compounds exhibited only weak affinity for Bcl-xL with a greater than 500-
fold window in affinity. As found in our previous studies, structural modifications on the
series that enhance the affinity for Mcl-1 had relatively little effect on improving binding
to Bcl-xL. As a result, the compounds became more selective against Bcl-xL as they
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became more potent for Mcl-1. A selective Mcl-1 inhibitor is preferred because it could
be more easily co-dosed with selective inhibitors of other family members to induce
34
11
apoptosis and to manage potential liabilities .
0
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0
Synthesis
All acylsulfonamides were prepared according to methods outlined in Scheme 1-4.
The synthetic route employed for the preparation of the indole-carboxylic acid cores was
13, 16
reported previously
. Experimental details are described in the supporting
information. In Scheme 1, the simplest acyl sulfonamides were synthesized using the
general coupling reaction condition from either commercially available sulfonamides or
sulfonamides prepared from the parent sulfonyl chloride by treatment with ammonia. The
more elaborate linked acylsulfonamides were prepared through sulfonamide
derivatization followed by general coupling from the commercially available carboxylic
acids or acid chlorides when possible in a one-pot procedure. Linked acylsulfonamides
with simple aliphatic R-groups were prepared utilizing the linear synthesis method due to
the difficulty of monitoring the formation of the sulfonamide intermediates. 7-Ar-indole-
2
-phenyl-acylsulfonamide derivatives 53-59 shown in Scheme 2 were prepared from the
corresponding ethyl 7-Br-indole-2-carboxylate using Suzuki coupling protocols followed
by the general coupling reaction with phenylsulfonamide. Scheme 3 describes synthesis
of the fluorescein probe 62. The 3,5-dimethyl-pyrazol-1-yl acetate was installed on the
indole core then the Boc protected di-aminohexane spacing unit was coupled.
Subsequent saponification liberated 2-indole carboxylic acid, which was converted to the
furanyl-acylsulfonamide 81. Sequence of deprotection reactions yield the zwitterionic
intermediate 82. The fluorescein label was then connected via an amide linkage to give
1
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isomeric mixture of probe 62. Finally, Scheme 4 summarizes syntheses of fully
elaborated Mcl-1 inhibitors shown in Table 5.
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59
60
CONCLUSION
Here we demonstrated a fragment-based strategy to improve the affinity of Mcl-1
inhibitors by accessing additional pockets on Mcl-1. We showed that by accessing the P4
pocket we could substantially improve affinity. These studies resulted in compounds that
exhibited a binding affinity increase of a hundred fold from the initial methyl
acysulfonamide lead 9 and clearly demonstrates that affinity gains can be achieved by
filling additional pockets in the BH3 groove. Structural studies were important to guide
and then verify the correct position of the fragments in the final linked compound. Initial
benchmark studies using a flexible linker were used to identify minimal P4 binding units.
These data were exploited in the next stage when we used rigid scaffolds as linkers to
reduce the number of rotatable bonds in the final compounds. The structural and SAR
data obtained on these linked ligands can now be used to design more potent Mcl-1
inhibitors with better pharmaceutical properties.
EXPERIMENTAL SECTION
Chemistry
General. All NMR spectra were recorded at room temperature on a 400 MHz AMX
1
Bruker spectrometer. H chemical shifts are reported in δ values in ppm downfield with
the deuterated solvent as the internal standard. Data are reported as follows: chemical
shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m =
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6
multiplet), integration, coupling constant (Hz). Low resolution mass spectra were
obtained on an Agilent 1200 series 6140 mass spectrometer with electrospray ionization.
All samples were of ≥95% purity as analyzed by LC−UV/vis-MS. Analytical HPLC was
performed on an Agilent 1200 series with UV detection at 214 and 254 nm along with
ELSD detection. LC/MS parameters were as follows: Phenomenex-C18 Kinetex column,
0
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0
5
0 x 2.1 mm, 2 min gradient, 5% (0.1% TFA/MeCN) / 95% (0.1% TFA/ H O) to 100%
2
(0.1% TFA/MeCN). Preparative purification was performed on a Gilson HPLC
(Phenomenex-C18, 100 x 30 mm, 10 min gradient, 5→95% MeCN/H O with 0.1% TFA)
2
or by automated flash column chromatography (Isco, Inc. 100sg Combiflash). Solvents
for extraction, washing, and chromatography were HPLC grade. All reagents were
purchased from chemical suppliers and used without purification.
