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New Discoveries in the Search for Mcl-1-Targeted Anticancer Agents

 

By: Carol A. Rouzer, VICB Communications
Published:  April 20, 2015

 

 

A combination of fragment-based discovery and structure-guided design leads to new high affinity 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 an important new lead in the development of small molecule inhibitors of Mcl-1 [J. P. Burke, et al. (2015) J. Med. Chem., published online April 6, DOI:10.1021/jm501984f].

 

Mcl-1 carries out its function primarily through protein-protein interactions that occur over a large binding surface. Consequently, Mcl-1 lacks the well-defined binding pockets typically found in proteins that interact with small molecule ligands. The absence of such binding pockets poses a challenge for the discovery of small molecules that will establish high affinity interactions with Mcl-1. To overcome these challenges, the Fesik lab used a combination of fragment-based discovery and structure-based design. Fragment-based discovery identifies small molecules that bind to the protein even if the affinity of binding is fairly low. Structure-based design then exploits structural information on how the small “fragment” molecules bind to the protein, providing key information enabling chemists to chemically link them together, yielding a much higher affinity ligand. Application of this approach had already yielded an Mcl-1 inhibitor with 55 nM binding affinity (Compound 1, Figure 1)( A. Friberg, et al. (2013) J. Med. Chem., 56, 15). However, the original screen of 15,000 small molecules that led to Compound 1 also revealed a second series of compounds based on a tricyclic indole 2-carboxylic acid scaffold (Compound 2, Figure 1) that exhibited Mcl-1 binding affinity. The Fesik lab decided to explore this series in an effort to identify more potent Mcl-1 inhibitors.

 

 


Figure 1. Structures of Compounds 1 and 2, and their affinities for Mcl-1.

 


Initial structure-activity relationship (SAR) studies focused on varying substituents at positions R1 and R2 and altering the C ring at position Z of Compound 2 as indicated on Figure 2. They found that compounds containing a methyl group at the R1 and R2 positions exhibited higher affinity for Mcl-1 in a fluorescence polarization anisotropy (FPA) assay than those lacking a substituent at that position. They also found that compounds containing a morpholine (Z = oxygen) or thiomorpholine (Z = sulfur) C ring were more potent than those containing a piperidine (Z = C) C ring.

 



Figure 2. Structure of Compound 2, indicating initial sites of modification.

 

 

To guide their further medicinal chemistry efforts, the investigators explored the binding of Compound 2 with Mcl-1 using nuclear magnetic resonance. The results identified amino acids of Mcl-1 that interact with Compound 2, allowing the researchers to propose a model for the binding site (Figure 3). The model placed Compound 2 in the upper region of the previously defined P2 binding pocket of Mcl-1. A key finding was the orientation of Compound 2 in this pocket, which the model predicted would point any substituent at the R1 position (Figure 2) into the deeper part of the pocket. Their earlier work with Compound 1 had demonstrated that joining a molecule that binds in the upper part of the P2 pocket with another that binds deep in the pocket produces a new molecule with much higher binding affinity than either of the two starting compounds. The work with Compound 1 had already identified a number of molecules that could bind deep in the P2 pocket. Thus, the investigators’ next task was to link these to Compound 2 or other molecules that share the Compound 2 scaffold.

 

 

 


Figure 3.
 NMR-based model of binding of Compound 2 to Mcl-1. (A) Structure of Compound 2 showing points of interaction with the designated amino acid residues of Mcl-1. (B) Model showing the orientation of Compound 2 in the Mcl-1 P2 binding pocket. Figure reproduced with permission from J. P. Burke, et al. (2015) J. Med. Chem., published online April 6, DOI:10.1021/jm501984f. Copyright 2015 American Chemical Society.

 

 

The chemists first linked a 1-naphthyl group to compound 2 analogs using a 3- or 4-carbon linker. They obtained new compounds with markedly improved affinity when they used the 4-carbon linker. They also found that they could modify the C ring of Compound 2, substituting sulfoxide or sulfone at the Z position (Figure 2), or even expanding the ring by adding an additional carbon atom. They also found that linking a 4-chloro-3,5-dimethyl-phenyl fragment to the Compound 2 scaffold worked as well as linking a 1-naphthyl group.

 

 

 

Figure 4. Structures of Compounds 20 and 24.



To further guide their optimization efforts, the researchers obtained co-crystal structures of Mcl-1 complexed with Compound 20 and Compound 24 (Figure 4). The structural data (Figure 5) revealed that both compounds adopted very similar binding poses in the P2 pocket of the protein. As expected, the tricyclic indole and naphthyl portions of the molecule were positioned in the upper and deep regions, respectively, of the P2 pocket. Of particular importance was the finding that there was space in the binding pocket in the region of the 6-position of the indole ring, suggesting that adding a substituent at that position could increase the binding affinity of the compounds even further. The medicinal chemists tested this hypothesis by synthesizing two compounds (34 and 35, Figure 6) containing a chloro group at this position. The result was a striking increase in affinity as indicated by Ki values of 3 nM and 9 nM for Compounds 34 and 35, respectively.

 

 

 

Figure 5. Co-crystal structures of Compound 20 (A) and Compound 24 (B) with Mcl-1. The arrows indicate empty space that was exploited for the design of more potent inhibitors. Figure reproduced with permission from J. P. Burke, et al. (2015) J. Med. Chem., published online April 6, DOI:10.1021/jm501984f. Copyright 2015 American Chemical Society.

 

 

 

Figure 6. Structures of Compounds 34 and 35.

 

 

Preclinical and clinical studies have shown that inhibition of Bcl-2 and Bcl-xL is associated with suppression of platelet formation leading to thrombocytopenia. Thus, the investigators tested their most promising inhibitors for activity against these proteins. In general, they found that high affinity binding to Mcl-1 was associated with poor affinity for Bcl-2 and Bcl-xL. Compounds 34 and 35 exhibited a >250 and >140 selectivity, respectively, for Mcl-1 over Bcl-2 and Bcl-xL (Figure 6).


For their routine binding affinity assays, the investigators used a truncated form of Mcl-1 (residues 172-327) to improve the stability and solubility of the protein. To verify that their inhibitors retained binding affinity for the native protein, they tested the ability of Compound 34 to inhibit the interaction of the biotin-labeled Mcl-1 binding peptide, MS-1, with Mcl-1 in lysates from human chronic myelogenous leukemia cells. In the absence of Compound 34, MS-1 could be used together with streptavadin-bound beads to pull down Mcl-1 from the cell lysates. The investigators found that addition of Compound 34, at concentrations of 1 to 100 μM, blocked the ability of MS-1 to pull down Mcl-1 in a concentration-dependent manner.


Compound 34 and others in this series clearly represent a promising new lead in the quest to find clinically useful inhibitors of Mcl-1. However, the researchers note that very recent new findings from investigators at AbbVie, Inc. (J. D. Leverson, et al., (2015) Cell Death and Disease, 6, e1590) suggest that even higher affinities will be required to achieve Mcl-1-specific inhibitory effects in vivo. Thus, the quest for even more potent inhibitors will continue.

 

 

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