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Progress in the Search for Mcl-1-Targeted Anticancer Agents

 

 

By: Carol A. Rouzer, VICB Communications
Published: March 7, 2016

 

 

Fragment-based discovery and structure-guided design yield new 2-indole-acylsulfonamide-based inhibitors of Mcl-1-dependent protein-protein interactions.

 

In cells responding to severe stress, the proapoptotic proteins Bax and Bak form oligomers at the outer mitochondrial membrane. These oligomers create a pore that allows cytochrome c to escape from the mitochondrion into the cytosol, resulting in caspase activation and, ultimately, death of the cell by apoptosis. This process is suppressed by antiapoptotic proteins, including Bcl-2, Bcl-xL, Bcl-A1, and Mcl-1, which bind to and sequester Bax and Bak. Whether or not the cell lives or dies depends on the balance between the pro- and antiapoptotic processes. Overexpression of antiapoptotic proteins, particularly in some cancer cells, can lead to inappropriate survival of abnormal or badly damaged cells. This is one mechanism by which cancer cells survive in the face of extensive genomic damage. It is also a mechanism by which cancer cells develop resistance to the toxic effects of chemotherapy. Mcl-1 is one of the most frequently overexpressed proteins in cancer, and for many types of cancer, Mcl-1 overexpression is a marker of aggressive disease and a poor prognosis. Thus, Mcl-1 is an important chemotherapeutic target. Now, Vanderbilt Institute of Chemical Biology member Steve Fesik and his laboratory report exciting progress in the development of small molecule inhibitors of Mcl-1 [N. F. Pelz, et al. (2016) J. Med. Chem., published online February 15, DOI:10.1021/acs.jmedchem.5b01660].

 

 

Figure 1.  Structures of Compounds 1 and 9 along with the two fragments with the highest affinity for Mcl-1.

 

 

Mcl-1 carries out its function primarily through protein-protein interactions that occur over a large binding surface. Proteins that bind to Mcl-1 do so through their BH3 domains, amphipathic helices that contain four hydrophobic residues, each of which binds to a corresponding hydrophobic binding pocket (P1 through P4) on Mcl-1. Through previous work, the Fesik lab had discovered Compound 1 (Figure 1)( A. Friberg, et al. (2013) J. Med. Chem., 56, 15), which exhibited high affinity binding to the P2 pocket of Mcl-1. Their plan was to identify molecules that bind to other regions of the protein and then link those molecules to Compound 1, thereby creating a new molecule with substantially higher affinity. To accomplish this goal, they first screened a 13,824 fragment library using two-dimensional NMR spectroscopy to search for small molecules that bind to Mcl-1 in the presence of Compound 1. The screen identified seven compounds, including Fragments 2 and 8 shown in Figure 1. These compounds all bound to Mcl-1 with affinities in the millimolar range. NMR data suggested that all of the compounds bound to Mcl-1 in the P4 binding pocket.

To provide a site on Compound 1 that could be used to link it to another molecule in P4, the investigators replaced the carboxylate with an acylsulfonamide, yielding Compound 9 (Figure 1). The intent was to retain a critical polar interaction with arginine-263 of Mcl-1 while also providing a site that could be used to add a linker to the second molecule. Replacement of Compound 1’s carboxyl group with the acylsulfonamide resulted in a substantial reduction in potency, but an X-ray crystal structure obtained on the Compound 9-Mcl-1 complex confirmed that it bound in the correct general orientation to enable the desired linkage. The structure also suggested that the loss in potency was due to a slight tilt of Compound 9’s indole ring relative to that of Compound 1.

 

To guide the design of a linker to connect Compound 9 with a P4-binding fragment, the investigators obtained crystal structures of ternary complexes of Mcl-1 with Compound 10 (a Compound 9 analog) and either Fragment 2 or Fragment 8 (Figure 2). The results confirmed that both fragments bound to P4 and suggested that a 3- to 4-atom linker should be optimal to connect them to Compound 9. Based on these results, the researchers constructed a series of compounds bearing a variety of linkers attached to a phenyl or cyclohexyl group, which served as a simple P4-binding fragment. The results of these efforts yielded Compound 19 (Figure 3), an analog of Compound 9 bearing a 4-atom amide linker connected to a cyclohexyl group. Compound 19 exhibited a 4-fold increase in affinity relative to that of Compound 9. Replacement of Compound 19’s cyclohexyl ring with a methyl group resulted in a substantial loss of affinity, confirming the importance of an interaction with the P4 pocket. Furthermore, an X-ray crystal structure of a complex of Mcl-1 and Compound 20, a close analog of Compound 19, confirmed that the linker was positioned as predicted, and the cyclohexyl group bound in the same general area as Fragments 2 and 8 (Figure 4).

