Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design

Abstract

Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified two chemically distinct hit series that bind to different sites on Mcl-1. Members of the two fragment classes were merged together to produce lead compounds that bind to Mcl-1 with a dissociation constant of <100 nM with selectivity for Mcl-1 over Bcl-xL and Bcl-2. Structures of merged compounds when complexed to Mcl-1 were obtained by X-ray crystallography and provide detailed information about the molecular recognition of small-molecule ligands binding Mcl-1. The compounds represent starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.

Figure 1

¹H-¹⁵N HMQC spectra of Mcl-1 with (red) and without (black) fragment 3. The NMR sample contained 50μM ¹⁵N-labeled protein and 800HM ligand.

Figure 2

Model structures of Mcl-1 complexed to two different classes of fragment hits obtained using NMR-derived distance restraints. Residues (labeled) with NOEs to the fragments (blue) are rendered as sticks (gray). R263 is also labeled. (A) Class I: benzothiophene 5, and (B) Class II: tethered aromatic 17.

Figure 3

Schematic illustrating the merging of fragment 2 and 17 to produce compound 60. The affinity of each molecule to Mcl-1 is shown as well as the ligand efficiencies.

Figure 4

X-ray structures of merged compounds bound to Mcl-1. Compounds incorporating different scaffolds (benzothiophene and indole) occupy the same binding pocket and adopt very similar binding poses (inset). (A) Compounds 53 and 60 interact with Mcl-1 in the BH3-peptide binding cleft between helices 3, 4, and 5. Surface depiction of Mcl-1 when complexed to (B) 60 and (C) 53 that illustrates how the merged compounds fill the pockets as predicted by NMR and interact with R263.

Figure 5

Several structural differences between peptide-bound (red) and compound-bound (gray) Mcl-1 complexes were identified. The Mcl-1-derived BH3-peptide is depicted in green and compound 60 in blue. (A) To accommodate the hydrophobic fragments into the deep pocket, helix 4 (α4) is slightly kinked in the structures of Mcl-1/compound complexes compared to the Mcl-1/peptide structures. This leads to further rearrangements of the loop, pulling it towards the merged compound. (B) Close-up of the small-molecule binding-site in Mcl-1. Compared to the peptide-bound state, our merged compounds induce changes in the position and/or in rotamer states of nearby residues. (C-D) The network of charge-charge interactions and polar contacts involving R263 are dramatically different in the two types of complexes. (C) While bound to the peptide, R263 of Mcl-1 interacts closely with D218 of the 16mer, a residue shown important by alanine-scanning. Moreover, R263 interacts with the side-chain of D256 and the backbone (bb) oxygen of V258 in Mcl-1. (D) When complexed with merged compounds (e.g. 60), this network is replaced by an interaction to the carboxylic acid of the small-molecule ligand and additional polar contacts to the backbone oxygen atoms of S255 and D256.

Figure 6

(A) Comparison of Mcl-1 (gray surface) when complexed to compound 60 (blue) and a 16mer BH3-peptide derived from Mcl-1 (green), (B) Overlay of the X-ray structures of Mcl-1/60 complex and Bcl-xL/ABT-737 (PDB: 2YXJ). The Mcl-1 protein is shown in gray, 60 in blue, and ABT-737 in green.

Scheme 1

Synthesis of benzothiophene- and benzofuran-containing compounds

Scheme 2

Synthesis of indole-based compounds

Scheme 3

Synthesis of compounds 69-72