Discovery of Potent Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors That Demonstrate in Vivo Activity in Mouse Xenograft Models of Human Cancer.

Overexpression of myeloid cell leukemia-1 (Mcl-1) in cancers correlates with high tumor grade and poor survival. Additionally, Mcl-1 drives intrinsic and acquired resistance to many cancer therapeutics, including B cell lymphoma 2 family inhibitors, proteasome inhibitors, and antitubulins. Therefore, Mcl-1 inhibition could serve as a strategy to target cancers that require Mcl-1 to evade apoptosis. Herein, we describe the use of structure-based design to discover a novel compound (42) that robustly and specifically inhibits Mcl-1 in cell culture and animal xenograft models. Compound 42 binds to Mcl-1 with picomolar affinity and inhibited growth of Mcl-1-dependent tumor cell lines in the nanomolar range. Compound 42 also inhibited the growth of hematological and triple negative breast cancer xenografts at well-tolerated doses. These findings highlight the use of structure-based design to identify small molecule Mcl-1 inhibitors and support the use of 42 as a potential treatment strategy to block Mcl-1 activity and induce apoptosis in Mcl-1-dependent cancers.

Small Molecule SOS1 Agonists Modulate MAPK and PI3K Signaling via Independent Cellular Responses.

Activating mutations in RAS can lead to oncogenesis by enhancing downstream signaling, such as through the MAPK and PI3K pathways. Therefore, therapeutically targeting RAS may perturb multiple signaling pathways simultaneously. One method for modulating RAS signaling is to target the activity of the guanine nucleotide exchange factor SOS1. Our laboratory has discovered compounds that bind to SOS1 and activate RAS. Interestingly, these SOS1 agonist compounds elicit biphasic modulation of ERK phosphorylation and simultaneous inhibition of AKT phosphorylation levels. Here, we utilized multiple chemically distinct compounds to elucidate whether these effects on MAPK and PI3K signaling by SOS1 agonists were mechanistically linked. In addition, we used CRISPR/Cas9 gene-editing to generate clonally derived SOS1 knockout cells and identified a potent SOS1 agonist that rapidly elicited on-target molecular effects at substantially lower concentrations than those causing off-target effects. Our findings will allow us to further define the on-target utility of SOS1 agonists.

Discovery and Structure-Based Optimization of Benzimidazole-Derived Activators of SOS1-Mediated Nucleotide Exchange on RAS.

Son of sevenless homologue 1 (SOS1) is a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on RAS. In its active form, GTP-bound RAS is responsible for numerous critical cellular processes. Aberrant RAS activity is involved in ∼30% of all human cancers; hence, SOS1 is an attractive therapeutic target for its role in modulating RAS activation. Here, we describe a new series of benzimidazole-derived SOS1 agonists. Using structure-guided design, we discovered small molecules that increase nucleotide exchange on RAS in vitro at submicromolar concentrations, bind to SOS1 with low double-digit nanomolar affinity, rapidly enhance cellular RAS-GTP levels, and invoke biphasic signaling changes in phosphorylation of ERK 1/2. These compounds represent the most potent series of SOS1 agonists reported to date.

Understanding the Species Selectivity of Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors.

To test for on target toxicity of a new chemical entity, it is important to have comparable binding affinities of the compound in the target proteins from humans and the test species. To evaluate our myeloid cell leukemia-1 (Mcl-1) inhibitors, we tested them against rodent Mcl-1 and found a significant loss of binding affinity when compared to that seen with human Mcl-1. To understand the affinity loss, we used sequence alignments and structures of human Mcl-1/inhibitor complexes to identify the important differences in the amino acid sequences. One difference is human L246 (F226 in rat, F227 in mouse) in the ligand binding pocket. Mutating rat F226 to a Leu restores affinity, but the mouse F227L mutant still has a ligand affinity that is lower than that of human Mcl-1. Another mutation of mouse F267, located ∼12 Šfrom the ligand pocket, to the human/rat cysteine, F267C, improved the affinity and combined with F227L resulted in a mutant mouse protein with a binding affinity similar to that of human Mcl-1. To help understand the structural components of the affinity loss, we obtained an X-ray structure of a mouse Mcl-1/inhibitor complex and identified how the residue changes reduced compound complementarity. Finally, we tested Mcl-1 of other preclinical animal models (canine, monkey, rabbit, and ferret) that are identical to humans in terms of these two residues and found that their Mcl-1 bound our compounds with affinities comparable to that of human Mcl-1. These results have implications for understanding ligand selectivity for similar proteins and for the interpretation of preclinical toxicology studies with Mcl-1 inhibitors.

Optimization of Potent and Selective Tricyclic Indole Diazepinone Myeloid Cell Leukemia-1 Inhibitors Using Structure-Based Design.

Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, has emerged as an attractive target for cancer therapy. Mcl-1 upregulation is often found in many human cancers and is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here, we describe a series of potent and selective tricyclic indole diazepinone Mcl-1 inhibitors that were discovered and further optimized using structure-based design. These compounds exhibit picomolar binding affinity and mechanism-based cellular efficacy, including growth inhibition and caspase induction in Mcl-1-sensitive cells. Thus, they represent useful compounds to study the implication of Mcl-1 inhibition in cancer and serve as potentially useful starting points toward the discovery of anti-Mcl-1 therapeutics.

Discovery and biological characterization of potent myeloid cell leukemia-1 inhibitors.

Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl-1 inhibitors. PDB ID CODES: Comp. 2: 5IEZ; Comp. 5: 5IF4.

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. 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.