Discovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods
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.
INTRODUCTION<!>Identification of Fragments that Bind to Additional Sites in the Mcl-1 BH3 Pocket<!>Fragment Linker Design<!>Optimization of the Fragment Linker<!>Selectivity profile over Bcl-xL<!>Synthesis<!>CONCLUSION<!>General<!>Protein expression and purification<!>Protein crystallization, data collection and structure refinement<!>Competition Assays<!>FPA saturation binding assays for 62<!>
<p>The ability of tumor cell populations to increase in number is not only dependent upon their rate of proliferation, but also upon their rate of attrition1, 2. Apoptosis, or programmed cell death (PCD), is a major source of cell attrition, and the evasion of apoptosis is a hallmark of cancer.3, 4 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, oncogene signaling, and cytotoxic drugs1. 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 caspase-dependent apoptosis5. 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 relatives resulting in the inhibition of apoptosis2, 6. Overexpression or up-regulation of the anti-apoptotic Bcl-2 family proteins enhance cancer cell survival and cause the resistance to a variety of anti-cancer therapies7. Consequently, targeting the Bcl-2 family of proteins represents an attractive strategy for cancer drug discovery8, 9. Indeed, this strategy was validated by the discovery of Navitoclax (ABT-263), a potent inhibitor of Bcl-xL, Bcl-w, and Bcl-210. In addition, a potent Bcl-2 selective inhibitor, Venetoclax (ABT-199), was recently discovered to avoid the thrombocytopenia observed by inhibiting Bcl-xL11. Venetoclax is currently in Phase III clinical trials12.</p><p>Early leads targeting another important member of the family, Mcl-1, have also recently been reported.13–18 Mcl-1 is of considerable interest because it is the major cause of resistance to Navitoclax, Venetoclax,19, 20 as well as the widely prescribed anti-cancer agents paclitaxel, vincristine21, and gemcitabine22. Also, Mcl-1 amplification is one of the most common genetic aberrations observed in human cancers23, and its overexpression24 is implicated in a variety of cancers, including leukemia, melanoma, lung, breast, prostate, pancreatic, ovarian, and cervical cancers.</p><p>The Bcl-2 family of proteins, including Mcl-1, are inherently difficult to target because they exert their effects through a large protein-protein interface25. The BH3 regions of pro-apoptotic proteins interact with Mcl-1 utilizing four hydrophobic pockets in Mcl-1's binding groove26, 27. The BH3 region of Bcl-2 family proteins is an amphipathic helix containing four key hydrophobic residues (H1-H4: L210, L213, V216, V220 in Mcl-1 BH3 PDB_ID: 3MK828 or ILIF in Bim BH3 PDB_ID: 2NL929) that are essential for binding to the corresponding active site pockets P1-P4 of the anti-apoptotic Bcl-2 family members27. 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 (V216A) or H4 (V220A) causes nearly a 100 fold drop in binding affinity to Mcl-128.</p><p>We previously described the discovery of small-molecule Mcl-1 inhibitors that primarily bind to the P2 pocket13, 16. 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.</p><!><p>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 Mcl-1 was saturated with compound 1. The 1H/15N HSQC spectrum of compound 1 (Ki= 55 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.</p><p>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 (Kd = 1.5 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 the P2 site30.</p><!><p>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 occupied by the H4 residue of BH3-peptides27. 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 parent acid (1). To explain this decrease in affinity, the co-crystal structure of 9 bound to Mcl-1 was obtained (Figure 2).</p><p>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 R263 and maintains critical charged-charged interactions13. 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.</p><p>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 3). 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.</p><!><p>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 contributions of the flexible linker, we made compound 18. This compound exhibited a 6-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 on our previously described SAR13. 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.</p><p>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 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.</p><p>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 (Ki = 118 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 Ki within the series (28 nM) with a 2-fold improvement compared to the benchmark cyclohexyl analog 19. These results suggest that a non-planar geometry at the linking position is preferred.</p><p>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 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 Ki than the analogs 14, 29 and 30. These results suggest that an H-bond donor at P4 is not beneficial, and 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.</p><p>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 (Ki = 430 nM) with the meta-fluoro isomer 37 being the most potent. 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 CF3 at the ortho-position (39, Ki = 18 nM) is four to six times more potent than the meta- (40) and para-isomers (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.</p><p>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 (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 (Ki = 10 nM) in this series was obtained by extending the branching point of 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 the Mcl-1 BH3 peptide28.</p><p>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 could improve permeability and other drug like properties of the molecules31. 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 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 6). The structure shows that the phenyl linker points to the P4 pocket, while the 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.</p><p>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 acids14. 