Protein expression and purification. A codon-optimized gene sequence encoding
residues 172-327 of human Mcl-1 (Uniprot: Q07820) was purchased (Genscript) and
cloned into a Gateway entry vector (pDONR-221, Invitrogen) using the protocols
provided. This construct was further sub-cloned into an expression vector (pDEST-
HisMBP) containing a maltose binding protein (MBP) tag for increased solubility, a
tobacco etch virus (TEV) protease recognition site for tag-removal and an N-terminal
His-tag to facilitate purification. The integrity of all plasmids was checked by
sequencing. Soluble Mcl-1 protein was expressed in Escherichia coli BL21 CodonPlus
(DE3) RIL (Stratagene) using ampicillin and chloramphenicol for selection.
In brief, a colony from a fresh transformation plate was picked to inoculate 100
mL of LB medium (37°C). The overnight culture was used to start a 10 L fermentation
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(
BioFlo 415, New Brunswick Scientific) grown at 37°C. When the cell density
corresponded to OD =2, the temperature was lowered to 20°C. After one hour, protein
600
expression was induced with 0.5 mM IPTG. Cells were harvested after 16 h by
centrifugation. Pellets were frozen and re-dissolved in lysis buffer (20 mM TRIS pH 7.5,
300 mM NaCl, 20 mM imidazole, 5 mM BME), approximately 100 mL / 10 g pellet,
before the cells were broken by homogenization (APV-2000, APV). Prior to application
to an affinity column (140 mL, ProBond, Invitrogen), lysate was cleared by
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centrifugation (18,000 rpm) and filtration (0.44 µm). Bound protein was washed on the
column and then eluted by a gradient (20 mM TRIS pH 7.5, 300 mM NaCl, 500 mM
imidazole, 5 mM BME). To enhance TEV protease cleavage, samples were buffer
exchanged (50 mM TRIS pH 7.5, 100 mM NaCl, 5 mM BME) on three serially
connected columns (HiPrep 26/10 Desalting, GE Healthcare). TEV protease was added to
a molar ratio of 1:10 (TEV:Mcl-1) and incubated at room temperature until cleavage was
complete. After adding 20 mM imidazole to the samples, they were passed over a
subtractive second nickel-column (120 mL, Ni-NTA Superflow, Qiagen) to remove the
MBP-tag, non-cleaved protein, and TEV protease. To achieve highly pure samples (e.g.
for crystal screening), a supplementary step of size-exclusion chromatography (HiLoad
2
6/60, Superdex 75, GE Healthcare) was implemented. The running buffer also acted as
the Mcl-1 storage and crystallography buffer (20 mM HEPES pH 6.8, 50 mM NaCl, 3
mM DTT, 0.01% NaN ). Purifications were done at 4°C, and concentration steps were
3
performed in stirred ultrafiltration cells (Amicon, Millipore).
To improve protein sample quality and to increase crystal diffraction, protein
mutants were created by site-directed mutagenesis (QuikChange, Agilent Technologies).
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Primers for a C-terminal deletion (∆5) were designed using their online tool and ordered
from Eurofins MWG Operon. Mutations were made on the entry vector above, analyzed
by in-house sequencing, and subsequently transferred into the pDEST-HisMBP
expression vector. Mutant proteins were purified in the same way as wild-type (WT)
proteins.
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Protein crystallization, data collection and structure refinement. Fresh batches of Mcl-
1 proteins, WT and ∆5, were concentrated to 600 µM (10.7 mg/mL) and 1 mM (17.4
mg/mL), respectively, and screened for crystallization conditions with a 1.2x excess of
ligand. Crystals were obtained by mixing 1 µL protein with 1 µL reservoir solution (25-
30% PEG 3350, 0.1 M Bis-TRIS pH 6.5, 0.2 M MgCl ) as a hanging drop at 4°C or
2
1
8°C. Crystals appeared within the first week and were flash frozen in liquid nitrogen
after cryoprotection using 10-20% glycol.
Data were collected on the Life Sciences Collaborative Access Team (LS-CAT)
2
1-ID-D beamline at the Advanced Photon Source (APS), Argonne National Laboratory.
3
5
Indexing, integration and scaling was performed with HKL2000 . Using a previously
determined ligand-bound structure (PDB: 4HW2), phasing was done by molecular
3
6
37
replacement with Phaser as implemented in CCP4 . Refinement of the structural
models were performed with Phenix and Refmac, and included rounds of manual model
3
8
39
building in COOT . Figures were prepared in PyMOL .