 

 


 

Figure 2. X-Ray co-crystal structures of (A) Fragment 2 bound to Mcl-1 in the presence of Compound 10 (D), and (B) Fragment 8 bound to Mcl-1 in the presence of Compound 10. (C) Superposition of a BH3 peptide with Fragments 2 and 8 confirms that the fragments bind in P4 of Mcl-1. Figure reproduced with permission from N. F. Pelz, et al. (2016) J. Med. Chem., published online February 15, DOI:10.1021/acs.jmedchem.5b01660. Copyright 2016 American Chemical Society.

 



Figure 3. Structures of selected compounds resulting from the exploration of various P4 binding groups using a short alkyl chain linker.

 

 

 

Figure 4. X-Ray crystal structure of a co-complex of Compound 20 with Mcl-1 overlaid with the structures of Fragment 2- and Fragment 8-Mcl-1 complexes. Figure reproduced with permission from N. F. Pelz, et al. (2016) J. Med. Chem., published online February 15, DOI:10.1021/acs.jmedchem.5b01660. Copyright 2016 American Chemical Society.

 

 

Having identified a linker that could effectively join Compound 9 with molecules in P4, the investigators next replaced Compound 19’s cyclohexyl group with various fragments. Examples of the more potent compounds are shown in Figure 3. Replacement of the cyclohexyl group with a 2-(R)-indoline enatiomer (Compound 28) resulted in a 2-fold improvement in affinity. The mode of attachment of the indoline group that resulted in the best affinity suggested that a nonplanar geometry at the linker position was optimal. The structure of Fragment 8 suggested a number of analogs bearing fluorinated alkyl chains. Of these, the compound that exhibited the highest affinity (Compound 39) achieved a 3-fold improvement over Compound 9. However, the best compound of the series (Compound 47) was notable for its relatively simple isobutyl P4 binding substituent. This compound achieved a 5-fold increase in potency relative to that of Compound 9.

 

Structural data suggested that the ethylene portion of the linker of Compound 9 could be replaced with a 5- or 6-membered ring. This type of substitution would reduce the flexibility of the molecule, increasing the likelihood of achieving the desired binding interaction with the protein. Indeed, replacement of the methyl group on the acylsulfonamide of Compound 9 with either a phenyl or a 1-furanyl group resulted in a 2-fold increase in affinity. An X-ray crystal structure of the phenyl-substituted compound in complex with Mcl-1 confirmed that the critical polar interaction with arginine-263 was retained, the indole ring had tilted back into the higher affinity pose observed with Compound 1, and the phenyl substituent was pointed in the correct direction to serve as a linker for P4-binding fragments.

 

The investigators next turned their attention to optimization of the indole core of the molecule that binds in the P2 pocket. They found that addition of a substituted pyridinyl or pyrazolyl group at position 7 and a chloro substituent at position 6 generated compounds with affinities too high to measure with their standard assay, which was based on displacement of a fluorescent-labeled BH3 peptide from MCl-1. To be able to measure the affinities of these compounds required a higher affinity probe. They achieved this goal with the discovery of Compound 62, (Kd = 0.46 nM), synthesized by the addition of a fluorescent probe to the 4-position of a pyrazole substituent at position 7 of the indole ring (Figure 5).

 

 

 

Figure 5. Figure 5. Structure of Compound 62, an Mcl-1-targeted fluorescent probe.

 

 

Finally, the investigators combined the information gained from all of their SAR studies. They synthesized compounds containing the optimized P2 binding unit with a rigid linker and a variety of P4 fragments. Examples of some of the compounds with the highest affinities are shown in Figure 6. The resulting compounds exhibited affinity constants in the range of 1 to 16 nM in the absence of serum and 42 to 266 nM in the presence of 1% serum. All compounds also exhibited a high selectivity for Mcl-1 as opposed to Bcl-Xl, an important property as Bcl-Xl inhibition has been associated with thrombocytopenia (a reduction in the number of platelets) in vivo. These results are a major advance over the binding affinity of Compound 1. However, prior experience with the discovery of inhibitors to other Bcl-2-family proteins suggests that affinities in the picomolar range and very low protein binding are necessary for in vivo efficacy. Thus, despite their quite high affinity for Mcl-1, these new molecules represent a step on the way to a new class of cancer chemotherapeutic agents, but not the final destination.

 

 

Figure 6. Structures of Compound 59, incorporating optimizations of the indole nucleus and a rigid linker, along with selected compounds built on the SAR derived from all of these studies.

 

 

 

View J Med Chem article: "Discovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods"

 

 

 

 

 

 

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