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 earlier indole acids14. 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 if 6-Cl analogs would give a further synergistic boost in affinity13. Compounds 57–59 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 peptide based FITC-Bak probe.</p><p>For compounds 56–59 (Table 4), with affinities higher than could be determined in our original assay, we measured their Ki's in the presence of 1% Fetal Bovine Serum (FBS). This approach assesses the effect of serum protein binding on the inhibitory activity of the ligand to Mcl-132, and affinities measured under the condition can be a good predictor of activity in cellular assays. This approach has been used in other studies of Mcl-114 and other Bcl-233 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.</p><p>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 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.</p><p>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 (Kd = 17 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 Ki's of 0.36 and 0.78 nM (Figure 8b), respectively, that were in good agreement with Kd of the labeled probe. The new probe has excellent sensitivity, aqueous 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.</p><p>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 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 activities beyond the detection limit of the Bak peptide probe, and had Ki's in the range 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 (Ki = 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.</p><p>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 (Ki = 2.4 nM) to 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 the 12fact, 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 4-pyridyl moiety.</p><p>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, 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 Ki's of 1 nM.</p><p>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 70, 72–75 have Pampa permeability < 3.0 10−6 cm/s) and these two factors likely contribute to their high micromolar EC50 in cell viability studies (data not shown). Although these compounds are potent Mcl-1 inhibitors, earlier work on Bcl-2 family inhibitors12 including Mcl-115 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).</p><!><p>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 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 apoptosis34 and to manage potential liabilities11.</p><!><p>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 reported previously13, 16. 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 isomeric mixture of probe 62. Finally, Scheme 4 summarizes syntheses of fully elaborated Mcl-1 inhibitors shown in Table 5.</p><!><p>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.</p><!><p>All NMR spectra were recorded at room temperature on a 400 MHz AMX Bruker spectrometer. 1H 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 = 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, 50 × 2.1 mm, 2 min gradient, 5% (0.1% TFA/MeCN) / 95% (0.1% TFA/H2O) to 100% (0.1% TFA/MeCN). Preparative purification was performed on a Gilson HPLC (Phenomenex-C18, 100 × 30 mm, 10 min gradient, 5→95% MeCN/H2O with 0.1% TFA) 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.</p><!><p>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.</p><p>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 (BioFlo 415, New Brunswick Scientific) grown at 37°C. When the cell density corresponded to OD600=2, the temperature was lowered to 20°C. After one hour, protein 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 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 26/60, 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% NaN3). Purifications were done at 4°C, and concentration steps were performed in stirred ultrafiltration cells (Amicon, Millipore).</p><p>To improve protein sample quality and to increase crystal diffraction, protein mutants were created by site-directed mutagenesis (QuikChange, Agilent Technologies). Primers for a C-terminal deletion (Δ5) 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.</p><!><p>Fresh batches of Mcl-1 proteins, WT and Δ5, 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 MgCl2) as a hanging drop at 4°C or 18°C. Crystals appeared within the first week and were flash frozen in liquid nitrogen after cryoprotection using 10–20% glycol.</p><p>Data were collected on the Life Sciences Collaborative Access Team (LS-CAT) 21-ID-D beamline at the Advanced Photon Source (APS), Argonne National Laboratory. Indexing, integration and scaling was performed with HKL200035. Using a previously determined ligand-bound structure (PDB: 4HW2), phasing was done by molecular replacement with Phaser36 as implemented in CCP437. Refinement of the structural models were performed with Phenix and Refmac, and included rounds of manual model building in COOT38. Figures were prepared in PyMOL39.</p><!><p>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, 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 IC50 determination, 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 IC50 (inhibitor concentration at which 50% of bound 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 constant (Ki) according to the formula40: 
Ki=[I]50/([I]50/Kd+[P]0/Kd+1) where [I]50 is the concentration of the free inhibitor at 50% inhibition, [L]50 is the concentration of the free labeled ligand at 50% inhibition, [P]0 is the concentration of the free protein at 0% inhibition and Kdpep represents the dissociation constant of the FITC-labeled peptide probe. Compounds were evaluated using replicate measurement, in duplicate; Ki values shown are the average of duplicate values.</p><!><p>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.</p><!><p>ASSOCIATED CONTENT</p><p>
          Supporting Information
</p><p>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.</p><p>
          Accession Codes
</p><p>Atom coordinates and structure factors for Mcl-1/ligand complexes have been deposited at the Protein Data Bank ID codes: 5FC4, 5FDO, 5FDR</p><p>The authors declare no competing financial interest.</p>