Competition Assays. A fluorescein isothiocyanate (FITC)-labeled BH3 peptide derived
from Bak (FITC-Bak-BH3; FITC-AHx-GQVGRQLAIIGDDINR-NH2) was purchased
from GenScript and used without further purification. For our most potent compounds a
new high affinity probe, 62 was used. FPA measurements were carried out in 384-well,
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black, flat-bottom plates (Greiner Bio-One) using the BioTek Cytation 3 plate reader. All
assays were conducted in assay buffer containing 20 mM TRIS pH 7.5, 50 mM NaCl, 3
mM DTT, and 5% final DMSO concentration. To measure displacement of the FITC-
Bak-BH3 peptide from Bcl-2 family members, 10 nM FITC-Bak-BH3 peptide was
incubated with either 15 nM Mcl-1 or 4 nM Bcl-xL. To measure displacement of 62 from
Mcl-1, 0.4 nM of 62, was incubated with 0.5 nM Mcl-1. For IC determination,
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60
5
0
compounds were diluted in DMSO in a 10-point, 3-fold serial dilution scheme, added to
assay plates, and incubated for 1.5 h at room temperature. The change in anisotropy was
measured and used to calculate an IC (inhibitor concentration at which 50% of bound
5
0
probe is displaced) by fitting the anisotropy data using XLFit (IDBS) to a four parameter
dose-response (variable slope) equation. This was converted into a binding dissociation
40
constant (K ) according to the formula :
i
K = [I] /([L] /K + [P] /K + 1)
i
50
50
d
0
d
where [I] is the concentration of the free inhibitor at 50% inhibition, [L] is the
5
0
50
concentration of the free labeled ligand at 50% inhibition, [P] is the concentration of the
0
pep
free protein at 0% inhibition and Kd represents the dissociation constant of the FITC-
labeled peptide probe. Compounds were evaluated using replicate measurement, in
duplicate; K values shown are the average of duplicate values.
i
FPA saturation binding assays for 62. A fluorescein labeled small molecule probe 62
was titrated with the Mcl-1 protein. FPA measurements were conducted under the same
condition as described above. To measure association of the probe to Mcl-1, 0.4 nM 62
was incubated with varying concentration of the Mcl-1 protein prepared by a 14-point, 2-
fold serial dilution with the top concentration equaling 100 nM.
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X-ray crystallography statistics, Dose-dependent blocking of Mcl-1 pulldown in cell
lysates. Synthesis details. This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
Atom coordinates and structure factors for Mcl-1/ligand complexes have been deposited
at the Protein Data Bank ID codes: 5FC4, 5FDO, 5FDR
AUTHOR INFORMATION
Corresponding Author
*
Phone: +1 (615) 322 6303. Fax: +1 (615) 875 3236. E-
mail:stephen.fesik@vanderbilt.edu.
Present Addresses
!
AbbVie Pharmaceuticals, North Chicago, IL
Bayer HealthCare, Berlin 13353, Germany
^
+ The institute of cancer research, Sutton, UK
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors thank co-workers at the High-Throughput Screening Core facility of
Vanderbilt University, TN, for compound management and providing the instrumentation
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to perform the binding assay. This research was supported by the U.S. National Institutes
of Health, NIH Director’s Pioneer Award DP1OD006933/DP1CA174419 to S.W.F., The
NCI Experimental Therapeutics (NExT) Program BOA29XS129TO22 under the Leidos
Biomed Prime Contract No. HHSN261200800001E, and a career development award to
S.W.F. from a NCI SPORE grant in breast cancer (Grant P50CA098131) to C. L.
Arteaga. The Biomolecular NMR Facility at Vanderbilt University is supported in part
by a NIH SIG Grant 1S-10RR025677-01 and Vanderbilt University matching funds. Use
of the Advanced Photon Source was supported by the U.S. Department of Energy, Office
of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.
ABBREVIATIONS
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Mcl-1, myeloid cell leukemia 1; Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma
extra large; BH3, Bcl-2 homology domain 3; Bax, Bcl-2-associated X protein; Bak, Bcl-2
homologous antagonist killer; FITC, fluorescein isothiocyanate; FPA, fluorescence
polarization anisotropy.
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